CN116622720A

CN116622720A - Upland cotton plant height regulating gene GhGA2ox3 and application thereof

Info

Publication number: CN116622720A
Application number: CN202310053192.1A
Authority: CN
Inventors: 李黎贝; 喻树迅; 冯震; 颜梦圆; 吴佳彦; 孙楠; 傅照彬; 孙东立
Original assignee: Zhejiang A&F University ZAFU
Current assignee: Zhejiang A&F University ZAFU
Priority date: 2023-02-03
Filing date: 2023-02-03
Publication date: 2023-08-22

Abstract

The invention discloses a landA cotton plant height regulating gene GhGA2ox3 and application thereof, relating to the technical field of genetic engineering; the nucleotide sequence of the regulatory gene GhGA2ox3 is shown as SEQ ID NO. 1. The invention screens out the gene GhGA2ox3 which can regulate the plant height from GhGA2ox family, and experiments prove that the GhGA2ox3 can respond to GA ₃ And MeJA; subcellular localization analysis of GhGA2ox3 in tobacco revealed that GhGA2ox3 was localized in the nucleus and cytoplasm. The invention also proves that the GhGA2ox3 can reduce the content of active gibberellin in plants, thereby reducing the plant height, and laying a foundation for cultivating dwarf and high-yield varieties and deeply researching the relevant mechanism of regulating and controlling the plant height of the GhGA2ox 3.

Description

Upland cotton plant height regulating gene GhGA2ox3 and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a upland cotton plant height regulating gene GhGA2ox3 and application thereof.

Background

Plant height is an important agronomic trait of cotton, not only affects cotton yield, but also plays an important role in the aspect of cotton fiber quality. The proper plant height can increase the planting density of crops, optimize field management, improve the yield of crops, and the dwarf is an important property of cotton mechanical harvesting. Cotton plant height is affected by the number of internodes and the spacing of each node. Plant height is related to genetic breeding of cotton, and more scholars have conducted intensive studies on traits such as plant height, internode number and internode spacing. In order to study the influence of plant types on cotton yield, liu Jiguo and the like are tested by taking Liaoning cotton No. 10 and No. 15 and insect-resistant cotton as study materials, and the positive influence of the number of fruit branches and the number of fruit branch nodes on cotton production and yield can be found through the ringing rate and the number of per mu yield. Cao Xinchuan and the like are tested by using 23 upland cotton materials with different genetic backgrounds at home and abroad, and the result shows that the first fruit branch position is closely related to the fiber quality factors such as the coat and branch, the single bell weight, the fiber length, the uniformity, the Mark value, the specific strength and the like, and can be indirectly used as the basis for evaluating the fiber quality. Alignment of The results of main component and multiple secondary regression analysis of 17 quantitative traits of 16 spring cotton varieties by Zijie and the like show that the cotton fruit branch initial node is relatively low, the branches are more, the plant height is relatively high, and the yield of ginned cotton can be improved. Shang Feiyu et al studied the effect of plant type on yield by environmental interaction with an additive-dominant genetic model, he selected 9 cotton varieties (or lines) and F ₁ The research is carried out on 20 combined results, and the result shows that the dominant genetic contribution rate of the plant height related property fruit node number to the ginned cotton yield is 57.8%; and the genetic contribution rate of the main stem pitch to the yield of ginned cotton is 17.7%. The contribution rate of cotton varieties to the yield of ginned cotton is between 0.1% and 11.1%. The plant height, main stem distance, fruit branch length and other plant height characters are statistically analyzed by utilizing a variety of salt cotton 48, and the plant height is considered to be properly reduced, so that close planting is facilitated, and the cotton yield is improved. Cheng Bei, etc. 20 popularization varieties are utilized to carry out the correlation analysis of the cotton properties, and the number of fruit branches, the number of internodes and the plant height are found to influence the cotton yield. The reported literature shows that the proper plant height can increase the lodging resistance and the planting density of cotton, is more suitable for mechanized management, and achieves the purposes of increasing the yield, reducing the cotton planting cost and increasing the income of cotton farmers. Therefore, the breeding of semi-dwarf varieties is particularly urgent.

Disclosure of Invention

The invention aims to provide a upland cotton plant height regulating gene GhGA2ox3 and application thereof, so as to solve the problems in the prior art, and the invention screens out the gene GhGA2ox3 which can regulate cotton plant height by carrying out bioinformatics and tissue expression analysis on GhGA2ox family, verifies the plant height regulating function of the gene GhGA2ox3 by genetic transformation of arabidopsis thaliana, rice, cotton VIGS and other experimental methods, and lays a foundation for cultivating dwarf and high-yield varieties and deeply researching relevant mechanisms of regulating the plant height of GhGA2ox3.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides a regulatory gene GhGA2ox3 for upland cotton plant height, and the nucleotide sequence of the regulatory gene GhGA2ox3 is shown as SEQ ID NO. 1.

The invention also provides a recombinant expression vector comprising the regulatory gene GhGA2ox3.

The invention also provides a recombinant microorganism strain which comprises the recombinant expression vector.

The invention also provides application of the regulatory gene GhGA2ox3, the recombinant expression vector or the recombinant microorganism strain in reducing the plant height of upland cotton.

The invention also provides a method for reducing the plant height of upland cotton, which comprises the step of up-regulating the expression quantity of the regulatory gene GhGA2ox3 in upland cotton.

Further, the expression level of the regulatory gene GhGA2ox3 in upland cotton is up-regulated by genetic transformation of the regulatory gene GhGA2ox3 into upland cotton plants to obtain transgenic plants.

The invention also provides application of the regulatory gene GhGA2ox3, the recombinant expression vector or the recombinant microorganism strain in reducing gibberellin activity in upland cotton.

Further, the gibberellin is GA ₁ And GA (GA) ₄ 。

The invention discloses the following technical effects:

according to the invention, through reported GA2ox family characteristics, 25 cotton GhGA2ox family members are identified, and according to public transcriptome data and the relative expression amounts of genes in CRI50 and TM-1, genes GhGA2ox3 which can regulate and control plant height are screened from the GhGA2ox family. Based on hormone-related cis-acting elements on the 2000bp sequence upstream of the GhGA2ox3 promoter, it was verified that GhGA2ox3 can respond to GA ₃ And MeJA. Subcellular localization analysis of GhGA2ox3 in tobacco revealed that GhGA2ox3 was localized in the nucleus and cytoplasm.

