芍药PlHSP70和PlDanJ基因的克隆及表达分析  

Cloning and expression analysis of PlHSP70 and PlDanJ genes of Paeonia

作　　者：贺丹[1,2] 华超 何松林 曹健康 张明星 张佼蕊 刘艺平 HE Dan;HUA Chao;HE Songlin;CAO Jiankang;ZHANG Mingxing;ZHANG Jiaorui;LIU Yiping(College of Landscape Architecture and Art,Henan Agricultural University,Zhengzhou 450002,China;College of Horticulture and Landscape Architecture,Henan Institute of Science and Technology,Xinxiang 453003,China;Henan High-quality Flower and Vegetable Seedling Engineering Research Center,Zhengzhou 450002,China)

机构地区：[1]河南农业大学风景园林与艺术学院,河南郑州450002 [2]河南科技学院园艺园林学院,河南新乡453003 [3]河南省优质花卉蔬菜种苗工程研究中心,河南郑州450002

出　　处：《河南农业大学学报》2022年第4期587-598,共12页Journal of Henan Agricultural University

基　　金：国家自然科学基金项目(31600568,31870698);河南省科技攻关项目(202102110234)。

摘　　要：【目的】探索芍药PlHSP70与PlDanJ基因在芍药属远缘杂交不亲和中的作用。【方法】利用RNA-seq数据库分析芍药不亲和转录组中的热激蛋白家族基因,以芍药品种‘粉玉奴’柱头为试验材料,采取RACE克隆的方法得到HSP70与DanJ基因,命名为PlHSP70与PlDanJ,并对其进行生物信息学分析及表达分析。【结果】从RNA-seq数据库筛选出20个HSP家族基因,HSP70在自交24 h的表达量都显著高于杂交24 h的表达量,而DanJ在杂交36 h的表达量都显著高于杂交24 h的表达量。根据芍药与拟南芥之间的系统发育关系,可将HSP家族基因进化树分为5个小簇。PlHSP70基因CDS区域全长为1539 bp,编码513个氨基酸。PlDanJ基因CDS区域全长为1269 bp,编码422个氨基酸。进化树结果分析表明,PlHSP70与TcHSP70同源性较高,PlDanJ与DcDanJ、PeDanJ同源性较高。RT-qPCR分析表明,PlHSP70和PlDanJ在‘粉玉奴’中的时空表达都具有组织特异性,PlHSP70在叶部相对表达量最高,PlDanJ在根部相对表达量最高。PlHSP70在自交和杂交授粉后(2~36 h)其相对表达量均呈现出多峰型变化趋势,在授粉后4 h达到最高。PlDanJ在自交授粉后(2~36 h)的相对表达量呈先升高后降低的单峰型变化趋势,授粉后4 h达到最高;在杂交授粉后呈现为先降低后升高再降低的多峰型变化趋势,授粉后2 h时表达水平最高。【结论】2个芍药HSP家族基因在授粉后的表达水平与花粉管的整体生长情况相吻合,推测PlHSP70和PlDanJ基因通过调控花粉在柱头上的生长来影响芍药与牡丹远缘杂交不亲和性。【Objective】To explore the role of Paeonia lactiflora PlHSP70 and PlDanJ in distant hybridization incompatibility of Paeonia lactiflora【Method】The RNA-seq database was used to analyze the heat shock proteins family genes in the transcription database of P.lactiflora,and the stigma of P.lactiflora‘Fenyunu’was used as the materials to clone the HSP70 and DanJ genes by RACE technology.These two genes were named PlHSP70 and PlDanJ,and bioinformatics analysis and expression analysis were performed.【Result】The results showed that 20 HSP family genes were screened from the RNAseq database.The expression levels of HSP70 at 24 h of selfing were all significantly higher than those at 24 h of hybridization,while the expression levels of DanJ at 36 h were all significantly higher than those at 24 h of hybridization.Based on the phylogenetic relationship between P.lactiflora and Arabidopsis thaliana,the evolutionary tree was divided into five subgroups.The CDS region of the PlHSP70 gene had a total length of 1539 bp,encoding 513 amino acids.The CDS region of the PlDanJ gene had a total length of 1269 bp encoding 422 amino acids.The results of evolutionary tree showed that PlHSP70 and TcHSP70 had higher homology,and PlDanJ had higher homology with DcDanJ and PeDanJ.RT-qPCR analysis showed that both PlHSP70 and PlDanJ had tissue expression specificity in‘Fenyunu’,and the expression levels of PlHSP70 and PlDanJ were the highest in leaves and roots respectively.The expression of PlHSP70 showed a multimodal trend after self-cross and hybridized pollination(2-36 h),reaching its highest level at 4 h after pollination.The expression level of PlDanJ after self-cross pollination(2-36 h)showed an unimodal change trend of firstly increasing and then decreasing,reaching the highest at 4 h after pollination,but showed a multimodal change trend of decreasing firstly and then rising and then decreasing after hybridization,with the highest expression level at 2 h after pollination.【Conclusion】The expression levels of th

关 键 词：芍药 热激蛋白 基因克隆 杂交不亲和 花粉发育 

分 类 号：S682.1[农业科学—观赏园艺]