CN112899284B - Rice drought-enduring gene segment and application thereof - Google Patents

Abstract

The invention discloses a rice drought-enduring gene segment and application thereof, belonging to the field of plant drought-enduring genes. The drought-enduring gene sequence is shown as SEQ ID NO.5, and is obtained by carrying out 3 continuous base deletion, 2 discontinuous base insertion and 1 single base substitution mutation on the basis of BGIOSGA033249 gene. Experiments prove that: the gene can obviously improve the drought resistance of rice and the yield of the rice in drought environments. The gene has important application value in the cultivation of new varieties of drought-tolerant rice.

Description

Rice drought-enduring gene segment and application thereof

Technical Field

The invention relates to the field of drought-enduring genes of plants.

Background

With the aggravation of climate conditions such as global warming and the like, the frequency and the range of drought occurrence in China are also increased, and especially, the high temperature and drought in the flowering and setting period of Chongqing paddy rice in the southwest high Wen Fu drought region of the land often lead to serious yield reduction of the paddy rice. Drought has become an important factor limiting rice production. Therefore, the development of drought-tolerant gene resources and cultivation of drought-tolerant rice varieties are a feasible way for resisting drought stress.

The rice genome is the largest plant genome for which genomic sequencing has been performed to date. The genome sequence of the subtype of indica rice (Oryza Sativa ssp. Indica) commonly completed by 12 scientific research units in China was published on Science, 4.5.2002, and the full length of the rice genome was 466×10 ⁷ A base pair containing 46022 to 55615 genes. Among the numerous genes in the rice genome, the functions of many genes have not been verified, or even predicted.

The BGIOSGA033249 Gene (the number BGIOSGA033249 is found in Gramen database http:// www.gramene.org) is a Gene of which one function is not predicted yet, and only a protein can be encoded by the Gene, and 2G 0 (Gene Ottolog) tags are known at present: negative gravitropism (negative gravity) and response to red or far red light (response to red and far-red light).

At present, reports on the gene and rice drought tolerance are not yet seen.

Disclosure of Invention

The invention mainly aims at providing a gene for improving drought resistance of rice; another object of the present invention is to provide a method for improving drought tolerance of plants.

The technical scheme of the invention comprises the following steps:

a drought-enduring gene has a sequence shown in SEQ ID NO.5.

A recombinant plasmid, the sequence of which comprises SEQ ID NO;5.

the recombinant plasmid as described above, further comprising SEQ ID NO.3, wherein SEQ ID NO.3 is located at the 5' end of SEQ ID NO.5.

The recombinant plasmid as described above, further comprising SEQ ID NO.6, wherein SEQ ID NO.6 is located at the 3' -end of SEQ ID NO.5.

The recombinant plasmid as described above, the sequence of which comprises SEQ ID NO.3, 5 and 6 simultaneously, and SEQ ID NO.3, 5 and 6 are arranged from 5 'to 3' end.

The recombinant plasmid as described above, which uses pBR322 vector as skeleton, inserts SEQ ID NO.3, 5, SEQ ID NO. 5-6 or SEQ ID NO.3, 5, 6.

A method for improving drought tolerance of plant features that the gene or recombinant plasmid is transferred to plant.

The method as described above, wherein the plant is rice; preferably, it is rice of the Nippon variety.

A method for identifying drought-enduring rice includes identifying whether rice has DNA with sequence shown in SEQ ID NO.5 or not, and judging whether rice is drought-enduring or not;

if the DNA with the sequence shown in SEQ ID NO.5 is provided, the rice drought tolerance is judged.

A pair of PCR primers for identifying DNA containing the sequence shown in SEQ ID No.5 has the sequence shown in SEQ ID No. 7-8.

