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Paddy rice Rhizoctonia solani effector gene AG1IA010188 and application thereof

A technology of AG1IA010188 and Rhizoctonia solani, applied in the field of genetic engineering, can solve the problem of unclear action sites of Rlm resistance genes, and achieve the effect of controlling the occurrence of sheath blight

Inactive Publication Date: 2012-06-27
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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  • Paddy rice Rhizoctonia solani effector gene AG1IA010188 and application thereof
  • Paddy rice Rhizoctonia solani effector gene AG1IA010188 and application thereof
  • Paddy rice Rhizoctonia solani effector gene AG1IA010188 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Cloning of predicted effector genes

[0038] The AG1IA strain was isolated and purified from rice sheath blight diseased plants by the Rice Institute of Sichuan Agricultural University. It is a common fungal strain 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 different proteins induced by Rhizoctonia solani AG1IA in maize, author Li Yun)

[0039] Under the ultra-clean workbench, use tweezers to pick a small amount of AG1IA mycelium, put it into 50ml PDA medium, mash the mycelium, culture at 28°C, 200r / m for two days, and collect the mycelium by filtering with four layers of gauze. Total RNA was extracted by triturating with liquid nitrogen. Using oligodT as a primer, cDNA was obtained by reverse transcription. Primers were designed according to the predicted sequence as follows:

[0040] Front primer BamHI5'GCC GGA TCC CCG GGC CCAGAAG...

Embodiment 2

[0041] Embodiment 2: Construction, transformation and expression of effector gene prokaryotic expression vector

[0042] The electrophoresis detection of the target gene obtained by PCR has no miscellaneous bands, and the target gene can be connected to the expression vector pEASY-E1 according to the instructions of TransGen Biotech Peasy-E1kit. As shown in SEQ ID NO.1. The transformation conditions are the same as the conventional E. coli transformation conditions. Positive transformants are picked, cultured at 37°C until the OD value is 0.6, then transferred to 28°C, induced by IPTG1mM for 7 to 11 hours, and the effector protein is expressed. SDS-PAGE detection picture of expressed protein image 3 .

Embodiment 3

[0043] Embodiment 3: Pathogenicity detection of expressed protein

[0044] The collected AG1IA010188-expressed cells were ultrasonically crushed to obtain crude protein, and care should be taken not to denature the protein during the crushing process. Select rice leaves that grow consistently in the field, and place them in a sterilized Petri dish with two layers of filter paper built in to keep the filter paper moist to keep the leaves hydrated. Use a sterilized toothpick to make a small hole in the center of the rice leaf, cover the small hole with three layers of sterilized 0.5cm×0.5cm filter paper, and inoculate 50 microliters of crude protein. The infected leaves were placed in a light incubator at 28 degrees Celsius, with light for 12 hours, dark for 12 hours, and RH (disease index) 80%. Observe the diseased condition of the leaves for several days and take pictures to record the course of the disease. 6 days after infection, the leaves infected with the control protei...

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Abstract

The invention provides a paddy rice Rhizoctonia solani effector gene AG1IA010188 which has the nucleotide sequence shown in SEQ ID NO.1. According to the invention, in practice, a molecular target of a novel pesticide can be designed according to the structure and functions of the gene; the acceptor protein gene of the effector protein in paddy rice and other host cells is deleted or mutated to obtain a durable resistant variety; the invention is helpful for establishing a molecule detection system of Rhizoctonia solani natural population pathogenicity variation, studying the distribution of the Rhizoctonia solani effector gene in field natural populations, revealing the composition and variation characteristics of microspecies in Rhizoctonia solani populations, and is help for disease resistance identification and reasonable distribution and rotation of the paddy rice varieties to effectively control the occurrence 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 AG1IA010188 and an application thereof. Background technique [0002] Rice sheath blight caused by Rhizoctonia solani is a fungal disease, and it is one of the three major rice diseases along with rice blast and bacterial blight. Rhizoctonia solani can infect a wide range of crops, including rice, corn, wheat, potatoes, pastures, soybeans, etc. Rhizoctonia solani can cause so many crop diseases is closely related to its secretion of effector (effector factor) molecules. This molecule can modulate host innate immunity and enhance parasitic infection. These effectors are now generally recognized as key pathogenic determinants of enhanced parasitic infection (Kamoun, 2007; Hogenhout et al., 2009). [0003] Rhizoctonia solani closely associates with its host plant and precisely manipulates plant cells by secreting effector molecules. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12Q1/68C07K14/37A01H5/00
Inventor 郑爱萍李平刘尧张丹华李双成邓其明王世全朱军
Owner SICHUAN AGRI UNIV
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