Analysis Of Full-length CDNA Library And Cloning Of Resistance Gene From Gossypium Barbadense Induced By Verticillium Dahliae

This study is based on the full-length cDNA expression library from Pima induced byVerticillium dahliae. We characterizate the ESTs from the library with the tools of theCOG、KOG、KEGG and GO and try to dig out the resistance related gene and specificresistance mechanism.The GbCRK and GbVPE were cloned and further analysed the expression, respectively.We constructed the eukaryotic vector of gene, and transformed into Arabidopsis thaliana.Futhermore, the VIGS vector was finished and transformed into cotton. The main resultsobtained are as follows:1. By SeqClean analysis,146192Reads and23162Unigenes were acquired in thelibrary. Among all the Unigenes,78.07%、59.71%、78.43%、57.88%、80.24%wasannotated into the databases of NR，Swiss-Prot，TREMBL,CDD and PFAM, respectively.And found that,3027homologous unigenes to known defense-related genes from otherplants,4936transcription factor,680new genes characteristic of Gossypium barbadense.Through the functional annotation found,12248unigenes were annotated into GOGdatabase,20397into GO database, which notes for the Biological Process has6893; notesfor the Cellular Component are11305; comments for Molecular Function has8383; allcomprising the three functional annotation of6184genes. We also found out289pathwaysrelated to Verticillium wilt resistance through KEGG.2. The size of GbCRK is1944bp, encoding a peptide of647amino acids, which has aconserved sequence of S-TK. Based the results of real-time PCR, the expression level ofCRK is diffenent between the resistant and susceptible cotton culivars. In the susceptiblecotton culivar cv “H208”, the expression level of CRK was much higher than its in theresistance cultivar cv “Pima90-53”. In addition, GbCRK could be induced obviously by V.dahliae both in above cultivars. We infered that GbCRK might be a functional gene relatedto Verticillium dahliae resistance.3. The ORF size of GbVPE is1946bp, encording a peptide of468amino acids. It hasa conserved sequence of C13, and it blongs to the C13family. The result of the real-timePCR showed that GbVPE has the highest expression level in the “H208.” After infection by V. dahliae, the expression of GbVPE was decreased firstly and then increased. Finally, ithas a high and stable expression level. It may associate with the resistance to V. dahliae.4. Construction the eukaryotic vector of pCAM-GbCRK and pCAM-GbVPE, andtransformed into Arabidopsis thaliana.The transgenic plants were obtained through theselection of kanamycin and PCR molecular detection.5. Construction the VIGS vector of pTRV2-GbCRK, pTRV2-GbCRK, verified thesilence effect and got gene silencing plants.