CN111072761A - EjSPL5 gene for promoting loquat flowering conversion and encoding protein and application thereof - Google Patents

Abstract

The invention belongs to the field of plant molecular biology, and particularly relates to an EjSPL5 gene for promoting loquat flowering conversion and application thereof. The full length of the coding region sequence of EjSPL5 gene cDNA is shown as SEQ ID No.1, and the amino acid sequence of the coding protein is shown as SEQ ID No. 2. The protein coded by EjSPL5 gene is located in nucleus and has typical transcription factor characteristic. The gene has the highest expression level in the flowering transition period from vegetative growth to reproductive growth of loquat. Further, the EjSPL5 gene overexpression vector is transferred into wild type Arabidopsis thaliana for overexpression, and the EjSPL5 gene is overexpressed in the transgenic wild type Arabidopsis thaliana, so that the flowering transformation of the Arabidopsis thaliana can be obviously advanced. The transgenic plant material obtained by utilizing the EjSPL5 gene overexpression vector can obviously promote the flowering conversion and the flowering time of plants, further advance the flowering and fruiting time of the plants, can be used for the directional breeding of early-flowering and early-maturing varieties of the plants, and has good application prospect.

Description

EjSPL5 gene for promoting loquat flowering conversion and encoding protein and application thereof

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to loquat EjSPL5 protein, and a coding gene and application thereof.

Background

Loquat (Eriobotrya japonica Lindl.) is an evergreen fruit tree in subtropical zone of the genus Eriobotrya in the family rosaceae and is one of important fruit trees in the south of China. It is popular with consumers because of its fresh and tender pulp, rich nutrition and medicinal value. At present, the loquat is used as one of the fresh fruits appearing on the market in spring, the earlier the fruit maturation period is, the higher the economic benefit is, and therefore, the breeding of the early flowering loquat variety is one of the important directions for loquat breeding. Meanwhile, advancing the flowering transition period is one of the factors promoting early flowering and early fruit ripening.

The SPL (Squamosa promoter binding protein-like) gene encodes a plant-specific class of transcriptional regulators. In Arabidopsis thaliana, the isolated SBP-box gene is named as SPL gene, and the SPL gene plays an important role in the flowering process. The arabidopsis thaliana SPL gene family has more than 30 members, including SPL3, SPL4, SPL5, SPL9, SPL15 and the like which encode smaller protein molecular weights. Among them, SPL5 is involved in inducing plants to flower under long-day conditions. In the floral development regulatory network, SPL5 integrates endogenous senescence and photoperiod signals into a protein complex that regulates flowering in arabidopsis. Meanwhile, the SPL5 gene is also regulated by SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1(SOC1) and Gibberellin (GA) signals. Under short day conditions, GA signals activate the SOC1 gene, whereas SOC1 directly induces SPL5 gene expression. In this regulatory network, the SPL5-SOC1 complex promotes Arabidopsis flowering transition and flowering time by integrating the photoperiod and GA signals. At present, the research on the SPL5 gene is mainly focused on the flower development regulation process of model plants, but the functional research and application of the loquat SPL5 homologous gene (EjSPL5) are not reported.

Disclosure of Invention

The invention aims to provide loquat EjSPL5 protein, and a coding gene and application thereof.

First, the present invention provides loquat EjSPL5 protein, which is:

1) a protein consisting of the amino acids shown in SEQ ID No. 2; or

2) Protein derived from 1) by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No.2 and having equivalent activity.

The invention also provides a gene for coding the loquat EjSPL5 protein.

The gene sequence is shown in SEQ ID No. 1.

The invention also provides an overexpression vector containing the gene, a host cell and an engineering bacterium.

The invention also provides the application of the gene in regulating and controlling the flowering transition and early flowering of angiosperms.

In one embodiment of the invention, the EjSPL5 gene is transferred into angiosperm plant genome and is over-expressed in transgenic plant, which can promote the transformation of transgenic plant into flower and further advance the flowering and fruiting.

The invention also provides a construction method of the transgenic plant, which adopts an agrobacterium-mediated method to transfer the overexpression vector containing the EjSPL5 gene into a plant genome and screen to obtain the transgenic plant.

Wherein, compared with the wild type, the transgenic plant remarkably promotes the flower-forming transformation and flowers in advance.