The inventor discovers that after the BGIOSGA033249 gene generates 1 single nucleotide polymorphism mutation, 3 continuous base deletion and 2 base insertion through the early-stage research result of the transcriptional analysis and the cluster team, the drought tolerance of rice can be improved. If the mutant gene is applied to improving the drought tolerance of plants, especially to culturing new rice drought tolerance varieties, the mutant gene has good prospect.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

Fig. 1:57054 and 805B, BGIOSGA033249 gene.

Fig. 2: relative expression levels of BGIOSGA033249 in each of the organizations 805B and 57054.

Fig. 3: and (3) carrying out PCR amplification identification on the T3 positive transgenic plants (Q9 family).

Fig. 4: and (5) drought tolerance identification of T3 positive transgenic plants (Q9 family).

Fig. 5: marker genotype performance of functional markers NH1 amplifications 805B and 57054.

Detailed Description

Experimental example 1 acquisition and verification of drought-enduring Gene of the invention

1. Reference material

Including rice material numbered 805B, barnyard grass, plus 9 (see Table 1 in particular) rice materials from the offspring 57013, 57054 of the distant hybridization of 805B/barnyard grass.

2. Drought tolerance identification

The test materials are planted in a plastic bucket and are placed in a paddy field greenhouse with ventilation around, and the top of the greenhouse is covered by a transparent agricultural film to prevent rainwater interference. The plastic barrels are 40cm in height and 30cm in diameter, a non-woven fabric planting bag is placed in each barrel, the plastic barrels are 40cm in height and 30cm in diameter, and rice field clay loam is filled in the non-woven fabric planting bags. The test materials were transplanted into barrels after 30d field seedling, 3 plants per barrel, and 6 barrels (3 barrels for A-drought stress group, 3 barrels for B-normal control group) were repeated for each treatment. The fertilizer and the prevention and control of the diseases, the weeds and the pests are the same as those of the field. And (3) the drought stress group (A) starts drought stress treatment at the main tillering (main stem) breach heading, integrally lifts the non-woven fabric planting bag during the treatment, pours out water in a plastic bucket and overturns and buckles the water in a paddy field on site, then integrally places the non-woven fabric planting bag on the bucket for rapid drainage, stops watering and naturally drought, and resumes normal water supply on the 16 th day of treatment. The normal control group (B) always maintained a water layer of 3cm-5cm in the bucket. The relative water content of the soil during drought stress was monitored, daily temperature and humidity. And (5) when the rice is mature, examining the data of the seed setting rate.

As shown in table 1, the fruiting rate of 57054 under drought stress conditions for 2 years is extremely remarkably higher than 805B and reaches about 35%, while the corresponding recipient parent 805B is only 7%, which indicates that 57054 has strong drought tolerance.

TABLE 1 fruit set rate performance of materials under drought stress

Note that: the different letters represent a significant at a probability level of 0.01.

Transcriptome analysis of 3.805B and 57054

Drought tolerance tests were performed at the Chongxi crop test station in 2018, 3-8, using 57054 and 805B, and sword-leaved as a sample for transcriptome analysis at the end of drought stress group stress treatment. And (3) taking 3 single plants respectively, and taking 3 tillered sword leaves from each single plant. Sampling time was fixed at 10:00-10:30 per day to reduce the effect of circadian temperature differences and rhythms on gene expression. After the sample is obtained, the sample is quickly frozen in liquid nitrogen by a freezing tube, and the sample is stored in a refrigerator at-80 ℃. After all material samples were taken, they were sent to the Megene company for transcriptome analysis. Transcriptome analysis revealed that 413 genes were expressed indifferently in a/B of 805B and up-or down-regulated in a/B of 57054 (some of the differentially expressed very significant genes are shown in table 2), with BGIOSGA033249 being expressed normally in a/B of 805B and a very large fold up-regulated in a/B of 57054, indicating that the gene plays an important role in drought tolerance of 57054.

