CN108866086B - Rice gene OsGDS L1 and application thereof in resisting rice blast - Google Patents
Rice gene OsGDS L1 and application thereof in resisting rice blast Download PDFInfo
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- CN108866086B CN108866086B CN201810837943.8A CN201810837943A CN108866086B CN 108866086 B CN108866086 B CN 108866086B CN 201810837943 A CN201810837943 A CN 201810837943A CN 108866086 B CN108866086 B CN 108866086B
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- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
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Abstract
The invention provides a rice geneOsGDSL1The open reading frame of the gene is 1119bp in length, codes GDS L-like lipase and consists of 372 amino acids, and the gene is obtained by constructing a rice overexpression vector pCXUN-HA-OsGDS L1 and by means of agrobacterium-mediated rice mature embryo transformation technologyOsGDSL1Over-expression of rice plants by wild type and over-expressionOsGDSL1The rice plant is carried out with rice blast germ inoculation experiment, and the result shows that the rice blast germ is over-expressedOsGDSL1The resistance of rice plants to rice blast germs is improved, and the gene can be used for cultivating rice blast resistant rice varieties.
Description
Technical Field
The invention belongs to the field of rice cultivation, and particularly relates to riceOsGDSL1The gene and the application of the over-expression vector thereof in rice blast resistance.
Background
Rice is one of important economic crops, more than half of the world population takes the rice as staple food and plays an important role in grain production, and the rice blast caused by rice blast germs is a main disease influencing the high quality and high yield of the rice. The disease has the characteristic of strong outbreak, and can cause the catastrophic consequences of rice yield reduction and even absolute harvest. Currently, as rice blast germs are found to infect gramineous crops such as wheat, barley, corn and the like, the disease attracts more attention in the scientific field. Many scientists have been working on finding effective methods of controlling rice blast to alleviate the potential problems of food shortage and safety. Practice proves that the cultivation and planting of disease-resistant rice varieties is the most economic, most effective, most green and environment-friendly measure for preventing and treating rice blast at present.
Most lipases contain signal peptide, take serine residue (Ser) as an active center, and can be divided into two categories, namely, GxSxG lipase and GDS L lipase according to the conserved sequence of the active center, wherein Ser is usually present in the active center and a catalytic site in the GxSxG lipase, while Ser is generally present in a region close to the N-terminal in the GDS L lipase.
The position of the gene influences the function of the lipase, and the distribution of the gene in the family is in the form of a gene cluster, for example, the OsGI L P gene of the lipase family is distributed on 12 chromosomes of rice, and most of the genes are distributed near a centromere at the end of a telomere.
Lipases are widely present in plants, animals, microorganisms and accumulate in plant tissues and organs, such as roots, stems, leaves, flowers, fruits, seeds. It is of various kinds and different functions. Although the research of the family gene starts late and the related function research reports are less, the related research shows that the family gene participates in oil metabolism, plant growth and development regulation and stress resistance.
The GDS L lipase also participates in the growth and development process of plants, the lipase RGE1 in Arabidopsis regulates the development of embryos during seed germination, in addition, another GDS L lipase EX L4 exists on the column head, the protein is pollen wall protein, can regulate and control hydration, promotes the formation of pollen tubes, is favorable for the combination of sperms and ovules, the tomato lipase GDS L1 is a key protein for controlling the precipitation of pericarp cutin, can determine the thickness of the fruit epidermis and also influences the growth and development of the embryosThe GDS L lipase gene of rapeBnLIP1Not only can participate in lipid metabolism, but also can be related to flowering and pod development.
The plant GDS L lipase can perform its function under abiotic stress, the pepper GDS L lipase GDS L-lipase 1 (CaG L1) is quickly up-regulated and expressed by methyl jasmonate (Me-JA) and artificial injury, but is insensitive to treatment of Salicylic Acid (SA), Ethylene (ET) or tobacco mosaic virus, the rice genome at least encodes 113 GDS L lipases (OsG L IPs), the large number of rice GDS L lipases benefits from large-scale gene tandem repeat events, the family of genes exhibit tissue-specific expression, 38 of whichOsGLIPThe s gene is specifically expressed in the stigma and germinating stage of the flower, and the rest genes are only expressed in the root. In addition to this, the present invention is,OsGLIPsthe gene is induced by various abiotic stresses, such as drought, salt, cold and heat stresses.OsGLIPsAlso in response to biotic stress, reportedOsGLIP1AndOsGLIP2negatively regulate and control the immune response of the rice. Plant hormone SA or pathogen infestation represses the transcriptional expression of both genes. Over-expressionOsGLIP1OrOsGLIP2All reduce the resistance of rice to bacteria and fungi and vice versa.
