普通小麦Trx59基因的克隆及功能验证

Cloning and Functional Verification of Trx59 Gene in Common Wheat

为进一步挖掘小麦春化相关基因,探明小麦春化发育调控机制,从不同春化发育特性小麦品种春化响应转录组高通量测序结果中筛选并克隆到1个EST序列,命名为Trx59,该基因的开放阅读框为372 bp,编码124个氨基酸,保守结构域分析显示,Trx59基因有1个Thioredoxin结构域。利用qRT-PCR分析了在春化处理过程中Trx59基因在不同春化发育特性小麦品种的表达特性,并利用VIGS技术进行基因功能的初步验证。结果表明:随着春化处理时间的延长,Trx59基因的表达量逐渐升高,从表达量和表达时间来看,Trx59基因的表达量和表达时间表现为春性品种高于且早于冬性品种。构建BSMV:Trx59重组载体并接种于小麦植株,14 d后出现明显的光漂白现象,Trx59基因的表达水平急剧降低,说明Trx59基因已被显著抑制;BSMV:Trx59接种的小麦植株穗分化明显晚于阴性对照组,初步判断Trx59基因与小麦发育有关。

In order to further excavate the related genes of wheat vernalization,explored the regulation mechanism of wheat vernalization,this study screened and cloned an EST sequence from a high-throughput RNA sequencing analysis comparing the transcriptomes with wheat varieties with different developmental characteristics under vernalization and non-vernalization treatment,and named Trx59. The open reading frame（ ORF） of Trx59 gene was372 bp,ecoding 124 amino acids,and contained one thioredoxin domain structure. Expression analysis was conducted by qRT-PCR and the gene function was verification by using VIGS technique. The result showed that the expression level of Trx59 was gradually up-regulated during vernalizaton process; the expression level and time of Trx59 gene in spring variety LC10 was higher and earlier than that in winter variety J841. BSMV： Trx59 recombinant vector was built and inoculated wheat plants of J841. After 14 days of inoculation,the leaves appeared visible light bleaching phenomenon,and the expression level of Trx59 reduced sharply,indicating that Trx59 gene had been significantly suppressed; the spike differentiation process of plants inoculated by BSMV： Trx59 was later than that of the negative control group,deducing that the Trx59 genes may be related to the wheat developmental characteristics.