Cotton GbSTK gene, encoding protein thereof and application for resisting verticillium wilt of plants

A gene coding and anti-Verticillium wilt technology, applied in the field of genetic engineering, can solve the problem that the variety and quantity cannot meet the needs of crop breeding and production

Inactive Publication Date: 2011-01-12
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, the types and quantities of genes with obvious resist...

Method used

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  • Cotton GbSTK gene, encoding protein thereof and application for resisting verticillium wilt of plants
  • Cotton GbSTK gene, encoding protein thereof and application for resisting verticillium wilt of plants
  • Cotton GbSTK gene, encoding protein thereof and application for resisting verticillium wilt of plants

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The cloning of embodiment 1GbSTK gene

[0030](1) Cotton planting and inoculation with Verticillium dahliae: the sea island cotton Pima90-53 seeds were develored with sulfuric acid, soaked at room temperature overnight, and the seeds that germinated uniformly were selected and sowed in a nutrient bowl equipped with frog stones. When the cotton seedlings grow to the first true leaf, they are inoculated with the spore suspension of Verticillium dahliae.

[0031] (2) RNA extraction: RNA extraction and reverse transcription use RNA extraction kit and reverse transcription kit (purchased from Beijing Aolibo Biotechnology Co., Ltd.), and operate according to the instructions to obtain double-stranded cDNA (dsDNA) materials.

[0032] (3) cDNA-AFLP: The obtained double-stranded DNA (dsDNA) is digested, ligated, and sequenced to obtain the target differential fragments, and then use the NCBI website expressed sequence tag (expressed sequence tag, EST) database to splice the targ...

Embodiment 2

[0091] Embodiment 2 GbSTK gene prokaryotic expression

[0092] (1) According to the cloned cotton GbSTK gene, design two pairs of primers and add enzyme cutting sites. The primer sequences are:

[0093] Forward primer: 5′-GTC GGTACC AGGAAGAATTCAAGCAAATCC-3′

[0094] (The underline indicates the Kpn I restriction site)

[0095] Reverse primer: 5′-TAC CTGCAG TCAAGGGGCCGTAGGTTGTG-3′

[0096] (The underline indicates the Pst I restriction site)

[0097] The recovered product obtained by PCR amplification (such as image 3 Shown is the amplification result of the open reading frame of the GbSTK gene of the present invention, 1% agarose gel, wherein 1 is the PCR amplification product, M is the DL2000 DNA marker, and a specific band of about 1.3kb is obtained in 1 lane). After recovering the amplified fragment, it was ligated with the pGEM-T vector, and the ligated product was transformed into Escherichia coli competent cells by heat shock method, and positive clones were scr...

Embodiment 3

[0112] Functional analysis and prediction of embodiment 3GbSTK protein

[0113] 1. Using onion epidermal cells as materials to study the distribution of the protein expressed by GbSTK in the cell (that is, the study of the subcellular localization of the protein expressed by the GbSTK gene):

[0114] (1) Design primer sequences: Utilize DNAMAN software to design PCR primers, the sequences are as follows:

[0115] C5-Y: 5′-GTCGACATGAAGAAGAAGCTTGTG-3′

[0116] C3-Y: 5′-CCCGGGAGGGGCCGTAGGTTGTGTAAC-3′

[0117] (2) Amplification of the open reading frame: use the plasmid as a template to amplify the open reading frame of the gene without the stop codon. White spot screening, sequencing, and positive clones containing pGEM-STK with correct open reading frame sequences were screened out for subsequent tests.

[0118] (3) Construction of fusion expression vector:

[0119] The intermediate vector pGEM-STK and the expression vector pCamE-GFP were digested with Sal I and ApaI, respec...

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Abstract

The invention relates to a cotton GbSTK gene and encoding protein thereof. Sea island cotton Pima90-53 is taken as a material, a cDNA-AFLP technology is used for screening to obtain differential fragments related to verticillium wilt resistance, and then, a reverse transcription-polymerase chain reaction (RT-PCR) and a RACE (rapid-amplification of cDNA ends) technology are used for amplifying to obtain a resistance gene of cotton verticillium wilt, namely a serine/threonine protein kinase (GbSTK) gene. The functional studies of the gene and the encoding protein thereof show that the GbSTK gene has certain effect on resisting verticillium wilt.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the cotton GbSTK gene, its encoded protein and its application in plant resistance to verticillium wilt. Background technique [0002] Verticillium wilt is one of the main diseases in crop production, and it is seriously harmful to the production of cotton, tomato, eggplant, rice, cucumber and sunflower. The world's annual loss of lint production accounts for about 10-20% of lint production, and the year of severe disease can reach 25-30%. Therefore, preventing the occurrence of Verticillium wilt has become an urgent problem to be solved in the field of crop production. Studies have reported that sea-island cotton and upland cotton are used for cross-breeding with high Verticillium wilt resistance materials, but after the F2 generation is separated, there is little gene exchange between the two, so it is difficult to directly use sea-island cotton to breed high Verticillium wi...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A01H5/00
Inventor 马峙英王省芬吴立柱周洪妹李懿义张桂寅吴立强李志坤
Owner HEBEI AGRICULTURAL UNIV.
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