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利用立枯丝核菌(Rhizoctonia solani)诱导胁迫下玉米高耐纹枯病自交系幼苗叶片所构建的cDNA文库中获得的EST序列,通过电子克隆和RT-PCR方法,结合cDNA末端快速扩增技术,从玉米叶片中分离克隆到衰老相关蛋白基因的全长cDNA序列,命名为ZmSAP(GenBank登录号:GU253311)。序列分析表明,ZmSAP全长1156bp,包含一个完整的603bp的开放阅读框(ORF),具有连续的Poly A尾和典型的加尾信号AATTAA。ProtParam程序预测表明该基因编码200个氨基酸,相对分子量为21.92kD,等电点为10.38。该氨基酸序列与豌豆、挪威云杉、寄生藻等有不同程度的同源性且在不同物种间具有一个保守结构域,染色体定位发现该基因位于玉米第1条染色体上。荧光定量PCR分析表明,在高耐纹枯病自交系R15和高感纹枯病材料478中ZmSAP基因在病菌胁迫初期呈诱导表达,可能参与了病原菌诱导的细胞程序性死亡过程,说明该基因与纹枯病菌胁迫响应机制密切相关。
The EST sequences obtained from the cDNA library of Rhizoctonia solani induced inbred lines with high resistance to Sheath Blight were rapidly cloned by cDNA cloning and RT-PCR Technology, the full length cDNA sequence of the senescence-related protein gene was cloned from maize leaves and named as ZmSAP (GenBank accession number: GU253311). Sequence analysis showed that the full length of ZmSAP was 1156 bp and contained a complete 603 bp open reading frame (ORF) with a continuous Poly A tail and a typical tailed AATTAA signal. ProtParam program predicted that the gene encoding 200 amino acids, the relative molecular weight of 21.92kD, isoelectric point of 10.38. The amino acid sequence has the same degree of homology with pea, Norway spruce and parasitic algae, and has a conserved domain among different species. Chromosomal mapping shows that the gene is located on the first chromosome of maize. Quantitative real-time PCR analysis showed that the ZmSAP gene was induced in the early stage of pathogen stress in R97 and R74 lines of susceptibilities to sheath blight, which may be involved in the process of pathogen-induced apoptosis, indicating that this gene It is closely related to the stress response mechanism of Rhizoctonia solani.