The GhGA2ox3 heterogenous transformation of the arabidopsis can reduce the plant height, root length, germination rate and in vivo active gibberellin content of the arabidopsis, and does not influence the thousand seed weight of the arabidopsis. The GhGA2ox3 heterologous transformation rice can reduce the plant height and root length of the rice at the seedling stage, and does not affect the grain length, grain width and hundred grain weight. VIGS silencing GhGA2ox3 in "CRI50" increases the "CRI50" strain height and active gibberellin content. The invention proves that the GhGA2ox3 can reduce the content of active gibberellin in plants, thereby reducing the plant height, and laying a foundation for cultivating dwarf and high-yield varieties and deeply researching the relevant mechanism of regulating and controlling the plant height of the GhGA2ox3.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of a subcellular localization vector; wherein A: genes downstream of EGFP; b: genes upstream of EGFP;

FIG. 2 is a candidate gene tissue expression profile analysis; wherein A: ghGA2ox3; b: ghGA2ox10; c: ghGA2ox14; d: ghGA2ox15; e: ghGA2ox19; different lowercase letters indicate significant differences (P < 0.05);

FIG. 3 is a height of 6 cotton varieties; different lowercase letters indicate significant differences (P < 0.05);

FIG. 4 shows the expression of TM-1 and CRI50 height and GhGA2ox3; wherein A: four leaf stage CRI and TM-1 strain height phenotype map; b: a pentaphyllic CRI and TM-1 strain height phenotype map; c: CRI and TM-1 strain height phenotype statistics in the four-leaf period; d: CRI and TM-1 strain height phenotype statistics in the penta-leaf period; ". X" means P <0.5; ", denotes P <0.001; the marked rule in A and B is 2cm;

FIG. 5 shows the change in GhGA2ox3 expression level within 24 hours after foliar hormone spraying; wherein A: GA is sprayed ₃ The method comprises the steps of carrying out a first treatment on the surface of the B: spraying MeJA; c: spraying ABA; "x" means P<0.5; "" means P<0.001；

FIG. 6 shows GhGA2ox3 sequence amplification, monoclonal positive bacterial fluid detection and recombinant vector verification; wherein A: PCR amplified sequence, M is Marker III, 1-5 is leaf, 6 is root, 7 is stem; b: detecting monoclonal positive bacterial liquid, wherein M is Marker III, and 1-8 are bacterial liquid PCR; c: verifying the recombinant vector, wherein M is Marker III, and 1-5 are recombinant vectors;

FIG. 7 is a view of transient expression of tobacco epidermal cells; the first column is green fluorescence, the second column is red fluorescence, the third column is a common bright field, and the fourth column is a superposition graph; the arrow in the figure indicates the cytoplasm; the scale bars in the figure are 20 μm;

FIG. 8 is a schematic representation of an expression vector; wherein A: an arabidopsis thaliana overexpression vector; b: a rice overexpression vector;

FIG. 9 is a recombinant vector PCR validation; wherein A: arabidopsis thaliana and rice recombinant vectors, M is Marker III, 1-5 is Arabidopsis thaliana overexpression recombinant vector, and 6-10 is rice overexpression recombinant vector; b: the VIGS recombinant vector, M is Marker III, and 1-4 are recombinant vectors;

FIG. 10 is the identification of mutants and transgenic Arabidopsis thaliana; wherein A: mutant detection, wherein M is Marker III, 1 and 2 are water, 3 and 4 are Arabidopsis wild type templates, and the primers of the Atga2ox8LP and the Atga2ox8RP and the Atga2ox8LB and the Atga2ox8RP are respectively used, 5 and 6 are Arabidopsis atga2ox8 templates and the primers of the Atga2ox8LP and the Atga2ox8RP and the Atga2ox8LB and the Atga2ox8RP are respectively used; b: detecting a transgenic Arabidopsis positive strain, wherein M is Marker III, 1 is water, 2 is cotton leaf cDNA, 3 is Arabidopsis wild type, 4-6 is overexpression Arabidopsis, 7 is Arabidopsis atga2ox8, and 8-10 is detection by using overexpression under the background of atga2ox8 as a template;

FIG. 11 is a transgenic Arabidopsis phenotype; wherein A: phenotype of bolting period; b: a flowering phase phenotype; c: a 7 week old phenotype; d: the relative expression quantity of different strains; E-G: gibberellin content; the same lowercase letters indicate significant differences (P < 0.05); the scales in A-C are all 2cm;

FIG. 12 is a transgenic Arabidopsis phenotyping; wherein A, B: bolting time; C. d: the number of rosette leaves during bolting; E. f: flowering time; G. h: total leaf number at flowering; I. j: height at flowering; K. l:7 week old; A. c, E, G, I, K: gibberellin is not sprayed, B, D, F, H, J, L: spraying 100 mu M gibberellin; different lowercase letters indicate significant differences (P < 0.05);

FIG. 13 is root length, germination and thousand kernel weight of transgenic Arabidopsis; wherein A: root long phenotype; b: root length statistics; c: a germination phenotype; d: germination statistics; e: thousand grain weight statistics; different lowercase letters indicate significant differences (P < 0.05); the marked rule in A and C is 1cm;

FIG. 14 shows the detection of the expression level of the GA-related gene of transgenic Arabidopsis; the same group of different lower case letters represents significant differences (P < 0.05);

FIG. 15 is a diagram showing identification of transgenic rice; wherein A: a plant height phenotype; b: the positive strain detection result is that M is Marker III, 1 is water as a template, 2 is cotton leaf cDNA as a template, 3 is a wild type of pseudo rice as a template, and 4-6 is over-expressed rice as a template; c: the relative expression quantity of different strains; ", denotes P <0.001;

FIG. 16 is a phenotype analysis of a rice positive line; wherein A: the length of the subsurface portion; b: length of the aerial parts; c: plant height; d: grain length; e: grain width; f: hundred weight; ". X" means P <0.5; "x" means P <0.01; ", denotes P <0.001;

FIG. 17 is a phenotype of VIGS cotton; wherein, A-C: a two-leaf stage to four-leaf stage phenotype; d: the relative expression quantity from the two-leaf stage to the four-leaf stage; ", denotes P <0.001; the scales in A-C are all 4cm;

FIG. 18 shows the measurement of gibberellin content of VIGS cotton; wherein A: GA (GA) ₁ The content is as follows; b: GA (GA) ₄ The content is as follows; c: GA (GA) ₁ And GA (GA) ₄ Total content; table P<0.05; ", x" represents P<0.01; "" means P<0.001。

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The nucleotide sequence of the GhGA2ox3 gene in the following examples is shown in SEQ ID NO. 1.

TABLE 1 GA2ox Gene family information in Arabidopsis, rice and upland cotton

All members of GhGA2ox family are identified from whole genome of upland cotton by bioinformatics analysis (see table 1 above), ghGA2ox3 is selected as a study object, and preliminary functional analysis is carried out by utilizing biochemical and molecular biological means, and the details are as follows:

example 1

1 materials and methods

1.1 plant Material

Cotton materials selected from "medium cotton No. 10" ("CRI 10"), "medium cotton No. 50" ("CRI 50"), "medium cotton No. 58" ("CRI 58"), "TM-1," "Yu cotton No. 21" ("Yu 21") and "Ningcotton No. 1" ("NM 1"). 6 varieties are planted in a Zhejiang agriculture and forestry university farm and are measured for the height of the spouting period; the materials used for tissue expression analysis, namely CRI50 and TM-1, are planted in a Zhejiang agriculture and forestry university farm and peace greenhouse; the external aid hormone spraying material is selected from CRI50, and is planted in an illumination incubator under the planting conditions of 16/8h,28/25 ℃, the illumination intensity of 15000Lux and the relative humidity of 50 percent. Subcellular localization using the tobacco (Nicotiana benthamiana) planted in a walk-in plant culture room under the conditions of 16/8h, 28/25deg.C and 300 μmol/m illumination intensity ² ·s ^-1 The relative humidity was 50%.

