Cloning And Functional Validation Of CPSGT1 And CPRAR1 In Carica Papaya L.

In recent years, research show that RAR1 and SGT1 take a very important role in plant disease resistance signal transduction. However, these studies were taken only on the traditional model plants and important crops, the similar study on carica papaya has not been taken, although which is a significant fruit.In this study, we built a local papaya databases, data of which come from genbank database. And the potential gene sequences of RAR1 and SGT1 in carica papaya were fund with blast analysis of RAR1 and SGT1 from tobacco or arabidopsis. We designed primers for the potential gene sequence and cloned the cDNA of the two genes in papaya (Hawaii) by RT-PCR successfully. Then we used reverse genetics methods and bioinformatics to research these functions. The main results as followed:1. The full-length cDNA of CpRARl and CpSGTl were cloned successfully. Using RT-PCR method we cloned two cDNA fragments from Hawaii leaves, the length of which was 684bp and 1086bp separately. Through Homology analysis with similar EST sequences of papaya and these from other species and analysis on amino acid sequences of the two fragments, we confirmed that the two cloned fragments are papaya RAR1 and SGT1 gene and were named CpRAR1, CpSGTl separately.2. CpRARl and CpSGTl transgenic arabidopsis. We have successfully cloned CpRARl and CpSGT1 into overexpression vector pBI121 and got four kinds of over-expression vectors derived from pBI121, which were pBI121+RAR1, pBI121+RAR1-GUS, pBI121+SGT1 and pBI121+SGT1-GUS. And then we transmited successfully the four vectors into arabidopsis thaliana (Columbia-0) by infecting inflorescence.3. Resistance of CpRAR1 and CpRAR1-GUS transgenic arabidopsis to Sclerotinia sclerotiorum. After obtaining transgenic arabidopsis successfully, we tested the change of resistance of transgenic arabidopsis to Sclerotinia sclerotiorum by inoculation experiment of detached leaves from CpRAR1 and CpRAR1-GUS transgenic arabidopsis. The results showed that CpRAR1 transgenic arabidopsis compared to the wild-type, its resistance to Sclerotinia sclerotiorum significantly was improved for its incidence delayed by 4 hours.4. Virus-induced gene silencing (VIGS) of papaya. In this study, we have been trying to comfirm the gene function of CpRARl and CpSGTl with experimental method of virus-induced gene silencing. And we have successfully constructed the silencing vectors(PTVOO+CpPDS and PTV00+CpSGT1). Unfortunately, after three attempts, we did not succeed finally in silencing of CpPDS.