甘薯NBS类抗病基因类似物的分离与序列分析

利用已克隆植物抗病基因NBS（Nucleotide binding site）序列中的保守模体（motif）“P-loop”和“GLPL”合成简并引物,以甘薯（Ipomoea batatas）栽培品种青农2号基因组DNA为模板进行PCR扩增,通过T/A克隆、测序和序列分析,共得到15条具有连续ORF的抗病基因类似物（Resistance gene analogues,RGAs）序列,它们之间核苷酸序列间的相似性系数在41.2%-99.4%之间,而相应推测的氨基酸序列间的相似性系数在20.6%-100%之间,同时对分离的RGAs的核苷酸和氨基酸序列进行系统发育树分析,表明甘薯RGAs可分为TIR（Drosophila Toll or human interleukin receptor-like）和nonTIR两类.对甘薯RGAs和5个已克隆植物NBS的氨基酸序列进行结构分析表明,它们包括“P-loop”、“Kinase-2”、“Kinase-3a”、“GLPL”4个抗病基因所共有的保守模体.这些表明甘薯与其它物种的NBS类RGAs可能具有同样的起源和进化机制.

Isolation and Sequence Analysis of NBS-type Resistance Gene Analogues in Sweet Potato (Ipomoea batatas (L.) Lam.)

Degenerate primers based on conserved motif (P-loop and GLPL) of the nucleotide binding site (NBS) region from the cloned plant disease resistance genes were used to isolate resistance gene analogues (RGAs) from genomic DNA of the sweet potato (Ipomoea batatas (L.) Lam.) cultivar Qingnong No. 2. The desired bands (~500 bp) were purified from the gel, and then cloned by T/A cloning. After sequencing and analyzing by alignment, 15 RGAs with uninterrupted open reading frames (ORFs) were obtained. Sequence identity among the 15 RGA nucleotide sequences ranged from 41.2% to 99.4%, while the 15 RGAs deduced amino acid sequences showed identity ranged from 20.6% to 100%. The phylogenetic analyses for RGA nucleotide sequences and the deduced amino acids showed that RGAs from sweet potato were divided into two groups, TIR (Drosophila Toll or human interleukin receptor-like) type and nonTIR type. The analysis of RGAs amino acid sequence structures suggested that they contained the domains such as P-loop, Kinase-2, Kinase-3a, and GLPL. These results showed that NBS type RGAs isolated from sweet potato might have the same origin and mechanism of evolution as that in other plants.