1.2 vectors and strains

Coli DH5 a (DL 1001), agrobacterium GV3101 (AC 1001) and P19 (AC 1003L) were all purchased from Shanghai Biotechnology Inc.; cloning vector TA/Blunt-Zero Cloning Kit (C601) was purchased from Northenan; GFP (8600612) was purchased from the Biovector plasmid vector strain cell protein antibody Gene preservation center; the nuclear co-localization vector p2300-35S-h2b-Mcherry was shared by the company New England Biolabs. The subcellular localization vector pCAMBIA1305: GFP-GhGA2ox3 is visualized as shown in FIG. 1.

1.3 Experimental methods

The short stalk material "CRI50" and the tall stalk material "TM-1" were screened. "CRI50" and "TM-1" plants Yu Pingshan greenhouse, which were measured for cotyledonary node to terminal bud height at four-leaf stage and five-leaf stage using ruler.

1.3.1 external aid hormone spraying

1,000100. Mu.M GA was prepared ₃ (gibberellin) and ABA (abscisic acid): 0.3849g GA is weighed ₃ 0.2935g of ABA are respectively dissolved in 200 mu L of absolute ethyl alcohol and then ddH is used ₂ O constant volume to 10mL; 1-fold 100. Mu.M MeJA (methyl jasmonate) was prepared: pipette to aspirate 229.22. Mu.L of constant volumeTo 10mL. Then, the mixture was filtered through 0.22 μm aqueous filters, and the hormones were diluted 1,000 times when used. When the CRI50 is grown to the three-leaf stage in the illumination incubator, spraying hormone until the leaf is dripped, and taking 0h, 6h, 12h and 24h leaf samples for real-time fluorescence quantitative analysis. 10 strains were taken for each biological repeat. Sampling, immediately placing into liquid nitrogen, and extracting RNA or placing at-80deg.C for use.

1.3.2 Total RNA extraction from Cotton

Taking roots, stems, leaves and terminal buds of 'CRI 50' in the trefoil stage, petals, pistils in the later growth stage, 3days (-3 days post anthesis, -3 DPA), 5DPA, 15DPA ovules and fibers, 20DPA ovules and fibers in the late growth stage, and taking stems of CRI50, TM-1 in the four-leaf stage and five-leaf stage in the Pingshan greenhouse. Three biological replicates were taken per sample, 10 strains per biological replicate. Sampling, immediately placing into liquid nitrogen, and extracting RNA or placing at-80deg.C for use.

Specific methods for extracting RNA refer to the polysaccharide polyphenol RNA extraction instructions in the norfirazan kit Plant Total RNA Isolation Kit (RC 401). The RNA obtained is stored at-20deg.C for use or is directly subjected to reverse transcription.

1.3.3 reverse transcription of cDNA

For specific procedures for reverse transcription of RNA, reference is made to the polymeric American kit M5 Super plus qPCR RT kit with gDNA remover (MF 166-plus) instructions. The obtained cDNA is subjected to gene cloning, real-time fluorescent quantitative PCR or placed at-20 ℃ for standby.

1.3.4 real-time fluorescent quantitative PCR

qRT-PCR primers were designed on NCBI based on the sequences of GhGA2ox3, ghGA2ox10, ghGA2ox14, ghGA2ox15 and GhGA2ox19, and the reference gene selection GhACin 4, qRT-PCR primers are shown in Table 2.

TABLE 2

Note that: lowercase letters are recombination sites.

cDNA concentration was determined using a Nanodrop 2 000 instrument with ddH ₂ O the cDNA was diluted to 100 ng/. Mu.L. Using Taq Pro Universal SYBR qPCR Master Mix (Nuo Wei ZanQ712) was subjected to qRT-PCR experiments, protected from light, the specific procedure was: mu.L of cDNA, 10. Mu.L of 2X Taq Pro Universal SYBR qPCR Master Mix, 0.5. Mu.L of Primer 1 (10. Mu.M upstream Primer), 0.5. Mu.L of Primer 2 (10. Mu.M downstream Primer), and 7. Mu.L of nucleic-free ddH were added to each well of a 96-well assay plate ₂ O. The reaction was set up on a real-time fluorescent quantitative PCR instrument: the dissolution curves of 95℃30s,95℃10s,60℃30s,95℃15s,60℃60s,95℃15s,40℃30s were run quantitatively for 40 cycles of 95℃10s and 60℃30s, and 95℃15s,60℃60s,95℃15s were run for 1 cycle.

1.3.5 cloning of the Gene GhGA2ox3

50 times mother liquor preparation of TAE electrophoresis liquid required by experiment: the disodium ethylenediamine tetraacetate dihydrate 33.6.21g and Tris 242g were weighed into a 1L volume beaker, 600mL of deionized water was added, stirred with a clean glass rod until fully dissolved, then 57.1mL of acetic acid was added, stirring was continued, then deionized water was used to determine the volume to 1L, and the mixture was stored at room temperature. Adding 1G agarose into 100mL TAE, heating in a microwave oven for 2min to prepare electrophoresis gel, cooling to 60deg.C, adding 10 μ LGelRed (Soy pal, G5560), mixing, making gel, and cooling at room temperature; LB medium formula: 1g peptone, 0.5g yeast and 1g NaCl (1.5 g agar powder is added to the solid) are added into each 100mL, and the mixture is sterilized for 15min at 121 ℃ in an autoclave; carna antibiotics and celebrate antibiotics are dissolved in ddH ₂ In O, stock mother liquor was configured at 50 mg/mL. The concentration of the working solution is 50 mug/mL; rifampicin antibiotic is dissolved in ddH ₂ In O, stock mother liquor was configured at 50 mg/mL. The working solution concentration was 25. Mu.g/mL. After the preparation, the mixture was filtered through a 0.22 μm aqueous filter membrane and stored at-20℃until use.

Cloning primers were designed on NCBI based on the sequence of GhGA2ox3, and were as shown in Table 2.

The cDNA of the stem, leaf and terminal bud of CRI50 was used as a PCR template, and KOD FX (TOYOBO, KFX-101) was used for PCR amplification. Add to 200. Mu.L PCR tube: 200ng cDNA, 25. Mu.L 2X PCR buffer for KOD FX, 5. Mu.L 2mM dNTPs, 2.5. Mu.L GhGA2ox3-F, 2.5. Mu.L GhGA2ox3-R, 1. Mu.L KOD FX, nucleic-free ddH ₂ O was replenished to 50. Mu.L. Reaction conditions on a PCR instrument: 94℃2min,98℃10s,60℃30s,68℃1min 10s,16℃heat preservation,wherein 98℃10s,60℃30s and 68℃1min 10s set 30 cycles and 60℃was reduced by 0.2℃per cycle.

