海南岛越南油茶种质CdS-RNase基因及其编码蛋白的多样性分析  

Diversity analysis of CdS-RNase genes and their encoding proteins in Camellia drupifera germplasm from Hainan island

作　　者：任家乐 郑道君 姚雨彤[3,4] 刘祖琪 孙秀秀 王春梅 申帅帅 陈业光[1,2] 戚华沙 陈加利 梁恒[1,2] 陈健妙 夏腾飞[1] REN Jiale;ZHENG Daojun;YAO Yutong;LIU Zuqi;SUN Xiuxiu;WANG Chunmei;SHEN Shuaishuai;CHEN Yeguang;QI Huasha;CHEN Jiali;LIANG Heng;CHEN Jianmiao;XIA Tengfei(Institute of Tropical Horticulture Research,Hainan Academy of Agricultural Sciences,Haikou 571100,Hainan,China;Sanya Institute,Hainan Academy of Agricultural Sciences,Haikou 571100,Hainan,China;School of Tropical Agriculture and Forestry,Hainan University,Haikou 570028,Hainan,China;School of Breeding and Multiplication(Sanya Institute of Breeding and Multiplication),Hainan University,Haikou 570028,Hainan,China)

机构地区：[1]海南省农业科学院热带园艺研究所,海南海口571100 [2]海南省农业科学院三亚研究院,海南海口571100 [3]海南大学热带农林学院,海南海口570028 [4]海南大学南繁学院(三亚南繁研究院),海南海口570028

出　　处：《中南林业科技大学学报》2024年第11期133-145,共13页Journal of Central South University of Forestry & Technology

基　　金：海南省重点研发项目(ZDYF2023XDNY026);海南省“南海新星”科技创新人才平台项目(NHXXRCXM202334);海南省农业科学院科研项目(HAAS2022KJCX01,HAAS2023RCQD13);海南省科技人才创新项目(KJRC2023C24);国家自然科学基金项目(32360414)。

摘　　要：【目的】为了分析海南岛本地越南油茶种质中CdS-RNase基因及其编码蛋白的多样性,探索CdSRNase蛋白类型与越南油茶自交坐果率之间的关系,为依据CdS-RNase蛋白类型合理搭配越南油茶田间种质和提高越南油茶坐果率,提供科学参考。【方法】选取60份海南岛本地越南油茶种质,以其花柱c DNA为模板,RT-RCR特异扩增CdS-RNase基因序列。琼脂糖凝胶电泳检测和切胶回收CdS-RNase基因特异性扩增条带,并把特异性扩增条带连接到平末端测序载体上。将测序载体转化到大肠杆菌感受态细胞中,用带有卡那霉素抗性的LB固体培养基筛选阳性单克隆,测序得到CdS-RNase基因序列。随后,60份越南油茶种质与“CV1”~“CV18”的CdS-RNase基因序列一起用于基因单体型划分、单体型聚类分析、获得CdS-RNase蛋白氨基酸序列、CdSRNase蛋白分型、CdS-RNase蛋白二级结构预测等。最后,根据越南油茶种质中Cd S-RNase蛋白类型,把越南油茶种质划分为三类:类型Ⅰ,只检测到正常CdS-RNase蛋白;类型Ⅱ,同时检测到正常CdS-RNase和突变CdSm-RNase蛋白;类型Ⅲ,只检测到CdSm-RNase蛋白。每类越南油茶种质挑选5份,开展自交坐果率统计试验。【结果】78份海南岛本地越南油茶种质中共鉴定到171个CdS-RNase基因单体型(Hap1~Hap171),编码35种正常CdS-RNase蛋白(CdS1-RNase~CdS35-RNase)和22种突变CdSm-RNase蛋白(CdSm1-RNase~CdSm22-RNase)。正常CdS-RNase蛋白和突变CdSm-RNase蛋白二级结构中α-螺旋比例差异不显著,但是突变CdSmRNase蛋白二级结构中β-转角和β-折叠的比例极显著或显著高于正常CdS-RNase蛋白,无规则卷曲比例显著低于正常CdS-RNase蛋白。类型Ⅰ越南油茶种质自交坐果率显著低于类型Ⅱ,类型Ⅱ越南油茶种质自交坐果率显著低于类型Ⅲ。【结论】海南岛本地越南油茶种质CdS-RNase基因及其编码蛋白多样性丰富,突变CdSmRNase蛋白可能通过增加其二级结构�【Objective】The study aimed to investigate the diversities of CdS-RNase genes and their coding proteins in Camellia drupifera germplasm from Hainan island,explore the relationship between CdS-RNase protein types and the self-pollination setting rate of C.drupifera,and provide scientific reference for a rational combination of C.drupifera germplasm in the field conditions based on CdS-RNase protein types and to improve the fruit setting rate of C.drupifera.【Method】Sixty local C.drupifera germplasm from Hainan island were selected,and their style cDNA was used as a template for RT-PCR-specific amplification of the CdS-RNase gene sequence.These CdS-RNase gene sequences were separated and re-purified with agarose gel electrophoresis,and further connected to the blunt sequencing vector.The constructed sequencing vector was transformed into E.coli competent cells and the positive clones were further screened with an LB solid plate with Kanamycin resistance.The resulting positive clones were sequenced to obtain CdS-RNase gene sequences.Subsequently,these CdS-RNase gene sequences were analyzed together with that from the“CV1”-“CV18”for gene haplotype classification,haplotype clustering analysis,obtain CdS-RNase protein amino acid sequences,categorize CdS-RNase protein types,and prediction of CdS-RNase protein secondary structure.Finally,based on the CdS-RNase protein types,these C.drupifera germplasm from Hainan island can be classified into three categories,including only detecting CdS-RNase protein(type I),co-detecting of CdS-RNase and CdSm-RNase proteins(type II),and only detecting CdSm-RNase protein(type III).Germplasms of each 5 of the three types were selected to do experiments of fruit setting percentage of self-pollination.【Result】A total of 171 CdS-RNase gene haplotypes(Hap1-Hap171),which encoding 35 normal CdS-RNase proteins(CdS1 RNase-CdS35 RNase)and 22 mutant CdSm-RNase proteins(CdSm1 RNase-CdSm22 RNase),were identified from these 78 C.drupifera germplasms from Hainan island.In the secondar

关 键 词：海南岛 越南油茶 自交不亲和 CdS-RNase基因 CdS-RNase蛋白 多样性分析 

分 类 号：S794.4[农业科学—林木遗传育种]