A Preliminary Study On The Artificial Evolution Of Disease Resistance Genes In Rice

Ultilizing resistance genes (R) to breed new lines is the most direct, environmental, economic and effective method to control rice disease and insect pests. Now there are only a few cloning R genes, of which many are alleles with different resistance spectrum, and pathogens evolve rapidly which is caused by excessive use of the resistance gene will lead to a loss of genetic resistance function. Therefore, we need to discover new resistance genes, enhancing resistance gene resources.At the same time, conducting research on function and structure characteristics of R genes, and understanding the characteristics of pathogen evolution, to create the ideal artificial gene in future.Researches on CC-NBS-LRR resistance genes function have shown that LRR usually participates in pathogens’recognition and NB-LRR protein function switch, and CC-NB-ARC structure domain usually appears the phenomenon of autoimmunity. At the same time, R genes interfamily transfer and PRRs chimeric proteins research has shown that structural domains between species and within species are conservative and fusional. Based on their conserve and fusion, we divided bacterial leaf blight Xa21D and rice blast resistance genes Pi36, Pbl, Pita into conservative functional domain and mutual fusion together to built new resistance genes. Besides, we also deleted a part of Xa2ID GC-Rich Region and constructed the incomplete Xa21D. Through the rice blast and bacterial leaf blight inculation,we can effectively explore Xa21D function key structures or components, and function characteristic and mechanism between LRR and CC-NB-ARC resistance gene or Signal Sequence GC-rich region, providing a research basis for further research. At the same time, we also want to explore the possibility that chimeric genes confer broad spectrum and efficient immune response to bacterial leaf blight and rice blast. Preliminary results obtained the following:1, According to results of web prediction and paper analysis, we divided Xa21D into Signal Sequence, the GC-rich region and LRR region three parts, Pb1, Pi36 and Pita into CC-NB-ARC, the Link/Spacer and LRR region three parts. Four genes according to the structure of authentication domain function and For the purposes to determine the function of the R genes domains and create genes confering broad spectrum resistance to bacterial leaf blight and rice blast, we designed eight chimeric genes which combine different domains derived from these four genes.2, According to the design requirements, We successful amplified the divided resistance genes fragments, got eight full-length chimeric genes by Overlap-PCR, and constructed these chimeric genes into the plant expression vector of PHB. Choosing japonica rice varieties TP309 as receptors materials, we conducted rice genetic transformation, have got RJLP2403, RiLP2406 and RiLP2407 transgenic To generation plants, and the other chimeric genes are still in genetic transformation.3,Incluating RiLP2403, RiLP2406 and RiLP2407 three chimeric gene transgenic plants with rice blast fungals, the results show that the three genes confer partial resistance to rice blast, and someone domain of three chimeric proteins functions in immunity.4, RiLP2403 showed different performance in rice blast strains inculation,which indicates that Pi36 LRR may participate in the rice blast effector recognition, but we cannot determine its recognition through indirect, or direct way. And Xa21D Signal Sequence and GC-Rich unknown region may also be involved in an important part of immune response, especially the GC-Rich regions, which will be helpful for us to find Xa21D co-parter protein.5, RiLP2406 and RiLP2407 incluation results can’t make us determine which domain participates in rice blast resistance. In particular, RiLP2406 consists of Pi36 CC-NB-ARC region, Xa21D GC-Rich unknown region and LRR region, with high possible resistance to bacterial leaf blight and rice blast, but this needs further experiments.In this work, based on resistance genes structure domains’conservative and fusion,we divided the bacterial leaf blight resistance genes Xa21D and blast resistance genes Pbl, Pi36 and Pita into different function domains and constructed chimeric genes combining different domains. We have got eight chimeric genes, some domains’functions are detemined by transgenic plants, and the results show that some domains of the chimeric genes confer rice blast resistance function, and have a broad spectrum resistance to bacterial leaf blight and rice blast with high possibility. This research results indicates that the method based on conservative and fusion of resistance genes structure domains is effective to design new resistance genes.