甘蔗属大茎野生种Ty1-copia反转录转座子逆转录酶序列克隆与特点分析  

Cloning and Characterisation of Reverse Transcriptase Sequences of Ty1-copia Retrotransposon in Saccharum robustum

作　　者：吴子莺 杨善 钱旺 吴嘉云 陈柯 张珂 李佩婷 邓祖湖[1,3] WU Zi-ying;YANG Shan;QIAN Wang;WU Jia-yun;CHEN Ke;ZHANG Ke;LI Pei-ting;DENG Zu-hu(National Engineering Research Center for Sugarcane,Fujian Agriculture and Forestry University,Fuzhou 350002;Guangdong Provincial Bioengineering Institute/Guangzhou Sugarcane Industry Research Institute,Guangzhou 510316;State Key Laboratory for protection and utilization of subtropical agro-bioresources,Guangxi University,Nanning 530004)

机构地区：[1]福建农林大学国家甘蔗工程技术研究中心,福州350002 [2]广东省生物工程研究所/广州甘蔗糖业研究所,广州510316 [3]广西大学亚热带农业生物资源保护与利用国家重点实验室,南宁530002

出　　处：《植物遗传资源学报》2020年第2期466-476,共11页Journal of Plant Genetic Resources

基　　金：国家自然科学基金(31771863,31571730,31401440);福建省重大科技项目(2015NZ0002-2);亚热带农业生物资源保护与利用国家重点实验室开放项目(SKLCUSA-b201606)。

摘　　要：为深入研究大茎野生种Ty1-copia反转录转座子逆转录酶序列(RT,reverse transcriptase)的特点,本研究使用简并引物将Ty1-copia RT序列克隆、测序,成功分离了60条Ty1-copia类RT序列,AT比例为46.0%~62.0%,长度为257~266bp,通过构建系统进化树分析,可将其分成6大类。将这些碱基序列翻译成氨基酸序列,通过ClustalX软件分析,发现存在上游TAFLHG、下游YVDDM以及中心SLYGLKQ保守结构域,并出现编码阅读框移码突变、终止密码子突变的现象。与其他禾本科物种的Ty1-copia RT序列一起构建系统进化树,结果发现可分成10类不同的进化谱系,60条大茎野生种Ty1-copia RT氨基酸序列中有12条序列归为Ⅰ类(Sre/Maximus)、有10条序列归为Ⅱ类(Retrofit/Ale)、有18条序列归为Ⅲ类(Ale)、有17条序列归为X类(Tork/TAR),仅有3条序列归为Ⅵ类(Tork/Angela);然而Bianca和Oryco/Ivana谱系未发现大茎野生种Ty1-copia RT序列,表明其与甘蔗杂交种、高粱的亲缘关系近。通过斑点杂交结果,可计算得到Ty1-copia RT序列在其基因组中拷贝数为6.75×103。FISH结果显示,Ty1-copia RT序列是弥散分布在染色体上,其中部分染色体端粒上的信号比较强。这些结果将有助于今后大茎野生种生物多样性研究、基因组研究,提供一定的参考依据。In order to investigate the characteristics of S.robustum E.W.Brandes&Jeswiet ex Grassl Ty1-copia retrotransposon reverse transcriptase(RT),the Ty1-copia RT sequences were isolated by PCR amplification with universal primers.Sixty Ty1-copia RT sequences were successfully isolated with a length of 257 to 266 bp.Through ClustalX software analysis,several conserved domains in the amino acid sequences were revealed,such as TAFLHG,YVDDM and SLYGLKQ,which were found at the upstream,downstream and central parts,respectively.Several frameshift mutation in the code reading box and at the termination codon mutation were found.The phylogenetic tree analysis of Ty1-copia RT sequences of S.robustum E.W.Brandes&Jeswiet ex Grassl and other Gramineae species suggested ten different evolutionary lineages.12 of S.robustum E.W.Brandes&Jeswiet ex Grassl Ty1-copia RT amino acid sequences were classified as class I(Sre/Maximus),10 were classified as ClassⅡ(Retrofit/Ale),18 were classified as ClassⅢ(Ale),17 were classified as Class X(Tork/TAR),and 3 of the sequences were classified as ClassⅥ(Tork/Angela).This analysis indicated that S.robustum E.W.Brandes&Jeswiet ex Grassl was closely related to the Saccharum spp.hybrid and Sorghum.Based on the result of dot blot hybridization,the copy number of the S.robustum E.W.Brandes&Jeswiet ex Grassl Ty1-copia RT sequence was calculated to be 6.75×103 in one genome.The FISH results showed that the S.robustum E.W.Brandes&Jeswiet ex Grassl Ty1-copia RT sequences were distributed on the chromosome,which the signal of telomere region was stronger than other regions in some chromosomes.These results would provide a reference for the biodiversity study and genome research of S.robustum E.W.Brandes&Jeswiet ex Grassl in the future.

关 键 词：大茎野生种 Ty1-copia反转录转座子 荧光原位杂交 进化分析 甘蔗 

分 类 号：S566.1[农业科学—作物学]