甜瓜STK类R基因同源序列的克隆与分析

以植物丝氨酸/苏氨酸蛋白激酶类( serine-threonine kinase,STK)抗病基因产物催化结构域I和Ⅸ的保守氨基酸序列( FGK/V/L/SVYK/RG,DY/IYSF/YGV/I/M)设计简并引物,对甜瓜(Cucumis melo L.)基因组DNA进行PCR扩增,得到大约500 bp的目的条带,通过重组质粒克隆并经PCR检测后得到12条不同的DNA序列,命名为tg1～tg12,其中tg2、tg5、tg9和tg12(Genbank登录号为JN646853 ～JN646856)可以编码完整的氨基酸序列.Blast分析结果显示:4条序列均具有ATP结合部位、底物结合部位和激酶结构域的活化环(A-loop)等,属于典型的蛋白激酶基因家族,可能是STK类R基因的同源序列片段；4条序列与蓖麻(Ricinus communisL.)的STK同源性均较高.氨基酸序列比对结果显示tg2、tg5、tg9和tg12均具有R基因的9个保守结构域,为STK类候选抗病基因类序列.分子系统树显示tg2、tg5、tg9和tg12与已知的R基因(Pto、Lr10和Lectin)在氨基酸水平上的相似性仅为33.5％ ～53.4％,且4个甜瓜同源序列的氨基酸相似性也较低,表明甜瓜RGAs标记可能具有较高的特异性.

Cloning and analysis on homologous sequences of STK-like R-gene from Cucumis melo

Using the conserved amino acid sequence of catalytic domain Ⅰ(FGK/V/L/SVYK/RG) and Ⅸ(DY/IYSF/YGV/I/M) of disease resistance genes belonging to plant serine-threonine kinase(STK) for designing degenerate primers,PCR of genomic DNA of Cucumis melo L.was performed and a target fragment about 500 bp was obtained.Twelve DNA fractions with different sequences named tg1-tg12 were obtained by cloning of recombinant plasmids and PCR detection,in which,tg2,tg5,tg9 and tg12 with the GenBank accession No.of JN646853-JN646856 can encode complete amino acid sequences.The blast analysis result shows that four sequences all possess ATP binding site,substrate binding site and A-loop of kinase domain,showing that they belong to a typical kinase gene family and may be homologous sequences of STK-like R-gene.And four sequences have higher homology with STK from Ricinus communis L.The alignment result of amino acid sequences reveals that tg2,tg5,tg9 and tg12 all contain nine conserved domains of R-gene,which are candidate analog sequences of STK-like disease resistance genes.The molecular phylogenetic tree shows that tg2,tg5,tg9 and tg12 only have 33.5%-53.4% similarity in amino acid level with known R-genes(Pto,Lr10 and Lectin),and also there is low amino acid similarity among four homologous sequences of C.melo,meaning that RGAs markers of C.melo may have high specificity.