TABLE 2 differential expression of partial genes found in transcriptome analysis

Drought tolerance verification of BGIOSGA033249 gene

4.1 BGIOSGA033249 gene sequence alignment

93 for downloading BGIOSGA033249 gene from Gramen11, designing primers to carry out high-fidelity amplification on the coding regions of 805B and 57054 respectively, and sequencing the corresponding products after agarose electrophoresis and rubber cutting recovery and then the sequencing of the products by Biotechnology company. Sequencing results using Clustal X alignment of parents compared to the 805B allele (BGIOSGA 033249 ^805B ) 57054 allele (BGIOSGA 033249 ⁵⁷⁰⁵⁴ ) 1 Single Nucleotide Polymorphisms (SNPs) sites, deletions of 3 consecutive bases and 2 base insertions were detected in the coding sequence of (fig. 1), and the inserted 2 bases directly caused the premature appearance of a terminator for the frame shift, thus leading to premature termination of translation.

4.2 Analysis of expression pattern of BGIOSGA033249 Gene

To analyze the expression pattern of BGIOSGA033249, RNA from roots, stems, sword leaves, and ears of 805B and 57054 was extracted using a high purity plant total RNA extraction kit that was healthy for century. The determination of the difference in expression of each organ of the gene in the parents (185 rRNA gene as reference) was performed on a fluorescent real-time quantitative RT-PCR instrument. The gene was found to be not expressed in the roots of rice, and expressed in stems, leaves and ears, and the expression level of BGIOSGA033249 in 57054 ears was significantly higher than 805B (fig. 2).

4.3 Transgenic full-length complementary verification of BGIOSGA033249 gene

To demonstrate the effect of the BGIOSGA033249 gene on drought tolerance, a vector comprising the BGIOSGA033249 gene (pBR 322 vector as backbone, inserted between Sau3A I and BamH I cleavage sites) was constructed, and the DNA fragment comprising the upstream promoter sequence (1.969 kb), full-length coding region (mutant 0.786 kb) and downstream sequence (0.958 kb) was transferred into japan (a rice variety).

T was amplified using primer LM1 (Forward primer (SEQ ID NO. 1): ACGGTGTCGTCCATCACAGTTTGCC; reverse primer (SEQ ID NO. 2): TTCCGGAAGTGCTTGACATTGGGGA) ₃ The genome of the plants of the family (T3 for two generations after the transgene; one family for the progeny from the same parent) was selfed to identify positive transgenic plants (Q9 family) (FIG. 3).

Upstream promoter sequence (SEQ ID NO. 3):

wild-type coding region (SEQ ID NO. 4):

mutant coding region (SEQ ID NO. 5):

full length of the downstream sequence (SEQ ID NO. 6):

the drought tolerance test was performed on non-transgenic Nippon Rice and Q9 families, and the treatment conditions were the same as those of group A of section 2.

As a result, Q9 family was found to have a significantly higher setting rate under drought stress than non-transgenic japan (nip.) (fig. 4). The mutant gene (SEQ ID NO. 5) of the invention can obviously improve the drought tolerance of rice.

Functional marker of BGIOSGA033249 gene

Based on the role of the BGIOSGA033249 gene in rice drought tolerance and the base sequence differences of the gene in 805B and 57054, the inventors utilized the 57054 allele (BGIOSGA 033249 ⁵⁷⁰⁵⁴ ) The 3 base deletion positions (108 th to 110 th bases of the coding region) designed 1 InDel marker NH1 (Forward primer (SEQ ID NO. 7): ATGCTTCAGAAGTTCGCGCT; reverse primer (SEQ ID No. 8): TAGGGCTCTGAACCGTGTCG) as BGIOSGA033249 ⁵⁷⁰⁵⁴ Is a functional marker (marker NH1 amplification 57054 allele length is 187bp,805B is 190 bp) (FIG. 5). The skilled person can use the functional marker to detect the gene sequence according to the invention as shown in SEQ ID NO.5.

In conclusion, the gene of the invention can obviously improve the drought tolerance of rice, enhance the yield of rice under drought stress and has great application prospect.

