云南3种野生稻中抗病基因同源序列的克隆及序列分析

Cloning and Sequence Analysis of Disease Resistance Gene Analogues from Three Wild Rice Species in Yunnan

根据已报道的NBS LRR类和STK类抗病基因结构中的氨基酸保守区域 ,设计简并引物 ,通过PCR扩增及克隆 ,从普通野生稻 (OryzarufipogonGriff.)、药用野生稻 (OryzaofficinalisWall.)、疣粒野生稻 (Oryzameye rianaBaill.)中共获得 14类NBS LRR类抗病基因同源序列 ,其中普通野生稻中的有 7类 ,药用野生稻中的有 2类 ,疣粒野生稻中的有 6类。药用野生稻中TO12代表序列与普通野生稻中TR19代表序列 ,同属一类 ,且具有 10 0 %的同源性 ,说明不同种野生稻中的同一类 (聚类 )抗病基因同源序列是完全一致的。同时 ,还获得 5类STK类抗病基因同源序列 ,其中普通野生稻中的有 4类 ;药用野生稻中的有 1类。通过氨基酸同源性比较分析 ,发现笔者克隆到的抗病基因同源序列与已克隆的抗病基因L6、N、Bs2、Prf、Pto、Lr10和Xa2 1等的氨基酸同源性都相当低 (均低于 2 5 % ) ,暗示了这些抗病基因同源序列可能是目前尚未报道的抗病基因的同源序列。

Two sets of degenerate oligonucleotide primers were designed according to amino acid conserved regions of reported plant disease resistance genes which encode proteins that contain nucleotide binding site and leucine rich repeats(NBS LRR),and the plant disease resistance genes which encode serine/threonine protein kinase(STK).By polymerase chain reaction(PCR),disease resistance gene analogues have been amplified from three wild rice species in Yunnan.The DNA fragments from amplification have been cloned into the pGEM T vector, respectively. Sequencing of the DNA fragments indicated that 7 classes, 2 classes and 6 classes NBS LRR disease resistance gene analogues from Oryza rufipogon Griff., Oryza officinalis Wall., and Oryza meyeriana Baill., respectively were obtained. The two representative fragments of TO12 from Oryza officinalis Wall. and TR19 from Oryza rufipogon Griff. belong to the same class and their sequences are totally identity. The result shows that the sequences of the same class disease resistance gene analogues have no difference among different species of wild rice. We also obtained five classes STK disease resistance gene analogues were obtained among which 4 classes were from Oryza rufipogon Griff., 1 class was from Oryza officinalis Wall.. By comparison analysis of amino acid sequences,it was found that the obtained disease resistance gene analogues have very low identity(lower than 25%) with the reported disease resistance gene L6, N, Bs2, Prf, Pto, Lr10 and Xa21 etc.The finding suggests that the obtained disease resistance gene analogues may be analogues of putative disease resistance genes that have not been isolated so far.