Haynaldia villosa CERK1-V gene, and encoded protein and application thereof

A CERK1-V, villous wheat technology, applied in the field of genetic engineering, can solve the problems of increasing manpower, material resources, environmental pollution, etc.

Active Publication Date: 2019-09-13
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although powdery mildew can be controlled by fungicides, chemical control will inevitably increase the input of manpower and material resources, and will also cause ecological problems...

Method used

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  • Haynaldia villosa CERK1-V gene, and encoded protein and application thereof
  • Haynaldia villosa CERK1-V gene, and encoded protein and application thereof
  • Haynaldia villosa CERK1-V gene, and encoded protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Cloning of CERK1-V gene and expression characteristics induced by powdery mildew

[0026] Homologous cloning primers P1 (ATGGAAGCTCCGCTCCTC, SEQ ID NO.3) and P2 (TCATCTCCCGGACATG, SEQ ID NO.4) were designed to clone the 1866bp sequence in the cDNA induced by Erysiphe spp. Shown in NO.1. The sequence encodes 621 amino acids, the sequence is shown in SEQ ID NO.2, and the gene is named CERK1-V.

[0027] The powdery mildew resistant wheat seeds (references: Qi Lili, Chen Peidu, et al., New source of resistance to powdery mildew in wheat—gene Pm21, Acta Crops, 1995, 21(3):257-262) were sown in a petri dish to germinate , transplanted to pots after dew whitening (isolated with cylindrical transparent plastic sheets around, and closed with filter paper at the top to form an environment without powdery mildew). 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...

Embodiment 2

[0029] Example 2 Construction of CERK1-V gene overexpression vector silencing vector

[0030] Using the above-mentioned CERK1-V gene cloning vector pMD18T-CERK1-V, the primer pair P5 (CG GGATCC ATGGAAGCTCCGCTCCTC, SEQ ID NO.7) and P6 (GA AGGCCT TCTCCCGGACATGAGGTTC, SEQ ID NO.8) for PCR amplification, and recovered amplified fragments. Insert the amplified target fragment into the vector pBI220 by double digestion with BamHI and StuI (references: Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: beta-glucuronidase as a sensitive and versatile genefusion marker in higher plants. EMBO J.1987 , 6:3901-3907) between the multiple cloning site BamHI and StuI behind the 35S promoter. Thus, the CERK1-V gene overexpression vector pBI220-CERK1-V (attached figure 2 ).

Embodiment 3

[0031] Example 3 Using single cell transient expression technology to transfer CERK1-V gene overexpression vector into wheat leaves

[0032] Single-cell transient expression technology is a reliable and rapid method for identifying gene functions (reference: Schweizer, Pokorny et al. A Transient Assay System for the Functional Assessment of Defense-Related Genes in Wheat Molecular Plant-MicrobeInteractions.1999,12:647 -654). In this study, the single-cell transient expression method was used to wrap plasmid DNA on the outer layer of metal particles, and the metal particles were bombarded to the epidermal cells of wheat leaves with the help of a gene gun, and then the powdery mildew haustoria index of the bombarded GUS cells was counted to determine whether the target gene It has powdery mildew resistance function.

[0033] The procedure for encapsulating carrier DNA and metal particles is as follows:

[0034] Preparation of tungsten powder: Weigh 30mg of tungsten powder into...

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Abstract

The present invention discloses a haynaldia villosa CERK1-V gene, and an encoded protein and an application thereof. A cDNA sequence of the CERK1-V is shown in SEQ ID NO.1 and an amino acid sequence encoded by the gene is shown in SEQ ID NO.2. A single cell transient expression technology transforms a CERK1-V gene overexpression vector pBI220-CERK1-V into a susceptible wheat variety Yangmai 158 and results show that transient expression of the CERK1-V can reduce haustorium indexes of the Yangmai 158. Expression amount of the CERK1-V in overexpression CERK1-V transgenosis plants is 2-12 times of the expression amount of the Yangmai 158 and shows middle and high resistance levels of wheat powdery mildew. Thus, the CERK1-V is expected to be used for gene engineering breeding and introductionof the overexpression vector pBI220-CERK1-V into the susceptible powdery mildew wheat variety is expected to improve the powdery mildew resistance of the wheat.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and discloses a tufting wheat CERK1-V gene, its encoded protein and its application. Background technique [0002] Wheat powdery mildew caused by the obligate parasitic fungus Blumeria graminis DC f.sp.tritici is one of the three major fungal diseases of wheat (Triticum aestivum L., 2n=6x=42, genome AABBDD) in my country. Threats to our safe production of wheat. The disease can occur during the whole growth period of wheat, and can infect all parts of aboveground wheat plants, mainly leaves and leaf sheaths, and its disease can cause 15-30% yield loss. [0003] With the impact of changes in cultivation system, farming methods and climatic conditions, especially the loss of resistance gene Pm8 contained in most cultivars in my country, the damage of wheat powdery mildew in my country has been increasing in recent years. Although powdery mildew can be controlled with fungicides, chemical control...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/12C12N15/82A01H5/00A01H6/46
CPCC12N9/1205C12N15/8282
Inventor 王秀娥樊安琪王宗宽刘佳韦璐阳王雅嘉张鑫袁春霞王海燕肖进
Owner NANJING AGRICULTURAL UNIVERSITY
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