利用CRISPR/Cas9技术编辑MODD增强水稻休眠性OA北大核心CSTPCD

[Objective]Dormancy is an important agronomic trait of rice.Proper dormancy can inhibit the preharvest sprouting of rice and is a key factor to ensure yield and quality.However,the genes and regulatory networks of rice dormancy regulation still need further study.The MODD encoded a protein with unknown function,and it negatively regulate rice abscisic acid signaling and drought resistance,but its function in regulating rice dormancy is unknown.Studying the function of MODD in regulating rice dormancy will help to improve the rice dormancy regulatory network,and at the same time provide a new theoretical basis and germplasm resources for genetic breeding of preharvest sprouting resistance.[Method]Based on the gene sequences published in the RGAP database,a CRISPR-Cas9 knockout vector for MODD was constructed,and the calli of Zhonghua 11 was transformed through agrobacterium mediated genetic transformation to obtain transgenic rice plants.The MODD knockout homozygous lines were screened and identified using PCR amplification,sequencing technology,and qRT-PCR technology.The amino acid sequences of the two mutant lines(KO-1 and KO-2)were obtained according to the CDS of the two mutant lines,and then the protein sequences of ZH11 and the two mutant lines(KO-1 and KO-2)were compared by DNAMAN.The homologous genes of MODD in rice were screened using Linux system.Take the seeds 35 days after heading and investigated the germination rate of ZH11 and knockout lines.The yeast hybridization and LUC experiments were used to verify the upstream gene of MODD.[Result]Six MODD homologous genes were found in rice,which were LOC_Os07g41160,LOC_Os03g30570,LOC_Os03g53630,LOC_Os04g35430,LOC_Os03g17050,LOC_Os06g01170.The knockout vector was successfully constructed and transferred it into ZH11,two homozygous mutant lines(KO-1 and KO-2)were obtained.The qRT-PCR results showed that the expression level of MODD in the two mutant line(KO-1 and KO-2)was significantly reduced.Protein sequence analysis showed that the frameshift mutations of KO-1 and KO-2 caused the early termination of protein translation.The germination rate of the two mutant lines(KO-1 and KO-2)was significantly lower than that of ZH11 by 15%and 15%respectively on the third day after water absorption;After that,the difference gradually expanded and reached the maximum on the 6th day,which was significantly lower than that of ZH11 by 35%and 35%respectively.The preharvest sprouting of two mutant lines(KO-1 and KO-2)was significantly lower than that of ZH11.The results of Y1H experiment showed that ABI5 could bind to the promoter region of MODD in yeast,and the binding range was further reduced to less than 300bp.LUC results showed that the fluorescence value of ABI5 was 2.6 times that of none alone,indicating that ABI5 could activate the expression of MODD.[Conclusion]Knocking out MODD could increase seed dormancy,and MODD may regulate seed dormancy through ABA signaling pathway.