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A tufted wheat lectin receptor kinase gene and its 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: 2019-08-13
王秀娥 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 environmental pollution. Therefore, discovering disease-resistant genes and cultivating disease-resistant varieties are effective measures to prevent and control wheat powdery mildew

Method used

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  • A tufted wheat lectin receptor kinase gene and its expression vector and application
  • A tufted wheat lectin receptor kinase gene and its expression vector and application
  • A tufted wheat lectin receptor kinase gene and its expression vector and application

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Experimental program
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Effect test

Embodiment 1

[0026] Implementation Example 1 Cloning of Hv-LecRK gene

[0027] In the previous research of our laboratory, it was found that Hv-CMPG gene plays an important role in wheat powdery mildew resistance. In order to better study the mechanism of Hv-CMPG gene resistance to powdery mildew, the yeast two-hybrid technique was used with Hv-CMPG gene as bait , Screening yeast two-hybrid library to obtain Hv-LecRK gene. Design primers P1: ATGGCCTTGGTCGTGTGCCCC (SEQ ID NO. 3) and P2: TCATCTTCCACCTGAG ATGTA (SEQ ID NO. 4), and clone the Hv-LecRK gene to obtain Hv-LecRK by using the 24h cDNA of P. tufts after being induced by powdery mildew as a template. The full length of the gene, the sequence is SEQ ID NO.1 ( figure 1 ).

Embodiment 2

[0028] Example 2 Expression characteristics of Hv-LecRK gene induced by powdery mildew

[0029] The high resistance to powdery mildew wheat line 91C43 (number: PI368886, cited from the British Botanic Garden) (reference: Qi Lili, Chen Peidu, etc., a new source of resistance to wheat powdery mildew-gene Pm21, Acta Agronomica Sinica, 1995, 21(3) ):257-262) sown in a petri dish to germinate, and then transplant it to a pot (the surrounding is isolated with a cylindrical transparent plastic sheet, and the top is sealed with a filter paper to form a powdery mildew-free environment). At the three-leaf stage, the fresh spores of Nanjing local mixed powdery mildew cultured on the susceptible variety Sumai No. 3 were gently shaken off the seedlings of P. tutus. After inoculation with powdery mildew, the leaves of Triticum aestivum were stored at 16℃. Samples were taken at 0h, 30min, 45min, 1h, 2h, 6h, 12h, 24h, 48h, 72h after inoculation, and stored in a refrigerator at -70℃ for later us...

Embodiment 3

[0031] Example 3 Construction of Hv-LecRK sense expression vector

[0032] Using the above-mentioned powdery mildew-induced Triticum aureus cDNA as a template, primer pairs P3 (CGGGATCCATGGCCTTGGTCGTGTGCC (SEQ ID NO. 5)) and P4 (GGGGTACCTCATCTTCCACCTGAGATG (SEQ ID NO. 6) that can amplify the protein coding region of the Hv-LecRK gene )) Perform PCR amplification and recover the amplified fragments. The amplified target fragment was inserted into the vector pBI220 (Jefferson RA, Kavanagh TA, BevanMW. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusionmarker in higher plants. EMBO J. 1987, 6: 3901) by double digestion with BamHI and KpnI. -3907) the multiple cloning site behind the 35S promoter between BamHI and KpnI. Thus, the target gene Hv-LecRK was cloned to the downstream of the strong promoter 35S, and the expression vector pBI220:LecRK ( image 3 ).

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Abstract

The invention discloses a haynaldia villosa agglutinin receptor-like kinase gene, its expression vector and an application thereof, and belongs to the field of gene engineering. According to the invention, cDNA sequence of Hv-LecRK is SEQ ID NO.1 and amino acid sequence coded by Hv-LecRK is SEQ ID NO.2. The gene is derived from Haynaldia villosa L.(2n=2x=14, genome VV), and can interact with powdery mildew resistance-related gene Hv-CMPG. Expression of the gene is enhanced when the gene is induced by powdery mildew in the powdery mildew resistant diploid haynaldia villosa. Through transient expression, the gene is transformed to a susceptible wheat variety Yangmai 158. It shows through results that overexpression of the Hv-LecRK gene can reduce haustorium index of Yangmai 158. Thus, the Hv-LecRK is expected to be used in genetic engineering breeding. By introducing the Hv-LecRK into a wheat variety susceptible to powdery mildew, powdery mildew resistance of wheat is expected to be raised.

Description

Technical field [0001] The present invention belongs to the field of genetic engineering, and discloses a lectin receptor kinase gene of Triticum acuminata and its expression vector and application. Background technique [0002] Wheat powdery mildew caused by the obligate parasitic fungus Blumeria graminis DC f.sp.tritici is one of the most serious leaf diseases in wheat (Triticum aestivum L., 2n=6x=42, genome AABBDD). It occurs widely in China and many other countries. The disease can occur throughout the growth period of wheat, and can invade all parts of the above-ground plant of wheat, 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 currently increasing. Powdery mildew can be controlled with fungicides, but chemical control will inevitably increase the input of manpower and material resources, and also cause ecological problems such as environmental poll...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/82A01H5/00A01H6/46
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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