CN103820470B - Maize sheath blight disease-resistant related gene GRMZM2G174449 and application thereof - Google Patents
Maize sheath blight disease-resistant related gene GRMZM2G174449 and application thereof Download PDFInfo
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Abstract
The present invention relates to field of plant genetic, be specifically related to functional verification and the application of a corn anti-banded sclerotial blight genes involved GRMZM2G174449, utilize strong promoter to drive transgenic technology, the overexpression carrier of GRMZM2G174449 gene is proceeded in rice varieties and spends 11.The resistance of genetic transformation paddy rice to sheath blight fungus that GRMZM2G174449 gene expression amount significantly improves obviously strengthens, and proves that GRMZM2G174449 gene plays a significant role in Rice Resistance banded sclerotial blight.
Description
Technical field
The present invention relates to field of plant genetic, provide a maize sheath blight disease-resistant related gene GRMZM2G174449 and functional verification thereof, contriver finds that the resistance of genetic transformation paddy rice to sheath blight fungus that GRMZM2G174449 gene expression amount significantly improves obviously strengthens.
Background technology
Plant, in the process of growth, is subject to the infringement of multiple pathogen.The phytopathy original of a great variety, comprises virus, bacterium, fungi and nematode etc.Pathogen invaded plants causes two kinds of results: (1) pathogenic agent is successfully bred in host plant, causes associated conditions; (2) host plant produces disease resistance response, kills pathogen or stops it to grow.Utilizing resistant gene resource to improve the disease resistance of plant, is the fundamental solution of pre-disease prevention protection of the environment again simultaneously.
The disease resistance response of plant is the complex process that polygene participates in regulation and control.The gene of involved in plant disease resistance response is divided into two classes: (1) disease-resistant gene, also known as R(resistance) gene and (2) disease-resistant related gene.
According to the understanding of current people's enantiopathy gene function, the product of this genoid is mainly as acceptor, and direct or indirect and pathogenic proteins interacts, and starts defense signaling path (Tang etc., 1996, the Science274:2060-2063 in plant materials; Baker etc., 1997, Science276:726-733; Jia etc., 2000, EMBOJ.19:4004-4014; Dangl and Jones, 2001, Nature411:826-833; Nimchuk etc., 2001, Curr.Opin.PlantBiol.4:288-294).The disease resistance response of disease-resistant gene mediation is strong, is good genetic resources.But due to following reason, make to utilize disease-resistant gene to improve plant resistance to environment stress to be restricted: the resource-constrained of (1) disease-resistant gene, the disease-resistant gene of the important disease bacterial leaf-blight of the opposing paddy rice as known at present is less than 30, and the disease-resistant gene resisting the important disease-rice blast of another paddy rice also only has about 40; (2) disease-resistant gene has cause of disease kind and cause of disease physiological strain specificity, is disease-resistantly limited in scope; (3) because the rapid mutation of cause of disease, the effect of a disease-resistant gene often just loses after several years or more than ten years.
Disease-resistant related gene refers to the gene of all participation disease resistance responses except disease-resistant gene, and their coded product participates in resistance signal molecule in synthesis plant materials, participates in intracellular signaling or participate in defense response etc.The common feature of this genoid be pathogeny evoked after their expression amount raise or reduce, therefore people can according to the difference of the expression amount of pathogeny evoked front and back gene plant identification disease-resistant related gene (Maleck etc. on a large scale, 2000, NatureGenet.26:403-410; Schenk etc., 2000, Proc.Natl.Acad.Sci.USA97:11655-11660; Zhou etc., 2002, ScienceinChina45:449-467).At present, the understanding of people's enantiopathy genes involved is limited.According to having been reported, resistance capacity during most disease-resistant related gene independent role may be less than disease-resistant gene.But according to following reason, they are genetic resourceses of worth Devoting Major Efforts To Developing: (1) does not need directly due to the product of most disease-resistant related gene and pathogen interacts, and this genoid is the genetic resources with durable resistance; (2) disease resistance response that most of disease-resistant related gene participates in does not have cause of disease specificity, and therefore they are the genetic resourceses with resistance of wide spectrum; (3) aboundresources of this genoid.
