Cloning And Quantitative Expression Of Resistance Genes Of Mulberry

Mulberry bacterial wilt caused by Ralstonia solanacearum is a kind of soilspread of devastating diseases. It is difficult to use chemical methods for effectiveprevention and control.Breeding and utilization of resistant varieties is the mosteconomical way to control this disease, effective and safe environment. However,useing conventional methods to attain the resistant varieties need a long breedingcycle and it has a low efficiency.Molecular breeding is an effective method by usingbiotechnology, and resistance gene cloning is the basis for molecular breeding.According to the acquired mulberry transcriptome sequences, we obtained thepart sequence fragments of NBS resistance gene and designed the specific primers.The method of RACE was used to obtain the full length sequence of resistance gene,which use cDNA as template. At the same time, Ralstonia solanacearum infectingmulberry resistance varieties, so we can analysis the resistance genes in different time,different varieties of expressing quantity changes. The main results are as follows:1. Mulberry NBS-type disease-resistance gene cloning: By looking for mulberrytranscriptome sequence database, we selected5NBS family genes. By designingprimers, we cloned the5NBS gene in the mulberry successfully （CL93、Unigene14278、Unigene26173、Unigene32704、Unigene31320）.2. Based on the partial sequence, we were cloned CL933’ and5’ fragment.So thecomplete sequence of CL93was obtained through RACE method. The full-length ofCL93was2109bp including a1848bp complete open reading frame encoding CL93protein of615amino acids. The deduced amino acids of CL93protein consisted ofnucleotide binding site (NBS) domain, and leucine-rich repeats (LRR) domain, whichwere the conserved domains of plant resistance genes.Based on the partial sequence, we were cloned Unigene142783’ and5’fragment.So the complete sequence of Unigene14278was obtained through RACEmethod. The full-length of Unigene14278was1647bp including a1170bpcomplete open reading frame encoding Unigene14278protein of390amino acids.3. By using the method of real time quantitative RT-PCR, We analysed the abovethe five NBS genes expression after infection in resistant varieties and sensitivevarieties.The results showed that CL93gene was induced expression by Ralstoniasolanacearum in sensitive varieties, but suppression in resistant varieties. It Suggest that CL93gene may be associated with disease resistance. Unigene26173gene was allinduced expression by Ralstonia solanacearum in resistant varieties and sensitivevarieties, and it was higher in resistant varieties. Suggest that Unigene26173genemay be related with the disease resistance. Unigene14278, Unigene32704,Unigene31320gene were also induced by Ralstonia solanacearum in resistantvarieties and sensitive varieties, but there’s no significant difference.