Cloning And Function Analysis Of CpRCI Gene Releated To Cold Resistance From Chimonanthus Praecox (L.) Link

Low temperature is one of the natural factors that cause severe loss in the yield of agriculture and forest.Many researches of molecular biology have recently been performed to try to improve plant cold resistence.However,the achievement about improving plant cold resistence by cold inducible gene is seldom reported.Besides the potential reasons of existing difficulty of gene transformation and the transgene silencing or inactivity,it is also because there are seldom qualitative genes interrelated with cold resistance and cold inducing genes are mainly from the pattern plants which annually grow,like Arabidopsis and so on.Chimonanthus praecox (L.) Link,a shrub originated from China,blossoms lordly in deep winter,possessing many advantages,such as resistance of coldness,drought,and secateur.Among them,chilling tolerance is its main characteristic.In our research,a cold acclimation gene CpRCI was cloned Ch.praecox flower, and the bioinformatics, Real-time quantitative PCR and ectopical expression in tabocco were analyzed.The main results are as follows:1. Cloning and bioinformatics analysis of CpRCI geneBased on the initial analysis of EST(Expressed Sequence Tag.EST)of cDNA library of Ch.praecox flower,a gene encoding rare cold inducible protein(RCIP) was isolated from Ch.praecox,named CpRCI. The fouther analysis showed that the full-length cDNA of CpRCI is969bp long, having180bp ORF,encoding a predicated protein of60amino acid. The predicated protein contains8.33%acidic amino acids and5%basic amino acids, with the predicated molecular weight of6.71KD and isoelectric point of4.23. The structure characteristics of CpRCI protein were analyzed with bioinformatics methods.The results showed that CpRCI protein contained two transmembrance domain and rich Hydrophobic domain. Secondary structure is mainly composed by Alpha helix (47.01%), Radom coil (33.90%) and Extend strand (5.08%).2. Gene Expression analysis of CpRCI gene in Chimonanthus praecoxReal-time quantitative PCR analysis showed that:The CpRCI gene exhibited different transcription levels in different tissues and had a significantly higher expression in flowers than other tissues. In the bloomed flower, the expression of CpRCI has the maximum at the outer petal and the minimum at the pistil.the CpRCI gene in the flower buds gradually increased in the early stages of flower development from sprouting stage to display-petal stage and then decreased at initiating bloom. We also studied the gene expression of the CpRCI under high salt stress(NaCl) and low temperature (4℃)treatment.The results showed that the CpRCI genes were general up-regulated by the NaCl and low temperature treatment, suggested that the DsbA-FrnE is a gene related to high salt and low temperature stress resistens.3.The construction of CpRCI plant over-expression vector and transformation of tobaccoThe plant over-expression vector of CpRCI named pCAMBIA2301G-CpRCI was constructed and transformed into tobacco plants using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens EHA105(including pCAMBIA2301G-CpRCI recombinant plasmid).27transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by GUS((3-glucuronidase)histochemical staining and PCR analysis4. Chlorophyll content analysis of transgenic tobacco under low tempurature.27plantles of transgenic tabacco and control,respectively,were treated under4℃.The leaf wilting started for both transgenic and controls after xx hour treatment.But during the whole course of cold treatments,the wilting process of transgenic tobacco was slower than that of non-transgenic. Furtherly,in order to validate the fuction of CpRCI in the course of cold treatment leaves of transgenic plantlets and controls under4℃for24h,were collected as sample to mensure and analyze the chlorophyll content concerning with cold injury.Using Ethanol measurement to determine the content of chlorophyll before and after treatment in leaf.we found the content of chlorophyll in all the transgenic and non-transgenic tobacco decreased,and the decreased extent of transgenic was slighly lower than that of control.These result indicated that CpRCI had some function on protecting enzyme system from oxidative modification.