CN112608938A - Application of OsAO2 gene in controlling drought resistance of rice - Google Patents

Abstract

The invention belongs to the field of rice genetic engineering, and particularly disclosesOsAO2The gene is applied to controlling the drought resistance of rice. Drought stress phenotype identification experiments in seedling stage and adult stage of CRISPR material prove that after the gene function is knocked out, the drought tolerance of rice is increased, and the function and application approach of the gene are verified. Thus, it was isolated from riceOsAO2The gene and the identification of the functions of the gene in improving the stress resistance of the rice have very important significance for cultivating new stress-resistant rice varieties.

Description

Application of OsAO2 gene in controlling drought resistance of rice

Technical Field

The invention relates to the field of rice genetic engineering. In particular to application of an OsAO2 gene in controlling drought resistance of rice, wherein a protein sequence coded by the OsAO2 gene is shown as SEQ ID NO. 2. The invention adopts a candidate gene screening method to clone the gene OsAO2 for controlling rice drought resistance, and the CRISPR technology shows that the OsAO2 mutant is closely related to the drought resistance phenotype, thereby confirming the function and application approach of the gene.

Background

Plants are affected by a plurality of environmental factors in the growing process, and drought, cold damage and high temperature can cause large-scale yield reduction of crops, and are bottlenecks in agricultural development in many areas. The cultivation of stress-tolerant crop varieties has always been one of the main targets of agricultural science and technology research. To combat or adapt to these adverse factors, plants sense changes in the extracellular environmental conditions and deliver them to cells via a variety of pathways, which induce the expression of response genes, produce functional proteins and osmolytes that protect cells from stress such as drought, high salinity, low temperature, etc. to adapt to adverse growth conditions (Xiong et al, Cell signaling reducing cold, stress and salt plant Cell 14(suppl), S165-S183, 2002). The correct expression of those functional genes in response to the environment is finely regulated by regulatory factors. The transcription factor is used as a regulating gene, and when an organism is subjected to adversity stress, the expression of a series of downstream genes can be regulated, so that the tolerance of a plant body to the adversity is enhanced, and the effect of resisting adverse environmental condition stress is achieved. Most types of transcription factors are involved in abiotic stress response in plants, including AP2/EREBP, bZip, HD-ZIP, MYB, MYC, NAC and Zinc finger-type transcription factors (Yamaguchi-Shinozaki K, Shinozaki K. transcription regulation networks in cellular responses and tolerance to depression and column protocols. Annu Rev Plant Biol,2006,57: 781-. Through genetic engineering, partial stress response transcription factors have been successfully applied to rice stress-resistant genetic breeding. The transgenic rice plant cultivated by the SNAC1 can improve the maturing rate by about 30% in a field drought environment, and the yield is not influenced and other phenotype changes are avoided under normal conditions. Transgenic plants are also significantly more resistant to drought and high salt during vegetative growth (Hu et al. overexpression a NAM, ATAF, and CUC (NAC) transformation factors resistance and salt tolerance in rice. Proc Natl Acad Sci U S A,2006,103: 12987-. These antiretroviral factors express their function by regulating the expression of a number of downstream genes. These downstream genes often contain regulatory proteins involved in signal transduction and gene expression, which in turn further form a secondary regulatory network. These downstream genes can also be used for genetic improvement of crop stress resistance. The downstream gene HsfA3 of the high temperature transcription factor DREB2A in Arabidopsis thaliana can also improve the resistance of transgenic over-expressed plants to high temperatures (Yoshida et al Functional analysis of an Arabidopsis heat-shock transcription factor HsfA3 in the transgenic plant down stream of the DREB2A stress-translation system. biochem Biophys Res Commun,2008,368: 515-21).

Ascorbic acid is a main antioxidant in plants, and plays an important role in clearing excessive generated active oxygen, maintaining redox balance in plants and regulating physiological and biochemical reactions of plants under abiotic stress. It can also improve tolerance to Abiotic Stress by promoting Plant growth, photosynthesis, transpiration, oxidative defense, etc. (Akram NA, et al. Studies have reported that increasing the level of ascorbic acid in various crops can enhance the stress tolerance of the plant. Ascorbate oxidase is localized to the cell wall and is a key enzyme that determines the redox status of apoplast and is involved in plant growth, stress sensing and subsequent signal transduction (Pasori G.M., Foyer C.H (2002). Common components, networks and pathways of cross tolerance to stress.the central roll of "redox" and available acid-mediated control. plant physiology.129, 460-468). The activity of ascorbic acid oxidases is associated with the tolerance of plants (Garchery C et al. A differentiation in ascorbyl oxidase activity enzymes carbon infection and improves yield in bottom water tolerance. plant Cell environ.2013,36, 159-175).