After the PCR reaction, the bands were observed by agarose gel electrophoresis migration experiments. After the gel was completely cooled, it was placed in an electrophoresis tank containing TAE working solution, and 1. Mu.L of Loading Buffer (Takara, 9157) reagent and 1. Mu.L of PCR amplification product were mixed and spotted into a spotting well. The time and voltage are set according to the gel size (typically 100-140v,20 minutes). After completion, the target strip is checked by a gel imager.

The PCR product was purified by electrophoresis using a MiniBEST DNA Fragment Purification Kit Ver.4.0 (Takara, 9761) kit after detecting the size of the cloned fragment at the correct position, for specific procedures. The obtained product is subjected to subsequent experiments or put into a refrigerator at the temperature of minus 20 ℃ for standby.

After purification, cloning vector connection is carried out, and then escherichia coli DH5 alpha is transformed, wherein the specific operation method is referred to the specification.

After transformation, the monoclonal which grows properly at 37 ℃ is subjected to bacterial liquid PCR detection. Taking a sterile 1.5mL centrifuge tube in a biosafety cabinet, adding a liquid LB culture medium containing the Kana resistance 1mL, selecting full monoclone, putting the monoclone into the centrifuge tube, sealing the tube by a sealing film, putting a shaking table at 37 ℃ for culturing at 200rpm until bacterial liquid is turbid, and detecting the size of a target fragment of the bacterial liquid by PCR. PCR was performed using 2X Specific Taq Master Mix (offshore protein technologies Co., ltd., E010), and the procedure was as follows: add to 200. Mu.L PCR tube: mu.L of the bacterial liquid, 10. Mu.L of 2X SpecificTM Taq Master Mix, 0.8. Mu.L of GhGA2ox3-F, 0.8. Mu.L of GhGA2ox3-R, 6.4. Mu.L of nucleic acid-free ddH ₂ O. Reaction conditions on a PCR instrument: incubation at 94℃1min 30s,94℃20s,60℃20s,72℃1min 10s,16℃was provided with 30 cycles of 94℃20s,60℃20s and 72℃1min 10s and a decrease of 60℃of 0.2℃per cycle. Then the size of the target fragment is detected by electrophoresis, the same method is adopted, bacterial liquid with the correct fragment size is sent to Zhejiang Kangshensu biotechnology Co., ltd for sequencing, the sequencing result is compared with the target gene sequence, and the bacterial liquid with the successful comparison is subjected to subsequent experiments or stored at the temperature of minus 80 ℃ in 20 percent glycerol.

1.3.6 construction of subcellular localization vectors

And adding the correctly sequenced cloning vector bacterial liquid into a liquid LB culture medium containing the kana antibiotics, shaking the bacteria to be turbid by a shaking table at 37 ℃, and extracting the cloning vector plasmid by using a TaKaRa rapid plasmid small extraction kit (DP 105). After obtaining the plasmid, the plasmid concentration was measured and used or stored in a-20℃refrigerator for further use.

Looking at subcellular localization vector restriction sites, and selecting single restriction site Bgl II and double restriction sites Spe I and Xba I to restriction pCAMBIA1305-35S-GFP vector. The recombination sites are introduced into the vector by PCR amplification. The primers were designed as shown in Table 2.

PCR amplification was performed using the primers and KOD FX in Table 2 as templates, followed by agarose gel electrophoresis detection, and purification was performed after the fragment size was correct, in the same manner as 1.3.5.

The vector pCAMBIA1305-35S-GFP was digested and the procedure was described with reference to TaKaRa QuickCut ^TM Bgl II(1606)、QuickCut ^TM Spe I (1631) and QuickCut ^TM Xba I (1634) Specification. Purification was carried out after cleavage, and the method was 1.3.5. After purification, the concentration is measured, and the subsequent experiments are carried out or stored in a refrigerator at the temperature of minus 20 ℃ for standby.

The purified PCR product was recombinantly ligated with the digested plasmid using Norflu ClonExpress II One Step Cloning Kit (C112).

mu.L of ligation product was used to transform DH 5. Alpha. And sequenced in the same manner as 1.3.5. And extracting plasmids from the bacterial liquid with correct sequencing comparison, and the method is the same as the above. The agrobacterium GV3101 was transformed with the plasmid, and the specific procedure for transformation was described in the specification.

The PCR method for identifying positive bacteria uses 20% glycerol bacteria to store at-80 ℃ with 1.3.5 bacteria liquid with correct band size.

1.3.7 tobacco subcellular localization

Osmotic resuspension configuration: 2.033g MgCl ₂ ·6H ₂ O and 1.952g of 2- (N-morpholino) ethanesulfonic acid were dissolved in 100mL ddH, respectively ₂ O, preparing mother liquor with 10 times concentration, sterilizing at 121 ℃ for 15min, and preserving at 4 ℃ in a refrigerator for later use; 19.62mg acetosyringone is dissolved in 1mL dimethyl sulfoxide to prepare 5 times mother liquor, and the mother liquor is preserved at the temperature of minus 20 ℃ for standby.

Subcellular localization method: firstly, preserving at-80 ℃ for 35S which is GhGA2ox3GFP, P19, P2300-35S-h2 b-Mchery and empty Agrobacterium strains were streaked on solid LB plates containing gentamicin, kanamycin and rifampicin, and the monoclonal was subjected to shaking in 1mL LB liquid medium containing kanamycin and rifampicin antibiotics, and cultured overnight at 200rpm at 28 ℃. The bacterial liquid after overnight culture is transferred to 25mL LB liquid medium containing kanamycin and rifampicin antibiotics, and cultured at 200rpm and 28 ℃ overnight until OD ₆₀₀ Centrifuging at 1.2 and 4000rpm for 10min, discarding LB medium, re-suspending Agrobacterium with osmotic re-suspension, and adjusting OD after re-suspension ₆₀₀ Until 0.6, the mixture was left to stand and suspend at room temperature for 3 hours. Then 35S of GhGA2ox3-GFP and P19, P2300-35S-h2b-Mcherry and P19 were mixed in a volume of 1:1 and injected. 1mL of the mixed bacterial liquid is sucked by a disposable injector, the lower part of a tobacco leaf (1 month old) is propped against by a finger, and bacteria in the injector are hydraulically delivered into leaf tissues by gentle force. And culturing the injected tobacco in dark at 25 ℃ for 24 hours, culturing normally for 48 hours, and observing tobacco leaves under a confocal microscope.

1.3.9 data measurement and statistics

Three biological replicates of 10 strains each were measured using a ruler to measure the height from the beginning node of the fruit branch and the cotyledon to the terminal bud. Two sample data significance tests were performed using the T test, and a single factor significance test was performed using the graph-based method. Use 2 ^-ΔΔCT The method calculates the gene expression level. Data were visualized and analyzed for significance using GraphPad Prism 5. Scales in the graph are labeled using ImageJ.