The invention separates 1 EjSPL5 gene which is closely related to loquat flowering conversion and flowering time regulation from loquat flower buds, and finds that the coded protein is positioned in a cell nucleus and has typical transcription factor characteristics. The real-time fluorescent quantitative PCR proves that the expression level of the gene is the highest in the loquat flowering conversion period, and the expression level is lower in the vegetative growth period, the bud white-exposed period and the full-bloom period, which shows that the expression level of the EjSPL5 gene has the function of promoting the loquat flowering conversion period and bud differentiation. A plant overexpression vector of EjSPL5 gene is constructed by using a genetic engineering means, and is transferred into wild arabidopsis thaliana for overexpression, so that the flowering transformation of the plant can be remarkably promoted, and the flowering and fruiting time of the plant can be further promoted. The invention provides good application prospect for the transformation of the florescence of the angiosperm.

Drawings

FIG. 1 is an electrophoretogram showing the 3'RACE, 5' RACE of the loquat EjsPL5 gene and the sequence verification of the coding region of the gene in example 1. Wherein, A is an electrophoresis picture of 3'RACE, M is DL2000 DNA marker, EjSPL5-3R is a PCR product of 3' RACE; b is an electrophoresis photograph of 5'RACE, M is DL2000 DNA marker, EjSPL5-5R is PCR product of 5' RACE; c is PCR electrophoresis photograph for verifying EjSPL5 gene ORF, M is DL2000 DNA marker, EjSPL5-ORF is PCR product of EjSPL5 gene ORF.

FIG. 2 is the comparison of the amino acid sequence of loquat EjSPL5 protein and the predicted protein sequences of apple and Arabidopsis thaliana, and the protein sequence has obvious sequence difference but conserved structure domain sequence compared with the sequences of the closely related species apple and the model plant Arabidopsis thaliana, which shows the specificity of the protein sequence and the conserved structure domain.

FIG. 3 shows that the loquat EjSPL5 gene is transiently expressed in tobacco leaves, and the expression product of the gene is localized to the nucleus. GFP: green fluorescent protein; DAPI: 4, 6-diamidine-2-phenylindole; BF: bright field imaging; merged: combined images of GFP, DAPI and BF.

FIG. 4 shows that the expression of the loquat EjsPL5 gene shows significant differences in different organs of loquat.

FIG. 5 shows the expression level changes of the loquat EjsPL5 gene in different developmental stages of loquat flowers.

FIG. 6 is a PCR identification of transgenic Arabidopsis plants. Wherein M is DNA marker III and 1-12 are EjSPL5 gene.

FIG. 7 is a photograph of flowering-time of wild type Arabidopsis thaliana before and after EjSPL5 transgene and EjSPL5 gene expression analysis. Wherein A is that compared with the non-transgenic wild type arabidopsis, the overexpression of EjSPL5 gene can lead the flowering transformation and flowering time of the transgenic arabidopsis to be advanced by about 15 days; b is the endogenous SPL5 gene expression level of the transgenic arabidopsis; c is EjSPL5 gene expression level of transgenic Arabidopsis thaliana.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular cloning: a laboratory manual,2001), or the conditions suggested by the manufacturer's instructions.

Example 1 cloning of cDNA sequence of EjsPL5 Gene of Eriobotrya japonica

Extraction of loquat flower bud total RNA

Collecting flower buds of loquat in the flowering conversion period with the fresh length of about 0.5cm, quickly sampling, putting into a freezing tube, putting into liquid nitrogen, quickly freezing for 2h, and then putting into an ultra-low temperature refrigerator at minus 80 ℃ for standby. Extracting total RNA in loquat flower buds by adopting an RNA extraction kit: taking out the collected flower bud material from an ultralow temperature refrigerator at-80 deg.C, placing into a mortar which is pre-frozen and added with 2mL of RLT lysate and 200 μ L of PLAntaid, and fully grinding at room temperature; sucking 1mL of the grinding fluid, transferring the grinding fluid into a 1.5mL eppendorf centrifuge tube, centrifuging at 13000rpm for 15min, sucking 600 mu L of supernatant, and transferring the supernatant into a new 1.5mL centrifuge tube; adding 300 μ L of anhydrous alcohol into the supernatant, sucking, mixing, adding into adsorption column, and placing into collection tube; adding 600 μ L deproteinized solution into adsorption column, centrifuging at 13000rpm for 1 min; adding 600 μ L of rinsing solution, centrifuging at 13000rpm for 1min, pouring off waste liquid in the collecting pipe, adding rinsing solution again, and centrifuging at 13000rpm for 2 min; the adsorption column is returned to empty for collectionCentrifuging at 13000rpm for 2min in a collecting pipe, removing residual rinsing liquid, and placing the adsorption column in a super clean bench for 2min to volatilize the residual rinsing liquid; the adsorption column was returned to the empty RNase-free centrifuge tube, and 50. mu.L of RNase-free H was added₂O, standing at room temperature for 2min, and then centrifuging at 13000rpm for 2 min; the first eluate is again added to the adsorption column and centrifuged again to increase the RNA concentration. 2. mu.L of the diluted RNA sample was aspirated, and the RNA concentration was detected by a trace nucleic acid concentration detector.

