Yunnan University Parsing Plant SSH Library

Researchers from the School of Life Sciences and Yunnan Agricultural University of Yunnan University used ordinary wild rice in Yunnan as material, using the suppression subtractive hybridization technique (SSH), to construct the differential expression of specific genes of Yunnan ordinary wild rice under the stress of bacterial leaf blight. The library was reduced, and its resistance-related genes were analyzed in depth. The research results were published in "Science in China: Life Science".

Rice bacterial leaf blight is a bacterial leaf spot disease caused by Gram-negative bacteria (Xanthomonas oryzae pv.oryzae, Xoo), which has become the most popular in the production of rice (Oryza sativaL.) One of the main diseases, especially in Asian rice areas, can cause a 20% to 30% reduction in rice production. In severe cases, it may even lead to a crop failure.

In recent years, the widespread cultivation of hybrid rice has greatly increased the total yield of rice. However, because hybrid rice is derived from male sterile lines and maintainers, the heritable variation is very small and it is more susceptible to bacterial blight. Therefore, many scholars have launched The study of the disease has been completed. The inheritance of rice resistance to bacterial leaf blight is manifested by the interaction between the host and the pathogen, that is, the specific interaction between the rice disease resistance gene (R gene) and the non-toxic gene of bacterial blight One of its resistance mechanisms and the most important resistance mechanism is R gene-mediated resistance.

The wide application of SSH (suppression subtractive hybridization) technology provides better technical support for the discovery of resistance genes. This technology can easily isolate genes that are differentially expressed at specific developmental stages and specific tissues and organs during plant development, to reveal The molecular mechanism during plant growth and development lays the foundation, and resistance-related genes induced by various biological stress conditions can also be isolated.

Common wild rice (Oryza rufipogon Griff) is highly resistant to bacterial leaf blight, and is rich in bacterial leaf blight resistance genes. Among them, the resistance gene Xa23 derived from wild rice was identified from ordinary wild rice by Zhang Qi et al. It comes out that this gene has the broadest resistance spectrum and is a fully dominant resistance gene for the whole growth period. Among the identified resistance genes of rice bacterial blight, the broad-spectrum resistance gene expressed throughout the growth period and easy to use is limited However, at present, there are few bacterial blight resistance genes unearthed from ordinary wild rice, and the molecular mechanism of resistance to ordinary wild rice is still unclear.

To this end, in this study, the researchers used SSH technology to construct a Yunnan common wild rice resistance to bacterial blight library, quickly obtain the resistance gene induced by bacterial leaf blight stress expression, and then use bioinformatics analysis methods to find participating white The resistance gene for leaf blight defense response provides a theoretical basis for the isolation and cloning of related bacterial leaf blight resistance genes from common wild rice in Yunnan, and provides functional genes for breeding new disease-resistant rice varieties. It also provides resistance to common wild rice in Yunnan The molecular mechanism of bacterial leaf blight was discussed, which laid the foundation for understanding the molecular mechanism of disease resistance of common wild rice in Yunnan from the perspective of gene and molecule.

The researchers sequenced all the positive monoclonals in the library and obtained 494 high-quality expressed sequence tags (EST) after cluster analysis. After BlastN analysis, there were 417 sequences with high homology with known functions; BlastX Analysis shows that 104 ESTs have high similarity to unknown functional proteins or putative proteins, 49 ESTs failed to find homology matches, and 341 ESTs had high homology to known functional proteins.

Preliminary analysis found that these genes are mainly involved in metabolic processes such as energy metabolism, protein metabolism, nucleic acid metabolism, defense and stress response, signal transduction, photosynthesis and membrane transport. Using semi-quantitative RT-PCR, seven possible The expression of EST sequences related to leaf blight resistance in common wild rice control in Yunnan and leaves treated with Xanthomonas oryzae pv. Oryzae, and the expression profiles of these genes were obtained.

It was found that the clone ESTs with clone numbers OR7, OR68 and OR826 were up-regulated under the stress of bacterial blight pathogens. OR826 EST has no homologous sequences in the protein database, which may be a new type of bacterial blight resistance genes The constitutively expressed OR143 EST could detect its mRNA expression in both control and inoculated leaves, but its expression level gradually increased after 48 hours of Xanthomonas oryzae strain stress. It is speculated that these genes are directly involved in ordinary wild rice in Yunnan Disease resistance defense response.

This study provided a theoretical basis for the discovery and cloning of new bacterial blight resistance genes from common wild rice in Yunnan, and laid the foundation for further research on the molecular mechanism of resistance to bacterial blight of common wild rice in Yunnan.

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