2. Results and analysis

2.1 Tissue expression analysis

The 5 genes GhGA2ox3, ghGA2ox10, ghGA2ox14, ghGA2ox15 and GhGA2ox19 were selected for tissue expression pattern analysis. Among them, transcriptome analysis of GhGA2ox3, ghGA2ox10, ghGA2ox15 and GhGA2ox19 showed that they were highly expressed in stems, these genes being candidate genes likely to regulate plant height, and GhGA2ox14 being paralogous genes of GhGA2ox 3. qRT-PCR analysis was performed on these 5 genes in roots, stems, leaves, petals, pistils, terminal buds, -3DPA, 5DPA, 15DPA and 20DPA ovules and fibers of CRI 50. The results show that: ghGA2ox10 was expressed high in petals, 5DPA and 15 DPA; the GhGA2ox14 has the highest expression level in the leaf; the GhGA2ox15 has the highest expression level in petals; tissue of GhGA2ox19 without specific expression; only GhGA2ox3 was expressed in high amounts in stems, terminal buds and leaves (FIG. 2). It is shown that GhGA2ox3 may be involved in the regulation of plant height.

2.2 screening of Cotton Material

According to the invention, 3 shorter varieties 'CRI 10', 'CRI 50', 'CRI 58', 3 higher varieties 'TM-1', 'Yu 21', 'NM 1' are selected. They were planted in a farm and the initial node height of the branches during the opening period was measured (fig. 3). The "TM-1" fruit branch is found to have the highest node position and has obvious difference with other materials; the "CRI50" has the lowest initial node level and has significant differences from both "TM-1" and "NM1".

The "CRI50" and "TM-1" to penta-leaf stage were grown in the greenhouse, the heights of cotyledonary nodes to top buds at the four-leaf stage and the penta-leaf stage were measured, and the stems were taken as tissues for qRT-PCR analysis. In the four-leaf and five-leaf phases, "TM-1" is significantly higher than "CRI50" (A-C in FIG. 4). Whereas the expression level of GhGA2ox3 in "TM-1" was significantly lower than "CRI50" (D in FIG. 4). Thus, "CRI50" and "TM-1" were selected as cotton varieties for the study of GhGA2ox 3.

2.3 response of GhGA2ox3 to exogenous hormone

Analysis of the GhGA2ox3 promoter cis-acting element found 3 hormone-related elements, GA respectively ₃ MeJA and ABA. Therefore, the invention sprays exogenous hormones in the trefoil period of CRI50 to study the response of GhGA2ox3 to the hormones. The relative expression level of GhGA2ox3 was measured by spraying 100. Mu.M GA ₃ Post MeJA and ABA changes are shown in fig. 5. Through GA ₃ After the treatment (A in FIG. 5), the relative expression amount of GhGA2ox3 reached a peak at 6 hours, and the relative expression amount was about 3 times that of the control group. The relative expression amount of 12h is reduced, but is obviously higher than that of a control group; after 24h, no significant difference from the control group was recovered. After MeJA treatment (B in fig. 5), the relative expression levels of 6h and 12h ghga2ox3 were not significantly different from that of the control group; the relative expression amount of 24h is obviously lower than that of the control group. In contrast, in ABA-treated "CRI50", the relative expression level of GhGA2ox3 was not significantly different from that of the control group within 24 hours (C in fig. 5). The above results indicate that GhGA2ox3 can respond to GA ₃ And MeJAThese two exogenous hormones. GA (GA) ₃ Can promote the expression of GhGA2ox3 in upland cotton, and MeJA has inhibiting effect on the expression of GhGA2ox 3.

2.4 cloning of GhGA2ox3 and verification of recombinant vector

PCR band amplification is carried out by using cDNA of root, stem and leaf of upland cotton 'CRI 50' as a template and utilizing cloning primers, and a PCR product is a fragment with the size of about 1083bp, which accords with CDS size of GhGA2ox3 gene (A in 6). And (3) purifying the target fragment of the obtained product, connecting with a TA/Blunt-Zero cloning vector to obtain recombinant plasmid transformed escherichia coli DH5 alpha, and carrying out sequencing by sending a bacterial liquid sample to Zhejiang Kagaku biotechnology Co Ltd after confirming that the selected positive clone is correct by bacterial liquid PCR (B in figure 6). The plasmid was transformed into GV3101 by extracting the correctly sequenced bacterial liquid, and the selected monoclonal was confirmed to be correct by bacterial liquid PCR on the grown monoclonal (C in FIG. 6) and stored at-80℃either for later use or using 20% glycerol.

2.5 subcellular localization of GhGA2ox3 in tobacco

GhGA2ox3-GFP and p2300-35S-h2b-Mcherry were simultaneously transiently expressed in tobacco, and observed in confocal microscopy to find 35S that GhGA2ox3-GFP was localized in the nucleus and cytoplasm; p2300-35S-h2b-Mcherry is located in the nucleus; intact cells were clearly seen in bright field, and the localization results of 35S: ghGA2ox3-GFP and p2300-35S-h2b-Mcherry could be combined and displayed as yellow light (FIG. 7). It is demonstrated that GhGA2ox3 is localized in the nucleus and cytoplasm where it interacts with other proteins to regulate plant growth and development.

Example 2

The atga2ox8 is a homologous gene of GhGA2ox3 in Arabidopsis thaliana.

1 materials and methods

1.1 plant Material

Arabidopsis thaliana used Columbia wild type (Clo-0) and atta 2ox8 (CS 850082) mutants. Mutants were purchased from TAIR-ABRC (https:// abc. Osu. Edu/stocks/number/CS 850082). Arabidopsis thaliana is planted in a walk-in plant cultivation room under the planting conditions of 16/8h,23/22 ℃ and 300 mu mol/m illumination intensity ² ·s ^-1 Relative humidity of 50%. The material used for genetic transformation of rice was "Zhonghua No. 11" ("ZH 11"). The rice is planted in a walk-in plant culture room in seedling stage, the planting condition is 16/8h, the planting temperature is 28 ℃, and the illumination intensity is 300 mu mol/m ² ·s ^-1 The relative humidity was 50%; later-stage planting in a greenhouse in the Pingshan. Virus-induced gene silencing (Virus induced gene silencing, VIGS) uses a material "CRI50". The VIGS plants are planted in a walk-in plant culture room under the planting conditions of 16/8h,25 ℃ and 300 mu mol/m of illumination intensity ² ·s ^-1 The relative humidity was 50%.

1.2 vectors and strains

Coli DH 5. Alpha. And Agrobacterium GV3101 were used as in example 1. Schematic diagrams of the Arabidopsis thaliana overexpression vector pBI121 and the rice overexpression vector pCAMBIA1300s are shown in FIG. 8A and B; the pTRV vector used in the VIGS was given by Liu Jibao teacher as a vector in the Nimble Cloning system.

1.3 Experimental methods

1.3.1 detection of Arabidopsis mutant

The three primer sequences used for the mutants were designed on T-DNA Primer Design (http:// signal. Salk. Edu/tdnaprimers.2. Html), parameters default, and sequence information as shown in Table 2.