3' RACE experiment of loquat EjSPL5 gene

The total RNA of loquat flower buds is used as a 3' RACE experiment template, and 3' RACE Adaptor is used as a joint primer for reverse transcription reaction to synthesize first-strand cDNA of the 3' RACE experiment. The specific operation is as follows: aspirate 1. mu.L of total RNA, 1. mu.L of 3' RACE Adaptor, DEPC-ddH₂O4.5 mu L, uniformly mixing, denaturing at 70 ℃ for 10min, and carrying out ice bath for 2 min; after the RNA denaturation reaction is finished, 0.25 mu L of RNase inhibitor, 1 mu L of 10mM dNTP, 2 mu L of 5 XM-MLV buffer and 0.25 mu L of M-MLV are sequentially added, mixed uniformly and placed at 42 ℃ for reaction for 90 min; then, reacting for 10min at 70 ℃; ice-bath for 2min, and storing at-20 deg.C.

According to conserved regions of the sequences of apple MdSPL5 homologous gene (HM122688.1) and Arabidopsis thaliana SPL5 gene (NM _112390.5) which are published by NCBI website and are predicted to be related closely, upstream specific primers 3REjSPL5F and 3REjSPL5F of 3' RACE experiment are directly designed: 5'-GACGGTGTCAGGCGGACAGGTGCAC-3' are provided. 3'RACE reverse transcription product was used as template, using high fidelity EX-taq enzyme, upstream Outer specific Primer 3REjSPL5F1 and 3' RACE Outer Primer: 5'-TACCGTCGTTCCACTAGTGATTT-3', carrying out PCR reaction at 94 ℃ for 5 min; 35 cycles of 94 ℃ for 40s, 56 ℃ for 40s and 72 ℃ for 40 s; 10min at 72 ℃. After the PCR reaction was completed, the PCR product was recovered by agarose gel DNA recovery kit according to the instructions by cutting the band of interest by electrophoresis on 1% agarose gel (FIG. 1A). And connecting the recovered PCR product to a pMD18-T vector, transferring the PCR product into escherichia coli competent cells, picking a monoclonal colony, and sequencing.

5' RACE experiment of loquat EjEjSPL5 gene

Specific primers 5REjSPL5-1 and 5REjSPL5-2 for 5' RACE experiments were designed based on homologous gene sequences, wherein 5REjSPL 5-1: 5'-CGCATACATCCTTGAACTGAGTCCCTG-3', 5REJSPL 5-2: 5'-GTCCTGCCAGACGCCTGCGACAACTCC-3' are provided. According to the 5' RACE experimental procedure: first Strand Synthesis Buffer Mix was prepared by adding 1.0. mu.L dNTP Mix (10mM), 2.0. mu.L 5 Xfirst-strand Buffer, and 1.0. mu.L DTT (20mM) in this order, mixing well, and standing at room temperature.

mu.L of total RNA, 1.0. mu.L of 5' -CDS primer A, and 1.75. mu.L of H were added to 200. mu.L of eppendorf tubes₂O, mixing uniformly, after instantaneous centrifugation, cooling to 42 ℃ for 2min at 72 ℃ for 3min, after cooling, centrifuging for 10s at 14000g, adding 1 μ L of SMARTER IIA oligo, 1.0 μ L of SMARTscrube Reverse transcriptase (100U), 4.0 μ L of Buffer Mix, 0.25 μ L of RNase inhibitor (400U/. mu.L), the total volume is 10 μ L, mixing uniformly, after instantaneous centrifugation, reacting at 42 ℃ for 90min, and denaturing at 70 ℃ for 10min to obtain control 5' -E-Ready cDNA.

