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Analysis of autonomous element of Tpnl family in the Japanese morning glory

日本牵牛花Tpnl家族自主元件分析

基本信息

批准号：
13640615
负责人：
NITASAKA Eiji
金额：
$ 2.05万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13640615/
关键词：
Transposable element Japanese morning glory Tpn Ipomoea AP2 Ipompea

项目摘要

Tpnl family, an En/Spm-related transposable element is a major mutagen of the Japanese morning glory (1pomoea nil). Structural feature of this Tpnl family is the following : Each end has 28bp of terminal inverted repeat (TIR) and a subterminal region including highly repetitive sequences of the inner side. Between these terminal sequences, there are group-specific sequences derived from host genes. For example, they are myosin, beta-galactosidase, and AP2B. By the molecular and phenotypic analysis of Tpn insertion mutations, it is thought that these non-autonomous Tpns are mobilized by an identified autonomous element coding transposase. In order to isolate autonomous Tpn element, some pairs of primers were designed using conserved motif of other En/Spm-related transposable element. Some homologous fragments were amplified using these primers and mRNA of mutable strains by the RT-PCR experiment. Since autonomous Tpn elements were thought to have more larger size, a cosmid library of genomic DNA was constructed and it was screened using RT-PCR products as probes. Although some clones isolated carried En/Spm-related sequences, no clone contain subterminal sequences which specify Tpnl family. There are some Tpn groups carrying longer ORFs which have no homology with other known genes. Therefore, one group of these elements is thought to be a deleted type from autonomous element. I am now searching for longer Tpn element using internal probe of these Tpns. Another strategy is also employed to compare Tpnl-related sequences among Ipomoea species since autonomous elements are thought to exist in all other sibling species, but normal Tpn capturing host genes might repeated generation-and-elimination cycle in relatively short period.

Tpnl家族是一个En/Spm相关的转座因子，是日本牵牛花的主要诱变剂。该Tpnl家族的结构特征如下：每个末端具有28 bp的末端反向重复序列（TIR）和包括内侧高度重复序列的亚末端区域。在这些末端序列之间，存在来自宿主基因的组特异性序列。例如，它们是肌球蛋白、β-半乳糖苷酶和AP 2B。通过对Tpn插入突变的分子和表型分析，认为这些非自主Tpn是由一个已鉴定的自主元件编码转座酶动员的。为了分离自主型Tpn元件，利用其它En/Spm转座因子的保守基序设计了几对引物。用这些引物和突变株的mRNA进行RT-PCR实验，扩增出一些同源片段。由于自主Tpn元件被认为具有更大的尺寸，因此构建了基因组DNA的粘粒文库，并以RT-PCR产物为探针进行筛选。虽然分离的克隆中有一些携带En/Spm相关序列，但没有克隆含有Tpnl家族的亚末端序列。有一些Tpn组携带较长的ORF，与其他已知基因没有同源性。因此，这些元素中的一组被认为是从自主元素中删除的类型。我现在正在寻找更长的TPN元素使用内部探测这些TPN。还采用另一种策略来比较牵牛属物种之间的Tpnl相关序列，因为自主元件被认为存在于所有其他兄弟物种中，但正常的Tpn捕获宿主基因可能会在相对较短的时间内重复产生和消除循环。