苹果PRE6-like基因的克隆及在花青素合成中的功能分析  

Cloning of PRE6-like gene and analysis of its function in anthocyanin synthesis in apple

作　　者：黄娟娟 黄亚萍 李文芳[1] 毛娟[1] 陈佰鸿[1] HUANG Juanjuan;HUANG Yaping;LIWenfang;MAO Juan;CEHN Baihong(College of Horticulture,Gansu Agricultural University,Lanzhou 730070,Gansu,China)

机构地区：[1]甘肃农业大学园艺学院,兰州730070

出　　处：《果树学报》2024年第5期812-823,共12页Journal of Fruit Science

基　　金：国家重点研发计划(2022YFD1602106);甘肃省教育厅“双一流”重点科研项目(GSSYLXM-02);甘肃省科技重大专项(22ZD6NA045)。

摘　　要：【目的】通过验证苹果非典型成员多效唑抗性蛋白基因(PRE6-like)的功能,探索其在花青素生物合成中的调控作用。【方法】基于俄矮2号芽变枝条果皮转录组测序结果,筛选获得花青素合成相关调节基因MdPRE6-like,对其进行生物信息学分析,克隆CDS并构建过表达载体,瞬时表达金冠苹果果实,遗传转化苹果愈伤和拟南芥进行功能验证。【结果】MdPRE6-like cDNA全长279 bp,编码92个氨基酸,相对分子质量10450.75 Da,理论等电点(pI)为6.41,含有HLH结构域,亚细胞定位预测结果为细胞核。组织特异性表达分析表明,MdPRE6-like基因在花、果皮中的表达量显著高于根和叶。瞬时表达金冠苹果表明,MdPRE6-like显著促进了金冠苹果果皮注射部位花青素的积累,并显著提高了花青素合成通路相关结构基因的表达水平。MdPRE6-like基因在苹果愈伤组织和拟南芥过表达表明,转基因苹果愈伤组织和拟南芥叶脉中的花青素含量显著高于野生型,且花青素合成通路结构基因表达水平上调。【结论】MdPRE6-like基因能够正向调控花青素的合成和积累,为后续MdPRE6-like基因参与改良苹果果实品质提供理论参考。【Objective】The PREs gene,also known as the polyoxazole resistance gene,belongs to the basic/helix-loop-helix(bHLH)transcription factor family.It lacks a DNA binding domain and typically forms homodimers or heterodimers to regulate the expression of target genes,thereby influencing plant morphology,cell size,pigment metabolism and response to abiotic stress.This study aimed to verify the function of the atypical member of the polyoxazole resistance protein gene(PRE6-like)in apple and explore its regulatory role in anthocyanin biosynthesis.【Methods】The Oregon SpurⅡ,which is highly prone to bud mutation,exhibits significantly improved fruit color compared to the original variety.A new mutant strain of the Oregon SpurⅡapple was discovered,which exhibits early and intense coloring,with a reddish fruit surface at maturity,and stable variation traits.Based on transcriptome sequencing of the skin of fruits on the bud mutation branch from the Oregon SpurⅡapple,a flower pigment synthesis-related regulatory gene,MdPRE6-like was identified.The coding sequence(CDS)of the Md-PRE6-like gene(MD14G1197600)was obtained from the apple genome website,and primers were designed using Primer 5.0 to amplify the cDNA from Oregon SpurⅡthe skin tissue of fruits on the bud mutation branch.Bioinformatics analysis software was used for biological information analysis,and tissue-specific expression was analyzed using the fluorescence quantitative PCR.The CDS region was cloned and ligated into the linearized expression vector pCAMBIA1301-GFP.Escherichia coli DH5αwas heat-shock transformed,and Agrobacterium tumefaciens GV3101 was transformed to construct the overexpression vector.Transient expression was performed in Golden Delicious apple fruit,and the color changes of the skin under intensive light conditions(12000 lx)were observed to preliminarily identify the gene’s role in anthocyanin synthesis.Further functional validation was conducted through Agrobacterium-mediated transformation of apple callus and by the floral dip m

关 键 词：苹果 MdPRE6-like 花青素合成 遗传转化 表达水平 

分 类 号：S661.1[农业科学—果树学]