5' RACE amplification System: 34.5 μ L of PCR-Grade water, 5.0 μ L of 10 × Advantage 2PCRbuffer, 1.0 μ L of 50 × Advantage 2polymerase Mix, 1.0 μ L of dNTP Mix, 2.5 μ L of control 5' -RACE-Ready cDNA, 1.0 μ L of 5REjSPL5-1 primer, 5.0 μ L of UPM (10 ×). The procedure for touchdown PCR was: 30s at 95 ℃, 3min at 72 ℃ and 5 cycles; 30s at 95 ℃ and 30s at 70 ℃ for 5 cycles; 3min at 72 ℃, 30s at 95 ℃ and 30s at 68 ℃ for 30 cycles; 5min at 72 ℃. After the PCR reaction is finished, taking a PCR reaction product of the 5' RACE of the first chain as a template, using high-fidelity LA-taq enzyme, an upstream outer side specific Primer 5REjSPL5-2 and a UPM Primer to carry out second chain PCR reaction, wherein the reaction program is 95 ℃ for 5 min; 30 cycles of 94 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 30 s; 10min at 72 ℃. After the PCR reaction was completed, the second strand of the PCR reaction was detected by electrophoresis on a 1% agarose gel (FIG. 1B). The band of interest was excised and the PCR product was recovered using an agarose gel DNA recovery kit. After being connected to pMD18-T vector, the vector is transferred into Escherichia coli competent cells, and a single clone is picked up for sequencing analysis.

Primers FEjSPL5F:5'-ATGAGTAAGTTGGACTTGAACAAGC-3' and FEjSPL5R:5'-TTATCTGATATGGAAATGCTTTGTG-3' are designed at both ends of the full-length loquat EjSPL5 gene sequence, and the reaction condition is 94 ℃ for 5 min; 30 cycles of 94 ℃ for 60s, 57 ℃ for 60s and 72 ℃ for 60 s; 10min at 72 ℃. After completion of the PCR reaction, the band of interest was cut out by 1% agarose gel electrophoresis (FIG. 1C), and the PCR product was recovered by using an agarose gel DNA recovery kit. After being connected to a pMD18-T vector, the vector is transferred into an escherichia coli competent cell, and a single clone is picked up and sequenced to verify the sequence of the coding region of the EjSPL5 gene.

And (3) carrying out sequence analysis and splicing on the PCR sequencing results of 3'RACE, 5' RACE and coding region sequence verification experiments by using DNAMAN software to obtain a coding region sequence (SEQ ID No.1) of the cDNA of the loquat EjsPL5 gene.

The sequence of the coding region of cDNA of the loquat EjsPL5 gene was translated into a protein sequence using primer 5 software (SEQ ID No. 2). Furthermore, the amino acid sequence of the loquat EjSPL5 gene and the coded protein is compared with the predicted sequences of apple, Arabidopsis and snapdragon, the amino acid sequence of the protein has obvious difference compared with the sequences of the kindred species and other angiosperms, and two conserved zinc finger domains and a nuclear localization signal domain exist at the same time, which shows the specificity of the protein sequence and the conservation of the domains (figure 2).

Example 2 subcellular localization analysis of the loquat Ej SPL5 Gene

The ORF sequence of EjSPL5 gene is subjected to enzyme cutting site analysis by using software Oligo7, enzyme cutting site primers at two ends are designed, EjSPL 5-SacI: 5' -cgagctcATGAGTAAGTTGGACTTG-3'；EjSPL5-BamHI：5'-cgggatccTCTGATATGGAAATGC-3'. And (3) amplifying by using a pMD18-EjSPL5 plasmid with correct sequencing as a template to obtain an EjSPL5 gene ORF sequence containing SacI and BamHI enzyme cutting sites. Respectively extracting target gene and modified vector pCAMBIA1300 plasmid, respectively carrying out double enzyme digestion reaction by using restriction enzymes SacI and BamHI, and recovering after agarose gel electrophoresis. By T₄The DNA ligase is used for connecting the target gene EjSPL5 subjected to double enzyme digestion with the modified pCAMBIA1300 vector, transferring the recombinant vector into an escherichia coli competent cell, and then carrying out bacterial liquid PCR and double enzyme digestion verification and sequencing to ensure the targetThe gene sequence was successfully ligated into the vector. The extracted and constructed vector plasmid is transferred into agrobacterium GV1301 competent cells by a freeze-thaw method.

A single colony of Agrobacterium was picked from the solid LB medium plate, inoculated into 10mL of liquid medium (containing Rif + kan), cultured at 28 ℃ and 250rpm until OD600 ═ 0.5. 5mL of culture solution is taken and centrifuged for 10min to collect thalli, then 2mL of penetrating fluid is added to resuspend the thalli, and then centrifugation is carried out for 10min to add 2mL of penetrating fluid to suspend the thalli. Finally, the tobacco leaves were diluted to an OD600 of 0.03 to 0.1, and the transformed tobacco leaves were transformed, and after culturing for 16 hours in a low light, normal growth was resumed, and after 3 to 4 days, GFP fluorescence was observed (fig. 3).

