Cloning And Genetic Transformation Of Blast Resistance Gene Piy43(t) From Japonica Yunyin

Yunyin is a Japonica cultivars from Yunnan Academy of Agricultural Sciences.Blast resistance of Yunyin was analyzed by injeced with multiple isolates in the field and sprayed isolate in the greenhouse.The results indicated that the Yuny was resistanct to 58 out of 60 isolates.The blast-resistance gene Piy43(t)was cloned with the map-based cloning technique.But the loci of Piy43(t)was same as the identified blast-resistance gene Pib.Thurtherly the loci still existed a pseudo gene,homology sequences of up to 96%for blast-resistance gene Pib.In order to further understand the relationship between the blast resistance gene Piy43(t)and Pib,we carried out the following researches:1.The characteristics of blast resistance were identified between the variety Yunyin and monogenic lines with Pib innoculated Sichuan 43 isolate.The results indicated that the variety Yunyin was blast resistant to isolate Sichuan 43,but the leaves of other varieties,such as Nipponbare,LTH and MG40,MG 13 had some lesions or necrosis.The phenotype of monogenic lines with Pib was moderate resistant to isolate Sichuan 43 after 5d inoculation.2.The expression of blast-resistance gene Piy43(t)was analyzed with qRT-PCR.The results indicated that the expression of blast-resistance gene Piy43(t)was the lowest between Yunyin and monogenic line with Pib at 12hai,up regulation at 24hai and up regulation to some degree at 72hai between Yunyin and monogenic line with Pib.The expression of blast-resistance gene Piy43(t)continued to up regulation for the variety Yunyin from72hai to 96hai,but down regulation for monogenic line with Pib from72hai to 96hai.Moreover,the expression of blast-resistance gene Piy43(t)for Yunyin was higher than that of monogenic line with Pib at 72hai.So we guessed that the gene Piy43(t)might be different to the blast-resistance gene Pib.3.The corresponding blast-resistance gene was cloned from the variety Yunyin and monogenic line with Pib based on the sequence of Pib gene.The sequence blasted results indicated the Pib(Piy43(t))sequence from yunyin was 7 base difference from the monogenic line with Pib.There were 3 amino acid difference between Pib(Piy43(t))from yunyin and from the monogenic line with Pib.4.The genome of pseudogene Pib2 also has been clond from the variety Yunyin and monogenic line with Pib.The results of sequence blasted indicated that there were a 4 bases difference between the variety Yunyin and monogenic line with Pib.But the differences of the promoter were base deletion and insertion between the variety Yunyin and monogenic line with Pib.5.The plant expression vectors including pC AMBIA1301-Piy43(t),pCAMBIA1301-Pib2,pCAMBIA1302-Piy43(t),pTCK303-Piy43(t)-RNAi were contructed.Meanwhile,the genome-editting vector of Piy43(t),Pib2 was contructed with CRISPR-Cas9 system respectively.But the genome-editting vector of Piy43(t)and Pib2 was contructed with CRISPR-Cas9.system.Furtherly,the vector was contructed with pseudogene Pib2 promoter driving expression of the gne GUS.The transgenic plants had been obtained for some constructs mediated with Agrobacterium transformation.