The Function Analysis Of The Candidate Cold Tolerance Gene OsRBCS3 Gene

China is a long-established agricultural country.Rice,as a major grain crops,has solved the problem of food and clothing for nearly 1.4 billion people.Cold damage is a extraordinary serious meteorological disaster,which seriously affects the production of rice,especially in the northeastern part of China.It is affected by low temperature and cold damage,and it is mostly obstacle cold damage,which causes huge losses to rice yield.Obstacle chills occur once every 4-5 years,seriously threatening rice production.In order to solve this contradiction,it is necessary to improve the cold tolerance of rice varieties.So we have to clone the resistance-tolerant chilling gene and use it to breed cold-tolerant varieties.In the early stage of this study,a cold-tolerant digital expression profile was screened to select a gene related to photosynthesis and up-regulated under low temperature stress,and the cold tolerance candidate gene OsRBCS3 was identified as the research object.In this study,after cloning the CDS region of this gene,sequence alignment was performed,and it was found that there was no difference between L11and K131 CDS regions.At the same time,under low temperature stress,Significant differences in expression levels between the two genes,and we thought that the expression level of the gene affected Cold tolerance of two varieties.It is cold-resistant when it is over-expressed.In order to prove this point,On the one hand,according to the existing OsRBCS3 gene over-expressing materials L-12 and L-18 strains in the laboratory,the positive strains of the offspring were screened by molecular means to verify whether the target gene was over-expressed.Cold tolerance;on the other hand,the CRISPR/Cas9 genome editing technology was used to create a knockout strain of the target gene in Kongyu 131,and it was confirmed whether the cold tolerance of the target gene was silenced.If the hypothesis is established,it means that the ability to increase the expression of this gene is more resistant to chills.The main findings are as follows:1.The optimal screening concentration of hygromycin resistance in transgenic rice was determined to be 15 mg/L.Using this concentration to screen the phenotype of T₁generation strains against hygromycin,a total of 11 strains with phenotypic resistance were obtained.After identification by exogenous gene PCR,60 plants of 11 strains were positive.The positive rate was 84.6%.Among them,there are 6 materials L-12,each material is 4⁶ strains of 33 strains,and the material L-18 is 5,and each material has 4⁶strains of 27 strains.2.Take the T₀ generation seeds of these 11 lines and screen the T₁ seedlings with hygromycin resistance medium.Different strains have different separation ratios for hygromycin resistance,among which 9 in T₁ generation.The isolate ratio of the lines was approximately 3:1,following the Mendelian inheritance model.It indicates that the foreign gene is a single copy insertion and is a single gene dominant inheritance.There are also some strains with a separation ratio lower than 3:1,which does not meet the Mendelian inheritance rule.3.The 9 lines of the seed,T₂ generation,were harvested.Each seedling was subjected to hpt-PCR amplification,and the expected target fragment size was 472 bp,and all were positive.Nine positive overexpressing lines were screened.There are 5strains of L-12 and 4 strains of L-18.4.The OsRBCS3 gene was cloned from cold-tolerant rice cultivar 131,and the knockout vector was constructed by CRISPR/Cas9 technology.The Agrobacterium tumefaciens-mediated method was used to genetically transform the air culture 131,and24 positive positive transgenic lines were obtained.Sequencing identified 18 mutant strains of target site editing,which were found by amino acid sequence alignment.Among them,5/18 strains caused frameshift mutation due to the insertion of bases,and the stop codon was generated in advance.The translation was terminated early.5.Over-expression materials and gene knockout materials were identified for cold tolerance index at booting stage,cold tolerance index identification and real-time PCR detection at seedling stage.The results showed that the cold tolerance and the expression of the target gene of the transgenic lines L-12 and L-18 overexpressing the OsRBCS3gene obtained by Gataway technology were significantly improved compared with the wild type;on the contrary,the cold tolerance was edited by the CRISPR/Cas9 method.The cold tolerance of the mutant CL of the OsRBCS3 gene and the expression level of the target gene were significantly lower than those of the wild type.6.The physiological indexes of over-expressed materials and gene knockout materials were detected under low temperature and cold damage.The results showed that the activity of Rubisco was higher than that of wild type under over-temperature treatment.The activity of Rubisco was lower than that of wild-type knockout materials.The type has dropped significantly.In contrast,the content of malondialdehyde in the low temperature treatment was lower than that of the wild type material in the overexpressed material,and the MDA content of the knockout material was higher than that of the wild type material.