摘要
【目的】对月季Ⅲ型聚酮合酶基因(RcPKSⅢ)进行生物信息学和原核表达分析,为进一步探究该基因的催化功能奠定基础。【方法】以‘月月粉’花瓣为研究材料,从其基因组中筛选月季PKS基因(RcPKSs)家族成员,并对其进行生物信息学分析;利用MEGA软件对RcPKSs与其他植物PKS氨基酸序列进行系统发育分析;利用DNAMAN软件对RcPKSs和紫花苜蓿查尔酮合酶2(CHS2)基因进行序列比对分析;对RcPKSs和紫花苜蓿CHS2活性位点上的氨基酸残基进行比较,分析其催化特征;利用SWISS-MODEL对RcPKSs进行同源建模,并与紫花苜蓿CHS2蛋白三维结构进行比较。利用实时荧光定量PCR方法,分析RcPKSs和间苯三酚甲基化酶编码基因在‘月月粉’花蕾期、半开期和盛开期的表达模式;克隆RcPKSs基因全长,构建其原核表达载体pET-32a-RcPKSs,进行诱导表达,并对重组蛋白进行纯化。【结果】从‘月月粉’基因组中共鉴定出6个RcPKSs,生物信息学分析表明,RcPKS1、RcPKS2和RcPKS3为CHS基因,RcPKS4、RcPKS5和RcPKS6为新型PKSⅢ基因。系统发育分析表明,RcPKS1、RcPKS2和RcPKS3编码CHS蛋白,RcPKS4、RcPKS5和RcPKS6编码新型的非CHS蛋白。序列比对分析表明,RcPKS1、RcPKS2和RcPKS3蛋白与紫花苜蓿CHS2相似度均为72.16%,RcPKS4、RcPKS5和RcPKS6与紫花苜蓿CHS2相似度分别为67.52%,33.85%和32.73%。活性位点上氨基酸残基比较发现,RcPKS1、RcPKS2和RcPKS3活性位点上氨基酸残基均保守,RcPKS4氨基酸残基在起始口袋和延伸口袋处发生替换,RcPKS5和RcPKS6氨基酸残基均在延伸口袋处发生替换。对RcPKSs蛋白质结构的分析结果显示,RcPKS4、RcPKS5和RcPKS6蛋白质采用了几乎相同的αβαβα三维整体折叠形式。实时荧光定量分析表明,RcPKS4、RcPKS5和RcPKS6表达量在‘月月粉’花朵盛开期最高,间苯三酚O-甲基转移酶基因(POMT)、苔黑酚O-甲基转移酶基因1(OOMT1)和OOMT2表达量在盛开期最低。聚丙烯酰�
【Objective】The bioinformatics and prokaryotic expression analysis on RcPKSⅢgenes in R.chinensis were conducted to lay information for further exploration of their catalytic function.【Method】The PKSⅢgenes were screened from R.chinensis‘Old Blush’genome and analyzed by bioinformatics.Phylogenetic analysis of amino acid sequences of RcPKSs and other plant PKSs was performed with MEGA software.Sequence alignment analysis of RcPKSs and alfalfa(Medicago sativa)CHS2 was performed with DNAMAN software.Amino acid residues on active sites of RcPKSs and alfalfa CHS2 were compared to their catalytic characteristics were analyzed.The protein homology modeling of RcPKSs was performed using SWISS-MODEL,and the three-dimensional structures were compared with alfalfa CHS2 protein.Real-time fluorescence PCR was employed to analyze the expression patterns of RcPKSs and genes encoding phloroglucinol methylase in flower bud period,half-open period and blooming period of R.chinensis‘Old Blush’.The full-length RcPKSs were cloned,and the prokaryotic expression vectors pET-32a-RcPKSs were constructed to induce and purify proteins.【Result】A total of 6 RcPKSs were identified from the R.chinensis‘Old Blush’genome.Bioinformatics analysis showed that RcPKS1,RcPKS2 and RcPKS3 were the genes encoding chalcone synthases.RcPKS4,RcPKS5 and RcPKS6 were novel PKSⅢgenes.Phylogenetic analysis showed that RcPKS1,RcPKS2 and RcPKS3 encoded CHS proteins,while RcPKS4,RcPKS5 and RcPKS6 encoded novel non-CHS proteins.The sequence alignment analysis showed that RcPKS1,RcPKS2 and RcPKS3 proteins were 72.16%similar to alfalfa CHS2,and RcPKS4,RcPKS5 and RcPKS6 proteins were 67.52%,33.85%and 32.73%similar to alfalfa CHS2,respectively.The comparison of amino acid residues on active sites showed that they were conserved in RcPKS1,RcPKS2 and RcPKS3,they were substituted in the initiation pocket and elongation pocket of RcPKS4,and they were substituted in the elongation pocket of RcPKS5 and RcPKS6.The protein structure analysis showed th
作者
汪王
吉乃喆
崔娇鹏
赵世伟
赵惠恩
WANG Wang;JI Naizhe;CUI Jiaopeng;ZHAO Shiwei;ZHAO Huien(College of Landscape Architecture,Beijing Forestry University,Beijing 100089,China;Beijing Institute of Landscape Architecture,Beijing 100020,China;Beijing Botanical Garden,Beijing 100089,China)
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2022年第12期97-107,共11页
Journal of Northwest A&F University(Natural Science Edition)
基金
北京市科技计划项目(Z201100008020004)。
