The Preliminary Function Analysis On OsNCED1 Gene In Rice(Orazy Sativa L.)

As one of the most popular food crop in China,rice(Oryza Sativa L.)maybe affect by undercooling,overheating or drought in unfevorable environment.Considering another important element during its life cycle,abscisic acid(ABA)can not only regulate plant growth and development,but also response to a variety of biotic or abiotic stress.9-cis-epoxidized carotenoid dioxygenase(NCED)is a key rate-limiting enzyme for biosynthesis of ABA.The purpose of the research is to study the molecular and physiological mechanism of OsNCED1.Orygza Sativa L.ssp japonica,var Nipponbare and indica,var E007 were taken as materials by the means of CRISP-Cas9,overexpression,GUS and other molecular biology,plant physiology and other technical methods.The main results are as follows:1?Obtaining Osnced1-Cas9 knockout mutant homozygous plant.The Osncedl-Cas9 knockout vector was constructed and was transform to Nipponbare and E007.Osncedl-Cas9 gene knockout homozygous plant were created.After analyzing the knockout loci of homozygous plants,it was found that a 1-base absent from the 928 bp of the genome target sequence was detected in Nipponbare and the two homozygous hybrids were detected in E007 1 base deletion and 1 base addition;2?Osncedl knockout mutants are sensitive to salt stress during seedling and third leaf age stage.Using different concentrations of salt stress during its seedling stage,the growth of mutants was significantly slower than wild-type.When the treatment of 150 mMNaCl was conducted in three-leaf age stage,the survival rate of Osncedl knockout mutant was 0%while that of wild type was 20%;3?The seed setting rate of knockout mutant will decrease under chilling condition stress.Using staging sowing to make the mutant undergo natural chilling condition during the panicle differentiation and flowering period,result shows that the seed set rate of knockout mutant is significantly lower than wild type.After chilling treatment of 18 C for 7 days during flowering period,the seed set rate of Osnced1 knockout mutant valued at 50.99%which was significantly lower than wild type recorded as 75.71%,same results were observed under artificial chilling treatment or natural chilling stress.It is indicated that knockouting the OsNCED1 would decrease the ability of chilling;4?Overexpression of OsNCED1 increase the ability of chilling injury resistant during flowering stage of rice.After over expression of OsNCED1 gene,PCR identification showed that the expression of NCED1 gene was obviously up-regulated in overexpressing plants.Seed set rate of OsNCED1 overexpressed transgenic seedlings was 85.22%while the value of wild type was 75.71%;5?QsNCED1 gene was expressed in root,coleoptile,leaf,stem,node and spikelet of rice.NCED1-GUS transgenic plants were Obtain by construct Pro:NCED1::GUS vector,GUS tissue staining showed that OsNCED1 was specifically expressed in roots,coleoptiles,leaves,stem,node and spikelet OsNCED1 semi-quantitative analysis showed consistent results with GUS tissue staining.OsNCED1 were expressed in root,young leaves,nodes,leaf sheath,flag leaf as well as spikelet,especially in leaf sheath and flag leaf then in spikelet.