‘红满堂’苹果MbbZIP43基因的克隆与功能研究  

Cloning and Functional Analysis of MbbZIP43 Gene in‘Hongmantang’Red-flesh Apple

作　　者：杨艳 胡洋 刘霓如 殷璐 杨锐 王鹏飞[1] 穆霄鹏[1] 张帅 程春振 张建成[1] YANG Yan;HU Yang;LIU Ni-ru;YIN Lu;YANG Rui;WANG Peng-fei;MU Xiao-peng;ZHANG Shuai;CHENG Chun-zhen;ZHANG Jian-cheng(College of Horticulture,Shanxi Agricultural University,Taigu 030801)

机构地区：[1]山西农业大学园艺学院,太谷030801

出　　处：《生物技术通报》2024年第2期146-159,共14页Biotechnology Bulletin

基　　金：山西省科技重大专项计划“揭榜挂帅”项目(202201140601027);山西省果树产业技术体系项目(2023CYJSTX07)。

摘　　要：【目的】为探究碱性亮氨酸拉链(bZIP)转录因子在‘红满堂’苹果花青素代谢中的调控作用。【方法】从‘红满堂’苹果克隆得到一个bZIP基因,分析了其基因及编码蛋白序列特性,利用洋葱表皮细胞瞬时转化确定其编码蛋白亚细胞定位,利用实时荧光定量PCR(RT-qPCR)研究了该基因在不同成熟时期‘红满堂’果实中的表达情况,基于烟草叶片、苹果叶片以及苹果果实瞬时转化研究了其对花青素积累和花青素合成相关结构基因表达的调控作用。【结果】该基因不含内含子,与MdbZIP43(XP_008393381.1)相似度最高,故命名为MbbZIP43。其编码序列长为522 bp,可编码由173 aa组成、含有bZIP_plant_GBF1结构域、定位于细胞核的亲水蛋白。RT-qPCR分析结果显示,随着果实成熟,MbbZIP43在‘红满堂’果实中的表达水平呈“先升后降”的变化趋势,在花后11周的果实中表达量最高。相关性分析显示MbbZIP43基因表达量与总黄酮和花青素含量正相关。瞬时过表达该基因的烟草叶片、苹果叶片和果皮中花青素含量分别提高了17.42%、25.66%和48.99%,过表达MbbZIP43的苹果叶片中花青素合成结构基因CHI、F3'H、DFR和UFGT分别提高了2.27倍、1.84倍、2.39倍和2.89倍;过表达MbbZIP43的苹果果皮中CHI、F3'H、DFR和UFGT分别提高了1.79倍、1.70倍、1.35倍和1.51倍,说明MbbZIP43可以通过上调这些结构基因的表达正调控苹果花青素合成。【结论】MbbZIP43可通过激活花青素合成相关结构基因CHI和UFGT等的表达进而促进花青素的积累。【Objective】To investigate the regulatory role of basic leucine zipper(bZIP)transcription factors in anthocyanin metabolism in‘Hongmantang’apple.【Method】We cloned a bZIP gene from‘Hongmantang’red-flesh apple,analyzed it and its sequence characteristics,studied the subcellular localization of its encoded protein using onion epidermis cell transient overexpression,and investigated its expression pattern in‘Hongmantang’red-flesh apple fruits at different ripening stages using quantitative real time PCR(RT-qPCR).Furthermore,we explored its regulatory roles in the expressions of genes related to anthocyanin accumulation and biosynthesis based on the transient overexpression experiments in tobacco leaves,apple leaves and fruits.【Result】This bZIP gene was intron-less and shared the highest similarity with MdbZIP43(XP_008393381.1),thus was named as MbbZIP43.Its coding sequence was 522 bp,which encoded a 173 aa hydrophilic nucleus-localized protein with conserved bZIP_plant_GBF1 domain.RT-qPCR analysis revealed that MbbZIP43 showed a‘rise-fall’expression change pattern in‘Hongmantang’fruits during ripening,with the highest expression in the fruits at 11 weeks post flowering.Correlation analysis results showed that MbbZIP43 expression was positively correlated with the total flavonoids and anthocyanin contents in‘Hongmantang’fruits,indicating that it might play regulatory roles in flavonoid biosynthesis.Consistently,transient overexpression of MbbZIP43 improved significantly the anthocyanin accumulations by 17.42%,25.66%and 48.99%in tobacco leaves,apple leaves and apple fruits,respectively.Moreover,RT-qPCR analysis results showed that,compared to the empty vector control,the expressions of anthocyanin biosynthesis related structural genes(CHI,F3'H,DFR and UFGT)in the apple leaves overexpressing MbbZIP43 were up-regulated by 2.27-,1.84-,2.39-and 2.89-fold,respectively.And their expressions in the peels of apple fruits overexpressing MbbZIP43 were up-regulated by 1.79-,1.70-,1.35-and 1.51-f

关 键 词：bZIP转录因子 苹果 花青素合成调控 基因克隆 瞬时转化 表达分析 亚细胞定位 

分 类 号：S661.1[农业科学—果树学]