| Xa7 is a dominant resistance gene,which has broad-spectrum,long-lasting and high resistance,and has good resistance in the whole growth period of rice.The cloning of Xa7 gene will lay a solid foundation for elucidating the molecular mechanism of disease resistance regulated by Xa7 gene.At the same time,by exploring its disease resistance function,it can show important application value in rice disease resistance molecular breeding.By analyzing the genome sequence of Xa7,it is found that there are big differences and complex sequences with other rice varieties.It is impossible to clone Xa7 gene by map based cloning.Therefore,in the previous study of our laboratory,through the radiation mutation of Zhenhui 084,a rice variety containing Xa7 gene,we screened out the susceptible mutants with 106 kb of chromosome deletion in the Xa7 location region,and there was 25 kb overlap with the previous 120 kb location region,thus Xa7 was located in the 25 kb region.In order to clone Xa7 gene,this study uses the method of constructing subclonal vector to further clone Xa7 gene.Four subclonal fragments(P1,P2,P3,P4)covering the 25 kb region were obtained by PCR using a positive monoclonal covering the 25 kb region.The four subclonal vectors covering the Xa7 region were constructed by connecting the fragments to the vector pCAMBIA 1300,and then transgenic to Zhonghua 11.Through primer identification of transgenic plants and PXO86 verification of positive transgenic plants,it was found that P2,P3 and P4 transgenic positive strains were all susceptible and did not complement the disease resistance phenotype of Zhenhui 084.In these clips.It was speculated that Xa7 gene was not in these fragments.Five resistant plants were identified in P1 transgenic line,and the length of disease spot was consistent with Zhenhui 084.The background of these transgenic plants is correct.In order to further analyze these resistant plants,P1-1 resistant plants were propagated to T1 generation,and primer identification and PXO86 test were carried out.It was found that the positive strains were resistant to disease,and the resistance spectrum of P1-1 resistant plants was consistent with that of Zhenhui 084 by comparing different physiological races.The above results show that Xa7 gene does exist in P1 subclonal fragment.Through the analysis of P1 fragment,it was found that there were three candidate genes.In order to determine Xa7,we also used P1 subclonal fragment to obtain four smaller subclonal fragments(C1,C2,C3,C4)covering P1 interval by PCR,and compared these fragments with vector pCAMBIA 1300.Four smaller subclonal vectors covering the P1 region were constructed,and then transgenic to Zhonghua 11.At present,the positive clones and transgenic seedlings have been obtained,and the positive transgenic lines have been screened out,and the field planting and bacterial inoculation verification will be carried out,so as to clone Xa7 gene finally. |
