cDNA文库与RACE方法结合克隆一个马铃薯病程相关蛋白基因cDNA

为克隆马铃薯晚疫病抗性相关基因，深入研究马铃薯晚疫病抗性机制，应用SMART LD—PCR技术，以晚疫病菌混合小种诱导48h的水平抗性马铃薯(Solanum tuberosum L．)(R—gene—free)叶片为材料，构建了一个富集晚疫病抗性相关基因的cDNA文库。为提高克隆全长cDNA的效率，将cDNA文库与RACE技术结合，依据本实验室得到的病原诱导表达片段测序结果，在其内部设计两个特异引物，与文库载体臂上的通用引物配对，以文库噬菌体DNA为模板，用高保真PCR分别扩增出cDNA5′端与3′端，从而简便、快捷地得到全长cDNA序列。采用此方法，在马铃薯中克隆了一个受晚疫病菌诱导表达的cDNA，该cDNA长904bp，5′端有29bp的非翻译区，3′端具有完整的polyA尾，包含一个678bp的完整开放阅读框架，编码226个氨基酸(GenBank登录号：AY 185207)。BLAST检索发现其氨基酸序列与烟草一个新的病程相关蛋白基因NtPRp27具有90％的同源性，在马铃薯中尚未发现与之同源的已知基因。Northern杂交结果表明，水杨酸(SA)、茉莉酸(JA)、茉莉酸甲酯(MeJA)、机械伤害和渗透胁迫都能诱导该基因表达。该基因可能是马铃薯一个新的病程相关蛋白基因。

Cloning of a Pathogenesis-related Protein Gene cDNA of Potato Using RACE Methods Combined with cDNA Library

For the purpose of cloning potato late blight resistance-related genes,a cDNA library,which enriched with the full-length resistance-related genes,was constructed by SMART LD-PCR techniques using R-gene-free potato (Solanum tuberosum L.) leaves infected with the mixed races of Phytophthora infestans for 48 h.To improve the efficiency of cloning full-length cDNA,strategies of RACE combined with cDNA library were used.Two specific primers were designed according to the sequence of a EST fragment that was derived from the pathogen induction.They were then combined with two universal primers of the vector arm to amplify the 5′ and 3′ end respectively using the phage DNA of the library as the template.The length of the cDNA obtained was 904 bp containing a complete open reading frame of 678 bp that encodes 226 amino acids,an untranslated region of 29 bp at its 5′ end and a polyA tail at the 3′ end (GenBank accession number AY 185207).The deduced amino acids shares 90% similarity with NtPRp27,a new member of pathogenesis-related protein genes of tobacco,but no similar genes have been reported in potatoes by the BLAST search.Northern blot results revealed that P.infestans,SA,JA,MeJA,mechanical wounding and osmotic stress could induce the expression of the potato NtPRp27-like gene.It was,therefore,postulated that it could be a new pathogenesis-related protein gene of the potato.