RNA Editing Of Cor8 Gene From Citrus Species And Low-temperature Identification Of The Promoter Of Trifoliate Orange Cor8 Gene

Citrus is the largest fruit crop in southern China, however the different degree of damage resulted from low temperature affects the citrus industry. Some cold resistant citrus resources had been discovered, but few studies of these species were conducted. A cold acclimation-related gene from the P.trifoliata, Ptcor8, had been cloned by our group. There exist highly homologous genes in Fortunella and C.limon, however, their cold-resistance ability are different, the P.trifoliata could survive from -26℃, while C.limon could not, which shows the possible reason of different cold resistance ability lies in the differences of RNA editing among different citrus types under low-temperature, rather than their genetic differences in DNA sequence. Besides, the existing study by our group revealed that the homology of the pPtcor8 and pClcor8 were 88.17%, and three special regulatory elements related to stress responsiveness in pPtcor8 were found. This study aims to analyze RNA editing of the cor8 gene in P.trifoliala, C.sinensis and C.limon between low-temperature and room temperature, meanwhile, construct plant expression vector containing cold-induced gene and their promoter, then transform into C.limon, finally to uncover the function of cold-induced gene and its promoter in citrus cold hardiness.The main results of this study were as follows:1. Cscor8 in C.sinensis and Clcor8 gene in C.limon were cloned respectively. The ClustalW2 software was employed to analyze their sequence characteristic. The results showed that the Clcor8 gene had five special editing sites including two invalid sites and three valid sites which will change amino acid. SMART and NPS@ online software were further used to predict their structure. We found that suffered from low temperature, a new trans-membrane domain arose in Clcor8 gene, and its composition of protein secondary structure also changed besides the editing sites, producing a new protein without WCOR413-like protein structure.2. The promoter fragment, pPtcor8 and pClcor8, containing EcoRI and SacI sites and the gene fragment, Ptcor8 and Clcor8, containing SacI and BamHI sites were respectively cloned in P.trifoliata and C.limon. Then pClcor8::Ptcor8::YFP, pPtcor8::Clcor8::YFP fusion expression vectors were successfully constructed by double endonuclease digestion.3. Four plant expression vectors (35S::Ptcor8::YFP, pPtcor8::Ptcor8::YFP, pClcor8::Ptcor8::YFP, pPtcor8::Clcor8::YFP) and the precursor expression vector p2A1(35S::YFP)were transient expressed in C.sinensis leaves mediated by Agrobacterium. The results showed that the fused gene was successfully expressed in C. sinensis leaves. But the fluorescence could not be detected under the low temperature (4℃), indicating that the transient expression analysis was not applicative in study of function of cold inducing gene and its promoter.4. The four fusion expression vectors above and the precursor expression vector p2Al were transformed into C.limon mediated by Agrobacterium.Two positive resistant shoots of 35S::Ptcor8::YFP and two positive resisitant shoots of pPtcor8::Ptcor8::YFP were gained. Further green fluorescence observation of YFP indicated that the pPtcor8 was low temperature inducible promoter.