Functional Verification Of The Rice Cold-tolerant 131 Cold Tolerance Candidate Gene OsRBCS3

Rice is one of the most important food crops in China and the Heilongjiang province.Heilongjiang province is located in the north of China.It is the northern limit of rice cultivation in the world.Low temperature injury can lead to a significant reduction in rice yield.The cold tolerance of different rice varieties were different,and the breeding of cold resistant varieties was the most effective measure to prevent and control the low temperature injury.Research and cloning of genes that related to cold tolerance in rice is good for cold tolerance breeding in rice.The Key Laboratory of molecular biology of Heilongjiang Province College established the digital expression profile of the cold tolerance of rice Kongyu 131 and cold sensitive rice Longjing 11.According to the study of the expression profile of rice digital low temperature stress,we selected the gene OsRBCS3 which related to photosynthesis and low temperature stress response.And analysis OsRBCS3 gene expression between cold tolerance rice Kongyu 131 and cold sensitive rice Longjing 11 under low temperature stress.The sequence differences of OsRBCS3 gene between Kongyu 131 and Longjing11 were compared;The over expression vector and CRISPR/Cas9 knockout vector of rice Kongyu 131 OsRBCS3 gene were constructed;The function of Kongyu 131OsRBCS3 gene was investigated by genetic transformation.The main results of the study include:1.The OsRBCS3 gene expression level of Kongyu 131 was significantly higher than that of Longjing 11 after low temperature stress at booting stage.The sequence of OsRBCS3 gene that from Kongyu 131 and Longjing 11 were same.The difference in cold tolerance of two rice lines may be caused by the different expression of OsRBCS3gene.2.Construction of Kongyu 131 OsRBCS3 gene over expression vector pGWB12-R3 and pGWB18-R3 by Gateway cloning technique.At the same time,the CRISPR/Cas9 knockout vector pVK-R3 was constructed.3.The expression vector containing Kongyu 131 OsRBCS3 gene was introduced into the cold sensitive rice Longjing 11 genome by Agrobacterium tumefaciens mediated method.The number of positive transgenic lines L-12 was 9.The number of L-18 was 10.Knockout of OsRBCS3 gene of cold tolerant rice Kongyu 131 using CRISPR/Cas9 gene editing technology.The number of positive transgenic lines KC was5.4.Identification of cold tolerance of Longjing 11 transgenic rice with OsRBCS3gene,the cold tolerance of rice lines overexpressing OsRBCS3 gene was higher than that of Longjing 11.5.The positive transgenic line KC was detected by molecular biology,and there was 1 line of T₀ that were edited at the target site.The cold tolerance of transgenic lines with Os RBCS3 gene that knocked down by CRISPR/Cas9 was lower than that of Kongyu 131.