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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
吴子莺1, 杨善1, 钱旺1, 吴嘉云2, 陈柯1, 张珂1, 李佩婷1, 邓祖湖1
0
(1.福建农林大学国家甘蔗工程技术研究中心;2.广东省生物工程研究所(广州甘蔗糖业研究所))
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(reverse transcriptase, RT)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 46.8 %~66.0 %,长度为 233~266 bp,通过构建系统进化树分析,可将其分成 4 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 8 类不同的进化谱系,大茎野生种 Ty1-copia RT 氨基酸序列有 20 %归为 I 类(Sre / Maximus)、有 16.7 %归为 II 类(Retrofit / Ale)、有 30 %归为 III 类(Ale)、有 28.3%归为 VIII类(Tork / TAR),仅有 5 %的序列归为 V 类(Tork / Angela);然而 Bianca 和 Oryco / Ivana 谱系类未发现大茎野生种 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。
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
DOI:10.13430/j.cnki.jpgr.20190506001
投稿时间:2019-05-06修订日期:2019-07-31
基金项目:国家自然科学基金(31401440,31571730,31771863);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)
Cloning and Characterisation of Reverse Transcriptase Sequences of Ty1-copia Retrotransposon in Saccharum robustum
WU Zi-ying1, YANG Shan1, QIAN Wang1, WU Jia-yun2, CHEN Ke1, ZHANG Ke1, LI Pei-ting1, DENG Zu-hu1
(1.National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University;2.Guangdong Provincial Bioengineering Institute, Guangzhou Sugarcane Industry Research Institute)
Abstract:
In order to investigate the characteristics of S. robustum 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 233 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 and other Gramineae species suggested eight different evolutionary lineages. 20 % of S. robustum Ty1-copia RT amino acid sequences were classified as class I (Sre / Maximus), 16.7 % were classified as Class II (Retrofit / Ale), 30 % were classified as Class III (Ale), 28.3 % were classified as Class VIII (Tork / TAR), and 5 % of the sequences were classified as Class V (Tork) / Angela). This analysis indicated that S. robustum 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 Ty1-copia RT sequence was calculated to be 6.75×103 in one genome. The FISH results showed that the S. robustum 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 in the future.
Key words:  Saccharum robustum  Ty1-copia retrotransposons  fluorescence in situ hybridization(FISH)  evolutionary analysis  sugarcane

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