马铃薯块茎花色素苷合成相关R2R3 MYB蛋白基因的克隆和功能分析  

Cloning and Functional Analysis of R2R3 MYB Genes Involved in Anthocyanin Biosynthesis in Potato Tuber

作　　者：谈欢 刘玉汇 李丽霞 王丽[2] 李元铭 张俊莲 TAN Huan;LIU Yu-Hui;LI Li-Xia;WANG Li;LI Yuan-Ming;ZHANG Jun-Lian(Gansu Key Laboratory of Crop Genetic Improvement and Germplasm Enhancement/College of Horticulture/Gansu Provincial Key Laboratory of Aridland Crop Science,Gansu Agricultural University,Lanzhou 730070,Gansu,China;College of Life Science and Technology,Gansu Agricultural University,Lanzhou 730070,Gansu,China)

机构地区：[1]甘肃省作物遗传改良与种质创新重点实验室/甘肃农业大学园艺学院/甘肃省干旱生境作物学重点实验室,甘肃兰州730070 [2]甘肃农业大学生命科学技术学院,甘肃兰州730070

出　　处：《作物学报》2018年第7期1021-1031,共11页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(31601356);甘肃省杰出青年科学基金项目(17JR5RA138);甘肃省农业生物技术研究与应用开发专项(GNSW-2015-15);甘肃省高等学校科研项目(2016A-030);甘肃农业大学"伏羲人才"计划项目(Gaufx-02Y04)资助~~

摘　　要：植物花色素苷的合成代谢受转录因子的调控,其中R2R3 MYB为最主要的转录调控因子。本研究以彩色四倍体马铃薯为试材,克隆了R2R3 MYB基因家族里调控马铃薯块茎花色素苷合成R2R3 MYB-St AN1的3个同源基因,并利用生物信息学分析、稳定烟草遗传转化、q PCR等方法对这3个同源基因的结构和功能进行分析和鉴定,结果表明,这3个同源基因均含有R2和R3保守结构域,其主要差别在于C端由10个氨基酸序列组成的重复结构(R)数目不同,根据R数目将其分别命名为St AN1-R0、St AN1-R1和St AN1-R3,其蛋白分子量分别为28 047.91、29 458.35和31 527.60 Da,等电点(p I)分别为6.14、6.90和8.39,均为亲水蛋白。通过转化烟草发现,转入St AN1-R0、St AN1-R1和St AN1-R3后,烟草叶片叶色变化明显,其中转St AN1-R1烟草叶色呈深红色,其叶片花色素苷含量最高。进一步利用q PCR分析表明,外源St AN1使烟草叶片花色素苷合成代谢途径的关键基因(Nt CHS、Nt CHI、Nt F3H、Nt F3’H、Nt DFR、Nt ANS、Nt UFGT)上调表达,同时烟草内源Ntb HLH基因的表达显著上升;St AN1-R1可以高效地调控烟草内源Ntb HLH基因和结构基因Nt DFR和Nt ANS的表达。结果表明,St AN1的3个同源蛋白均可以调控花色素苷的合成,而只含一个重复序列R的St AN1调控花色素苷合成的能力最强。The anthocyanins biosynthesis is regulated by transcription factors,and R2R3 MYB is the most important transcriptional regulator in plant.In this study,three homologous genes were isolated from tetraploid potato,belonging to R2R3 MYB gene family.The structure and function analysis of the three homologous genes were characterized by bioinformatics analysis,stable tobacco genetic transformation and qPCR assays.The three homologous genes contained R2 and R3 conserved domains,differed in the number of repeats(R)consisting of 10 amino acid sequences,and named as StAN1-R0,StAN1-R1,and StAN1-R3 according to the number of R.The coding proteins are hydrophilic with molecular weight of 28 047.91,29 458.35,31 527.60 Da,and isoelectric points(pI)of 6.14,6.90,and 8.39,respectively.The accumulation of anthocyanin was significantly increased in StAN1-R0,StAN1-R1 and StAN1-R3 overexpressed plants.The leaf color of StAN1-R1-overexpressed plants was dark red with the highest anthocyanin content among the three transgenic events.The qPCR assays showed that exogenous StAN1 genes enhanced the expression of endogenous NtbHLH transcription factor as well as NtCHS,NtCHI,NtF3H,NtF3’H,NtDFR,NtANS,and NtUFGT,involved in anthocyanin biosynthesis in transgenic tobacco leaves.The overexpression of StAN1-R1 resulted in higher expression of NtDFR,NtANS,and endogenous NtbHLH in transgenic tobacco.The results showed that three homologous genes of StAN1 can regulate anthocyanin biosynthesis,among them StAN1-R1 containing one R has the strongest regulatory capacity.

关 键 词：马铃薯 花色素苷 R2R3 MYB 基因克隆 生物信息学分析 烟草转化 基因表达分析 

分 类 号：S532[农业科学—作物学]