A primer set, kit and method for detecting pathogenic variants of avirulent gene avrpi9 of oryzae oryzae

An avirulent gene, rice blast fungus technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc., to achieve the effect of controlling rice blast and improving breeding efficiency

Active Publication Date: 2022-05-10
CHINA NAT RICE RES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, more than 40 avirulent genes of Magnaporthe grisea have been identified, but only 19 avirulent genes have been successfully cloned

Method used

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  • A primer set, kit and method for detecting pathogenic variants of avirulent gene avrpi9 of oryzae oryzae
  • A primer set, kit and method for detecting pathogenic variants of avirulent gene avrpi9 of oryzae oryzae
  • A primer set, kit and method for detecting pathogenic variants of avirulent gene avrpi9 of oryzae oryzae

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Isolation of Magnaporthe grisea and Extraction of DNA:

[0048] 1. Moisturize the panicle and stem nodes collected from fields all over the country overnight, and then use a fine needle to pick a single blast fungus conidia under a microscope to tomato oat medium (tomato juice 150mL, oatmeal 40g, CaCO 3 0.6g, dilute to 1000mL). After 7 days of culture, collect conidia for observation to confirm whether the blast fungus has been successfully isolated. The successfully isolated blast fungus was transferred to a new tomato oat medium, and sterilized filter paper was added to culture for 7 days. After the filter paper was collected, it was stored in Standby at -20°C.

[0049] 2. Cultivate the mycelium of Magnaporthe grisea, after the test strain of Magnaporthe grisea is activated on the tomato oat medium for several days, pick an appropriate amount of mycelia, and use liquid medium (proportioning per liter of liquid medium: KH 2 PO 4 0.5g, K 2 HPO 4 0.5g, MgSO 4 0...

Embodiment 2

[0052] Amplification and sequencing of AvrPi9 sequence:

[0053] Primer pairs were designed according to the sequence of the cloned AvrPi9:

[0054] AvrPi9W1: 5'-CAGGATTCCAGCTATTCGACAAC-3';

[0055] AvrPi9W2: 5'-CACTCGCATTATCGCATAATTGC-3',

[0056] Use primers AvrPi9W1 / W2 to amplify the genome sequence of AvrPi9 gene. The amplification system is 20 μL, including 10 μL 2×PCR mix, 0.5 μL 100 ng / μL DNA template, 10 μM primers AvrPi9W1 / W2, 1 μL each, and the balance is ddH 2 O. The reaction conditions were: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, amplification for 35 cycles, and final extension at 72°C for 5 min. The amplified products were subjected to two-way sequencing analysis (entrusted to Hangzhou Youkang Biotechnology Co., Ltd.).

[0057] From the sequencing results of 300 isolated strains, the coding region CAA of 8 strains of AvrPi9 was mutated into TAA, resulting in premature ter...

Embodiment 3

[0059] Rice inoculation: Take the filter paper piece with rice blast hyphae on OA medium, culture on OA medium at 27°C for 3 days, take a piece of blast fungus with a diameter of about 5mm in the ultra-clean workbench, and transfer it to On the PA medium, cultivate for another 7 days, wash the surface hyphae with sterile water under aseptic conditions, filter through double-layer filter paper, collect them into a 1.5mL centrifuge tube, and adjust the final concentration of the spore liquid to 5×10 5 cells / mL for in vitro inoculation. Select the tender rice leaves of suitable size and cut them into leaf segments with uniform length and length, and then evenly prick the wounds with needles, place the leaf segments on the filter paper moistened with sterile water, and press the two sides of the leaf segment with wet filter paper. To prevent the leaves from curling and causing the bacterial solution to be unable to suspend on the surface, and finally affix corresponding labels for...

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Abstract

The invention discloses a primer set, a kit and a method for detecting the pathogenic variation of the rice blast fungus avirulence gene AvrPi9. SNP changes in the avirulent gene AvrPi9 of Magnaporthe grisea lead to premature termination of the coding of the AvrPi9 gene and transformation into a virulent gene. The primer set includes two pairs of primers, the first pair of primers is used to amplify the AvrPi9 gene fragment; the second pair of primers is used to identify whether there is a SNP that causes AvrPi9 to transform into a toxic gene after mutation. The invention provides two pairs of molecular markers of the avirulent gene AvrPi9 of Magnaporthe grisea. The molecular markers can be used to quickly establish a molecular detection system for the pathogenic variation of AvrPi9 in the natural population of Magnaporthe grisea. The distribution and dynamic changes in the population are used to guide rice blast resistance breeding in various regions and the rational distribution and rotation of varieties, so as to improve breeding efficiency and control the occurrence of rice blast more effectively, so as to design rice blast comprehensive new strategies for prevention.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer set, a kit and a method for detecting the pathogenic variation of the rice blast fungus avirulent gene AvrPi9. Background technique [0002] Rice is the main food crop for more than half of the world's population, and it is also an important food crop in my country. Maintaining stable rice production is of great significance to meet the demand for rice production from the growing global population. Rice blast is a fungal disease caused by the rice blast fungus (Magnaporthe oryzae / Piricularia oryzae), which occurs in all rice-growing areas in the world, and generally causes rice yields to decrease by 10% to 30%, and even crop failure in severe cases. [0003] At present, the means of controlling rice blast are mainly the application of pesticides and planting of resistant varieties. The use of pesticides has a series of unfavorable factors such as polluting the environment,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
Inventor 寇艳君邱结华沈浙南时焕斌
Owner CHINA NAT RICE RES INST
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