The disease resistance response of plant can be divided into two large classes.Investigators give different names to this two large class disease resistance response for a long time, as vertical resistance and horizontal resistance (VanDerPlank etc., 1968, DiseaseResistanceinPlants, Academic, NewYork), Qualitative resistant and Quantitative (Ou etc., 1975, Phytopathology65:1315-1316), complete resistance and partial resistance (Parlevliet, 1979, Annu.Rev.Phytopathol.1:203-222).Qualitative resistant (or vertical resistance or complete resistance) is the disease resistance response of disease-resistant gene mediation.Quantitative (or horizontal resistance or partial resistance) is by quantitative trait locus (quantitativetraitlocus, QTL) disease resistance response regulated and controled, it is considered to without cause of disease specificity, and resistance lasting (Roumen, 1994, RiceBlastDisease, Zeigler etc. write, CABInternational, Cambridge, UK, pp.245-265).At present, the gene essence of people to plant disease-resistant QTL is not clear.Therefore, although identified a large amount of disease-resistant QTL, as rice bacterial blight resistance QTL(Li etc., 1999, Mol.Gen.Genet.261:58-63), blast resisting QTL(Wang etc., 1994, Genetics136:1421-1434; Chen etc., 2003, Proc.Natl.Acad.Sci.USA100:2544-2549), anti-banded sclerotial blight QTL(Li etc., 1995, and viral diseases QTL(Albar etc. Theor.Appl.Genet.91:382-388), 1998, Theor.Appl.Genet.97:1145-1154) etc., but these Resistance QTLs are not favourably utilised for the improvement of disease resistance of plant.
Research in recent years finds that the chromosome position of a lot of disease-resistant related gene is corresponding with disease-resistant QTL, points out these disease-resistant related genes may be exactly corresponding QTL.Chromosome position this phenomenon corresponding with disease-resistant QTL of disease-resistant related gene is all observed in various plants, comprises paddy rice (Xiong etc., 2002, Chinese science 45:518-526; Ramalingam etc., 2003, Mol.Plant-MicrobeInteract.16:14-24; Wen etc., 2003, Mol.Gen.Genomics269:331-339; Chu etc., 2004, Mol.Gen.Genomics271:111-120), wheat (Faris etc., 1999, Theor.Appl.Genet.98:219-225), beans (Geffroy etc., 2000, Mol.Plant-MicrobeInteract.13:287-296) and potato (Trognitz etc., 2002, Mol.Plant-MicrobeInteract.15:587-597).These results are adopt candidate gene strategy to be separated, to clone and utilize the gene of disease-resistant QTL to provide foundation.
Corn and paddy rice are food crop important in the world, but banded sclerotial blight usually causes the decline of its yield and quality, and corn and Rhizoctonia solani Kuhn can infect mutually.Therefore, understand the pathogenesis of banded sclerotial blight, contribute to utilizing high effective way to improve the resistance of corn and rice varieties, control the generation of disease, the loss reducing or avoid Plant diseases to bring.Separating clone disease-resistant related gene is the prerequisite to corn and paddy disease-resistant study mechanism.Meanwhile, compared with the application of disease-resistant gene, the application of disease-resistant related gene can provide plant more wide spectrum and long-acting resistance.Carried out the improvement of corn and rice varieties by overexpression disease-resistant related gene, the disease resistance of plant will be strengthened further, widen the anti-spectrum of plant.These aspects are that employing conventional plant breeding and improving technology institute are inaccessiable.Therefore how to utilize the clone of corn disease-resistant gene to obtain disease-resistant plant and become one of problem demanding prompt solution.
Summary of the invention
The present inventor is for the situation of above-mentioned prior art, provide the DNA fragmentation of the disease-resistant related gene GRMZM2G174449 complete coding region section that clones and isolates from corn, its improvement corn and paddy rice is utilized to resist the ability of disease, the sequence of its gene is as shown in sequence table SEQ IDNO.1, and its encoding amino acid sequence is as shown in sequence table SEQ IDNO.2.The resistance of genetic transformation paddy rice to sheath blight fungus that GRMZM2G174449 gene expression amount significantly improves obviously strengthens, and can give plant and produce disease resistance response to the disease caused by sheath blight fungus, obtain high disease-resistant plant after visible employing overexpression.
