One resistance gene analog fragment named RGA-CIN14 was isolated from TcLr19 wheat,which contains kinase-2,kinase-3a,and the GLPL motif of the NBS-spanning region,using degenerated primers according to the nucleotide binding site (NBS) conserved domain.Based on the RGA-CIN14,a full-length cDNA,CIN14,which was 2 987 bp encoding 880 amino acids,was obtained by using the method of the rapid amplification cDNA ends (RACE).Bioinformatics analysis showed that the deduced amino acids of CIN14 protein consisted of a NB-ARC conserved domain and many leucine-rich repeats (LRR) domains.The phylogenetic tree analysis indicated a considerable identity of the protein encoded by CIN14 with that of wheat leaf rust resistance gene Lr1,but a lower similarity with Lr21.The expression profile of the CIN14 gene detected by semi-quantitative RT-PCR showed that the CIN14 gene was not induced by Puccinia triticina and it was a constitutive gene with low abundance in the wheat leaf tissue.The resistance homology sequence was successfully obtained,which provides the shortcut for cloning of the resistance gene in TcLr19 wheat.
【目的】研究叶锈菌诱导小麦叶片的基因表达情况,从分子水平阐明小麦的抗病机制。【方法】以小麦抗叶锈病近等基因系TcLr41为材料,利用抑制差减杂交技术,构建叶锈菌诱导的小麦叶片cDNA文库,随机挑取文库中的阳性克隆测序,并对测序结果进行功能注释和分类。【结果】成功构建了叶锈菌诱导的小麦叶片抑制差减杂交文库,共获得3 456个阳性克隆。挑取165个阳性克隆进行测序,获得160条高质量EST。对EST序列进行聚类后,共获得107条非重复序列(Unigene),与GenBank进行BLASTx和BLASTn同源比对,其中70条非重复序列与已知基因同源性很高,占全部非重复序列的65.4%。已知功能的EST涉及植物的能量代谢、信号转导、转录调控及抗病防卫等多方面。【结论】通过对功能已知的基因分析,推测促分裂原活化蛋白激酶(mitogen activated protein kinase)、丝氨酸/苏氨酸蛋白激酶(serine/threonine protein kinase)、GTP结合蛋白(GTP-binding protein)、钙网蛋白(calreticulin)、过氧化氢酶(catalase)、谷胱甘肽-S-转移酶(glutathione-S-transferase)、多聚泛素基因(polyubiquitin)、锌指蛋白(zinc-finger protein)、肉桂醇脱氢酶(cinnamyl alcohol dehydrogenase)、转脂蛋白(lipid transfer protein)、类甜蛋白(thaumatin-like)、几丁质酶(chitinase)等可能参与了小麦与叶锈菌的非亲和互作过程。