The green fluorescent protein in the tobacco epidermal cells of the control group (the expression vector containing no EjSPL5) is expressed in cytoplasm and nucleus; the green fluorescent protein in the experimental group (the expression vector containing EjSPL5 gene) in the tobacco epidermal cells is only expressed in the cell nucleus, and has typical transcription factor characteristics.

Example 3 real-time fluorescent quantitative PCR analysis of the EjsPL5 Gene of Eriobotrya japonica

Respectively extracting total RNA of stems, leaves, leaf buds, sepals, petals, filaments, anthers, pistils, ovaries and young fruits of the loquats; simultaneously, respectively extracting RNA (S1) of the loquat flower development materials in 8 different periods (physiological differentiation period of flower buds, S2) flowering conversion period (differentiation period of flower bud form from vegetative growth to reproductive growth), S3. inflorescence main shaft differentiation period, S4. inflorescence fulcrum shaft differentiation period, S5. inflorescence lateral growth fulcrum shaft rapid elongation period, S6. small flower differentiation period, S7. flower bud white exposure period and S8. full flowering period). After removing a small amount of DNA from the total RNA, the cDNA is reverse transcribed. Real-time fluorescent quantitative PCR primers qEjSPL5F:5'-CACGGCTGATCTGAGTGAAGA-3' and qEjSPL5R:5'-TCGTTCATTGTGTCCTGCCA-3' were designed using oligo 7.0 software based on loquat cDNA as a template. Taking loquat actin gene as reference gene, the primer is qRTEjactinF: 5'-AATGGAACTGGAATGGTCAAGGC-3' and qRTEjactinR: 5'-TGCCAGATCTTCTCCATGTCATCCCA-3', detecting the specificity by PCR, and carrying out real-time fluorescent quantitative PCR experiment under the premise of ensuring PCR specific amplification, wherein each reaction is provided with 3 biological repeats. The PCR reaction program is pre-denaturation at 94 ℃ for 5 min; 94 ℃ 20s, 55 ℃ 20s, 72 ℃ 20s, 41 cycles, then, a dissolution curve was taken: adjusting the temperature to 60 ℃ for 90s, and pre-dissolving; then the temperature is increased at the speed of 1.0 ℃/s, and the temperature is kept at 1 ℃ per liter for 5s until the temperature reaches 95 ℃. The results show that: EjsPL5 was expressed in different tissues and organs of Eriobotrya japonica, and the expression level was significantly different (FIG. 4). The expression level in sepals and young fruits is higher; while the expression level was low in petals, ovaries and filaments (FIG. 4), indicating that the EjSPL5 gene expression level was significantly different. In the different stages of flower development, EjSPL5 expression is mainly concentrated in the first 6 stages of flower development, EjSPL5 expression level shows a trend of increasing first and then decreasing, and the expression level is highest in the flowering transition stage (FIG. 5), which shows that EjSPL5 gene is closely related to the flowering transition of the differentiation from the terminal bud to the flower bud of loquat.

Example 4 construction of plant transgenic vector pBI121-EjsPL5 for loquat EjsPL5 Gene

And introducing enzyme cutting sites at two ends of a CDS region of the loquat EjSPL5 gene by adopting a PCR amplification method. Taking cDNA reverse transcribed by total RNA of loquat flower buds as a template, and taking TEJSPL 5F:5' -TCTAGAATGAGTAAGTTGGACTTG-3' (introduction of Xba I cleavage site) and TEJSPL 5R:5' -CCCGGGTTATCTGATATGGAAATGC-3' (incorporating SmaI cleavage sites) as primers, PCR amplification was carried out using Ex-taq enzyme. PCR reaction procedure: 5min at 94 ℃; 30 cycles of 94 ℃ for 40s, 56 ℃ for 40s and 72 ℃ for 40 s; 10min at 72 ℃. After the PCR reaction was completed, the PCR product was subjected to 1% agarose gel electrophoresis, and the PCR product was recovered using an agarose gel DNA recovery kit. And connecting the recovered PCR product with a pMD18-T vector, transferring into an escherichia coli competent cell, picking a monoclonal, and sequencing. And (5) extracting the plasmid according to the analysis of the sequencing result. The pMD18-EjSPL5 recombinant plasmid and pBI121 vector were double-digested with Xba I and SmaI restriction enzymes, respectively, detected by 1% agarose gel electrophoresis, and recovered using an agarose gel DNA recovery kit. Using T₄The EjSPL5 gene after double enzyme digestion is connected with pBI121 by DNA ligase, and then transferred into escherichia coli competent cells to obtain a plant transgenic expression vector pBI121-EjSPL 5.

