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Rice Rhizoctonia solani effector gene AG1IA10434 and application thereof

A technology of AG1IA10434 and Rhizoctonia solani, applied in the field of genetic engineering, can solve problems such as unclear action sites of Rlm resistance genes, and achieve the effect of controlling the occurrence of sheath blight

Inactive Publication Date: 2013-08-07
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it is clear that they have avirulent activity, the mechanism of AvrLm recognition, the corresponding Rlm resistance genes and their sites of action remain unclear

Method used

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  • Rice Rhizoctonia solani effector gene AG1IA10434 and application thereof
  • Rice Rhizoctonia solani effector gene AG1IA10434 and application thereof
  • Rice Rhizoctonia solani effector gene AG1IA10434 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Cloning of the predicted effector gene

[0037] The AG1IA strain was isolated and purified from rice sheath blight disease-infected plants in the field by the Rice Research Institute of Sichuan Agricultural University. It is a fungal strain that is ubiquitous on rice plants and is widely distributed in rice cultivation areas. (AG1IA strain has been published in Sichuan Agricultural University, Master's thesis, Analysis of Rhizoctonia solani AG1IA-induced corn differential protein, author Li Yun)

[0038] Under the ultra-clean workbench, use tweezers to pick out a small amount of AG1IA hyphae, insert them into 50ml PDA medium, mash the hyphae, culture at 28°C, 200r / m for two days, filter and collect the hyphae with four layers of gauze. Triturate with liquid nitrogen to extract total RNA. Use oligodT as primer and reverse transcription to get cDNA. The primers are designed according to the predicted sequence as follows:

[0039] 5’ATGCGCGGATTTACATCGTACCTTGTTGCAGCA...

Embodiment 2

[0042] Example 2: Construction, transformation and expression of prokaryotic expression vector of effector gene

[0043] The target gene obtained by PCR has no miscellaneous bands detected by electrophoresis. You can connect the target gene to the expression vector pEASY-E1 according to the instructions of TransGenBiotech Peasy-E1kit. After sequencing, the gene AG1IA10434 is 57 bases longer than the predicted sequence. The base sequence is as SEQID Shown in NO.1. The transformation conditions are the same as those of conventional Escherichia coli. The positive transformants are picked, cultured at 37°C to an OD value of 0.6, then transferred to 28°C, IPTG 1mM is induced for 7 to 11 hours, and the effector protein is expressed.

Embodiment 3

[0044] Example 3: Pathogenicity detection of expressed protein

[0045] Collect the cells expressed by AG1IA10434 and ultrasonically break them to obtain crude protein. Be careful not to denature the protein during the crushing process (the crushing time is 5S, the power should not exceed 200W, and the sample is always kept under ice bath conditions during the crushing process). Select rice leaves at the three-leaf stage cultured in a greenhouse with consistent growth and place them in a sterile petri dish with two layers of filter paper to keep the filter paper moist to keep the leaves moisturizing. Use a sterilized toothpick to make a small hole in the center of the rice leaf, and cover the small hole with a three-layer sterilized 0.5cm×0.5cm filter paper. The amount of crude protein inoculated is 50 microliters. The infected leaves were placed in a 28 degree Celsius light incubator with 12 hours of light and 12 hours of darkness, and RH (sickness index) 80%. Observe and take ...

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Abstract

The invention provides a rice Rhizoctonia solani effector gene AG1IA10434, which has a nucleotide sequence as represented by SEQ ID NO. 1. To research the gene provided in the invention, a molecular target of a novel pesticide can be designed according to the structure and the function of the gene in practice; and acceptor protein genes of effector protein in cells of hosts like paddy rice can beknocked out or be subjected to mutation so as to obtain a species with durable disease-resistance. The invention is beneficial for establishment of a molecular detection system for pathogenicity variation of natural Rhizoctonia solani population, for research of distribution situations of the Rhizoctonia solani effector gene in field natural population, for reveal of composition and variation characteristics of microspicies in the Rhizoctonia solani population and for resistance identification and reasonable arranging and replacing of rice species so as to realize effective control of sheath blight.

Description

Technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a Rhizoctonia solani effector gene AG1IA10434 and its application. Background technique [0002] Rice sheath blight caused by Rhizoctonia solani is a fungal disease, and it is the three major diseases of rice alongside rice blast and bacterial blight. Rhizoctonia solani infects a wide range, including rice, corn, wheat, potatoes, pasture, soybeans and other crops. The ability of Rhizoctonia solani to cause diseases of so many crops is closely related to its secretion of effector molecules. This molecule can regulate the host's innate immunity and enhance parasitic infection. It is now generally accepted that these effectors are the key pathogenic determinants of enhanced parasitic infection (Kamoun, 2007; Hogenhout et al., 2009). [0003] Rhizoctonia solani secretes effector molecules to closely contact its host plants and precisely manipulate plant cells. Effector targe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C07K14/37C12N15/63C12N1/21C12N5/10C12Q1/68C12N15/09A01H5/00C12R1/645
Inventor 郑爱萍张丹华李平王世全邓其明李双成朱军
Owner SICHUAN AGRI UNIV
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