SEQUENCE LISTING

<110> Chongqing market agricultural academy of sciences

<120> a rice drought-enduring gene fragment and application thereof

<130> GY752-2019P018364CCR4

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 25

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 1

acggtgtcgt ccatcacagt ttgcc 25

<210> 2

<211> 25

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

ttccggaagt gcttgacatt gggga 25

<210> 3

<211> 1969

<212> DNA

<213> Rice (Oryza sativa)

<400> 3

cacatcaaac ggggaatttt gatgtgtttc gtgcattagg acccgtttta acattcagct 60

ccaaatttgt gtcatttaca aaaactagca tggtagcccg cgcagattgc gcagctaaca 120

tcattatatt ttctctcata taatagcata tatgttttct cattatatta ttcaaatata 180

ttaaaatttt atatttaaac ataattttaa attttgaaat aactttacaa cactactaat 240

gtgtcatatt catattgtat tttatatatg tattagttat taattatttt taatatcata 300

tattcctata tggactctag actcgtcttt taatatttct ttttttaatt ccgaattttc 360

tgtaaattgt atttctttat agactctatg ctcttcttcc aatattattt atgtttattt 420

ctgaattttt attatttcta attgtatttc tatatgtgga ctctaaactc atctttcaat 480

attctttaat ttttaatttc gaatttcagt tacttctaaa ttgtattcct atattgactt 540

taaactttct tcccatgttt ttcttatttt caaattttat ttatttataa attgtatatt 600

tatacggact ctaaactcta cttttaattt tattatgttt atttcaaatt ttagttagtt 660

ttaaattcct atatagactc taaactctac ttctaatatt ctttattttt aaattccgaa 720

tttctatttt ttttcttaat tttatttcta tatggactct atactctact tctaatattc 780

tttatttttt aattccgaat ttctattttt tttcttaatt gtatttctat atggactcta 840

tactctactt ctaatattcc ttatttttaa ttccgaattt catctatttc ttaattgtat 900

ttctatatgg actctagtct cctcttctaa tattccttat tttttaattc cgaatttcat 960

ctatttctaa attgtatttc tatatggact ctagtctcgt cttctaatat tccttctttt 1020

ttaattccga atttcagcta tttctaaatt gtattcctat atggactctg ccttttcttt 1080

ttctccgatt aatgtgggaa tttttagacc atgagagcga acgtggaggc tcctttttct 1140

attcctttaa taaaataata gataatatac atagaaatat ttcttacaaa aatcaaataa 1200

atgtttataa taattaataa tactcgatta accgtaagct aaacgaccta tctcgtttca 1260

cgtgccacga aaactaaacc tctatcttca acgatagaac acagccttgt cttggtgcaa 1320

aagttgtgca aatcttttac acttcattta ttttttcatg caccaaggct taacaaaaga 1380

ctgcgaagtg agcgtaggct aactagtttg gttcttaaac tgtaactagc tcacctgggt 1440

tcaaatccta gatttgatat gagtgctcgc atttacggct aattattctt tcagtggtag 1500

acgacgtacc cctcgataac gagacgcctg tggtgatttc gtcgattagt actgtttgtc 1560

gaaaagaaaa aaaaaagctt aacatagact ataactgtca tcctctcttc aaattaaata 1620

ctcctacaca aatttacagg tagactagta acaaaatcaa aacaatcacg agattagaag 1680

catcttttag taacagacac ctaccccctg aatcccggta attccaattc caattccaat 1740

tccaattcat ctcttctccc cctcccctcc cctctcttct cgacggagac acgcactcac 1800

tctctctctc gcgtcagatc ccctctcctc tctcgccatc tccacctcct ccattccacc 1860

tccactcccg cttcctcccc cccacgattc gccggaattc gagctcggcg cggcggcggc 1920

tgcggcggcg gcggggtttg ttgtgggggg cgagcggatc ggcgcagcc 1969

<210> 4

<211> 1413

<212> DNA

<213> Rice (Oryza sativa)