At present, there is no reference to riceOsGDSL1The application of the gene in rice blast resistance is reported. Modification of rice by biotechnological meansOsGDSL1The expression level of the rice can improve the disease resistance of rice, and the rice has obvious value in practical application. Therefore, the genetic improvement of crops can be carried out through the over-expression of the gene, and the rice blast resistant transgenic rice can be cultivated.
Disclosure of Invention
The invention aims to provide riceOsGDSL1The application of the gene in rice blast resistance can be used for carrying out crop genetic improvement through over-expression of the gene and cultivating the rice blast resistance transgenic rice.
In order to realize the purpose, the following technical scheme is adopted:
rice and its production processOsGDSL1The nucleotide sequence of the gene is shown in SEQ ID NO. 1.
The rice geneOsGDSL1The amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.
Contains riceOsGDSL1An overexpression vector of a gene, said vector comprising a strong 35S promoter.
Rice (Oryza sativa L.) with improved resistance to stressOsGDSL1The gene or the over-expression vector constructed by the gene is applied to rice blast resistance of rice. Transforming the overexpression vector into agrobacterium tumefaciens EHA105 by adopting a cold shock method, and obtaining the overexpression vector by virtue of an agrobacterium-mediated rice mature embryo transformation technologyOsGDSL1Over-expressing rice plants, and carrying out spray inoculation on the rice blast resistance identification. The resistance identification result shows that the over-expression is carried outOsGDSL1The gene obviously enhances the resistance of rice to rice blast.
The invention has the following beneficial effects:
the rice of the inventionOsGDSL1The over-expression of the gene in rice obviously improves the rice blast resistance compared with wild rice, and the gene can be used for cultivating rice blast resistance rice varieties.
Drawings
FIG. 1 map of pCXUN-HA vector.
FIG. 2 verification of pCXUN-HA-OsGDS L1 overexpressing rice.
FIG. 3 analysis of genes by qRT-PCROsGDSL1In thatOsGDSL1The expression level in the over-expressed plants.
FIG. 4OsGDSL1And (5) determining the resistance of the transgenic rice to the rice blast.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
Examples
The invention is realized by constructionOsGDSL1The over-expression vector pCXUN-HA-OsGDS L1 is transferred into mature rice embryos by an agrobacterium-mediated genetic transformation method, the background variety of the transformed rice is wild type rice Nipponbare, and the transformed rice is obtained by screening progenyOsGDSL1And (4) inoculating rice blast germs to the over-expressed strain to determine the disease resistance of the rice.
The method specifically comprises the following steps:
OsGDSL1obtaining gene and constructing overexpression vector pCXUN-HA-OsGDS L1, searching and obtaining from rice genome databaseOsGDSL1Coding sequence ofOsGDSL1The full-length coding sequence of the gene is used as a template to carry out primer design, a forward primer OsGDS L1-HA-F: 5'-ATGAGGCTCTCGGTCTCCGTCG-3' and a reverse primer OsGDS L1-HA-R: 5'-TCAGTGGTGGCGGGTCTCGAG-3' are obtained, wild type rice Nipponbare (NPB) cDNA is used as the template, and KODplus high fidelity enzyme amplification is used for obtaining the geneOsGDSL1After the gel is cut and recovered, the A tail is added by rTaq polymerase. By usingXcmThe method comprises the steps of firstly, linearizing a pCXUN-HA vector by using I enzyme, removing a ccdB gene fragment, connecting an A tail product with the linearized pCXUN-HA, converting escherichia coli competent cells, coating an L B plate (containing 50 mg/L Kan) for culture, carrying out inverted culture at 37 ℃ for 18h to form a single colony, screening out positive clone by using colony PCR, adding 15% of glycerol into bacterial liquid after the sequencing is correct, mixing uniformly, and storing in a refrigerator at-80 ℃.
The specific steps of obtaining the agrobacterium strain of pCXUN-HA-OsGDS L1 are that positive clone plasmids of a recombinant vector pCXUN-HA-OsGDS L1 are transformed into competent cells of agrobacterium tumefaciens EHA105 by a cold shock method, a L B plate (containing 50 mg/L Kan and 50 mg/L Rif) is coated for culture, inverted culture is carried out for 2d at 28 ℃, a single colony is formed, and agrobacterium carrying target genes is screened by colony PCR.
