甘蔗Ty3-gypsy类逆转座子RT基因的克隆及分析  

Cloning and Analysis of Reverse Transcriptase Gene of Ty3-gypsy-like Retrotransposons in Sugarcane

作　　者：刘俊仙 刘菁 阳太亿 高轶静 段维兴 雷敬超 刘丽敏 刘红坚 张荣华 何为中 李松 熊发前 LIU Junxian;LIU Jing;YANG Taiyi;GAO Yijing;DUAN Weixing;LEI Jingchao;LIU Limin;LIU Hongjian;ZHANG Ronghua;HE Weizhong;LI Song;XIONG Faqian(Sugarcane Research Institute,Guangxi Academy of Agricultural Sciences,Nanning,Guangxi 530007,China;Cash Crops Research Institute,Guangxi Academy of Agricultural Sciences,Nanning,Guangxi 530007,China)

机构地区：[1]广西壮族自治区农业科学院甘蔗研究所,广西南宁530007 [2]广西壮族自治区农业科学院经济作物研究所,广西南宁530007

出　　处：《福建农业学报》2021年第9期989-998,共10页Fujian Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31960416、31960409);广西自然科学基金项目(2018GXNSFDA294004、2018GXNSFDA281027、2020GXNSFAA297081);广西农业科学院科技发展基金项目(桂农科31960416)。

摘　　要：【目的】从甘蔗基因组中分离Ty3-gypsy类逆转座子的RT基因序列,分析序列的特点、差异及系统进化关系,为研究其转录活性和调控功能奠定基础。【方法】根据Ty3-gypsy类逆转座子RT基因序列的保守区设计简并引物,对甘蔗品种新台糖22的基因组DNA进行PCR扩增,目的条带经回收、克隆和测序后,对所获得序列进行生物信息学分析。【结果】目的条带大小约430 bp,成功分离到36条RT基因序列,只有1条序列的长度为430 bp,其余序列的长度均为432 bp,AT所占比为56.71%~64.81%,AT与GC比值为1.31~1.84;核苷酸序列间相似性为46.2%~99.3%。聚类分析后,36条RT基因序列被划分为5个家族,家族Ⅰ和家族Ⅳ是包含序列最多的家族。翻译后,有6条序列发生了无义突变;氨基酸序列间相似性为10.1%~100%,呈现高度异质性;36条RT基因序列中的34条序列的保守基序完全一致,呈现高度保守性。各家族中代表序列的蛋白质三级结构整体构型基本类似,但在氢键数和转角数上存在着较大差别,呈现一定异质性和多态性。系统进化树显示,所有RT基因序列被分为7类,Ⅰ类中的18条甘蔗RT基因序列与拟南芥的BAB40828.1具有较高相似性,Ⅶ类中的SoRT3-26与大豆的BAB40834.1、菠菜的BAB40833.1和粳稻的BAB40824.1的亲缘关系最近,暗示在进化过程中甘蔗与这些物种植物之间可能发生了Ty3-gypsy类逆转座子的横向传递;通过比对甘蔗EST数据库,发现了10条具有转录活性的甘蔗品种新台糖22的Ty3-gypsy类逆转座子。【结论】获得甘蔗Ty3-gypsy类逆转座子RT基因序列及其具有转录活性序列,为下一步分离Ty3-gypsy类逆转座子全长序列及研究Ty3-gypsy类逆转座子转录转座活性和功能提供序列基础,也为开发甘蔗LTR逆转座子的分子标记奠定基础。【Objective】 The reverse transcriptase(RT) gene sequences of Ty3-gypsy-like retrotransposons were isolated from the sugarcane genome, and their characteristics, differences, and phylogenetic relationships analyzed to study the transcriptional activities and regulatory functions associated with the retrotransposons. 【Method】 Degenerate primers were designed according to the conserved region of the RT gene sequences of Ty3-gypsy-like retrotransposons. The genomic DNA of sugarcane variety, ROC22, was amplified by PCR. The recovered, cloned, and sequenced target band was analyzed by bioinformatics. 【Result】 Thirty-six RT gene sequences were successfully isolated. Only one sequence of the target bands was430 bp in length, while the remainders were 432 bp. The proportions of AT in the sequence ranged from 56.71% to 64.81%,and the ratio of AT to GC from 1.31 to 1.84. A high heterogeneity existed among the nucleotide sequences, as the similarity was shown to be 46.2% to 99.3%. The cluster analysis divided the 36 RT gene sequences into 5 families. Family I and Family IV contained most of the sequences. After translated into amino acids, 6 sequences displayed nonsense mutations. The similarity between amino acid sequences ranged from 10.1% to 100% exhibiting a high degree of heterogeneity. The conserved motifs of 34 of the sequences were identical, indicating a highly conservative property. The overall configuration of the tertiary structure of the representative proteins in each family was basically similar, however, there were significant differences in the number of hydrogen bonds and turns, suggesting a certain degree of heterogeneity and polymorphism. The phylogenetic tree showed 7 distinct groups among the RT gene sequences. Of which, 18 in Group I were highly homology with BAB40828.1 of Arabidopsis thaliana, and SoRT3-26 in Group VII was genetically closest to the BAB40834.1 of soybean, BAB40833.1 of spinach, and BAB40824.1 of japonica rice. It indicated that a horizontal transfer of Ty3-gypsy-like retrot

关 键 词：甘蔗 Ty3-gypsy类逆转座子 逆转录酶 新台糖22 异质性 

分 类 号：S566.1[农业科学—作物学]