龙眼胚性愈伤组织NBS-LRR类抗性基因的同源克隆及序列分析

摘要

Longan(Dimocarpus longan Lour)resistance gene analog(RGA)was conducted using the longan embryogenic callus (EC)cDNA as material. 29 sequences in 51 positive clones were found homology to known R genes. Via sequences multiple alignment, the typical NBS motifs were found in these 29 sequences. 26 sequences contained intact NBS domain, including p-Loop/Kinase-la[GGV(I/M)GKTT], kinase-2[LVDDVW(D)], kinase-3a(GSRIIITTRD) and GLPL [GL (F)PL (F)AL], and 3 sequences seem like truncated NBS sequences and 2 of them contained stop condon. Phylogenetic analysis showed that 29 sequences could be devided into TNL group and non-TNL group, 2 sequences were devided into TNL group, and other 27 sequences were devided into 4 non-TNL groups, non-TNL-1 and non-TNL-2 subgroup showed homology to BED-NBS-LRR genes, non-TNL-1 subgroup kinase-2 [LLMDGLW]motif showed high difference to known R genes. Cloned non-TNL-1 member NBS39 full length cDNA by GeneRacer kit, NBS39 protein sequence N -end detected an obvious coiled -coil structure, pfam analysis detected NBS and LRR8 domains, but did not detected BED-finger structure.%以龙眼胚性愈伤组织cDNA为材料进行龙眼抗性基因同源克隆(RGA).结果表明:在51个阳性克隆中有29条没有重复的序列与已知抗性基因具有同源性.经多重序列对比发现,29条序列均发现典型的NBS结构,其中26条序列具有完整的NBS结构域基序,包括p-Loop/Kinase-1a[GGV (I/M) GKTT],kinase-2[LVDDVW(D)],kinase3a (GSRⅢTTRD)以及一个疏水的[GL(F)PL(F)AL];3条序列表现为截断的NBS序列,其中有2条出现终止信号.进化树分析发现,29条序列可分为明显的2个类群,仅2条序列归为TNL类群,其余27条均为non-TNL类群.non-TNL类群可以进一步分为4个亚类群,non-TNL-1、non-TNL-2类群与毛果杨BED-NBS-LRR (BNL)基因有较高同源性,non-TNL-1类群kinase-2[LLMDGLW]基序在组内保守,但与已知R基因存在较大差异.通过RACE克隆non-TNL-1成员NBS39全长发现,NBS39氨基酸残基序列N-端具有明显的coiled-coil结构,pfam分析具有NBS及LRR8功能域,但不具有BED-finger结构.