Cloning And Functional Analysis Of Two Cold Induced Genes OrCr1and OrCr2in Chaling Wild Rice

Chaling wild rice （Oryza rufipogon Griff.） is one of two northernmost wild rice groups in the world. It is very cold tolerant as it can survive over winter in Chaling county （26°50′N） and Changsha city （28°13’N）, Hunan province, China. To understand mechanism underlying its cold response and discover new cold-tolerance genes in it,we performed a global genome expression profiling of it subjected to cold stress. Expression profiles were obtained from plants under cold stress and no stress as control using the GeneChip Rice Genome Array （Affymetrix） representing51279transcripts from japonica and indica rice. We identified a number of genes, highly induced or repressed under the cold stress. Two genes, OrCrl （Oryza rufipogon cold responsive gene1）and OrCr2, highly induced ones among them,were selected and cloned for further analysis.Sequence analysis showed that the cDNA of OrCrl encodes a protein of492amino acid residues with theoretical M.W.=523.207kD and p1=6.21. Using the deduced aa sequence coded by the OrCrl as query to perform BLAST search in GenBank, the result show that the aa sequence is99.18%similar to that of （BAD01231.1, putative COSTANS-like B-box zinc finger protein） encoded by nipponbare gene AK099722.1.The cDNA of OrCr2encodes a protein of169amino acid residues with theoretical M.W.=17.467kD and pI=10.06.Searching sequence databases showed that the aa sequence is identical with that of the protein（NP₀01172191.1, putative leucine-rich repeat receptor-like kinase2precursor） encoded by nipponbare gene AK108484.1.Analysis of their putative promoter regions for cis-regulatory elements using PlantCAREsoftware（http://bioinformatics.psb.ugent.be/webtools/plantcare/html/） identified3and2matches to known cis-elements related to stress responses, respectively.In order to find their functions in cold tolerrance or others, we constructed a plant binary vector pCAMBIA1300-OrCrl/2,driven by strong constitution promoter2xCaMV35S. After that, pCAMBIA1300-OrCrl/2were transformed into rice Zhonghuall through agrobacterium-mediated method. Twenty-six OrCrl and twenty-one OrCr2independent transgenic rice plant were obtained. The cold tolerance of transgenic rice plants were assessed for their TO generations seedlings. The results suggest that the transgenic TO generation seedlings free proline content accumulates more than that of controls after cold treatment. Additionally, in the normol condition, the OrCr2TO transgenic plants were lower than those of wild-type. Further phenotypic identification is in progress.