Example 5 transfer of the transgenic expression vector pBI121-EjSPL5 into Arabidopsis thaliana

Taking 1 mu g of pBI121-EjSPL5 plasmid, adding 50 mu L of agrobacterium tumefaciens competent cells, and uniformly mixing; performing ice bath for 10min, transferring into liquid nitrogen, rapidly freezing for 2min, rapidly placing at 37 deg.C, and performing water bath for 10 min; adding 800 μ L LB liquid culture medium, oscillating at 28 deg.C and 250rpm for 5 h; the bacterial liquid is transferred to LB (50mL LB + 50. mu.g/mL Kan + 50. mu.g/mL Rif) solid selection medium, evenly coated and inversely cultured for 48h at the temperature of 28 ℃.

Agrobacterium containing pBI121-EjSPL5 positive clones were streaked on 25mL solid plate medium (containing 25. mu.g/mLKan + 25. mu.g/mL Rif), and cultured for 48h at 28 ℃ in an inverted state; selecting a single clone, and inoculating the single clone into 10mL of liquid LB culture medium (containing 10 mu g/mL Kan +10 mu g/mL Rif); the cells were cultured overnight at 28 ℃ and 250rpm with shaking until OD was 0.7-0.8. Uniformly coating 1mL of culture solution on a 25mL solid LB medium plate (containing 25 mu g/mL Kan +25 mu g/mLRif), and carrying out inverted culture at 28 ℃ for 48 h; agrobacterium on solid medium was scraped off using a sterilized glass triangle rod, and the pellet was resuspended in 1/2MS liquid medium containing 5% sucrose and 3% Silwet L-77 to an OD of 0.2 for arabidopsis transgenesis.

Placing Arabidopsis seeds on wet filter paper, placing the filter paper at 4 ℃ for 48h, then sowing the seeds into nutrient soil (perlite: vermiculite: nutrient soil: 1:4:5), and culturing the seeds under the conditions of temperature of 22 ℃, humidity of 70% and 14h light/10 h dark; before transgenosis, arabidopsis thaliana (purchased from arabidopsis thaliana mutant library) plants are watered thoroughly; cutting off existing siliques on an arabidopsis plant to be used during dip dyeing, and soaking flower buds into the PBI121-EjSPL5 agrobacterium tumefaciens dip dyeing solution for about 90 s; covering a black sealing film, maintaining a high-temperature and high-humidity environment in the film, and uncovering the film after dark culture for 2 d; the method is used for infecting 4 times with the interval time of 7 d.

Example 6 transgenic Arabidopsis thaliana screening and phenotypic characterization of the loquat EjsPL5 Gene

And (4) collecting EjSPL5 transgenic arabidopsis mature seeds, and cleaning the seeds. Performing vernalization treatment in a refrigerator at 4 deg.C for 14 d; placing Arabidopsis seeds into a collecting pipe, adding 800 μ L of absolute ethyl alcohol into the seeds, and shaking for 6 min; centrifuging at 5000rpm for 2 min; pouring off alcohol in the collecting pipe, adding 800 μ L70% ethanol into the collecting pipe, and shaking for 5 min; centrifuging at 5000rpm for 2 min; airing the seeds; the suspension was spread evenly on 1/2MS medium (pH 5.8, 50. mu.g/mL Kan, 3% sucrose and 0.8% agar) plates. Putting the inoculated flat plate into a refrigerator at 4 ℃ for vernalization for 2 d; and (4) placing the vernalized seeds in an artificial climate box for normal culture. After 6 true leaves grow, the leaves are moved into nutrient soil, and after hardening and strengthening seedlings, the seedlings are managed according to conventional water and fertilizer until the flowers bloom.

Extracting EjSPL5 transgenic Arabidopsis DNA, placing 1 piece of Arabidopsis leaf in 1.5mL eppendorf tube, placing in liquid nitrogen for quick freezing, and grinding; adding 600 μ L of extraction buffer solution, vortex shaking, and placing on ice; after all samples are treated, placing the samples in a water bath at 65 ℃ for 25 min; taking out the sample from the water bath, placing the sample to room temperature, adding 340 mu L of potassium acetate solution after cooling to the room temperature, carrying out vortex oscillation and carrying out ice bath for 20 min; 13000rpm, high speed centrifugation for 5min, transfer the supernatant to a new eppendorf tube; adding equal volume of isopropanol, centrifuging at 4 deg.C and 13000rpm for 10min, removing clear liquid, and rinsing with ice anhydrous ethanol (anhydrous ethanol is placed in a refrigerator at-20 deg.C 2h in advance); rinsing the precipitate with 70% and 100% ethanol in sequence; after the precipitate was blown dry, it was dissolved in 50. mu.L of sterile water.