The rice is an important grain crop and a model plant, and the cultivation of the rice with enhanced stress resistance is of great significance today under frequent extreme climatic conditions. The invention separates the OsAO2 gene from rice and identifies the function of the gene in improving stress resistance of the rice, and has very important significance for breeding new stress-resistant rice varieties.

Disclosure of Invention

The invention aims to provide application of an OsAO2 gene in controlling improvement of drought resistance of rice, wherein an amino acid sequence coded by the OsAO2 gene is shown in SEQ NO. 2.

In order to achieve the purpose, the invention adopts the following technical measures:

the application process of the OsAO2 gene in regulating and controlling the drought resistance of rice comprises the steps of controlling the drought resistance of rice by controlling the expression of the Os AO2 gene by utilizing the conventional scheme of the invention, wherein the sequence of the OsAO2 gene is shown as SEQ ID NO.1, and the sequence of the encoded protein is shown as SEQ ID NO. 2;

in the above application, preferably, the gene is knocked out by selecting a target site in the OsAO2 gene by a CRISPR/Cas9 method, and the obtained rice mutant is drought-tolerant rice;

in the above-mentioned application, it is preferable that the drought-sensitive rice is obtained by overexpressing the OsAO2 gene in rice by a transgenic method.

In the above applications, the drought-enduring rice preferably comprises a nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6.

The expression vector carrying the OsAO2 gene of the present invention can be introduced into Plant cells by conventional biotechnological methods using Ti plasmid, Plant viral vector, direct DNA transformation, microinjection or electroporation (Weissbach,1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey,1998, Plant Molecular Biology (2nd Editi on).

The host which can be transformed by the expression vector of the OsAO2 gene is various plant hosts such as rice and is used for cultivating new drought-tolerant plant varieties.

Compared with the prior art, the invention has the following advantages:

the invention firstly provides that the gene for coding the protein shown by SEQ ID NO.2 can control the drought resistance of rice, and the drought stress phenotype identification of the seedling stage and the adult stage shows that the drought stress resistance of the rice is enhanced when the gene fragment is deleted, thereby confirming the function and the application approach of the gene

Drawings

FIG. 1 shows the results of genotype detection of OsAO2 CRISPR mutant strain OsAO2-11 and OsAO2-13 in T0 generation.

FIG. 2 Rice OsAO2 CRISPR plant seedling stage drought stress phenotype.

FIG. 3 Rice OsAO2 CRISPR mutant OsAO2-11, OsAO2-13 and OsAO2-14 plants maturity drought stress phenotype;

wherein WT is a wild type, and the phenotype before stress of each plant is listed on the left; the right column lists the phenotype of each plant under severe stress.

Detailed Description

The following examples define the present invention and describe the methods of the present invention in constructing CRISPR material of OsAO2, cloning a DNA fragment comprising the complete coding segment of the OsAO2 gene, and verifying the function of the OsAO2 gene. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Example 1:

construction of OsAO2 gene CRISPR vector

The CRISPR-Cas9 gene editing vector constructed by the invention uses a tRNA-gRNA tandem editing system. Two suitable targets, "GAGCAGCCCGAGCCGTTCCG" and "ACCAGGAGAAGT GCCTGAGG", were designed on the genomic sequence of the conserved domain of OsAO2 protein, and the fragments of interest containing these two targets were ligated into the GEpRB 32 vector according to the materials method of Xie, Kabin et al, boosting CRISPR/Cas9 multiplex editing capability w ith the endogenic tRNA-processing system, proceedings of the National Academy of Sciences of the U National States of America vol.112,11 2015 (3570-5. doi: 10.1073/pnas.1420294112). The positive plasmids were screened by PCR using pairs of primers UGW-U3-F (GACCATGATTACGCCAAGCTTAAGGAA TCTTTAAACATACG) and UGW-gRNA-R (GGACCTGCAGGCAGGCACGCGCATACGAAACGGACTAGC), and the resulting recombinant plasmid vector was named OsAO2-pRGEB32, and the sequence of interest on the vector was:

GATCCGTGGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGAGCAGCCCGAGCCGTTCCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAACCAGGAGAAGTGCCTGAGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTT。

example 2:

construction of the OsAO2 gene overexpression vector:

a DNA fragment containing the entire coding region of the OsAO2 gene (said DNA fragment comprising the sequence shown in SEQ ID NO. 1) was amplified using the primers OXAO2-FL-F (sequence specific primers plus linker containing KpnI cleavage recognition site, 5'-TACGAACGATAGCCGGTACCATGGCGGCCGCCGTGCAGCTGCT-3') and OXAO2-FL-R (sequence specific primers plus linker containing BamHI cleavage recognition site, 5'-TTGCGGACTCTAGAGGATCCTCATGGCGCCGGCGCCGGCGACGGA-3') using the DNA extracted from the leaves of the rice cultivar "Nipponbare" as template, and the fragment was ligated in one step into the vector pCAMBIA U (Xiaoing, Yong et al., propagation of OsbP 23 a key layer of the basic leucosine zipper thinning factor for transforming the coding amplification sensitivity and purity nutrition gene, Escherichia coli, 1932, 19352, 1938, 19352, and 1938. coli DH 52, and further transformed with the primers OXAO2-FL-F (sequence specific primers plus linker containing KpnI cleavage recognition site, 5'-TACGAACGATAGCCGGTACCATGGCGGCCGCCGTGCAGCTGCT-3') and OXAO 3878-FL-R (5'-TTGCGGACTCTAGAGGATCCTCATGGCGCCGGCGCCGGCGACGGA-3'), the recombinant plasmid vector obtained by enzyme digestion screening of positive clones and sequencing was named OsAO2-OX-p 1301U.

Example 3:

genetic transformation of the construction vector

The overexpression vector and the gene knockout vector are respectively transferred into a rice variety 'Zhonghua 11' (a publicly used rice variety provided by Chinese rice research) by an agrobacterium-mediated rice genetic transformation method, and a transgenic plant is obtained by pre-culturing, infecting, co-culturing, screening a hygromycin-resistant callus, differentiating, rooting, training and transplanting. The above Agrobacterium-mediated genetic transformation method (system) for rice (Zhonghua 11) was carried out on the basis of the method reported by Hiei et al (Hieiet al, Efficient transformation of rice, Oryza sativa L., medium d by Agrobacterium and sequence analysis of the nucleic acids of the T-DNA, Plant J, 6:271-282, 1994).

The specific genetic transformation steps of this example are as follows:

(1) and (3) electric conversion: the final target vector was transformed into Agrobacterium EHA105 strain at 1800v, spread on LA medium with corresponding resistance selection and screened for positive clones for transformation callus as described below.

(2) Callus induction: removing husk from mature rice seed medium flower 11 (a publicly used rice variety provided by the Chinese Rice research institute), treating with 70% ethanol for 1min, and sterilizing the surface of 0.15% mercuric chloride (HgCl2) seed for 15 min; washing the seeds with sterilized water for 4-5 times; placing the sterilized seeds on an induction medium; and (3) placing the inoculated callus induction culture medium in a dark place for culturing for 4 weeks at the temperature of 25 +/-1 ℃.

(3) Callus subculture: the bright yellow, compact and relatively dry embryogenic calli were selected and placed on subculture medium for 2 weeks in the dark at 25 + -1 deg.C.

(4) Pre-culturing: compact and relatively dry embryogenic calli were selected and placed on pre-culture medium for 2 weeks in the dark at 25 + -1 deg.C.

(5) And (3) agrobacterium culture: pre-culturing agrobacterium EHA105 (from CAMBIA, a commercial strain carrying the final destination vector) on LA medium with corresponding resistance selection for two days at 28 ℃; transferring the agrobacterium to a suspension culture medium, and culturing for 2-3 hours on a shaking table at 28 ℃.

(6) Infection of agrobacterium: transferring the pre-cultured callus into a sterilized bottle; adjusting the suspension of Agrobacterium to OD 6000.8-1.0; soaking the callus in agrobacterium tumefaciens suspension for 30 minutes; transferring the callus to sterilized filter paper and sucking to dry; then, the cells were cultured on a co-culture medium at a temperature of 19 to 20 ℃ for 3 days.

(7) Callus washing and selective culture: washing the callus with sterilized water until no agrobacterium is visible; soaking in sterilized water containing 400ppm Carbenicillin (CN) for 30 min; transferring the callus to sterilized filter paper and sucking to dry; the calli were transferred to selection medium for 2-3 selection 2 weeks each (carbenicillin concentration 400ppm for the first selection, 250ppm after the second selection, hygromycin concentration 250 ppm).