Infecting rice to obtain an OsGDS L1 overexpression strain and verifying:
the agrobacterium containing the pCXUN-HA-OsGDS L1 recombinant vector is transferred into rice callus, positive over-expressed plants are obtained after screening, differentiation and rooting, the authenticity of the plants is identified by PCR amplification of hygromycin resistance gene (HPH) fragments, the result is shown in figure 2, the selected plants all carry hygromycin resistance labels, rice leaf RNA is extracted, the cDNA is obtained through reverse transcription, and the detection of the hygromycin resistance in transgenic rice is carried outOsGDSL1The results of basal expression of the gene are shown in FIG. 3, which shows that the gene is tested in transgenic rice plants compared with wild riceOsGDSL1All up-regulated expression, the expression quantity is increased by 10-100 times, which indicates thatOsGDSL1Overexpression was obtained.
Determination of disease resistance of over-expressed plants: NPB of rice andOsGDSL1culturing over-expressed transgenic rice to IIIIn the leaf-heart stage, the spore liquid of the rice blast fungus Guy11 is inoculated in a spraying way, and the concentration of the spore liquid is 5 × 104Then, after overnight dark culture, the cells were transferred to light conditions for moist culture, and after 7 days, the disease grade was examined. As shown in FIG. 4, the control NPB leaf lesions were of grade 5, most of the lesions were connected, and the leaf tissue was severely necrotic.OsGDSL1The over-expression plants showed disease-resistant phenotype, the lesion size was 2-3 grades, and no serious necrotic lesion was observed, indicating that the over-expression plants were over-expressedOsGDSL1Can improve the rice blast resistance of rice.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> rice gene OsGDS L1 and application thereof in rice blast resistance
<130>4
<160>4
<170>PatentIn version 3.3
<210>1
<211>1119
<212>DNA
<213> Artificial sequence
<400>1
atgaggctct cggtctccgt cgccgcggtg ctcgtcgttc ttgcggcgct ccggccgccg 60
cgggtggcgg tggcgcagaa gtacgcggcc atcttcaact tcggggactc gctggtggac 120
gccggcaacc tcgtcgtgga tggcatcccg gactacctcg ccacggcgcg gctgccgtac 180
gggatgacgt acttcgggta ccccaccggg cgttgctccg acggccgcct cgtcgtcgac 240
ttcatcgcgc aggaggtggg gttgccgctg ctgccgccgt cgaaggcgaa gaacgcgacg 300
ttccaccgcg gcgccaactt cgccatcacc ggcgcgacgt cgctggacac gccgtacttc 360
caggggcgcg ggctggggca cacggtgtgg aactccggct ctctgcacac ccagatcaag 420
tggttccagg acatgaaggc ctccatctgc aagtcccctc aagagtgcag ggacctgttc 480
cggcgatctc tgttcatcgt gggggagttc ggcggcaacg actacaactc gccgctgttc 540
gcgttccggc cactggagga ggtgcacacc ttcgtccccg acgtcgttga ctccatcggc 600
aagggcatcg agaagctgat cgaggaaggg gcggtggagc tggtggtgcc cggggtgctg 660
ccgatcgggt gcttcccggt gtacctctcc atcttccgga agcagccgga gatgtacggt 720
cgccggagcg gctgcatcag ggacctcaac acgctgtcct gggtgcacaa cgctgcgctg 780
cagcggaaga tcgccgagct ccggctcaag caccccggcg tccgcatcat gtacgccgac 840
tactacaccc cggcgatcca gttcgtcctc cacgccgaga aatacgggtt tttgaggcag 900
acgccgcggg cgtgctgcgg ggctccgggg gtcggggagt acaacttcaa cctgacgtcc 960
aagtgcgggg acccggggtc gtacgcgtgc gacgacccgt cgaaccactg gagctgggac 1020
ggcatccacc tcaccgaggc gtcctacggc cacatcgcca agggctggct ctacggcccg 1080
ttcgccgacc ctcccatcct cgagacccgc caccactga 1119
<210>2
<211>372
<212>PRT
<213> Artificial sequence
<400>2
Met Arg Leu Ser Val Ser Val Ala Ala Val Leu Val Val Leu Ala Ala
1 5 10 15
Leu Arg Pro Pro Arg Val Ala Val Ala Gln Lys Tyr Ala Ala Ile Phe
20 25 30
Asn Phe Gly Asp Ser Leu Val Asp Ala Gly Asn Leu Val Val Asp Gly