PCR analysis was performed using nontransgenic wild type Arabidopsis leaves (negative control) and the selected transgenic Arabidopsis leaves DNA as templates, and pBI121-EjSPL5 plasmid as a positive control template, using TEjSPL5F and TEjSPL5R as primers, respectively, and it was shown that 12 positive EjSPL5 transgenic wild type Arabidopsis plants were obtained in total (FIG. 6). Among these transgenic lines, 3 transgenic lines were randomly selected for flowering-time phenotypic identification analysis, and the flowering-time phenotypes of the 3 representative transgenic Arabidopsis thaliana were observed, counted, and photographed (FIG. 7A). Further using real-time fluorescent quantitation primers qEjSPL5F2: 5'-TGCTTGTCTCTGGCCTCCAC-3' and qEjSPL5R2: 5'-TGAATTTCCTGGAAGCTGTCATC-3', as well as the Arabidopsis thaliana SPL5 gene and reference gene as controls, Arabidopsis thaliana actin TUB 2-F: 5'-ATCCGTGAAGAGTACCCAGAT-3', respectively; TUB 2-R: 5'-AAGAACCATGCACTCATCAGC-3', respectively; qAtSPL 5-F: 5'-CGACTGTTGCAGGGGTCAGG-3', respectively; qAtSPL 5-R: 5'-GAGTTGGTCATAGGAAGTTTCCT-3' are provided. The expression amounts of EjSPL5 gene and endogenous SPL5 gene were analyzed using cDNA of non-transgenic wild type Arabidopsis as a control and 3 transgenic lines as experimental groups.

The results show that: overexpression of the EjSPL5 gene resulted in a flowering transition and flowering time in transgenic Arabidopsis approximately 15 days earlier than in non-transgenic wild Arabidopsis (FIG. 7A). Analysis of the expression of the SPL5 and EjSPL5 genes in transgenic arabidopsis thaliana revealed that the endogenous SPL5 gene expression level of these arabidopsis thaliana themselves was not significantly changed (fig. 7B), while the EjSPL5 gene in transgenic arabidopsis thaliana, which promoted the flowering transition and flowering time, was significantly highly expressed (fig. 7C), compared to that in non-transgenic wild-type arabidopsis thaliana. Thus, the results show that: the EjSPL5 gene expression leads to the transformation of flowering and the advance of flowering of Arabidopsis, and the transgenic Arabidopsis material of the EjSPL5 gene can be used for the transformation of plant flowering time, so that the transformation of flowering and the advance of flowering of plants are promoted, the fruit maturation time of the plants is effectively promoted to be advanced, and the breeding of early-flowering and early-maturing varieties is facilitated.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> university of southwest

<120> EjSPL5 gene for promoting loquat flowering conversion, and encoding protein and application thereof

<160>22

<170>SIPOSequenceListing 1.0

<210>1

<211>778

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>1

acatgggatc cattgatgga gaggatgagt aagttggact tgaacaagca gatgagggag 60

aagccgcggg tggtggcggt ggtgaagaag gaggaggagg attttgatga tgagctgcaa 120

gtggacagga agaaaaaagg aggcgtgaag agatcgttgt cctcgtcctc gtcatccggt 180

ggaggaggag gcggcgcaat gagacggtgt caggcggaca ggtgcacggc tgatctgagt 240

gaagaaaagc agtatcatag aaagcataag gtttgtgacc ttcattccaa gtctcaggtt 300

gtgcttgtct ctggcctcca ccaaaggttt tgccagcaat gcagcagatt tcatcagcta 360

ccagaattcg acgacaccaa aaggagttgt cgcaggcgtc tggcaggaca caatgaacga 420

cgaaggaaga atccagcgga gtctcatgca gtagaaggct caagccgaaa tgttggtgca 480

gggactcagt tcaaggatgt atgcgggcag gtcgatgaca gcttccagga aattcaactc 540

acaaagcatt tccatatcag ataagattta gagcaagcat gctcactctc ttctgtcagc 600

ttaattagag atgcataatt attagatgtt ggatatttgg cttgtaacaa aatcaatttc 660

atggagtgtg tgtgctagga ttgctgttaa ccggctgtag gaaggtatca aaactaccaa 720

gtgatttagc tagctctata actaatataa tttgcaagat taaaaaaaaa aaaaaaaa 778

<210>2

<211>182

<212>PRT

<213> loquat (Eriobotrya japonica)

