百合ATP合成酶家族基因ATPase3的克隆和生物信息学分析  

Cloning and bioinformatics analysis of ATP synthase family gene ATPase3 in Lilium

作　　者：贾文杰[1] 马璐琳[1] 段青[1] 杜文文[1] 王祥宁[1] 李想 崔光芬[1] JIA Wen-jie;MA Lu-lin;DUAN Qing;DU Wen-wen;WANG Xiang-ning;LI Xiang;CUI Guang-fen(Flower Research Institute,Yunnan Academy of Agricultural Sciences/National Engineering Research Center for Ornamental Horticulture,Kunming 650205,China)

机构地区：[1]云南省农业科学院花卉研究所/国家观赏园艺工程技术研究中心,昆明650205

出　　处：《西南农业学报》2023年第4期796-804,共9页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31960614);云南省创新人才培养对象项目(2018HB083)。

摘　　要：【目的】为有效解决百合花粉污染问题,利用分子生物学技术培育雄性不育的无花粉百合,从基因克隆和生物信息学角度研究百合ATP合成酶家族基因ATPase3在导致百合雄性不育过程中的作用,为应用该基因进行百合无花粉分子育种提供一定的理论基础。【方法】以百合可育系与不育系花粉母细胞时期花药为试验材料,在百合可育系与不育系转录组测序结果中筛选出1个花粉母细胞时期显著上调表达的ATPase3基因,利用荧光定量qRT-PCR技术检测ATPase3基因在百合可育系与不育系花粉母细胞时期不同部位的相对表达量。结合qRT-PCR与Race技术扩增得到百合花药ATPase3基因cDNA全长序列,并通过Prot Param、Prot Scale、NCBI Conserved Domain、SWISS-MODEL等在线生物信息学数据库对百合ATPase3同源性、蛋白结构、理化性质等进行生物信息学分析。【结果】百合ATPase3基因在花器官中表达量最高,其中在可育系花药中的表达量是不育系的7倍。通过克隆得到百合ATPase3基因,该基因全长327 bp,编码109个氨基酸,含有1个典型的保守ATPase3结构域;编码蛋白相对分子量约为12.14 KD,理论等电点为7.71,为疏水性蛋白,存在跨膜结构,并预测蛋白二级和三级结构。生物信息学分析说明该预测蛋白具有典型ATP合成酶特征。同源序列分析表明,该蛋白与拟南芥质膜ATPase3同源性最为接近,与拟南芥H[+]ATPase1,H[+]ATPase2,H[+]ATPase3,H[+]ATPase8同源性也较为接近。推测该蛋白属于百合中提供能量的质膜离子转运蛋白,它在百合不育系花药中表达量下调,提供能量不足,导致雄性不育。【结论】本研究验证了ATPase3基因与百合雄性不育相关,并成功克隆得到了ATPase3基因,对该基因进行了生物信息学分析和功能预测,为百合ATPase3基因编码的质膜离子转运蛋白功能缺失导致百合雄性不育现象的发生提供一定的理论基础。【Objective】The paper aimed to effectively solve the problem of pollen pollution and cultivate male sterile Lilium using molecular biology technology.The role of ATPase3 gene,a member of the ATPase family,in the male sterility Lilium was studied,which would provide a theoretical basis for the application of this gene in the pollen-free molecular breeding of Lilium.【Method】Anthers of fertile lines and sterile lines of Liliums were used as experimental materials,and a significantly up-regulated ATPase3 gene was screened from the results of transcriptome sequencing of fertile lines.Relative expression of ATPase3 gene in different parts of Lilium was detected by fluorescence quantitative qRT-PCR during the period of microsporocyte.Full-length cDNA sequence of anther ATPase3 gene of Lilium was amplified by qRT-PCR and Race technology,and the structure and physicochemical properties of Lilium ATPase3 were analyzed by various online bioinformatics software(Prot Param,Prot Scale,NCBI Conserved Domain,SWISS-MODEL).【Result】The expression of ATPase3 gene in Lilium was the highest in flower organs,and the expression in anthers of fertile lines was 7 times higher than that of sterile lines.Full-length ORF of Lilium ATPase3 gene was 327 bp,encoding 109 amino acids and containing a typical conserved ATPase3 domain;Relative molecular weight of the protein was 12.14 KD and theoretical isoelectric point was 7.71.It was a hydrophilic protein with transmembrane structure,and the secondary and tertiary structures of the protein were predicted.Bioinformatics analysis showed that the predicted protein had typical characteristics of ATP synthase.Homologous sequence analysis showed that the protein had the closest homology with Arabidopsis thaliana plasma membrane ATPase3 and H[+]ATPase1,H[+]ATPase2,H[+]ATPase3,H[+]ATPase8.This protein was a plasma membrane ion transporter that provided energy in plants.It was speculated that the expression level of this protein was down-regulated in the anthers of Lilium sterile lines,which l

关 键 词：百合 基因克隆 雄性不育 ATPase3基因 生物信息学 

分 类 号：S644[农业科学—蔬菜学]