利用CRISPR-Cas9技术编辑OsBADH2基因改良优质恢复系桂恢852的香味品质  

Improving the aroma quality of high-quality restorer line Guihui 852 by editing OsBADH2 gene with CRISPR-Cas9 technology

作　　者：齐金岗 陈颖 刘开强[1] 王小姣 李林娟 赵丽冰 李孝琼[1] 郭嗣斌[1] QI Jin-gang;CHEN Ying;LIU Kai-qiang;WANG Xiao-jiao;LI Lin-juan;ZHAO Li-bing;LI Xiao-qiong;GUO Si-bin(Rice Research Institute,Guangxi Academy of Agricultural Sciences/Guangxi Key Laboratory of Rice Genetics and Breeding/State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,Nanning,Guangxi 530007,China)

机构地区：[1]广西农业科学院水稻研究所/广西水稻遗传育种重点实验室/亚热带农业生物资源保护与利用国家重点实验室,广西南宁530007

出　　处：《南方农业学报》2024年第7期2058-2068,共11页Journal of Southern Agriculture

基　　金：国家自然科学联合基金项目(U20A2031);广西自然科学基金面上项目(2022GXNSFAA035494);广西农业科学院科技发展基金项目(桂农科2022JM23)。

摘　　要：【目的】利用CRISPR-Cas9技术编辑桂恢852的甜菜碱醛脱氢酶基因(OsBADH2),以期快速改良广西优质恢复系桂恢852的香味品质,为广西香稻新种质的创制提供亲本资源及技术参考。【方法】以广西优质非香型水稻桂恢852为材料,对其OsBADH2蛋白的序列特征、保守结构域、保守基序、系统发育进化等进行分析,再根据CRISPR-GE网站信息挑选合适的靶位点,分别构建OsBADH2基因第1、2外显子的双突载体pLM62和第7外显子的单突载体pLM63。利用CRISPR-Cas9技术和水稻遗传转化技术靶向敲除桂恢852的OsBADH2基因,获取不同类型的osbadh2突变体。以桂恢852为对照,通过咀嚼法鉴定突变体osbadh2是否具有香味。最后,通过农艺性状测定和单因素方差分析,判断OsBADH2基因突变是否影响农艺性状。【结果】OsBADH2基因序列全长为6268 bp,包含15个外显子和14个内含子,其编码区(CDS)为1512 bp,编码503个氨基酸残基,具有1个典型的Aldedh结构域(第16~485位氨基酸),属于醛脱氢酶,其蛋白序列与结构相对保守。通过OsBADH2氨基酸序列的BLAST比对分析,分别获得水稻的3个高相似性蛋白和拟南芥的4个高相似性蛋白,其中水稻OsBADH1、OsBADH2、OsALDH2B2、OsALDH2C1及拟南芥BADH1、BADH2、AL2C4均属于ALDH-SF(Aldehyde dehydrogenase superfamily)超家族成员。OsBADH2与OsBADH1为旁系同源基因,与拟南芥中BADH1和BADH2为直系同源基因。在桂恢852的遗传背景下成功获得4种突变类型且不含T-DNA的纯合突变体株系(osbadh2-1~osbadh2-4)。这4种突变类型均会使OsBADH2基因发生移码突变,导致翻译过程提前终止,破坏OsBADH2蛋白的原有结构和功能,导致4个osbadh2突变体株系的稻米产生香气。4个osbadh2突变体株系的株高、有效穗数、穗长、结实率、千粒重、长宽比与桂恢852野生型间无显著差异(P>0.05),但osbadh2-2突变体与osbadh2-4突变体的结实率存在极显著差异(P<0.01)。【结论】�【Objective】CRISPR-Cas9 technology was used to edit the betaine aldehyde dehydrogenase gene(OsBADH2)of Guihui 852,in order to quickly improve the aroma quality of the high-quality restorer line Guihui 852 in Guangxi,and to provide parental resources and technical references for the creation of new fragrant rice germplasm in Guangxi.【Method】The sequence characteristics,conserved domains,conserved motifs and phylogenetic evolution of the OsBADH2 protein of Guihui 852,a high-quality non-fragrant rice in Guangxi,were analyzed.According to the information on the CRISPR-GE website,appropriate target sites were selected to construct the double mutation vector pLM62 for the first and second exons of the OsBADH2 gene and the single mutation vector pLM63 for the seventh exon.The OsBADH2 gene of Guihui 852 was targetedly knocked out using CRISPR-Cas9 technology and rice genetic transformation technology to obtain different types of osbadh2 mutants.Guihui 852 was used as a control,and the chewing method was used to identify whether the mutant osbadh2 had aroma.Finally,agronomic traits were determined and one-way ANOVA was performed to determine whether the mutation of OsBADH2 gene affected agronomic traits.【Result】The full length of OsBADH2 gene sequence was 6268 bp,including 15 exons and 14 introns.Its coding region(CDS)was 1512 bp,encoding 503 amino acid residues.It had a typical Aldedh domain(amino acids positions 16-485),belonging to aldehyde dehydrogenase,and its protein sequence and structure were relatively conservative.Through BLAST analysis of OsBADH2 amino acid sequence,three highly similar proteins in rice and four highly similar proteins in Arabidopsis were obtained.OsBADH1,OsBADH2,OsALDH2B2,OsALDH2C1 of rice,BADH1,BADH2 and AL2C4 of Arabidopsis thaliana all belonged to the ALDH-SF(Aldehyde dehydrogenase superfamily)members.OsBADH2 and OsBADH1 were paralogous genes,and were orthologous genes to BADH1 and BADH2 in A.thaliana.Four homozygous mutant lines(osbadh2-1-osbadh2-4)without T-DNA were successfull

关 键 词：水稻 恢复系 香味 OsBADH2 CRISPR-Cas9 

分 类 号：S511.035.3[农业科学—作物学]