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Plant disease-resistance regulating gene SlASN2 and application thereof

A technology for regulating genes and genes, applied in plant products, genetic engineering, botanical equipment and methods, etc., can solve the problems of long breeding cycle and limited range of disease-resistant resources, and achieve short breeding cycle and available disease-resistant resources Wide range, relatively simple and convenient operation

Inactive Publication Date: 2016-04-06
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to natural genetic isolation, traditional disease-resistant breeding has significantly limited the available range of disease-resistant resources. Only disease-resistant resources with close genetic relationships can be used, and multiple crosses and backcrosses are required. Therefore, the breeding cycle is long and requires A lot of manpower and material resources

Method used

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  • Plant disease-resistance regulating gene SlASN2 and application thereof
  • Plant disease-resistance regulating gene SlASN2 and application thereof
  • Plant disease-resistance regulating gene SlASN2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The present invention establishes a set of high-quality disease-resistant regulation gene SlASN2 cloned by the inventor, and adopts genetic engineering technology to create and obtain disease-resistant plant materials with a wide range of disease-resistant objects. The main steps include:

[0029] 1) Cloning and preservation of tomato (Solanumlycopersicum) S1ASN2 gene

[0030] The tomato (Solanumlycopersicum) S1ASN2 gene provided by the present invention is cloned through the following steps. First, primers SlASN2-F (5'-ccgagctcatgtgtggaatacttgcaatt-3', the italic part is the SacⅠ restriction site) were designed according to the ASN2 sequence in the tomato genome database (sequence shown in SEQID: 3), and SlASN2-R (5'- caggtacctcatagttctttaacctcata-3', the part in italics is the KpnI restriction site) (sequence shown in SEQID: 4). The total RNA of Nicotiana benthamiana leaves was extracted with TRIZOL reagent, the SlASN2cDNA was amplified by RT-PCR method, the PCR pro...

Embodiment 2

[0042] Example 2 Acquisition of broad-spectrum disease-resistant transgenic S1ASN2 gene tobacco plants

[0043] The main operation steps include:

[0044] 1) Construction and acquisition of SlASN2 gene expression structure

[0045] SacI / KpnI double enzyme digestion was performed on the vector carrying the SlASN2 gene sequence owned by the inventor's laboratory, and the ORF sequence of the SlASN2 gene was recovered by electrophoresis and gel tapping, and subcloned into the strong promoter of the plant expression vector pCHF3 - cauliflower mosaic virus ( After the CaMV)35S promoter, a plant expression construct pCHF3::SIASN2 capable of strongly expressing the SIASN2 gene was obtained.

[0046] 2) Acquisition of Agrobacterium transformed with SlASN2 gene expression construct

[0047] Take 1-2 μl of SlASN2 gene expression construct pCHF3::SlASN2, add it to 40 μl of EHA105 Agrobacterium competent cells, mix quickly, and use Bio-RadGenePulserII electric shock instrument under the ...

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Abstract

The invention provides a plant disease-resistance regulating gene. The gene has an open reading frame length of 1680bp, is used for encoding asparagine synthetase, and consists of 559 amino acids. The gene encoded product comprises a glutamine aminotransferase structural domain and an asparagine synthetase structural domain. The broad spectrum effect of the SlASN2 gene on plant disease resistance is stated in the invention. The gene is a resource of high-quality disease-resistance regulating genes, and a disease-resistance material acquired by utilizing the gene has the advantage of wide disease resistance spectrum. The gene is used for widely influencing the resistance of multiple diseases caused by various pathogens by regulating nitrogen metabolism. The host resistance of nicotiana tabacum on tobacco mosaic virus, pseudomonas tabaci and sclerotinia and non-host resistance on xanthomonas oryzae can be remarkably enhanced by means of overexpression of the SlASN2 gene. The plant disease-resistance regulating gene is applicable to development and selection of disease-resistance plant materials and varieties disease-resistance object with wide disease-resistance objects.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a plant disease resistance regulation gene (SIASN2) and its application. Background technique [0002] 1. Plant gene cloning technology [0003] There are many methods for plant gene cloning. With the increase of plant species whose genome sequence has been determined, the method of plant gene cloning based on database sequence has been more and more widely used. The operation steps of the method mainly include primer design based on conserved sequences, RNA extraction from target plant tissue, reverse transcriptase-polymerase chain reaction (RT-PCR), connection of PCR product and vector, bacterial transformation of the connection product, and plasmid extraction. , enzyme digestion test, sequencing analysis, etc. [0004] 2. Plant transgenic technology [0005] It is used to introduce exogenous genes into target plants, so as to obtain transgenic plants carrying exogenous ge...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00C12N15/82A01H5/00
CPCC12N9/93C12N15/8281C12N15/8282C12N15/8283C12Y603/05004
Inventor 蔡新忠吕琳慧
Owner ZHEJIANG UNIV
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