Contriver provide firstly the DNA fragmentation of the disease-resistant related gene GRMZM2G174449 complete coding region section that clones and isolates from corn, the sequence of its gene is as shown in sequence table SEQ IDNO.1, and its encoding amino acid sequence is as shown in sequence table SEQ IDNO.2.
After obtaining this fragment, it is connected with suitable carrier, vegetable cell can be proceeded to, produce transgenic plant, find that the resistance of genetic transformation paddy rice to sheath blight fungus that GRMZM2G174449 gene expression amount significantly improves obviously strengthens, prove that GRMZM2G174449 gene plays a significant role in Rice Resistance banded sclerotial blight, plant can be given after therefore confirming this gene of overexpression disease resistance response is produced to the disease caused by sheath blight fungus, obtain high disease-resistant plant.
This gene fragment derives from corn B73, it is obtained by the primer amplification corn B73cDNA of particular design, wherein said primer comprises primer 1, its sequence of 5 '-ATGGATGCTCATGCCGTG-3 ' is as shown in sequence table SEQ IDNO.3, primer 2, its sequence of 5 '-CTAAGGATTTGATGTGTAAGCC-3 ', as shown in sequence table SEQ IDNO.4, utilizes the transgenic technology of strong promoter driving principle afterwards, is proceeded in rice varieties by the overexpression carrier of GRMZM2G174449 gene and spends 11.The resistance of genetic transformation paddy rice to sheath blight fungus that GRMZM2G174449 gene expression amount significantly improves obviously strengthens, prove that GRMZM2G174449 gene can play a significant role in Rice Resistance banded sclerotial blight, the resistance of the transfer-gen plant obtained as utilized overexpression to sheath blight fungus obviously strengthens.
Why adopt above-mentioned method, mainly due to the disease-resistant related gene of clone is proceeded to susceptible plant, contribute to producing new disease-resistant plants.Particularly can use genetic transfoumation cumulative multiple resistant gene in plant, and can not produce in traditional breeding technology with the linked gene group sequence occurred.And the clone of disease-resistant related gene overcomes the prerequisite that traditional breeding method can not shift disease-resistant related gene problem between plant species.
In sum, the present inventor demonstrates the function of a corn disease-resistant related gene in the world first, utilize its function finally to find to adopt and can give plant after overexpression and produce disease resistance response to the disease caused by sheath blight fungus, obtain high disease-resistant plant.
Accompanying drawing explanation
Fig. 1. the detection of expression of GRMZM2G174449 gene after inoculation sheath blight fungus YWK62 and YWK196;
Result shows, after inoculation sheath blight fungus, GRMZM2G174449 is induced to express, and reaches peak value and reduce gradually subsequently after inoculation 8h;
Fig. 2 is GRMZM2G174449 overexpression T1 for genetic transformation plant inoculation sheath blight fungus result signal gray-scale map after YWK1967 days;
Fig. 3 is the histogram of result in Fig. 2;
In figure, overexpression T1 obviously contrasts WT than flower 11(in rice varieties for the scab that formed afterwards for PXO997 days of genetic transformation plant (pCXUN::GRMZM2G174449-10,11 and 12) inoculation sheath blight fungus) short;
In spend 11 for genetic transformation acceptor material.
Embodiment
The present invention is defined further in following examples, according to above description and these embodiments, those skilled in the art can determine essential characteristic of the present invention, and when not departing from spirit and scope of the invention, various change and amendment can be made, to make its applicable various uses and condition to the present invention.Except special indicating, be of the present inventionly state of the art;
Expression pattern after embodiment 1:GRMZM2G174449 gene inoculation sheath blight fungus
In order to detect GRMZM2G174449 gene by sheath blight fungus abduction delivering pattern, expression after utilizing qRT-PCR to have detected this gene inoculation pathogenic bacteria, the primer is primer 3, its sequence of 5 '-TTCTGCATCTGCTGCACATG-3 ' is as shown in sequence table SEQ IDNO.5, primer 4, its sequence of 5 '-GGTGCCTACGGTCGATCGT-3 ' is as shown in sequence table SEQ IDNO.6.
Analytical results shows, the induction (as shown in Figure 1) of hard to bear YWK196 and YWK62 of GRMZM2G174449 gene energy, illustrates that its gene take part in the Resistant reaction of corn to banded sclerotial blight.
