小麦雄性不育育性转换相关基因TaG3BP的克隆与表达分析

为揭示YS型小麦温敏雄性不育的育性转换基础,以该类型不育系A3017不育幼穗和可育幼穗为材料构建正、反杂交SSH-cDNA文库,从可育文库中筛选出一个与G3BP(Ras-GTPase activating protein SH3 domain-binding protein)基因同源的EST序列(GenBank登录号为DY543200)。以该EST序列的同源性比对和拼接结果为依据设计引物,在可育幼穗中扩增出一条1230bp的cDNA序列。该片段含有与G3BP相似的由409个氨基酸组成的结构域,与水稻G3BP的氨基酸序列同源性为79%,被命名为TaG3BP(GenBank登录号为GU475149)。利用Real-time PCR检测TaG3BP基因在YS型小麦温敏雄性不育系花药发育各时期中的表达模式,发现该基因在育性转换的关键时期上调表达,且可育条件下的表达量高于同时期不育条件下的表达量。进一步对TaG3BP在3种不同类型的小麦K型雄性不育材料的不育系及其保持系的幼穗中的表达模式进行半定量RT-PCR分析,结果该基因在保持系幼穗中表达量较高,在不育系中表达量较低。表明该基因可能在其育性转换中具有重要作用。

Cloning and Expression Analysis of TaG3BP Gene Related to Fertility Conversion in Male Sterile Lines of Wheat

Thermo-photoperiod sensitive male sterile (TMS) wheat (Triticum astivum L.) is a valuable material for heterosis study and utilization by means of two-line system. YS-type TMS lines are applicable in hybrid wheat in major wheat growing areas in northern China. The objective of this study was to reveal the molecular basis of fertility conversion in YS-type TMS wheat. YS-type TMS line A3017 was grown in a phytotron with controlled temperatures in favor of male sterility and fertility, respectively. Two suppression subtractive hybridization (SSH) cDNA libraries were constructed using cDNA from the male sterile and fertile young spikes of the same individuals that were orderly treated with temperature cycle (day/night) of 16℃/12℃ and 25℃/16℃. Using a pair of primers designed on the basis of aligned homological sequence, RT-PCR amplification was carried out and an EST with 1230 bp in length (GenBank accession number: DY543200) was obtained from the fertile SSH-cDNA library, which encodes 409 amino acid residues. The cDNA fragment contained the typical NTF2 and RRM domains of G3BP, and designated TaG3BP (GenBank accession number: GU475149). The deduced amino acids had 79% of similarity with G3BP (Ras-GTPase activating protein SH3 domain-binding protein) gene from Oryza sativa (BAD07751). The expression pattern of TaG3BP was analyzed with Real-time PCR using anthers from the TMS line in various developmental periods under male sterile and fertile conditions. In fertile anthers, TaG3BP gene was expressed in a higher level at the critical stage of fertility conversion (between uninucleate and binucleate stages) as compared with those in sterile anthers. In the K-type male-sterile lines and their maintainers of 1B/1R, 1B and non 1B/1R types, TaG3BP gene showed higher expression levels in the young spike of maintainers than in that of the male-sterile lines. These results suggest that the expression of TaG3BP gene is related to fertility conversion in male-sterile wheat lines.