(8) Differentiation: transferring the resistant callus to a dark place on a pre-differentiation culture medium for culturing for 5-7 weeks; transferring the pre-differentiation cultured callus to a differentiation culture medium, and culturing under illumination at 26 ℃.

(9) Rooting: cutting off roots generated during differentiation; then transferred to rooting medium and cultured for 2-3 weeks under illumination at 26 ℃.

(10) Transplanting: residual medium on the roots was washed off and seedlings with good root system were transferred to the greenhouse while keeping the water moist for the first few days.

Example 4:

method for identifying genetic material

And (3) detecting the genotype of the CRISPR mutant: extracting small DNA samples from leaves of T0 transgenic material of seedlings, amplifying genome DNA containing a target sequence by PCR, detecting whether a target band exists or not by gel electrophoresis to judge the genotype, sequencing a PCR product and comparing a Nipponbare reference sequence to judge whether a target site is edited or not (figure 1), selecting a material with the target sequence shifted for breeding, and finally reserving three families and naming the three families as osa 2-11 (containing a sequence shown in SEQ ID NO. 4), osa 2-13 (containing a sequence shown in SEQ ID NO. 5) and osa 2-14 (containing a sequence shown in SEQ ID NO. 6). The primer for detecting the gene sequence is OsAO 2-seq-F: TGGGCGGACGGGACGGCAT, respectively; OsAO 2-seq-R: TCTTACCCCTTGAATCTTGACG are provided. The PCR procedure was: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30S, annealing at 57 ℃ for 30S, extension at 72 ℃ for 1min, carrying out 33 cycles in the amplification process, finally extension at 72 ℃ for 5min, and keeping the temperature at 25 ℃.

And (3) detecting the copy number and expression amount of the overexpression material: the copy number and expression quantity of the T0 generation of the material are detected by a fluorescent quantitative PCR method. Firstly, the DNA effect of the extracted leaf is used for detecting the copy number, RNA is extracted from a single plant with the copy number of single copy and is reversely transcribed into cDNA to detect the gene expression quantity, the copy number and the expression quantity are detected again after the T1 generation, and the single plant with the copy number of double copy and the expression quantity more than 10 times of the wild control is selected for breeding. The primer for detecting the copy number is Hpt-qHGF: GATGCAATAGGTCAGGCTCTCG and Hpt-qHGR: GATGTAGGAGGGCGTGGATATG, respectively; the internal reference gene primer is SPS-qHGF: CCTCTTCTAGCATCGAGGTCAC and SPS-qHGR: CTCCCCGACGATCAGATACATG are provided. The primer for detecting the gene expression level is OsAO 2-qF: GGACAGGATGCACGAGCTC and OsAO 2-qR: ATGCTGAGGAGGAGCTCATG, respectively; the primer of the internal reference gene is Ubq-qF: AACCAGCTGAGGCCCAAGA and Ubq-qR: ACGATTGATTTAACCAGTCCATGA.

Example 5:

identifying the CRISPR material seedling stage drought stress phenotype:

in order to identify the phenotype of CRISPR material in seedling stage, homozygous CRISPR material (osao2-14) with well-identified genotype and Wild Type (WT) are germinated and directly sown into the same small round barrel, 10-12 strains on both sides. The soil used for the test is formed by mixing the rice soil in south China and the coarse sand according to the volume ratio of 2:3, the equal amount of uniform sand soil and the equal amount of water are added in each barrel, the water automatically leaks to ensure that the compactness of the soil is consistent, and the test is repeated for 3 times. The plants in 4-leaf stage with healthy growth are subjected to water-cut drought stress for 6-10 days (according to the weather conditions), then are subjected to rehydration for 5-7 days, and the survival rate of the plants is photographed and investigated. Plants homozygous for CRISPR material showed a drought tolerant phenotype compared to the wild type control (figure 2).

Example 6:

identifying CRISPR material maturity stage drought stress phenotype:

the material is germinated and tested in the same seedling stage, the material is planted in a bread basin after one week, and the material is transplanted into a medium blue barrel after about three weeks of seedling age. Each pedigree was replicated for 6 blue buckets, 3 of which were used for normal growth and the other 3 for drought stress treatment. When the rice plant enters the young ear differentiation V phase to carry out drought stress treatment, the mutant and the wild type are photographed for one time. Thereafter, water cut-off for stress was initiated. Observing the leaf rolling condition, and rehydrating the leaves when the leaves are fully rolled. The mutants were more drought tolerant than the wild type as judged from leaf rolling during the stress when the phenotype of the mutant and wild type was significantly different from the stress period to the rehydration period as recorded by photographs (FIG. 3).