35 40 45
Ile Pro Asp Tyr Leu Ala Thr Ala Arg Leu Pro Tyr Gly Met Thr Tyr
50 55 60
Phe Gly Tyr Pro Thr Gly Arg Cys Ser Asp Gly Arg Leu Val Val Asp
65 70 75 80
Phe Ile Ala Gln Glu Val Gly Leu Pro Leu Leu Pro Pro Ser Lys Ala
85 90 95
Lys Asn Ala Thr Phe His Arg Gly Ala Asn Phe Ala Ile Thr Gly Ala
100 105 110
Thr Ser Leu Asp Thr Pro Tyr Phe Gln Gly Arg Gly Leu Gly His Thr
115 120 125
Val Trp Asn Ser Gly Ser Leu His Thr Gln Ile Lys Trp Phe Gln Asp
130 135 140
Met Lys Ala Ser Ile Cys Lys Ser Pro Gln Glu Cys Arg Asp Leu Phe
145 150 155 160
Arg Arg Ser Leu Phe Ile Val Gly Glu Phe Gly Gly Asn Asp Tyr Asn
165 170 175
Ser Pro Leu Phe Ala Phe Arg Pro Leu Glu Glu Val His Thr Phe Val
180 185 190
Pro Asp Val Val Asp Ser Ile Gly Lys Gly Ile Glu Lys Leu Ile Glu
195 200 205
Glu Gly Ala Val Glu Leu Val Val Pro Gly Val Leu Pro Ile Gly Cys
210 215 220
Phe Pro Val Tyr Leu Ser Ile Phe Arg Lys Gln Pro Glu Met Tyr Gly
225 230 235 240
Arg Arg Ser Gly Cys Ile Arg Asp Leu Asn Thr Leu Ser Trp Val His
245 250 255
Asn Ala Ala Leu Gln Arg Lys Ile Ala Glu Leu Arg Leu Lys His Pro
260 265 270
Gly Val Arg Ile Met Tyr Ala Asp Tyr Tyr Thr Pro Ala Ile Gln Phe
275 280 285
Val Leu His Ala Glu Lys Tyr Gly Phe Leu Arg Gln Thr Pro Arg Ala
290 295 300
Cys Cys Gly Ala Pro Gly Val Gly Glu Tyr Asn Phe Asn Leu Thr Ser
305 310 315 320
Lys Cys Gly Asp Pro Gly Ser Tyr Ala Cys Asp Asp Pro Ser Asn His
325 330 335
Trp Ser Trp Asp Gly Ile His Leu Thr Glu Ala Ser Tyr Gly His Ile
340 345 350
Ala Lys Gly Trp Leu Tyr Gly Pro Phe Ala Asp Pro Pro Ile Leu Glu
355 360 365
Thr Arg His His
370
<210>3
<211>22
<212>DNA
<213> Artificial sequence
<400>3
atgaggctct cggtctccgt cg 22
<210>4
<211>21
<212>DNA
<213> Artificial sequence
<400>4
tcagtggtgg cgggtctcgag 21
Claims (2)
1. Rice geneOsGDSL1The application in rice blast resistance, the nucleotide sequence of the gene is shown as SEQ ID NO. 1; the rice geneOsGDSL1The amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.
2. Rice geneOsGDSL1The overexpression vector contains a 35S strong promoter, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1.
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| CN201810837943.8A CN108866086B (en) | 2018-07-26 | 2018-07-26 | Rice gene OsGDS L1 and application thereof in resisting rice blast |
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| CN112941050B (en) * | 2021-03-22 | 2022-09-13 | 西南大学 | Chimonanthus nitens GDSL lipase gene CpGLIP1 and application thereof |
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|---|---|---|---|---|
| CN107446946A (en) * | 2016-05-30 | 2017-12-08 | 中国科学院上海生命科学研究院 | Negative regulatory factor and its application in grass resistance signal's approach |
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| Publication number | Priority date | Publication date | Assignee | Title |
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Non-Patent Citations (4)
| Title |
|---|
| Multifunctionality and diversity of GDSL esterase/lipase gene family in rice (Oryza sativa L.japonica) genome: new insights from bioinformatics analysis;Chepyshko等人;《BMC Genomics》;20121231;第13卷(第309期);参见全文 * |
| NCBI Reference Sequence: XM_015792652.1.PREDICTED: Oryza sativa Japonica Group GDSL esterase/lipase At5g45910 (LOC4325258),.《GenBank》.2016, * |
| PREDICTED: Oryza sativa Japonica Group GDSL esterase/lipase At5g45910 (LOC4325258),;NCBI Reference Sequence: XM_015792652.1;《GenBank》;20160301;参见序列和相关信息 * |
| 水稻ARAB-1类似基因的电子克隆及生物信息学分析;鲍思元等;《安徽农业科学》;20151231;第43卷(第31期);参见第41页 * |
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