The size of the atga2ox8LP+atga2ox8RP product is 1088bp, and the size of the atga2ox8LB+atga2ox8RP product is 544-844bp. After 18 days of arabidopsis growth, rosette leaves are selected for rough extraction of DNA, and then PCR amplification and gel electrophoresis detection are carried out. Crude extraction of DNA detection Arabidopsis thaliana was subjected to M5 super-light speed mix (Polymert, MF 848), following specific procedures: 40. Mu.L of the optimal partner for amplification was added to a 1.5mL centrifuge tube, followed by 2mm addition ² Grinding the left and right samples, boiling for 10 minutes; then centrifuging at 12000rpm for 2min, taking 2 mu L as a follow-up PCR template; PCR amplification reaction system: mu.L of the DNA obtained by the above-described step was roughly extracted in a 200. Mu.L PCR tube, 10. Mu.L of 2 XM 5 Hiper super-optical speed mix (with blue dye), 0.5. Mu.L of GhGA2ox3-F, 0.5. Mu.L of GhGA2ox3-R, and 7. Mu.L of nucleic-free ddH were added ₂ O, post-short PCR. The PCR procedure was: incubating at 95℃for 3min,94℃for 25s,60℃for 25s,72℃for 1min 10s,72℃for 5min,16℃with 30 cycles of 94℃for 25s,60℃for 25s and 72℃for 1min 10s and a decrease of 60℃of 0.2℃per cycle; finallyGel electrophoresis was performed in the same manner as in 1.3.5 of example 1. The correct strain for the band was the homozygous atga2ox8 mutant.

1.3.2 over-expression vector and VIGS vector construction

Arabidopsis thaliana overexpression vector 35S GhGA2ox3 double cleavage sites XbaI and SacI (TaRaKa, 1627) were selected; the rice overexpression vector 35S was selected for the single cleavage site XbaI by GhGA2ox 3.

The primers used to construct the over-expression vector and the VIGS vector are shown in table 3.

TABLE 3 Table 3

Note that: lowercase letters are recombination sites

The method for constructing the overexpression vector was the same as in 1.3.6 of example 1.

VIGS vector construction using Nimble Cloning kit (henna your biotechnology limited, NC 001), specific procedures: firstly, NC linker is introduced by PCR, namely, a cloning vector is used as a template, pTRV-GhGA2ox3-F and pTRV-GhGA2ox3-R are used as primers, KOD FX is used for PCR amplification, agarose gel electrophoresis is carried out for detection, and purification is carried out after the fragment size is correct, wherein the PCR cycle number is 27.pTRV2 vector plasmid was extracted in the same manner as in 1.3.6 of example 1; then, 50ng of the PCR purified product obtained in the above step and 100ng of the pTRV2 vector obtained in the above step were added into 200. Mu.L of PCR tube, and then 5. Mu.L of Nimble Mix was added thereto, followed by pipetting 20 times with a pipette, mixing well, reacting at 50℃for 30min on a PCR instrument, and incubating at 4 ℃. Finally, the products of the above steps are transformed into E.coli, sequenced, extracted and GV3101 transformed, and PCR is performed in the same manner as in example 1, 1.3.6. The GV3101 with correct detection was placed in 20% glycerol and kept at-80℃until use.

1.3.3 genetic transformation of Arabidopsis thaliana

1/2MS permeate configuration per 100mL Arabidopsis: 1/2MS culture mediumSucrose-free and agar) 0.22g, sucrose 5g, dissolved in 100mL ddH ₂ And sterilizing at 121 ℃ for 15min for standby. 20. Mu.L Silwet L-77 was added prior to padding.

The method for dip-dyeing the arabidopsis inflorescence comprises the following steps: cutting inflorescences and pods of arabidopsis thaliana 1 day before dip dyeing, and pouring a proper amount of water to keep the substrate moist; then the bacterial liquid for identifying the correct positive colony is added into 200mL LB liquid medium containing kanamycin and rifampicin, and cultured on a constant temperature shaking table at 28 ℃ and 200rpm until the bacterial liquid reaches OD ₆₀₀ At 1.2; pouring the bacterial liquid into a 50mL centrifuge tube, symmetrically putting the centrifuge tube into a large-sized centrifuge, centrifuging at 4000rpm for 10min, pouring the supernatant, and reserving bacterial cells; then using 1/2MS penetrating fluid to suspend the thalli, vortex oscillating until the thalli is completely dissolved in the penetrating fluid, and then regulating the penetrating fluid to OD ₆₀₀ Between 0.6; finally, immersing the arabidopsis bud in the permeate for 45s, and culturing in the dark for 24 hours and then culturing normally; dip-dyed together at intervals of one week for 2 times.

1.3.4 genetic transformation of Rice

The genetic transformation method of rice refers to "woody army. Agrobacterium-mediated transgenic research of rice [ D ]. University of agriculture in China, 2001".

1.3.5 detection of Positive strains

The harvested Arabidopsis (or rice) seeds were cultivated on a 1/2MS solid medium containing a resistance to Kana (or Hn) and subjected to preliminary screening, the procedure being referred to in example 1.1. Transplanting the surviving seedlings, taking young leaves to carry out rough extraction DNA after the seedlings grow to a proper size, and carrying out PCR amplification and gel electrophoresis detection, wherein the method is the same as that of the example 1.3.1, and the band size is correct to be a positive strain.

1.3.6 cotton VIGS experiment

The VIGS osmotic resuspension was the same as 1.3.7 of example 1. TRV1, TRV2 empty and TRV: ghCA 1 and TRV: ghGA2ox3 Agrobacterium preserved at-80℃were plated on LB medium containing Kana, rifampicin and celebrate 5 days in advance, and resuscitated at 28℃for 2 days. Then picking single bacteria to shake at 28 ℃ in LB liquid medium containing kanamycin and rifampicin antibiotics for overnight; then transferring the activated bacterial liquid to 25mL LB liquid medium containing kanamycin and rifampicin antibiotics, shaking the bacterial liquid at 28 ℃ at 200rpm for overnight,culturing to OD ₆₀₀ To 1.2; pouring the shaken bacterial liquid into a 50mL centrifuge tube, centrifuging at 4000rpm for 10min, removing supernatant, adding about equal volume of heavy suspension into the centrifuge tube, swirling until the bacterial body is completely melted into the heavy suspension, and regulating OD by the heavy suspension ₆₀₀ To 0.6. After standing for 2 hours at the dark room temperature, starting to inject cotton with flattened cotyledons; TRV1 was mixed with equal volumes of TRV: ghCA 1, TRV2 empty and TRV: ghGA2ox3 Agrobacterium, respectively, prior to injection. The needle of the injector is used for scratching the lower epidermis of the cotyledon when in injection, but the cotyledon is not penetrated by holes, and the injector is used for sucking mixed heavy suspension and injecting the mixed heavy suspension into the cotyledon to reach more than 90 percent of the total area of the cotyledon; dark treatment at 25℃overnight after injection, and final normal culture at 25 ℃.

1.3.7 Total RNA extraction from Arabidopsis, rice and VIGS cotton

RNA was extracted using Arabidopsis and rice leaves. Specific procedures for RNA extraction in arabidopsis and rice were referred to the norwegian kit Plant Total RNA Isolation Kit general plant extraction instructions. The method for extracting RNA using the VIGS cotton stems was the same as 1.3.2 of example 1.

1.3.8 reverse transcription of Arabidopsis, rice and VIGS Cotton into cDNA

The method for synthesizing cDNA by reverse transcription is the same as that of 1.3.3 of example 1.