<400>2

Met Glu Arg Met Ser Lys Leu Asp Leu Asn Lys Gln Met Arg Glu Lys

1 5 10 15

Pro Arg Val Val Ala Val Val Lys Lys Glu Glu Glu Asp Phe Asp Asp

20 25 30

Glu Leu Gln Val Asp Arg Lys Lys Lys Gly Gly Val Lys Arg Ser Leu

35 40 45

Ser Ser Ser Ser Ser Ser Gly Gly Gly Gly Gly Gly Ala Met Arg Arg

50 55 60

Cys Gln Ala Asp Arg Cys Thr Ala Asp Leu Ser Glu Glu Lys Gln Tyr

65 70 75 80

His Arg Lys His Lys Val Cys Asp Leu His Ser Lys Ser Gln Val Val

85 90 95

Leu Val Ser Gly Leu His Gln Arg Phe Cys Gln Gln Cys Ser Arg Phe

100 105 110

His Gln Leu Pro Glu Phe Asp Asp Thr Lys Arg Ser Cys Arg Arg Arg

115 120 125

Leu Ala Gly His Asn Glu Arg Arg Arg Lys Asn Pro Ala Glu Ser His

130 135 140

Ala Val Glu Gly Ser Ser Arg Asn Val Gly Ala Gly Thr Gln Phe Lys

145 150 155 160

Asp Val Cys Gly Gln Val Asp Asp Ser Phe Gln Glu Ile Gln Leu Thr

165 170 175

Lys His Phe His Ile Arg

180

<210>3

<211>25

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>3

gacggtgtca ggcggacagg tgcac 25

<210>4

<211>23

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>4

taccgtcgtt ccactagtga ttt 23

<210>5

<211>27

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>5

cgcatacatc cttgaactga gtccctg 27

<210>6

<211>27

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>6

gtcctgccag acgcctgcga caactcc 27

<210>7

<211>25

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>7

atgagtaagt tggacttgaa caagc 25

<210>8

<211>25

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>8

ttatctgata tggaaatgct ttgtg 25

<210>9

<211>25

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>9

cgagctcatg agtaagttgg acttg 25

<210>10

<211>24

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>10

cgggatcctc tgatatggaa atgc 24

<210>11

<211>21

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>11

cacggctgat ctgagtgaag a 21

<210>12

<211>20

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>12

tcgttcattg tgtcctgcca 20

<210>13

<211>20

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>13

aatggaactg gaatggtcaa 20

<210>14

<211>26

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>14

tgccagatct tctccatgtc atccca 26

<210>15

<211>24

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>15

tctagaatga gtaagttgga cttg 24

<210>16

<211>25

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>16

cccgggttat ctgatatgga aatgc 25

<210>17

<211>20

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>17

tgcttgtctc tggcctccac 20

<210>18

<211>23

<212>DNA

<213> loquat (Eriobotrya japonica)

<400>18

tgaatttcct ggaagctgtc atc 23

<210>19

<211>21

<212>DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400>19

atccgtgaag agtacccaga t 21

<210>20

<211>21

<212>DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400>20

aagaaccatg cactcatcag c 21

<210>21

<211>20

<212>DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400>21

cgactgttgc aggggtcagg 20

<210>22

<211>23

<212>DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400>22

gagttggtca taggaagttt cct 23

Claims (10)

1. Loquat EjsPL5 protein, which is:

1) a protein consisting of the amino acids shown in SEQ ID No. 2; or

2) Protein derived from 1) by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No.2 and having equivalent activity.

2. A gene encoding the loquat EjsPL5 protein of claim 1.

3. The gene of claim 2, having the sequence shown in SEQ ID No. 1.

4. A vector containing the gene according to claim 2 or 3.

5. A host cell comprising the vector of claim 4.

6. An engineered bacterium comprising the gene of claim 2 or 3.

7. Use of the gene of claim 2 or 3 for regulating flowering transition and early flowering in angiosperms.

8. The use according to claim 7, wherein the EjSPL5 gene of claim 2 or 3 is transferred into angiosperm genome and over-expressed in transgenic plant to promote the flower transformation of transgenic plant and advance flowering and fruiting.

9. A construction method of transgenic plant, adopting Agrobacterium mediated method, transferring the over-expression vector containing the gene of claim 2 or 3 into plant genome, and screening to obtain transgenic plant.

10. The method of claim 9, wherein said transgenic plant exhibits substantially improved flowering and premature flowering compared to the wild type.

Images