Embodiment 2: separating clone GRMZM2G174449 gene
Contriver is according to database GRMZM2G174449 gene order design primer (primer 1, its sequence of 5 '-ATGGATGCTCATGCCGTG-3 ' is as shown in sequence table SEQ IDNO.3, primer 2, its sequence of 5 '-CTAAGGATTTGATGTGTAAGCC-3 ' is as shown in sequence table SEQ IDNO.4) be used for obtaining GRMZM2G174449 gene.Extract corn B73RNA and obtain cDNA by reverse transcription, using cDNA as template, carrying out pcr amplification;
Response procedures is as follows: denaturation 94 DEG C of 5min, sex change 94 DEG C of 40s, and anneal 54 DEG C of 40s, extends 72 DEG C of 1.5min, reacts 35 circulations, rear extension 72 DEG C of 7min;
After end, reclaim test kit with the DNA of Kang Wei company to reclaim and purified amplified fragments, then the DNA fragmentation of purifying is connected to (Promega company) in carrier pGEM-T, Transformed E .coliDH5 ɑ competent cell, select positive colony upgrading grain, order-checking is completed by Hua Da gene, obtains the GRMZM2G174449 fragment that length is 1526bp, has the DNA sequence dna of sequence table SEQ IDNO.1.
The functional verification of embodiment 3:GRMZM2G174449 gene
The cDNA fragment of above-mentioned purifying is connected into overexpression carrier pCXUN by TA clone, after heat-shock transformed E.coliDH5 α competent cell, bacterium colony PCR is carried out to recon, agarose gel electrophoresis detects, and includes the band identical with object clip size in recon.Plasmid is extracted by after the recon enlarged culturing identified, with former sequence alignment after plasmid order-checking, the fragment be connected into is full-length cDNA and does not have base mutation and disappearance, proves that GRMZM2G174449 overexpression carrier pCXUN::GRMZM2G174449 successfully constructs.
Adopt agrobcterium-mediated transformation (Lin and Zhang, Optimisingthetissuecultureconditionsforhighefficiencytra nsformationofindicarice, 2005, PlantCellRep.23:540-547) spend 11 by overexpression vector introduction rice varieties.The genetic transformation plant obtained is named as pCXUN::GRMZM2G174449.The present invention obtains independent transformation plant 20 strain altogether, and wherein positive plant is 13 strains.Respectively to T1 generation 3 transgenic line pCXUN::GRMZM2G174449-10,11,10 individual plants of 12 carry out the inoculated identification of Rhizoctonia solani Kahn.Result shows, inoculation sheath blight fungus is after YWK1967 days, transfer-gen plant average scab compared with wild-type of overexpression GRMZM2G174449 shortens 3.12cm, 3.36cm and 3.79cm(table 1, shown in Fig. 2), illustrate that GRMZM2G174449 take part in the Resistant reaction of paddy rice to banded sclerotial blight, visible GRMZM2G174449 improves in sharp eyespot resistance can be widely used in crop improvement.
The scab length of table 1GRMZM2G174449 overexpression plant inoculation after YWK1967 days
Claims (1)
1. corn anti-banded sclerotial blight genes involved GRMZM2G174449 improves the application in sharp eyespot resistance in crop improvement, it is characterized in that: described gene order is as shown in SEQIDNO.1.
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| CN104498503A (en) * | 2014-11-28 | 2015-04-08 | 安徽拜森生物科技有限公司 | Corn sheath blight anti-disease gene GRMZM2G127328 and application thereof |
| CN104498507B (en) * | 2014-12-25 | 2017-08-04 | 安徽拜森生物科技有限公司 | Maize sheath blight disease-resistant gene GRMZM2G456997 and application |
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| the resistance to banded leaf and sheath blight in maize of 282 inbred lines;wensheng chen et al.;《african journal of agricultural research》;20130502;第8卷(第16期);1547-1552 * |
| zea mays hypothetical protein(LOC100273307),mRNA,NM_001147748.1;genbank;《genbank》;20130421;origin * |
| 玉米纹枯病抗性相关基因序列克隆与分析;曾兴;《中国优秀硕士学位论文全文数据库 农业科技辑》;20090215(第2期);全文 * |
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