1.3.9 analysis of Arabidopsis, rice and VIGS Cotton qRT-PCR

AtUBQ10 was used as an internal reference for the Arabidopsis qRT-PCR analysis. OsActin1 was used as an internal reference for the qRT-PCR analysis of rice. Primers used for the Arabidopsis and rice qRT-PCR are shown in Table 3. The primers used for cotton qRT-PCR are as in Table 2.

The qRT-PCR analysis of Arabidopsis, rice and VIGS cotton was the same as that of example 1, 1.3.4.

1.3.10 determination of gibberellin content of Arabidopsis thaliana and VIGS Cotton

The samples of Arabidopsis for gibberellin content select aerial parts during flowering, and the samples of VIGS plants for gibberellin content select four-leaf stage stems. After sampling, the sample was pre-cooled in liquid nitrogen, and then the sample was transported to Jiangsu enzyme non-real company, inc., using plant gibberella 1 (GA ₁ ) ELISA kit (MM-1189O 1) and plant gibberellin 4 (GA) ₄ ) ELISA kit (MM-1532O 1) and BCA protein assayThe quantitative kit (MM-9227A) was used for gibberellin content substitution measurement.

1.3.11 trait determination and data statistics

Counting bolting time by arabidopsis thaliana; the number of rosette leaves before bolting; flowering time; total leaf number at flowering; height of aerial parts during flowering; 7 week old aerial height; germination rate for 7 days; root length 10 days; thousand grain weight. The length and plant height of the overground and underground parts at the time of tillering of the rice are counted; grain length; grain width; hundred weight. Cotton VIGS plants were counted for cotyledonary node to terminal bud height in two-leaf, three-leaf and four-leaf stages. The data difference significance test method is the same as 1.3.7 of example 1.

2 results and analysis

2.1 PCR verification of recombinant vectors

Recombinant vector coated plate for transforming GV3101, bacterial liquid PCR is performed on the grown monoclonal, and the successful transformation of the selected monoclonal is confirmed. The PCR product size of the arabidopsis and rice over-expression recombinant vector is about 1113bp (A in FIG. 9), and the PCR product size of the VIGS recombinant vector is about 390bp (B in FIG. 9). And (5) expanding and shaking bacterial liquid with correct fragment size for subsequent infection.

2.2 functional verification of 2GhGA2ox3 in Arabidopsis thaliana

2.2.1 detection of mutants and positive strains

The transgenic positive strain of arabidopsis is transplanted to a small square basin for growth after surviving on a 1/2MS solid culture dish containing the resistance to the carbarn, the rosette leaves of the arabidopsis are taken for detection, DNA is extracted in a rough way, and the size of the band is determined by PCR. Each generation is tested until T ₃ Subsequent experiments were performed after the generation of homozygosity. The detection results are shown in FIG. 10.

Arabidopsis thaliana atta 2ox8 mutant detection was performed using primers atta 2ox8LP and atta 2ox8RP and primers atta 2ox8LB and atta 2ox8RP. Taking water as a template, and no strip exists; using wild-type DNA as a template, primers atta 2ox8LP and atta 2ox8RP were used, with a band size of approximately 1088bp; primers, atga2ox8LB and atga2ox8RP, were used without bands; the mutant DNA was used as a template, primers, atta 2ox8LP and atta 2ox8RP, were used without bands, primers, atta 2ox8LB and atta 2ox8RP, were used with bands within 544-844bp, and were single and matched to the size of the mutant detection band (A in FIG. 10). The results demonstrate that the atga2ox8 mutant is homozygous and can be used directly in subsequent experiments.

Wild type Arabidopsis (wild type, WT) over-expression positive lines and mutant re-complement positive lines are transplanted into a small square basin for growth after surviving on a solid culture dish containing a Kana resistance 1/2MS, arabidopsis rosette leaves are taken for detection, DNA is extracted in a rough way, and the size of a band is determined by PCR. Each generation is tested until T ₃ And carrying out subsequent experiments instead of the previous step. Positive line T ₃ The generation detection result is shown as B in FIG. 10. Taking water as a template, wherein the water is free of strips; cotton cDNA is taken as a template strip with the length of about 1083 bp; wild-type and atga2ox8 mutant DNA was used as template, without bands; DNA of strain lines of OE1, OE2, OE3, atta 2ox8-OE1, atta 2ox8-OE2 and atta 2ox8-OE3 is used as a template, the size of the band is about 1083bp, the band is single, and the transgenic strain meets the experimental requirement.

2.2.2 phenotypic analysis of transgenic Arabidopsis thaliana

The phenotypic differences of wild type, over-expressed arabidopsis, mutant and over-expressed arabidopsis in the mutant background were observed under the same culture conditions. Evaluating bolting time and the number of rosette leaves before bolting comprehensively; flowering time and total leaf number at flowering were assessed for flowering. qRT-PCR analysis of the expression levels of different strains shows that the relative expression level of the OE3 strain is highest and the relative expression level of the OE1 strain is lowest in the over-expression strains; of the Arabidopsis lines overexpressed in the mutant background, the atta 2ox8-OE1 line had the highest expression level and the atta 2ox8-OE3 line had the lowest expression level (D in FIG. 11). Gibberellin content assay to find GA in OE3 ₁ Minimal content, significantly lower than other strains, GA in mutants, WT and atga2ox8-OE3 ₁ The content is significantly higher than other strains (E in fig. 11); GA in OE3 and atga2ox8-OE1 ₄ Minimal content of GA in mutant and WT ₄ The content is significantly higher than other strains (F in fig. 11); GA in OE3 ₁ And GA (GA) ₄ The total content of GA in the mutant is minimal, significantly lower than other strains ₁ And GA (GA) ₄ The total content is significantly higher than other strains, GA in WT ₁ And GA (GA) ₄ The total content of (a) is also significantly higher than that of other transgenic lines (G in fig. 11); the total content of active gibberellin of each strain is consistent with the result of the relative expression amount.

Time for bolting mutantShort, minimal rosette leaves; the bolting time of the over-expression strain and the re-complement strain is fast, and the number of rosette leaves is large; WT bolting time was also significantly faster than OE2, OE3, atta 2ox8-OE1 and atta 2ox8-OE2, rosette number significantly less than OE1, OE2, OE3, atta 2ox8-OE1 and atta 2ox8-OE2 (FIG. 11A, FIGS. 12A and C); GA is sprayed ₃ The postphenotype was completely restored (a in fig. 11, B and D in fig. 12). The flowering time of the mutant is shortest and the total leaf number is small; the flowering time of the anaplerotic strain atga2ox8-OE1 is longest; OE2, OE3, atta 2ox8-OE1 and atta 2ox8-OE2 have the highest total leaf numbers; WT flowering time was significantly faster than OE2, OE3, atta 2ox8-OE1 and atta 2ox8-OE2, with total leaf numbers also less than OE2, OE3, atta 2ox8-OE1 and atta 2ox8-OE2 (FIG. 11B, E and G in FIG. 12). GA is sprayed ₃ The phenotype of the later part of the flowering (B in FIG. 11, F and H in FIG. 12); the mutant and WT are obviously higher than other strains at flowering and the mutant is highest at 7 weeks of age, the WT is obviously higher than the over-expressed strain, and GA is sprayed ₃ The postphenotype was completely restored (C in FIG. 11, I-L in FIG. 12). The result shows that the overexpression of GhGA2ox3 can delay the development of the Arabidopsis, dwarf the plant height and allow the exogenous GA to be sprayed to partially or completely restore the phenotype of the Arabidopsis. The expression level and gibberellin content of each strain tend to be consistent with bolting, flowering and plant height phenotype, which shows that GhGA2ox3 reduces the in vivo active gibberellin GA of arabidopsis ₁ And GA (GA) ₄ To affect the arabidopsis phenotype.