<400> 4

atgcttcaga agttcgcgct cgcgttcaag accaagacga tcgagttctt cgcggaggag 60

gaggaggacg aggatgctga tggcggtgtc tccgcggcgg cggcggcggc ggtgggggtg 120

ggggaaggtg gcgtgctggc tggtcagcgg gtggtggtgc tcaagcccga cacggttcag 180

agccctaacc ctagcggggg ggtgggggtg ggggtggtgg tgggtgaggc ggctgcggtg 240

gaggcggcgc tcgcgaccgc gtcgtcgttc caggcggcgt acctccacct gcaggcggcc 300

cacgcgccgt tcctcccgga tgccgcggcg gccgccgacg ccgccgcggt gtcgcacctg 360

cggaggctgt cggaggtgaa gcggctcgcg cgggatcccg gcgtcggcgg cggcgcgctc 420

acggcgcacc tcgaggccca ggtgcgcgag aaccaggcgc tgctgcggtc gttcgacgcc 480

gtggtgaacc gcctccaggc cgcgctcgac ggcaaggacg ccgcggcggc ctccctgcgg 540

agggaccacg ccgagctcgc cgacggcaac gcgcggctcg gggcgaggct cgaccgcgca 600

ctcgcgccgc ctccaggcgc cggcggcgac gacgccctcg gcgccatgct ctccgccggc 660

gtcttcgact ccgtcctccg cgacgcgctc cgcgtcgccc accgcttcac ccgctcgctc 720

gccgatctcc tccgatgcgc cgggtgggat cttgctgccg cggcggcggc ggtctacccc 780

ggtgtagcct actccaggcc tggtcactgc cgctacgcgc tcctctcccg ggtttgccta 840

tccatgtttg atggcttcga ttcataccaa tttggtggtt caactgatgc caccacactg 900

gaaggaatcg atctcgcaat ccgaaggaat gagtcactgc agcaattcat cgagcactca 960

gatgcagacc caatggagct tatcaattca agcccagact gcgaattcgc ccaattctgt 1020

gaccggaagt acaagcagct cattcatcct ggcattgagt cctcactgtt tgggaattct 1080

gattgtggca aattgccagt gttgggcgtg gccggaccac tctacgagct gttcgttgca 1140

atggcaagct caatatggac acttcataga ttggcttggg cgtatgatcc ggcagtcggc 1200

atattccaga ttggccaggg agcggagtac tcggtggttt acatggagaa cattgtgcgt 1260

tcaaaaggat tttcaggaag caaggagctc ggaaagatga tgcgaccgaa ggtcggtttc 1320

acggtggtgc cgggatttcg gctcggtggg acggtgatcc agtgtagggt gtacctagat 1380

tgtggcaaga gggaaggaat tataggtgaa tga 1413

<210> 5

<211> 786

<212> DNA

<213> Rice (Oryza sativa)

<400> 5

atgcttcaga agttcgcgct cgcgttcaag accaagacga tcgagttctt cgcggaggag 60

gaggaggacg aggatgctga tggcggtgtc tccgcggcgg cggcggcggt gggggtgggg 120

gaaggtggcg tgctggctgg tcagcgggtg gtggtgctca agcccgacac ggttcagagc 180

cctaacccta gcgggggggt gggggtgggg gtggtggtgg gtgaggcggc tgcggtggag 240

gcggcgctcg cgaccgcgtc gtcgttccag gcggcgtacc tccacctgca ggcggcccac 300

gcgccgttcc tcccggatgc cgcggcggcc gccgacgccg ccgcggtgtc gcacctgcgg 360

aggctgtcgg aggtgaagcg gctcgcgcgg gatcccggcg tcggcggcgg cgcgctcacg 420

gcgcacctcg aggcccaggt gcgcgagaac caggcgctgc tgcggtcgtt cgacgccgtg 480

gtgaaccgcc tccaggccgc gctcgacggc aaggacgccg cggcggcctc cctgcggagg 540

gaccacgccg agctcgccga cggcaacgcg cggctcgggg cgaggctcga ccgcgcactc 600

gcgccacctc caggcgccgg cggcgacgac gccctcggcg ccatgctctc cgccggcgtc 660

ttcgactccg tcctccgcga cgcgctccgc gtcgcccacc gcttcacccg ctcgctcgcc 720

gatctcctcc gatgcgccgg gtgggatctt cgctcgccgc ggcggcggcg gtctaccccg 780

gtgtag 786

<210> 6

<211> 958

<212> DNA

<213> Rice (Oryza sativa)

