Screening And Mutant Identification Of Eleven Rice Cold Response Related Genes

Rice is an important monocotyledon model crop and an important staple foodcrop in China.During the rice growth and development,chilling injury is a common and worldwide problem.Therefore,it is great significance for rice production and food safety to study the molecular physiological mechanism of rice cold tolerance in China.In order to study the mechanism of the low temperature tolerance in rice,the most direct and effective tool is to construct rice mutants,especially and gene knockout mutant for this goal.In this study,11 genes were induced by low-temperature stress to choose from low-temperature expression microarrays,and 8 gene deletion mutants using CRISPR/Cas9 technology and 3 RISD TDNA Mutant lines(Rice T-DNA Insertion Sequences database)were created.The main results obtained are as follows:1.Screening of low-temperature response genes in rice: Eleven low-temperature specific response genes(Metall,UL,WRKY45,WI12,ADT,RBP,OS2,POT,PRP,CP12 and RCI2-6)were screened based on the gene expression profile of japonica variety Nipponbare seedlings.The qRT-PCR technique was used to detect the inducible expression levels of these 11 genes at 4°C stress,and it was found that the expression inducibility of PRP gene was the most significant,and the expression of WRKY45 and WI12 genes was also evident at 4°C,ADT,RBP,OS2 and POT gene was induced by low temperature,while Metall,UL,CP12 and RCI2-6 genes had some changes under the 4°C stress,suggesting that these genes had a certain functional relationship with cold stress response.2.Creation of CRISPR/Cas9 deletion mutants: CRISPR/Cas9 dual target site vectors for WI12,ADT,RBP,OS2,POT and PRP genes were constructed using CRISPR/Cas9 technology.In addition,one target site was selected for the CP12 and RCI2-6 genes each and a common dual target site vector for these two genes was constructed.A total of 52 mutants with gene editing were obtained using Agrobacterium genetic transformation.Among them,homozygous mutant lines have been screened for the WI12,RBP,OS2,POT and PRP transgenic lines,and heterozygous mutants have been screened for the ADT gene,and heterozygosity of these two genes has been screened for the genes CP12 and RCI2-6.Among dual target sites,the POT transgenic line P-56 was edited by a special editor with deletions of large fragments that were not edited at the target site(outside the two target sites)occurred.3.Subcellular localization of Metall,UL and WRKY45 proteins: In order to further analyze the gene functions,subcellular localization vectors of three genes(Metall,UL and WRKY45)were constructed to transform rice protoplasts.Under confocal laser scanning microscopy,it was found that Metall is into the cytoplasm,UL is into the chloroplast,and WRKY45 is into the nucleus.4.The investigation of agronomic traits of the Metall,UL and WRKY45 T-DNA homozygous mutant lines: T-DNA homozygous mutants were screened by co-segregation analysis.T-DNA homozygous mutant target gene expression is lower in qRT-PCR than wild-type.Analysis of agronomic traits of Metall,UL and WRKY45 T-DNA homozygous mutant lines revealed that seed-set rate,spike length,1-4 internode lengths,plant height,and effective tillernumbers were significantly lower than the wild-type DJ,indicating that the lack of expression of these three genes affects rice growth and development.5.Identification of cold stress tolerance of T-DNA homozygous mutants: Metall homozygous mutants were treated with 4°C stress for three-leaf-old rice seedlings of mutants and wild-type.Metall homozygous mutants were treated at 4°C for 4 days while UL and WRKY45 homozygous mutants were stressed at 4°C for 5 days.After regeneration,it was found that the Metall,UL and WRKY45 T-DNA mutant were all sensitive to cold stress.