Haynaldia villosa agglutinin receptor-like kinase gene and expression vector and application

A kind of receptor kinase, expression vector technology, applied in the field of genetic engineering, can solve the problems of increasing manpower, material resources, environmental pollution and so on

Active Publication Date: 2016-08-03
王秀娥 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Powdery mildew can be controlled by fungicides, but chemical control will inevitably increase manpower and material resources, and will also cause ecological problems such as ...

Method used

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  • Haynaldia villosa agglutinin receptor-like kinase gene and expression vector and application
  • Haynaldia villosa agglutinin receptor-like kinase gene and expression vector and application
  • Haynaldia villosa agglutinin receptor-like kinase gene and expression vector and application

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Experimental program
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Effect test

Embodiment 1

[0026] The cloning of implementation example 1Hv-LecRK gene

[0027] In the previous research of our laboratory, it was found that the Hv-CMPG gene played an important role in wheat powdery mildew resistance. In order to better study the mechanism of Hv-CMPG gene resistance to powdery mildew, yeast two-hybrid technology was used to use Hv-CMPG gene as bait , Screening yeast two-hybrid library to obtain Hv-LecRK gene. Primers P1: ATGGCCTTGGTCGTGTGCCCC (SEQ ID NO.3) and P2: TCATCTTCCACCTGAGATGTA (SEQ ID NO.4) were designed, and the Hv-LecRK gene was cloned using the cDNA of A. villosa 24 hours after being induced by Erysiphyllum as a template to obtain the full length of the Hv-LecRK gene. The sequence is SEQIDNO.1( figure 1 ).

Embodiment 2

[0028] Embodiment 2Hv-LecRK gene is subjected to the expression characteristic that powdery mildew induces

[0029] The high resistance to powdery mildew wheat strain 91C43 (number: PI368886, cited from the British Botanic Garden) (references: Qi Lili, Chen Peidu, etc., new resistance to powdery mildew in wheat - gene Pm21, Acta Crops, 1995, 21 (3 ): 257-262) sow in the petri dish to germinate, and transplant to the pot (surroundings are isolated with cylindrical transparent plastic sheets, and the top is closed with filter paper to form an environment without powdery mildew) after dew white. At the three-leaf stage, shake off the fresh spores of Nanjing local mixed powdery mildew cultured on the susceptible variety Sumai No. 3 on the seedlings of A. The leaves of T. villosa inoculated with powdery mildew were stored at 16°C. Samples were taken at 0h, 30min, 45min, 1h, 2h, 6h, 12h, 24h, 48h, and 72h after inoculation, and stored in a -70°C refrigerator for later use. TRIZOL ...

Embodiment 3

[0031] Embodiment 3Hv-LecRK sense expression vector construction

[0032] Utilizing the above-mentioned C. villosa cDNA induced by powdery mildew as a template, PCR was carried out with the primer pair P3 (CGGGATCCATGGCCTTGGTCGTGTGCC (SEQ ID NO.5)) and P4 (GGGGTACCTCATCCTCACCTGAGATG (SEQ ID NO.6)) capable of amplifying the Hv-LecRK gene protein coding region Amplify and recover the amplified fragment. Insert the amplified target fragment into the multiple cloning site BamHI behind the 35S promoter of the vector pBI220 (JeffersonRA, KavanaghTA, BevanMW.GUSfusions:beta-glucuronidaseassensitiveandversatilegenefusionmarkerinhigherplants.EMBOJ.1987,6:3901-3907) with BamHI and KpnI double digestion and KpnI. Thus the target gene Hv-LecRK is cloned to the downstream of the strong promoter 35S to obtain the expression vector pBI220:LecRK( image 3 ).

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Abstract

The invention discloses a tufting wheat lectin receptor kinase gene, its expression carrier and application, and belongs to the field of genetic engineering. The cDNA sequence of Hv-LecRK is SEQ ID NO.1 and the encoded amino acid sequence is SEQ ID NO.2. The gene comes from diploid W. villosa (Haynaldia villosa, L., 2n=2x=14, genome VV), and can interact with powdery mildew resistance-related gene Hv-CMPG, and is resistant to powdery mildew in diploid W. villosa Enhanced expression induced by powdery mildew. The gene was transformed into the susceptible wheat variety Yangmai 158 by transient expression, and the results showed that the overexpression of Hv-LecRK gene could reduce the haustoria index of Yangmai 158. Therefore, Hv-LecRK is expected to be used in genetic engineering breeding, and introducing it into wheat varieties susceptible to powdery mildew is expected to improve the powdery mildew resistance of wheat.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and discloses a lectin-like receptor kinase gene, an expression carrier and application thereof. Background technique [0002] Wheat powdery mildew caused by the obligate parasitic fungus Blumeria graminis DCf.sp.tritici is one of the most serious leaf diseases of wheat (Triticum aestivum L., 2n=6x=42, genome AABBDD), in China and many other countries Occurs widely. The disease can occur throughout the growth period of wheat, and can infect all parts of aboveground wheat plants, mainly leaves and leaf sheaths. [0003] With the influence of cultivars, cultivation conditions and climatic conditions, the harm of wheat powdery mildew in my country is increasing. Powdery mildew can be controlled with fungicides, but chemical control will inevitably increase the input of manpower and material resources, and will also cause ecological problems such as environmental pollution. Therefore, discovering...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/12C12N15/82A01H5/00
Inventor 王秀娥程江月李颖波肖进王宗宽王海燕曹爱忠邢莉萍祝燕飞穆罕默德 法希姆袁春霞郝永利赵佳陈炜陈忠明
Owner 王秀娥
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