栽培种花生Ty3-gypsy类逆转座子逆转录酶基因序列的分离与分析  

Isolation and Characterization of Reverse Transcriptase Gene Sequences of Ty3-gypsy-like Retrotransposons from Cultivated Peanut(Arachis hypogaea)

作　　者：熊发前[1] 刘俊仙[2] 刘菁 阳太亿 贺梁琼[1] 蒋菁[1] 韩柱强[1] 唐秀梅[1] 钟瑞春[1] 吴海宁[1] 黄志鹏[1] 唐荣华[1] Xiong Faqian;Liu Junxian;Liu Jing;Yang Taiyi;He Liangqiong;Jiang Jing;Han Zhuqiang;Tang Xiumei;Zhong Ruichun;Wu Haining;Huang Zhipeng;Tang Ronghua(Cash Crops Research Institute,Guangxi Academy of Agricultural Sciences,Nanning,530007;Sugarcane Research Institute,Guangxi Academy of Agricultural Sciences/Key Laboratory of Ministry of Agriculture and Rural Affairs for Sugarcane Biotechnology and Genetic Improvement(Guangxi)/Guangxi Key Laboratory of Sugarcane Genetic Improvement,Nanning,530007)

机构地区：[1]广西农业科学院经济作物研究所,南宁53007 [2]广西农业科学院甘蔗研究所/农业农村部广西甘蔗生物技术与遗传改良重点实验室/广西甘蔗遗传改良重点实验室,南宁530007

出　　处：《基因组学与应用生物学》2022年第4期862-876,共15页Genomics and Applied Biology

基　　金：国家自然科学基金项目(31960409);广西自然科学基金项目(2018GXNSFDA281027);广西农业科学院科技发展基金项目(桂农科2018YM06,桂农科2017JZ13,桂农科31960409)共同资助。

摘　　要：植物长末端重复(long terminal repeat,LTR)逆转座子具有普遍性、高拷贝、高异质性和插入多态性,适合开发成分子标记。本研究从栽培种花生(Arachis hypogaea)基因组中获得Ty3-gypsy类逆转座子逆转录酶基因序列,分析其特点和差异,为基于LTR逆转座子的分子标记开发奠定基础。利用根据Ty3-gypsy类逆转座子逆转录酶基因保守区设计的简并引物对栽培种花生‘桂花1026’品种的基因组DNA进行PCR扩增,目的条带经回收、克隆和测序后,对获得序列进行生物信息学分析。克隆到27条逆转录酶基因序列,序列长度皆为432 bp,AT所占比例为55.32%~66.90%,AT与GC比值为1.24~2.02,核苷酸序列间相似性为44.90%~98.80%,呈现较高异质性;聚类分析后,27条基因序列分为3个家族,家族Ⅰ是主要成分;27条编码蛋白序列的基因中有10条发生了无义突变;基因编码蛋白序列间相似性为34.50%~97.90%,呈现高度异质性;除AhRT2-4外,各基因编码蛋白序列间保守基序完全一致;对栽培种花生和其他物种植物的Ty3-gypsy类逆转座子逆转录酶序列构建系统进化树,进化树显示所有序列被分为6类,其中,Ⅰ类包含19条其他植物物种和1条栽培种花生的序列,Ⅱ类包含8条其他物种和7条栽培种花生的序列,表明栽培种花生逆转录酶序列具有较高的保守性,栽培种花生逆转录酶基因编码蛋白序列与绿豆(Vigna radiata)、枣(Ziziphus jujuba)、黄瓜(Cucumis sativus)、油棕(Elaeis guineensis)、火龙果(Hylocereus undatus)等植物的逆转录酶序列亲缘关系较近,表明不同植物的Ty3-gypsy逆转座子之间可能存在着水平基因转移;通过比对花生EST数据库,发现了4条具有转录活性的栽培种花生Ty3-gypsy类逆转座子。本研究所获得的逆转录酶基因序列可为栽培种花生Ty3-gypsy逆转座子的分子标记开发利用及分子育种奠定一定基础。Plant LTR(long terminal repeat)retrotransposons have the characteristics of universality,high copy number,high heterogeneity and insertion polymorphism,which are suitable for the development of mole-cular markers.In this study,the reverse transcriptase gene sequences of Ty3-gypsy-like retrotransposons were acquired from the genome of cultivated peanut(Arachis hypogaea),and their characteristics and differences were analyzed,which will lay a foundation for the development of LTR retrotransposons based molecular markers.A pair of degenerate primers were designed according to the conservative region of reverse trans-criptase gene of Ty3-gypsy-like retrotransposons.Then the genomic DNA of cultivated peanut variety‘Guihua1026’was used for amplification.After that the target band was recovered,cloned and sequenced,the obtained sequences were analyzed by bioinformatics.Finally,twenty-seven reverse transcriptase gene sequences were obtained successfully.All gene sequences were 432 bp.The proportion of AT content ranged from 55.32%to 66.90%.The ratio of AT to GC ranged from 1.24 to 2.02.The similarity of nucleotide sequences ranged from 44.90% to 98.80%.These sequences existed higher heterogeneity.Twenty-se-ven gene sequences can be divided into three families by cluster analysis.FamilyⅠwas the main component.Ten of twenty-seven gene coding protein sequences showed nonsense mutations.The similarity of gene coding protein sequences was 34.50%~97.90%.These indicated that the coding protein sequences of reverse transcriptase gene existed high heterogeneity.Except for AhRT2-4,the conserved motifs of the protein sequences encoded by the reverse transcriptase gene sequences of Ty3-gypsy-like retrotransposons are completely identical.The phylogenetic tree was constructed from the amino acid sequences of reverse transcriptase gene of Ty3-gypsy-like retrotransposons in cultivated peanut and some other plant species.The phylogenetic tree showed that all sequences were classified into six categories.Among them,Class Ⅰ contai

关 键 词：花生 Ty3-gypsy类逆转座子 逆转录酶 异质性 

分 类 号：S565.2[农业科学—作物学]