Analysis Of Cloning And Bioinformatics Of Rice Pathogenesis-related Genes Induced By Rhizoctonia Solani

In order to obtain the rice genes which are induced by Rhizoctonia solani and further understand the gene expression profile during the process that the rice resistant to sheath blight,our laboratory used the EST sequences which have been gained by SSH as information poke to e-clone three pathogenesis-related genes including intact ORF, and then analyse these genes through the bioinformatics method and designe their primers according to their conservative regions.By the way of RT-PCR and Digoxin hybridization,we cloned and identified these genes.1. Silico cloning and bioinformatics analysis of rice Ras geneA 926bp sequence which include a intact ORF about 609bp was obtained by the way of silico cloning which was translated into a protein which was predicted that it was located in cytoplasm and water-soluble stable protein. Starting codon of the ORF is ATG, its stop codon is TGA,5'UTR is 71bp long,3'UTR is 249 bp long.The protein with a conservative region contains 202 amino acids,its molecular weight is 12.7 kDa, its isoelectric point is 8.93. its instability index is 29.33, the protein belong to GTPase Rab1 family. In order to confirm the truth of sequence, prmer was designed according to its conservative sequence,and then the Ras gene was amplified through RT-PCR,the similarity between the real sequence and the e-cloning sequence is about 98%.The result demonstrated that the primer was rational.2. Silico cloning and bioinformatics analysis of rice eIF-1 geneA 656bp sequence which include a intact ORF about 348bp was obtained by the way of silico cloning which was translated into a protein which was predicted that it was located in cytoplasm and water-soluble instable protein. Starting codon of the ORF is ATG, its stop codon is TGA,5'UTR is 63bp long,3'UTR is 245 bp long.The protein with a conservative region contains 115 amino acids.its molecular weight is 22.56 kDa, its isoelectric point is 5.55. its instability index is 19.48, its average hydrophobicity is-0.434,the protein belong to translation initiation factor SUI1 family.In order to confirm the truth of sequence,a prmer was designed according to its conservative sequence,and then the eIF-1 gene was amplified through RT-PCR. the similarity between the real sequence and the e-cloning sequence is about 99%.The result demonstrated that the primer was rational.3. Silico cloning and bioinformatics analysis of a rice WRKY geneA 1189bp sequence which include a intact ORF about 609bp was obtained by the way of silico cloning which was translated into a protein which was predicted that it was located in cell nucleus and water-soluble instable protein. Starting codon of the ORF is ATG, its stop codon is TAG,5'UTR is 33bp long,3'UTR is 556 bp long.The protein with a conservative region contains 202 amino acids,its molecular weight is 22.03 kDa, its isoelectric point is 8.62. its instability index is 53.23,its average hydrophobicity is-1.010,the protein belong to WRKY protein superfamily family.In order to confirm the truth of sequence, prmer was designed according to its conservative sequence,and the WRKY gene was amplified through RT-PCR. the similarity between the real sequence and the e-cloning sequence is about 96%.The result demonstrated that the primer was rational.