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Application of rice blast resistance related gene OsCOL9

A resistance-related, rice blast technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve problems such as no reports, and achieve the effect of great application value

Active Publication Date: 2016-04-27
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of transcription factor plays an important role in regulating plant perception of circadian rhythm changes and flowering. Studies have shown that banana MaCOL1 is involved in regulating plant responses to biotic and abiotic stress responses, but whether CO and COL proteins are involved in regulating rice blast resistance? The relevant research has not been reported

Method used

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  • Application of rice blast resistance related gene OsCOL9
  • Application of rice blast resistance related gene OsCOL9
  • Application of rice blast resistance related gene OsCOL9

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Cloning and sequence analysis of rice OsCOL9 gene

[0028] 1) Extraction of total RNA from rice

[0029] Take Zhongerruanzhan (in the literature "High-quality and high-yield new rice variety Zhongerruanzhan [J]. Chinese Agricultural Sciences,2001,(06):49." and its rice high-resistant rice blast lines H4 (near isogenic line H4) (in the document "Rice blast resistance protein Pik 2 -H4 gene cloning and interaction protein screening[J].Guangdong Agricultural Sciences,2014,(04):156-160."Open in) Four-leaf stage rice seedlings, frozen and ground with liquid nitrogen in a ceramic mortar Into a powder, transfer to a 1.5mL centrifuge tube, and add Trizol reagent (Invitrogen) at the ratio of 1mL extraction reagent per 100mg material, and mix well; add chloroform at the ratio of 200μL chloroform per 100mg material, mix well 10000g, 4 Centrifuge at ℃ for 15min, discard the middle and lower organic phases, collect the upper aqueous phase and transfer to a new centrifuge tube;...

Embodiment 2

[0035] Example 2 Analysis of OsCOL9 expression trend of different rice after inoculation with Magnaporthe grisea

[0036] The expression pattern of OsCOL9 gene after inoculation with Magnaporthe grisea was analyzed by quantitative RT-PCR technology. Inoculation of Magnaportheoryzae GD0193 in resistant variety H4 (in the literature "Evaluation of resistance to rice blast and resistance genetic analysis of 3 spatially mutagenized rice lines[J].Journal of South China Agricultural University,2012,33(03) : 273-276.”) at different time points (0h, 6h, 12h, 24h, 36h, 48h, 72h) after the leaves were collected and their total RNA was extracted, and the reverse transcription kit ReverTraAce was used to reverse transcribe the cDNA. Synthesis of a chain. Then follow the instructions of the SYBRPremixExTaq kit to analyze the expression of OsCOL9 with ABStepOnePlus fluorescent quantitative PCR detector, with Actin as the internal reference gene.

[0037] The primers used are:

[0038] OsCOL9-RT...

Embodiment 3

[0043] Example 3 Construction of Rice OsCOL9 Overexpression Vector and Obtaining Transgenic Seedlings

[0044] The OsCOL9 cDNA fragment amplified by OsCOL9-F and OsCOL9-R was digested with KpnI (NEB) and BamHI (NEB), and ligated to the overexpression vector pOX (in the document "Cloning and functional analysis of rice SDG711 and SDG723 [D ]. South China Agricultural University, 2012” disclosed in the corresponding site, the ligation product was transformed into E. coli DH5α competent cells, and the product recovered after digestion of the overexpression vector pOX was also transformed into E. coli DH5α receptor Cells (as a control); select positive clones to extract plasmid DNA, and perform restriction digestion ( Figure 4 , 1, 3, 4 are the correct positive pOX-OsCOL9 plasmids), and finally get the OsCOL9 overexpression vector pOX-OsCOL9 driven by the rice Ubi promoter. The pOX-OsCOL9 was transformed into Agrobacterium by electric shock method, and according to the method of Hie...

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Abstract

The invention discloses application of a rice blast resistance related gene OsCOL9and belongs to the technical field of plant gene engineering. The expression level of the gene OsCOL9 is increased after the gene OsCOL9 is induced by rice blast pathogenic bacteria. The expression of the gene is up-regulated after the gene is induced by Magnaporthe oryzae of Oryza Sativa, and the rice blast resistance of the Oryza Sativa can be obviously improved through overexpression. A plant overexpression vector is constructed by OsCOL9 and a Ubi promoter so as to convert the Oryza Sativa, and a result shows that the rice blast resistance can be remarkably improved through the overexpression of the gene. An expression vector can be constructed by using the gene disclosed by the invention and is applied to the rice blast resistant breeding of the Oryza Sativa. According to the application, better understanding of an action mechanism of OsCOL9 is facilitated, a foundation for further understanding the interaction of Oryza Sativa-Magnaporthe oryzae and a disease resisting signal conduction path is laid through OsCOL9cloning, and the application value in breeding is relatively high.

Description

Technical field [0001] The invention belongs to the technical field of plant genetic engineering, and relates to the application of a rice disease resistance gene, in particular to the application of a rice blast resistance-related gene OsCOL9. Background technique [0002] Rice (Oryza Sativa) is one of the most important food crops in the world. It feeds more than half of the world's population (Khush, 2005), and is also the main food source for nearly half of my country's population (Liu Guoquan et al., 2004). Rice blast caused by Magnaportheoryzae infection is an important limiting factor in rice production (Liuetal, 2013). It has the characteristics of rapid spread, wide range of occurrence, and serious damage. In epidemic years, rice blast severely affected areas generally reduce rice yield by 10%-20%, sometimes by 40%-50%, and some fields or even grains have no harvest, which seriously threatens rice production (Pennisi, 2010). Aggregating multiple major disease resistance...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8282
Inventor 王加峰刘浩董双玉陈志强王慧
Owner SOUTH CHINA AGRI UNIV