Sequence listing

<110> university of agriculture in Huazhong

Application of <120> OsAO2 gene in controlling drought resistance of rice

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Glu Ser Tyr Ser Val Leu Leu Lys Ala Asp Gln Lys Pro Ala Ser Tyr

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Gln Leu Pro Ala Gly Glu Pro Pro Val Thr Pro Ala Trp Asn Asp Thr

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tgcaaattgt agacgatctt gatcaatggg agaggacagt tcgagtgcac gctggggcca 540

gcgaggaaga gctttgagaa gctcctcaac gagaacgtgg agacctgcgt cgacgaccag 600

aagatgtgca gcgaccagga gaagtgccta ggaggagcga gtgcgggccg tactgcccca 660

ggagccagtg cgcccctgtc gtgttcaatg tcgagcaggg gaagacttac cgccttagga 720

tcgccagcac cacctccctt tctctcctca acgtcaagat tcaaggggta agataattca 780

atgtttttta tggattgtat ttttagatgt acaaa 815

<210> 6

<211> 828

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

actacggtcg agacgttcgt ctacagttcg tcgccgacaa gccgggcacc tacttctacc 60

acggccactt cgggatgcag cgcgccgcgg gcctgtacac gtccctcatc gtcctcgact 120

cgccggaggc ccgagccgtt ccgccaccag tacgacgacg gcggcgagct ccccatgatg 180

ctcctcagcg actggtggca ccagaacgtc tacgcccagg ccgccggact cgacggcaag 240

gacaggcact tcgagtggat cggcgagccc caggtaaata aaaaaacaca tcgccgccgt 300

cgtcatcgtc gccgccatct ccggtgatag agaaccatgt cgataaagaa ggcacgatgg 360

gtcacccgtg ccttcaggcc ggcacggcac ggctcgactc gcctcgggcc gtgcctagcc 420

cgtgtcgggc ggcccttatg gccatctata ccggtgagtg ctaacagtta gttttttgca 480

aattgtagac gatcttgatc aatgggagag gacagttcga gtgcacgctg gggccagcga 540

ggaagagctt tgagaagctc ctcaacgaga acgtggagac ctgcgtcgac gaccagaaga 600

tgtgcagcga ccaggagaag tgccaaggag gagcgagtgc gggccgtact gccccaggag 660

ccagtgcgcc cctgtcgtgt tcaatgtcga gcaggggaag acttaccgcc ttaggatcgc 720

cagcaccacc tccctttctc tcctcaacgt caagattcaa ggggtaagat aattcaatgt 780

tttttatgga ttgtattttt tagattgtac gaagaaccgc acgtataa 828

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gagcagcccg agccgttccg 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

accaggagaa gtgcctgagg 20

<210> 9

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gaccatgatt acgccaagct taaggaatct ttaaacatac g 41

<210> 10

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggacctgcag gcatgcacgc gctaaaaacg gactagc 37

<210> 11

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tacgaacgat agccggtacc atggcggccg ccgtgcagct gct 43

<210> 12

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ttgcggactc tagaggatcc tcatggcgcc ggcgccggcg acgga 45

<210> 13

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgggcggacg ggacggcat 19

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tcttacccct tgaatcttga cg 22

<210> 15

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gatgcaatag gtcaggctct cg 22

<210> 16

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gatgtaggag ggcgtggata tg 22

<210> 17

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cctcttctag catcgaggtc ac 22

<210> 18

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ctccccgacg atcagataca tg 22

<210> 19

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ggacaggatg cacgagctc 19

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

atgctgagga ggagctcatg 20

<210> 21

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

aaccagctga ggcccaaga 19

<210> 22

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

acgattgatt taaccagtcc atga 24

Claims (4)

1.OsAO2Application of gene in controlling drought resistance of rice, and the geneOsAO2The protein sequence of the gene code is shown in SEQ ID NO. 2.

2. The use as claimed in claim 1, ofOsAO2The sequence of the gene is shown as SEQ ID NO. 1.

3. The use according to claim 1, wherein the application is carried out by CRISPR/Cas9 methodOsAO2Knocking out genes to obtain the drought-resistant rice.

4. The use according to claim 3, wherein the drought-resistant rice contains a sequence shown by SEQ ID No.4, SEQ ID No.5 or SEQ ID No. 6.

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