芍药胼胝质合成酶基因家族鉴定及PlCalS5功能分析  

Identification of Callose Synthetase Gene Family and Functional Analysis of PlCalS5 in Paeonia lactiflora

作　　者：贺丹[1,2] 尤啸龙 何松林 张明星[1] 张佼蕊 华超 王政 刘艺平 HE Dan;YOU XiaoLong;HE SongLin;ZHANG MingXing;ZHANG JiaoRui;HUA Chao;WANG Zheng;LIU YiPing(College of Landscape Architecture and Art,Henan Agricultural University,Zhengzhou 450002;College of Horticulture Landscape Architecture,Henan Institute of Science and Technology,Xinxiang 453003,Henan)

机构地区：[1]河南农业大学风景园林与艺术学院,郑州450002 [2]河南科技学院园艺园林学院,河南新乡453003

出　　处：《中国农业科学》2023年第16期3183-3198,共16页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31600568,31870698);河南省自然科学基金(232300420006);河南省高等学校重点科研项目(23A220001)。

摘　　要：【目的】CalS家族在调控植物胼胝质合成方面具有重要作用,鉴定芍药CalS家族成员,并进行生物信息学和表达模式分析,为芍药属远缘杂交不亲和研究提供依据。【方法】通过荧光显微镜观察自交、杂交柱头内花粉管生长过程及花粉萌发情况;测定柱头所含胼胝质、内源脱落酸含量(ABA)以及β-1,3-葡聚糖酶活性;分段克隆8个PlCalS并进行序列分析;使用Expasy、MEME、TBtools、MEGA 7.0等软件和在线工具预测PlCalS家族成员蛋白质基本理化性质、保守基序并构建系统发育进化树;利用qRT-PCR技术检测8个PlCalS在自交24 h、杂交24 h、杂交36 h的相对表达水平;对CalS5进行多序列比对并构建系统进化树,分析PlCalS5响应不同浓度ABA处理的表达特征。【结果】花粉管荧光显微观察发现杂交柱头内发生较为严重的胼胝质堵塞从而影响花粉管的正常生长及花粉的萌发。测定柱头内胼胝质含量发现多数时期自交柱头均低于同时期杂交柱头的含量,柱头内β-1,3-葡聚糖酶活性、ABA含量变化均具有一定的规律性。通过对结构域的完整性分析鉴定芍药PlCalS基因家族成员,后经同源性比对分析命名8个CalS,均含有15个保守基序且在PlCalS基因家族中的分布类似。多物种系统进化关系表明,CalS家族可分为3个分支,PlCalS家族仅分布在2个分支,其中PlCalS5与牡丹、拟南芥和番茄的CalS5亲缘关系较近。生物信息学分析结果表明8个家族成员编码1745—1951个氨基酸,原子总数为28583—31870个,等电点为7.99—9.13。对转录组FPKM值的分析表明,PlCalS家族成员在相同时期杂交处理中有较高表达,相同处理情况下在杂交36 h时高表达;荧光定量PCR表明,8个基因的相对表达量在自交24 h时均低于杂交24 h和杂交36 h。经两个浓度的ABA处理,发现PlCalS5对高浓度的ABA更为敏感。【结论】芍药CalS家族有8个基因成员且具有较高的保守性,8个家族【Objective】The CalS family plays an important role in regulating callose synthesis in plants.In this study,the members of the CalS family were identified and their bioinformatics and expression patterns were analyzed,which provided an evidence for distant cross incompatibility of Paeonia lactiflora(P.lactiflora).【Method】The pollen germination and tube growth in self-and cross-pollinated stigma were observed by fluorescence microscope.The callose content,endogenous Abscisic acid(ABA)content andβ-1,3-glucanase activity in stigma were measured.Eight members of the CalS family were cloned,and Expasy,MEME,TBtools,MEGA 7.0 and so on were used to predict the basic physicochemical properties and conserved motifs of proteins of the PlCalS family members and to construct a phylogenetic evolutionary tree.The expression of eight PlCalS in stigma at 24 h of self-pollination,24 h of cross-pollination and 36 h of cross-pollination were analyzed by quantitative real-time polymerase chain reaction(qRT-PCR).Multiple sequence alignment of CalS5 was performed,a phylogenetic tree was constructed,and the expression characteristics of PlCalS5 in response to ABA treatment at different concentrations were analyzed.【Result】Fluorescence microscopic observation of pollen tube showed that callose plugging occurred in the stigma of hybrid,which restricted the pollen germination and tube growth.Callose content in cross-pollinated stigma was found to be higher in most periods than that in self-pollinated stigma.Then,the callose activity ofβ-1,3-glucose and ABA content in stigma with different pollination affinity showed regular differences.The members of the PlCalS family of P.lactiflora were identified and named by integrity analysis of the structural domains and sequence matching,and 15 conserved motifs with stable distribution in the PlCalS family were encoded by every eight genes.Multi-species phylogenetic relationships showed that the CalS family could be divided into three branches,with the PlCalS family distributed in only

关 键 词：芍药属 远缘杂交不亲和 CalS基因家族 表达分析 

分 类 号：S682.12[农业科学—观赏园艺]