利用CRISPR/Cas9技术编辑MODD增强水稻休眠性

CRISPR/Cas9-Mediated Editing of MODD Enhances Rice Dormancy

【目的】休眠是水稻重要的农艺性状。适当的休眠可以抑制水稻的穗发芽现象,是确保产量和品质的关键因素。然而,水稻休眠调控的基因及其调控网络仍需进一步研究。已知基因MODD编码未知功能的蛋白,负向调控水稻脱落酸信号和抗旱性,但其调控水稻休眠的功能未知。研究MODD在调控水稻休眠中的功能,有助于完善水稻休眠调控网络,同时为抗穗发芽遗传育种提供新的理论基础和种质资源。【方法】根据RGAP数据库公布的基因序列,构建MODD的CRISPR-Cas9敲除载体,通过农杆菌介导的遗传转化方法转化中花11(ZH11)愈伤组织,从而获得水稻转基因植株;利用PCR扩增、测序技术及qRT-PCR技术筛选并鉴定MODD敲除纯合系;根据2个突变系(KO-1和KO-2)的CDS得到突变系的氨基酸序列,然后,用DNAMAN对比ZH11和2个突变系(KO-1和KO-2)的蛋白序列;利用Linux系统筛选出MODD在水稻中的同源基因;取开花后35d的种子,调查ZH11和敲除系的发芽率;利用酵母单杂和LUC试验验证MODD的上游基因。【结果】查找到水稻中有6个MODD的同源基因,分别为LOC_Os07g41160、LOC_Os03g30570、LOC_Os03g53630、LOC_Os04g35430、LOC_Os03g17050和LOC_Os06g01170;成功构建了敲除载体,并转入ZH11中,获得2个纯合突变系(KO-1和KO-2);qRT-PCR结果表明,2个突变系(KO-1和KO-2)的MODD表达量显著降低;蛋白序列分析表明,KO-1和KO-2的移码突变造成了蛋白翻译的提前终止;发芽率结果显示,2个突变系(KO-1和KO-2)的发芽率在吸水第3天比ZH11分别显著降低了15%和15%;之后差异逐渐扩大,在第6天差异达到最大,比ZH11分别显著降低35%和35%;2个突变系(KO-1和KO-2)的穗发芽现象显著低于ZH11;酵母单杂试验结果表明,在酵母中,ABI5可以结合MODD的启动子区域,并且进一步把结合范围缩小至300 bp以内;LUC结果显示,加入ABI5的荧光值是单独加NONE空载荧光值的2.6倍,说明ABI5可以激活MODD的表达。【结论】敲除MODD可以增加种子的休眠,MODD可能通过ABA信号途径调控种子休眠。

【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.