桑树花青素合成相关基因MnANS和MnFLS的克隆及其在番茄中过表达分析  

Cloning of anthocyanidin⁃related genes MnANS and MnFLS from mulberry and analysis of overexpression in tomato

作　　者：代洁 唐清霞 曾益春[1] 刘江 何琴 危玲[1] 佟万红[1] 姚永权[1] 郑继川[1] 李永远[1] 刘刚[1] 黄盖群[1] DAI Jie;TANG Qing-xia;ZENG Yi-chun;LIU Jiang;HE Qin;WEI Ling;TONG Wan-hong;YAO Yong-quan;ZHENG Ji-chuan;LI Yong-yuan;LIU Gang;HUANG Gai-qun(Sericulture Research Institute of Sichuan Academy of Agricultural Sciences/Institute of Special Economic Flora and Fauna of Sichuan Academy of Agricultural Sciences,Nanchong,Sichuan 637000,China;Sichuan General Station of Horticultural Crops Technology Extension,Chengdu 610000,China)

机构地区：[1]四川省农业科学院蚕业研究所/四川省农业科学院特种经济动植物研究所,四川南充637000 [2]四川省园艺作物技术推广总站,成都610000

出　　处：《西南农业学报》2024年第12期2602-2608,共7页Southwest China Journal of Agricultural Sciences

基　　金：四川省财政自主创新专项(2022ZZCX085);四川省重点研发计划项目(2024YFCY0009);四川省农业科学院“1+9”揭榜挂帅科技攻关项目(1+9KJGG004);国家现代农业产业技术体系四川蚕桑创新团队项目(SCCXTD-2024-17);国家现代农业产业技术体系项目(CARS-18-SYZ19);南充市研发资金项目(23YYJCYJ0041)。

摘　　要：【目的】探究桑树(Morus notabilis)花青素生物合成途径中花青素合酶(Anthocyanidin synthase/Leucoanthocyanidin dioxygenase,ANS/LDOX)和黄酮醇合酶(Flavonol synthase,FLS)基因的功能,为桑椹品质改良提供理论基础。【方法】克隆2个桑树花青素合成途径的关键基因MnANS和MnFLS,将这2个基因构建至过表达载体中,并通过农杆菌介导的遗传转化将2个载体转化至番茄Micro-Tom中。【结果】基因和蛋白序列分析表明,桑树MnFLS基因编码含335个氨基酸残基的蛋白,预测分子量为38.3 kDa,等电点为5.91;MnANS基因编码含358个氨基酸残基的蛋白,预测分子量为40.9 kDa,等电点为5.62。MnFLS蛋白和MnANS蛋白均含有Fe2OG-dioxygenase保守结构域。经转基因阳性鉴定,获得含MnFLS和MnANS基因的转基因番茄植株。表型观察结果显示,转MnANS基因番茄植株的茎秆呈深紫色,表明MnANS蛋白对花青素的积累具有潜在影响。转MnFLS基因番茄植株并未表显出明显的表型变化。【结论】通过对转基因番茄植株表型和基因表达量的综合分析,初步揭示MnANS基因在桑树花青素合成中的作用,并为桑树品质改良提供重要的分子生物学信息和进一步研究桑果颜色和花青素代谢工程奠定基础。[Objective]In the study,the functions of ANS and FLS genes in the anthocyanin biosynthesis pathway of mulberry(Morus notabi-lis)were investigated to provide a theoretical basis for the improvement of mulberry quality.[Method]Two key genes in the anthocyanin bio-synthesis pathway in mulberry,MnANS and MnFLS,were cloned.In order to investigate the biological functions of these two genes,these two genes were constructed into overexpression vectors,and the two vectors were transformed into the tomato Micro-Tom by agrobacterium.[Result]Sequence analysis showed that MnFLS encoding a 335 amino acid protein with a predicted molecular weight of 38.3 kDa and an isoelec-tric point of 5.91,while MnANS encoded a 358 amino acid protein with a predicted molecular weight of 40.9 kDa and an isoelectric point of 5.62.Both proteins contained the conserved domain of Fe2 OG-dioxygenase.The transgenic plants of MnFLS and MnANS were obtained after transgene positive identification.Phenotypic observations showed that plants with overexpressing the MnANS showed a dark purple colour at the stalk,indicating its potential effect on anthocyanin accumulation,while MnFLS gene overexpression plants did not show significant pheno-typic changes.[Conclusion]Through the comprehensive analysis of the phenotype and gene expression of transgenic plants,the study prelimi-narily reveales the role of MnANS gene in anthocyanin synthesis and provides important molecular biological information for mulberry quality improvement,which lays the foundation for further research on mulbery fruit colour and anthocyanin metabolic engineering.

关 键 词：桑树 花青素生物合成 MnANS基因 MnFLS基因 基因克隆 番茄过表达分析 

分 类 号：S888.3[农业科学—特种经济动物饲养]