蚬壳花椒POD同工酶基因在种子萌发过程中的功能分析  

Functional Analysis of Zanthoxylum dissitum Hemsl POD Isoenzyme Gene during Seed Germination

作　　者：白磊 王恒 孙吉康[1] 周韬[1] 杜哲 吴艳 Bai Lei;Wang Heng;Sun Jikang;Zhou Tao;Du Zhe;Wu Yan(College of Life Science and Technology,Central South University of Forestry and Technology,Changsha,410004)

机构地区：[1]中南林业科技大学生命科学与技术学院,长沙410004

出　　处：《分子植物育种》2024年第23期7684-7694,共11页Molecular Plant Breeding

基　　金：长沙市科技局科技计划项目(kq2014157);湖南省教育厅科学研究项目(19A531);湖南省自然科学基金项目(2021JJ31149);湖南省重点研发计划项目(2022NK2006);中南林业科技大学引进人才科研启动基金项目(2019YJ035)共同资助。

摘　　要：本研究克隆表达了过氧化物酶同工酶基因家族中两个与蚬壳花椒(Zanthoxylum dissitum Hemsl.)种子萌发密切相关的基因ZdPOD1、ZdPOD2,对比分析了两者在蚬壳花椒种子萌发过程中调控功能的异同。根据蚬壳花椒种子转录组测序结果,筛选得到两个重要的过氧化物酶基因,对其进行生物信息学分析、酵母表达体系蛋白表达分析,利用荧光定量PCR分析其在蚬壳花椒萌发种子及幼根中的表达变化,同时进行原生质体亚细胞定位分析。克隆获得蚬壳花椒ZdPOD1、ZdPOD2基因全长序列,分别为987和982 bp,蛋白表达产物分别为38.24、37.57 kD。酵母表达实验表明相对于ZdPOD1,ZdPOD2具有加固酵母细胞壁功能。荧光定量PCR实验表明ZdPOD2的表达在种子萌发前期较低,萌发后期显著升高,而ZdPOD1表达情况与之相反。亚细胞定位显示ZdPOD1主要分布于小麦原生质细胞核附近,而ZdPOD2在细胞膜附近表达较多。ZdPOD1主要与蚬壳花椒种子萌发初期细胞抗氧化性相关,其功能在于祛除种子萌发时因油脂代谢产生的大量的活性氧,避免细胞生物膜氧化损伤,以防止萌发失败;ZdPOD2主要与细胞壁形成相关,其功能是在蚬壳花椒种子萌发后期具有加固细胞壁的作用。This study cloned and expressed two genes,ZdPOD1 and ZdPOD2,from the peroxidase isozyme gene family that are closely associated with seed germination in Zanthoxylum dissitum.The regulatory functions of these two genes during seed germination in Zanthoxylum dissitum were compared to elucidate their similarities and differences.Base on the results of the transcriptome sequencing of Z.dissitum,two important peroxidase genes were screened.Bioinformatics analysis,protein expression analysis using a yeast expression system,and fluorescent quantitative PCR were conducted to analyze their expression changes in germinating seeds and young roots of Zanthoxylum dissitum.Meanwhile,subcellular localization analysis in protoplasts was performed.The full length sequences of ZdPOD1 and ZdPOD2 genes of Zanthoxylum dissitum were obtained,which were 987 and 982 bp,respectively,the molecular weights of the proteins were respectively 38.24 and 37.57 kD.Compared with ZdPOD1,ZdPOD2 was able to strengthen the cell wall of yeast.Fluorescence quantitative PCR showed that the expression level of ZdPOD2 was significantly lower in the early stage of germination but higher in the late stage of germination,while the expression level of ZdPOD1 was opposite.The subcellular localization showed that ZdPOD1 was mainly distributed near the wheat protoplasmic nucleus,while ZdPOD2 was more expressed near the cell membrane.Zd-POD1 is mainly related to antioxidant activity of cells in the initial germination of Z.dissitum seeds,which remove excess reactive oxygen species caused by lipid metabolism during seed germination to avoid oxidative damage that leads to germination failure.ZdPOD2 is mainly related to cell wall formation,and its function is to strengthen cell wall in the late stage of seed germination.

关 键 词：蚬壳花椒 过氧化物酶 基因克隆 表达分析 

分 类 号：S567.19[农业科学—中草药栽培]