In addition to delaying development and reducing plant height, transgenic arabidopsis thaliana also affects root length and germination. The mutant Arabidopsis root length became longer and the root length became shorter after overexpression (FIGS. 13A and B). The germination rate of transgenic arabidopsis thaliana was significantly reduced for 7 days (C and D in fig. 13). Thousand kernel weights were not significantly different in transgenic lines OE1, OE2, atta 2ox8-OE2 and atta 2ox8-OE3 (E and F in FIG. 13). The results show that GhGA2ox3 can reduce the plant height and affect the root length and germination of Arabidopsis, but the thousand seed weight of Arabidopsis can not be changed.

2.2.3 determination of the expression level of the transgenic Arabidopsis gibberellin-related Gene

According to previous studies, it was reported that in Arabidopsis, the related gene, atga2ox8, in gibberellin metabolic pathway can reduce Arabidopsis height and gibberellin content in vivo; the atta 20ox1, the atta 20ox2, the atta 3ox1 and the atta 3ox2 increase the height and the in vivo gibberellin content of Arabidopsis; the gibberellin signal transduction related genes include AtGID1A, atGAI, atRGA, atRGL1 and the like, and the proteins of the genes can regulate the steady state of gibberellin in plants and are subjected to feedback regulation of gibberellin content. Thus, expression analysis of these genes was performed in transgenic Arabidopsis thaliana. Wherein the relative expression level of atga2ox8 is significantly reduced in the over-expressed strain, and hardly detected in the mutant and over-expressed strain in the mutant background; the expression levels of atga20ox1, atga20ox2, atga3ox1 and atga3ox2 increased; atGID1A, atGAI, atRGA and RGL1 (FIG. 14) were reduced in expression level. The results were consistent with gibberellin homeostasis, and also with phenotype, again confirming that GhGA2ox3 overexpression of Arabidopsis could reduce the amount of active gibberellin in vivo.

2.3 functional verification of GhGA2ox3 in Rice

2.3.1 detection of Rice Positive plants and qRT-PCR analysis

T ₀ The rice positive lines with correct generation detection are cultivated on a solid medium without hygromycin resistance 1/2MS until tillering, rice leaves are used for detection, DNA is extracted in a rough way, and the size of the bands is determined by PCR. T (T) ₁ The generation detection result is shown as B in FIG. 15. Taking water as a template, and no strip exists; cotton cDNA is used as a template, and the band is about 1083 bp; taking wild rice DNA as a template, and having no band; and DNA of OE1, OE2 and OE3 strains are respectively used as templates, the size of the bands is about 1083bp, and the bands are single, so that the transgenic strains meet the experimental requirements.

Leaves were taken for qRT-PCR analysis. The highest relative expression of OE1 and lowest relative expression of OE2 compared to the wild type, was consistent with the observed phenotypic outcome (a and C in fig. 15).

2.3.2 phenotypic analysis of transgenic Rice

The positive lines were identical to the wild-type culture conditions and phenotypic differences were observed. The length and plant height of the overground and underground parts of wild and transgenic rice at tillering are counted. Statistics T ₀ Seed grain length, grain width and hundred grain weight of the harvested seeds. The length of the overground and underground parts of the transgenic rice in seedling stage is significantly shorter than that of the wild type (A in FIG. 15, A and B in FIG. 16); the plant height was significantly lower than the wild type (a in fig. 15, C in fig. 16); t (T) ₀ The dwarf phenotype partially reverts upon growth to the later stage. The OE2 positive lines had no significant differences in grain length, grain width and hundred grain weight from the wild type (D-F in fig. 16). The result shows that the heterologous transformation of GhGA2ox3 in rice can reduce the plant height of the rice, and the yield of the rice can not be changed.

2.4VIGS inhibits GhGA2ox3 expression

TRV GhCLA1 plants developed albino phenotype at the beginning of the two-leaf stage (A in FIG. 17), and albino phenotype was evident in the three-leaf stage and four-leaf stage (B and C in FIG. 17), indicating that the VIGS system was functioning. The relative expression level of TRV: ghGA2ox3 plants was significantly lower than that of TRV:00 empty plants in the two-leaf, three-leaf and four-leaf periods (D in FIG. 17); the TRV: ghGA2ox3 plants were significantly higher than the TRV:00 empty plants in the two-leaf stage (A and E in FIG. 17), the three-leaf stage (B and E in FIG. 17) and the four-leaf stage (C and E in FIG. 17), indicating that inhibiting expression of cotton GhGA2ox3 could increase cotton plant height.

The invention detects gibberellin content in the cotton stems of VIGS in the four-leaf stage and discovers that TRV: ghGA2ox3 plants activate gibberellin GA in cotton in the four-leaf stage ₁ And GA (GA) ₄ GA (GA) ₁ And GA (GA) ₄ The total content of (C) was significantly higher than that of the TRV 00 empty plants (FIG. 18). It was demonstrated that inhibition of GhGA2ox3 expression increased the content of active gibberellin in cotton, thereby promoting elongation of cotton stems. The reverse direction demonstrates that GhGA2ox3 can reduce cotton plant height by reducing the content of active gibberellin in cotton.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. The regulatory gene GhGA2ox3 for upland cotton plant height is characterized in that the nucleotide sequence of the regulatory gene GhGA2ox3 is shown as SEQ ID NO. 1.

2. A recombinant expression vector comprising the regulatory gene GhGA2ox3 of claim 1.

3. A recombinant microbial strain comprising the recombinant expression vector of claim 2.

4. Use of the regulatory gene GhGA2ox3 of claim 1, the recombinant expression vector of claim 2 or the recombinant microorganism strain of claim 3 for reducing upland cotton plant height.

5. A method for reducing the plant height of upland cotton, comprising the step of up-regulating the expression level of the regulatory gene GhGA2ox3 of claim 1 in upland cotton.

6. The method of claim 5, wherein the expression level of the regulatory gene GhGA2ox3 in upland cotton is up-regulated by genetic transformation of the regulatory gene GhGA2ox3 into upland cotton plants to obtain transgenic plants.

7. Use of the regulatory gene GhGA2ox3 of claim 1, the recombinant expression vector of claim 2 or the recombinant microorganism strain of claim 3 for reducing gibberellin activity in upland cotton.

8. The use as claimed in claim 7 wherein the gibberellin is GA ₁ And GA (GA) ₄ 。