2022年7月7日 星期四
甜瓜 STK 类 R 基因同源序列的克隆与分析
Cloning and analysis on homologous sequences of STK-like R-gene from Cucumis melo
2012年 第21卷 第3期 页码[8-12]    下载全文[0.7MB]  
摘要

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 communis L.)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。

Abstract

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.

关键词甜瓜; 丝氨酸/苏氨酸蛋白激酶; 抗病基因同源序列; 氨基酸序列; 克隆; 同源性;
Key wordsCucumis melo L.; serine-threonine kinase ( STK); resistance gene analogs ( RGAs); amino acid sequence; cloning; homology
作者张志忠, 孙志浩, 蓝茂锋
所在单位福建农林大学园艺学院, 福建 福州 350002)
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基金项目国家“十一五”科技支撑计划项目子课题(2007BAD07B03); 福建省重大专项项目(2008NZ0002-1); 福建省自然科学基金资助项目(2011J01083);