红仁核桃自然杂交后代不同表型叶片差异表达CHS基因的鉴定及生物信息学分析  

Identification and bioinformatics analysis of CHS genes in different phenotypic leaves of natural hybrid progenies of red-kernel walnut

作　　者：赵伟 李琳 刘永辉[1] 章露露 杨莹 孟海军[1] 王磊 吴国良[1] ZHAO Wei;LI Lin;LIU Yonghui;ZHANG Lulu;YANG Ying;MENG Haijun;WANG Lei;WU Guoliang(College of Horticultural Science,Henan Agricultural University/Henan Key Laboratory of Fruit and Cucurbit Biology,Zhengzhou 450002,Henan,China)

机构地区：[1]河南农业大学园艺学院·河南省果树瓜类生物学重点实验室,郑州450002

出　　处：《果树学报》2021年第2期179-191,共13页Journal of Fruit Science

基　　金：国家重点研发计划(2018YFD1000604);河南省二〇一九年科技发展计划(科技攻关)(192102110041);河南省高校科技创新团队支持计划(19IRTSTHN009)。

摘　　要：【目的】查尔酮合成酶(CHS)是植物黄酮类化合物合成途径中的第一个关键酶,探究关键CHS基因在红仁核桃花青苷生物合成过程中的功能,为探明红仁核桃花青苷合成的分子机制提供理论基础。【方法】以红仁核桃自然杂交后代不同表型(红叶和绿叶)株系3个发育时期的叶片为试材,观察颜色表型变化并测定其花青素含量,进一步通过转录组测序结果构建核桃CHSs基因表达图谱,筛选获得4个显著差异表达的JrCHS基因JrCHS1~JrCHS4,并利用生物信息学方法对相关序列、理化性质及结构功能进行预测分析。【结果】两种叶片在不同发育时期的颜色表型及花青素含量均存在显著差异;4个JrCHS基因在不同表型叶片的各发育时期均有表达,且均在时期1红叶中的表达量显著高于绿叶,与颜色表型变化及花青素含量呈正相关。生物信息分析表明,4个JrCHS基因分别位于不同染色体上,均只含有1个内含子结构,分布差异较小;各基因编码蛋白主要分布于细胞质中,蛋白保守结构域数量及位置、CHS功能结构域数量均相同,属于亲水酸性蛋白质,不存在跨膜结构;二级、三级结构预测显示该蛋白以α螺旋为主;通过与其他物种花青苷合成相关CHS蛋白进化分析发现,JrCHS1和JrCHS2与金花茶CnCHS、杜鹃花RsCHS及蓝莓VaCHS亲缘关系较近,JrCHS3与毛果杨PtCHS、荔枝LcCHS以及龙眼DlCHS亲缘关系较近,JrCHS4与木薯MeCHS及石榴PgCHS1、PgCHS2亲缘关系较近,表明其功能也较为相似;分析启动子顺式作用元件发现,4个JrCHS基因启动子序列中均包含多个MYB、MYC转录因子结合位点。【结论】4个JrCHS基因及其编码蛋白的结构与功能等较为相似,可能为红仁核桃自然杂交后代不同表型叶片花青苷代谢相关差异表达基因。【Objective】Anthocyanin is a widely distributed flavonoid metabolite,which plays important roles in setting color in plant tissues and organs.Chalcone,the basic skeleton of flavonoids,is the key position in the whole flavonoids synthesis pathway,and its synthetase(CHS)is the first key enzyme in anthocyanin synthesis pathway,which determines the anthocyanin synthesis direction and the final product numbers.In this research,the function of JrCHS genes in the anthocyanin biosynthesis was studied to provide a theoretical basis in the molecular mechanism of anthocyanin synthesis in red-kernel walnut.【Methods】By comparing the results of transcriptome sequencing with walnut genome database,four CHS genes,JrCHS1-JrCHS4,and corresponding Chalcone synthetase as functional annotation were selected from different phenotypes(red leaf and green leaf)of natural hybrid progenies of red-kernel walnut in three developmental stages.The corresponding sequences,physicochemical properties and structural functions were analyzed by bioinformatics.The basic information of CHS genes was obtained from NCBI database.The isoelectric points and molecular weights of CHS proteins were predicted by ExPASy tool,and the subcellular localization of CHS proteins was analyzed by Cell-PLoc online software.Tbtools software was used to identify the structure of CHS genes and chromosomal position.MEME online software was used to analyze the conserved motifs of CHS proteins.ProtScale and TMpred software were used to analyze hydrophobicity and protein transmembrane,and SOPMA and SWISS-MODEL were used to predict protein secondary and tertiary structures.The sequence of multi species CHS proteins was analyzed by MEGA 7.0 software,and NJ method was used to construct phylogenetic tree.The upstream 2000 bp fragment of CHS genes was selected to analyze the cis-acting elements of promoter by the PlantCARE software.The RNA-seq data of different phenotypes of natural hybrid progenies of red-kernel walnut were analyzed,and the CHS gene expression map was cons

关 键 词：红仁核桃 花青苷 CHS基因 差异表达 生物信息分析 

分 类 号：S664.1[农业科学—果树学]