四倍体野生种花生Ty3-gypsy类反转录转座子RT基因的分离及序列分析  

Isolation and Sequence Analysis of Reverse Transcriptase of Ty3-gypsy-like Retrotransposons from Arachis monticola

作　　者：蔡铁城 张冲 刘菁 阳太亿 陈华 蒋菁[2] 贺梁琼[2] 韩柱强[2] 唐荣华[2] 庄伟建 刘俊仙[3] 熊发前[2] Cai Tiecheng;Zhang Chong;Liu Jing;Yang Taiyi;Chen Hua;Jiang Jing;He Liangqiong;Han Zhuqiang;Tang Ronghua;Zhuang Weijian;Liu Junxian;Xiong Faqian(College of Plant Protection,Fujian Agriculture and Forestry University,Fuzhou,350002;Cash Crops Research Institute,Guangxi Academy of Agricultural Sciences,Nanning,530007;Guangxi Key Laboratory of Sugarcane Genetic Improvement,Key Laboratory of Sugarcane Biotechnology and Genetic Improvement(Guangxi),Ministry of Agriculture and Rural Affairs,Sugarcane Research Center,Chinese Academy of Agricultural Sciences,Sugarcane Research Institute,Guangxi Academy of Agricultural Sciences,Nanning,530007)

机构地区：[1]福建农林大学植物保护学院,福州350002 [2]广西农业科学院经济作物研究所,南宁530007 [3]广西农业科学院甘蔗研究所,中国农业科学院甘蔗研究中心,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：《分子植物育种》2023年第21期6972-6981,共10页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31960409;32072103;31960416);广西自然科学基金项目(2018GXNSFDA281027;2018GXNSFDA294004;2020GXNSFAA297081)共同资助。

摘　　要：本研究从四倍体野生种花生中分离了Ty3-gypsy类反转录转座子的反转录酶(RT)基因序列,分析了其序列特点和差异,为研究其转录活性和功能奠定基础。根据Ty3-gypsy类反转录转座子RT基因的保守区设计简并引物,PCR扩增四倍体野生种花生(Arachis monticola)(PI468199)的DNA,目的条带经回收、克隆和测序后,进行生物信息学分析。结果显示,目的条带大小约为430 bp,共有29条AmRT2-X基因序列被分离出,这些序列长度变化范围为397~433 bp,AT所占比例范围为55.66%~65.97%,核苷酸序列间相似性在45.1%~97.2%之间,存在较高异质性;29条AmRT2-X基因序列被聚类划分为8个家族,家族A是包含序列最多的家族;翻译成氨基酸后,有10条AmRT2-X基因序列发生了无义突变;氨基酸序列间相似性在22.3%~98.6%之间,呈现高度异质性;绝大部分AmRT2-X基因序列的保守基序一致;系统进化树显示,所有RT基因序列被分为6类,其中,Ⅰ类包含20条AmRT2-X基因序列,Ⅱ类中的4条Am RT2-X基因序列与来自拟南芥、多花黑麦草、火龙果、牡丹、果梅、枣、山桑、白皮松等物种的序列之间亲缘关系较近;通过比对花生EST数据库,发现2条具有转录活性的四倍体野生种花生Ty3-gypsy类反转录转座子。本研究为Ty3-gypsy类反转录转座子全长序列的分离及其功能的研究提供一定基础。The reverse transcriptase sequences of Ty3-gypsy-like retrotransposons were isolated from tetraploid wild peanut species,and their sequences characteristics and differences were analyzed,which will lay a foundation for studying their transcriptional activity and function.The genomic DNA of tetraploid wild peanut species(Ara-chis monticola)(PI468199)was amplified using a pair of degenerate primers designed according to the conservative region of reverse transcriptase of Ty3-gypsy-like retrotransposons.Then the target band was recovered,cloned and sequenced.Finally,the obtained sequences were analyzed through bioinformatics analysis.The results showed that the size of the target band was approximately 430 bp.Twenty-nine AmRT2-X sequences were obtained successfully.The length of sequences ranged from 397 bp to 433 bp.The proportion of AT content ranged from 55.66%to 65.97%.The similarity between nucleotide sequences ranged from 45.1%to 97.2%.These indicated that the nucleotide sequences of reverse transcriptase existed higher heterogeneity.The twenty-nine AmRT2-X sequences were clustered into eight families.Family A was the main component of Ty3-gypsy-like retrotransposons.After being translated into amino acids,ten AmRT2-X sequences had nonsense mutations.The similarity between amino acids ranged from 22.3%to 98.6%.These indicated that the amino acids sequences of reverse transcriptase existed high heterogeneity.The conservative motifs of most AmRT2-X sequences were identical.The evolutionary tree showed that all RT gene sequences were classified into six categories.Among them,classⅠcontained twenty AmRT2-X sequences.ClassⅡcontained four AmRT2-X sequences,which had close relationship with Arabidopsis,ryegrass,pitaya,peony,mume,jujube,mulberry and pine.Two Ty3-gypsy-like retrotransposons from tetraploid wild peanut species with transcriptional activity were found by aligning with peanut EST database.The reverse transcriptase sequences obtained in this study will provide a basis for further isolating full length

关 键 词：花生 Ty3-gypsy类反转录转座子 反转录酶 异质性 四倍体野生种 

分 类 号：S565.2[农业科学—作物学]