含CBS结构域的小麦TaCDCP1基因的克隆及其表达分析

Cloning and Expression Analysis of a CBS Domain Containing Protein Gene TaCDCP1 from Wheat

利用电子克隆和RT-PCR技术,在本实验室前期构建的非亲和互作的SSH文库基础上,从条锈菌侵染的小麦水原11叶片中首次分离出一个含CBS结构域蛋白的基因,暂命名为TaCDCP1（Triticum aestivum CBS domain containing protein 1）。TaCDCP1包含一个完整的654 bp的开放阅读框,编码217个氨基酸。推测该基因拟编码的蛋白具有2个CBS保守结构域,不含跨膜区且无信号肽,定位在叶绿体基质内;经过同源比对,小麦TaCDCP1氨基酸序列与大麦、水稻和玉米等的同源序列的相似性较高;该基因表达量在小麦叶中显著高于在根和茎中;在小麦与条锈菌的非亲和、亲和组合中,TaCDCP1基因均受到条锈菌诱导,分别在接种后18 h和96 h达到表达高峰,非亲和组合表达量在侵染前期（接种后1848 h）高于亲和组合,而在侵染后期（接种后96120 h）低于亲和组合;外源植物激素脱落酸诱导该基因上调表达,苄基腺嘌呤,乙烯,赤霉素,茉莉酸甲酯和水杨酸处理后其表达量在不同程度上受到抑制;TaCDCP1在低温和干旱条件下表达量上升,在机械伤害和高盐处理下表达量无明显差异。表明TaCDCP1可能通过脱落酸等信号途径参与小麦对条锈菌的防御反应,同时参与低温和干旱环境下的信号转导途径。这些结果对于明确CBS结构域的功能以及CBS结构域蛋白尤其是TaCDCP1在小麦与条锈菌互作中的作用奠定了基础。

To elucidate the defense response of wheat（Triticum aestivum L.） to Puccinia striiformis f.sp.tritici（Pst）,we constructed the incompatible interaction SSH cDNA library of wheat（cv.Suwon 11） leaves infected by Pst CYR23.A total of 652 unigenes were identified and 424 genes were annotated. On the basis of previous study,according to cDNA sequence LWSRP2502（GenBank accession No.EV254338）,a full-length sequence of the CBS domain containing protein gene,tentatively designated as TaCDCP1（Triticum aestivum CBS domain containing protein 1）,was isolated and characterized from wheat leaves infected by Pst through in silico cloning and reverse transcription PCR（RT-PCR） approaches. The open reading frame of TaCDCP1 was 654 bp in length and predicted to encode 217 amino acids protein which contained two conserved cystathionine beta-synthase（CBS） domains and was without transmembrane domain or signal peptide sequence.The deduced protein was predicted existing in chloroplast stroma.The amino acid sequence of TaCDCP1 shares 92%,72%,and 63% identify with the homologs in barley（Hordeum vulgare）,rice（Oryza sativa） and maize（Zea mays）,respectively.The TaCDCP1 gene was highly expressed in leaves than in roots and stems.Challenged by Pst,TaCDCP1 was induced by this fungus in both incompatible and compatible interactions,with the maximal expression at 18 h post inoculation（hpi） and 96 hpi,respectively.Its transcript accumulation was much higher in the incompatible interaction than in the compatible interaction at the early stage of infection（18-48 hpi）,but much lower at the late stage（96-120 hpi）.The expression of TaCDCP1 was also up-regulated after treated by phytohormones such as abscisic acid（ABA）,and down-regulated by benzyladenine,ethylene,gibberellins,methyl jasmonate and salicylic acid to a certain degree.And it was obviously up-regulated by various abiotic stresses,such as low temperature and drought. However,mechanical wound and high salinity stress could not induce the expression of TaCDCP1.These results suggest that TaCDCP1 is probably involved in the disease resistance and defense response in wheat to Pst through ABA,and also participate in the signal transmission pathways under low temperature,and drought conditions.