甘蓝型油菜R2/R3型MYB转录因子BnPAP2a介导花青素累积研究  

R2/R3 type MYB transcription factor BnPAP2a mediated anthocyanin accumulation in Brassica napus L.

作　　者：李芳 唐秀华 张彦锋[2] 安然 黄淑华 谢长根 LI Fang;TANG Xiuhua;ZHANG Yanfeng;AN Ran;HUANG Shuhua;XIE Changgen(College of Life Sciences,Northwest A&F University,Yangling,Shaanxi 712100,China;Hybrid Rapeseed Research Center of Shaanxi Province,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]陕西省杂交油菜研究中心,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2023年第7期35-44,共10页Journal of Northwest A&F University(Natural Science Edition)

基　　金：陕西省重点研发计划一般项目-农业领域(2018NY-097);陕西省创新能力支撑计划项目(2020TD-051);陕西省油菜分子设计育种科技资源共享平台项目(2021PT-036)。

摘　　要：【目的】探讨甘蓝型油菜R2/R3型MYB转录因子BnPAP2a对花青素累积的作用,为富含花青素油菜的培育提供理论指导。【方法】在全基因组水平系统鉴定甘蓝型油菜PAP1、PAP2亚家族R2/R3型MYB转录因子同源基因。利用BnTIR网站、BRAD网站和GSDS等软件对鉴定所得基因的结构、蛋白序列和组织表达谱进行分析。采用RT-PCR方法从“中双11”cDNA中扩增BnPAP2a基因,并构建35S启动子驱动的植物表达载体35S-pHZM27-BnPAP2a-mGFP,将其转染拟南芥,用显微成像法进行转基因株系种子与幼苗花青素累积表型分析;采用分光光度法测算转基因株系幼苗花青素水平;采用荧光成像技术考察转基因株系中BnPAP2a的亚细胞定位。【结果】成功地在甘蓝型油菜中鉴定到9个PAP1、PAP2亚家族R2/R3型MYB转录因子同源基因,分别为BnPAP1a,BnPAP1b,BnPAP1c,BnPAP1d,BnPAP1e,BnPAP1f,BnPAP1g,BnPAP2a,BnPAP2b。基因结构分析结果显示,拟南芥、白菜、甘蓝和甘蓝型油菜的MYB转录因子同源基因大都含有3个外显子和2个内含子。基因组织表达谱分析表明,BnPAP2a在叶片、花、花蕾、果荚中均有表达但其表达水平不高。成功构建了35S-pHZM27-BnPAP2a-mGFP载体,转染后获得了转基因拟南芥植株。过量表达BnPAP2a基因可使拟南芥种皮由黄褐色转变成紫黑色,幼苗叶片由绿色变为紫色,花青素含量极显著增加。激光共聚焦显微镜下观察发现,过表达BnPAP2a植株GFP荧光信号主要集中在细胞核中。【结论】BnPAP2a为功能性的细胞核定位R2/R3型MYB转录因子,具有促进花青素形成的作用,可利用其培育富含花青素的油菜新材料。【Objective】This study investigated the role of R2/R3 MYB transcription factor BnPAP2a in anthocyanin accumulation in rapeseed(Brassica napus L.)to provide guidance for cultivation of rape varieties rich in anthocyanin.【Method】Orthologues of PAP1 and PAP2 subfamily members of Brassica napus L.were identified via genome-wide annotation.The gene architecture,protein motif arrangement and expression profiles of orthologues were determined via on-line tools,such as GSDS,BRAD and BnTIR.The full length cDNA of BnPAP2a was obtained from Zhongshuang 11 by RT-PCR.The overexpression vector of BnPAP2a(pHZM27-BnPAP2a-mGFP)driven by 35S promoter was obtained through gateway technology.Transgenic lines overexpressing BnPAP2a were obtained via floral dip method.The phenotype of seeds and seedlings was photographed under a microscope.The quantity of anthocyanins in the transgenic lines was measured by spectrophotometric method.The subcellular localization of BnPAP2a was determined using a confocal microscope.【Result】A total of 9 orthologues of PAP1 and PAP2 subfamily members including BnPAP1a,BnPAP1b,BnPAP1c,BnPAP1d,BnPAP1e,BnPAP1f,BnPAP1g,BnPAP2a and BnPAP2b were obtained.Most orthologues in Arabidopsis thaliana,Brassica rapa,Brassica oleracea and Brassica napus contained three exons and two introns.Low expression of BnPAP2a was detected in leaves,flower,bud and silique.The 35S-pHZM27-BnPAP2a-mGFP vector was obtained through gateway technology and the transgenic line overexpressing BnPAP2a was obtained via floral dip method.Overexpression of BnPAP2a significantly increased anthocyanin accumulation,converted seed color from tan to purple black,and converted seedling leaves from green to purple.The fluorescence signal of GFP in BnPAP2a overexpression lines was localized exclusively in the nucleus.【Conclusion】BnPAP2a is a functionally nuclear localized R2/R3 type MYB transcription factor.It plays important roles in anthocyanin biosynthesis and can be used for artificial breeding of rapeseed germplasm rich in anthocy

关 键 词：甘蓝型油菜 花青素 BnPAP2a MYB转录因子 油菜育种 

分 类 号：S565.403.2[农业科学—作物学]