Regulation Analysis Of Rice Blast Resistance Gene Pi63 Promoter And Study On The Resisatnce Of Pi63 Homologous Genes

Rice blast is one of the most serious diseases in rice.Genetic improvement using the host resistance provides a cost-effective and environment-friendly solution to rice blast diseases.Due to highly frequent variation in the Magnaporthe oryzae population,resistance of new rice varieties simply with only a major resistance gene could be weakened or even lost quickly.Therefore,further studies on the mechanism of rice blast resistance gene and the development of new broad-spectrum resistance genes are needed to provide new resources for durable resistance breedingThe rice blast resistance gene Pi63 studied here has a good field resistance.In the previous research,the expression level of Pi63 is higher in resistant variety and its resistance is positively correlated with its expression,suggesting that this characteristic is closely related to the regulation of the promoter.By bioinformatics analysis of the cis-acting elements in the promoter region of Pi63,four fragments containing different cis-acting elements were constructed.These four constructs were linked to GUS gene and Pi63 full-length gene,respectively,for genetic transformation to analyze expression regulation.In addition,Pi63 is the first gene cloned in the blast resistance gene cluster in the region of 31 Mb on chromosome 4 of rice.In order to study the evolution mechanism of Pi63 and explore resistant resources,Pi63 homologous genes were cloned in cultivar and wild rice and the resistance of the cloned homologous genes transgenic plants were analyzed.The results are as follows:1)Four constructs ligated with Pi63 full-length gene were transferred into vector p MDC99(named as p MDC99-P0 fl to P3 fl,respectively)and then transformed into susceptible variety Nipponbare.The q PCR method was applied to identify the copy number of the T1 plants.The plants with single copy were inoculated with M.oryzae race 007 and the expression level of Pi63 gene was detected before and after inoculation.The results showed that,compared with before inoculation,after inoculation P1f1 had the highest expression level,P0f1 followed,the changes of P2f1 and P3f1 were not significant.We speculated that the cis-acting element related to resistance response is located in the region of P1 to P2,and a negative regulation or feedback regulation element exists in the region of P0 to P1.Meanwhile,expression vectors(named as p CAMBIA1391Z-P0 to P3,respectively)of four promoter constructs as described above ligated with GUS gene were constructed and genetic transformation of the rice Nipponbare was carried out.The results of GUS staining and GUS assay of T1 plants showed that the p CAMBIA1391Z-P0,p CAMBIA1391Z-P1 and p CAMBIA1391Z-P3 were expressed in the roots,stems and leaves of Nipponbare,and strongest in leaves.p CAMBIA1391Z-P1 had the deepest GUS staining color and the strongest GUS activity,suggesting that there were negative regulatory elements in the region of P0 to P1,which was consistent with the result of expression.2)The homologous genes of Pi63,O3,M4,Hg3,Hg5 and Hg7,were successfully cloned from Owarihatamochi,Morobereban,and three common wild rice,respectively.These five homologous genes were liagted with Pi63 promoter,transferred into vector p MDC99 and transforamed into Nipponbare.The T0 positive plants were inoculated and Hg5 showed resistance to M.oryzae race 007.The resistance of other genes need to be further identified.