<400> 6

tcctttgctt ccttcagtat aagatcagat gctgcacttt gtcatctgtt gtggaaatgc 60

aaacttgcca ttgtaccctg tttgttcatt tgtaactagg gttgttccct gtttgttcaa 120

gtagaatggt tcagaacaac agttcaagtt tcctttcact gatcgatgtt tcttattctg 180

aggacatgct taatgtttgt ctgaattctg aaatgattgg gattcagtat tcgattccga 240

attcagtcag gtggcacaca cactttctgt cttgaggaaa atgcccaacg cctccaaaaa 300

cttgtgtgtt gtgtggcagc atccaggttt catgtcagtc aggtgacttt ctgtctatgg 360

gacagtatca tttctgtgtc ctttgctgca tctttttcgt tctctcctgt agcccgtcaa 420

cacagtttgt cgtcaaagtt cagagctact actgaactct tttgcactgt aaaaagcttt 480

gtactccata atactctagt aaaactaatg aaactacctg gaattaattc ctaatgactg 540

ctgctgcctt tgcaattctt catagttggt ccatgttccc ccatcaatta tgaagggcct 600

cgtggctaat aaattccaca tcccggttct taccaattcc aaactaacta cagcttgtca 660

cctgtactgc cagcaaacta gtaggagact cctagcacac tcacaatgct cttaccagaa 720

aaaaagaccc catatattca atgggtgatc tggtcataac ttgctcaaag gccagcattt 780

tgctgtcaat cttgcacctc agagattaag cctaaccaaa tcattgcctc catcttgtga 840

cagtagttaa ctgccactgt tttagctgaa gaacaagctg acatagtagc agctgcggca 900

ttaagaatgc gtgcctgcca ctttaccagt ggggttgggc atgttccttg cttcggct 958

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 7

atgcttcaga agttcgcgct 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

tagggctctg aaccgtgtcg 20

Claims (9)

1. A drought-enduring gene, characterized in that: the sequence is shown as SEQ ID NO.5.

2. A recombinant plasmid, characterized in that: the sequence of the plasmid comprises SEQ ID NO.5.

3. The recombinant plasmid of claim 2, wherein: the sequence also comprises SEQ ID NO.3, wherein SEQ ID NO.3 is positioned at the 5' end of SEQ ID NO.5.

4. The recombinant plasmid of claim 2, wherein: the sequence also comprises SEQ ID NO.6, wherein SEQ ID NO.6 is positioned at the 3' end of SEQ ID NO.5.

5. The recombinant plasmid of claim 2, wherein: the sequence comprises SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.6 at the same time, and SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.6 are arranged from the 5 'end to the 3' end.

6. The recombinant plasmid according to any one of claims 2 to 5, wherein: the plasmid takes a pBR322 vector as a framework.

7. A method for improving drought tolerance of plants, which is characterized by comprising the following steps: transferring the gene of claim 1 or the recombinant plasmid of any one of claims 2-6 into a plant; the plant is rice.

8. The method of claim 7, wherein: the plant is rice of Japanese sunny variety.

9. A method for identifying drought-tolerant rice is characterized by comprising the following steps: it judges whether the rice has drought tolerance by identifying whether the rice has DNA with sequence shown as SEQ ID NO. 5;

if the DNA with the sequence shown in SEQ ID NO.5 is provided, the rice drought tolerance is judged.

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