摘要
用 2对根据已知植物抗病基因 (R基因 )编码的蛋白质的NBS保守区而设计的特异简并引物 ,对荔枝、龙眼、芦柑、柚子及银杏的基因组DNA进行体外扩增。其中一对引物 (P1/P2 )在荔枝、龙眼、芦柑和柚子中获得的扩增产物均表现为一条大小约 50 0bp的明亮谱带 ,而银杏扩增产物的亮带则在约 750bp处 ,且弥散在 40 0～ 10 0 0bp之间。另一对引物在荔枝、龙眼、芦柑和柚子中没有特异扩增带 ,而在银杏中获得了一条 40 0～ 60 0bp的较亮的宽带。将荔枝、龙眼、芦柑和柚子的 50 0bp特异扩增带以及银杏的 40 0～ 60 0bp条带回收克隆 ,并分别筛选几类阳性克隆进行测序。共获得 15个片段的序列。经比较发现 ,来自荔枝、龙眼、芦柑和柚子的 12个片段均属于NBS -LRR类抗病基因同源序列 (RGA) ,与已知R基因相应区段的氨基酸序列的一致性为 15 5%～47 1% ;而 3个来自银杏的片段均不是RGA ,其中 1个与反转录酶有较高的同源性 ,另 2个在GeneBank中没有找到同源序列。该结果显示 ,应用同源扩增技术克隆果树RGA是可行的 ,但银杏作为古老的裸子植物 。
Degenerate oligonucleotide primers based on conserved motifs in the NBS region of known resistance proteins were used to amplify resistance gene analogs (RGAs) from genomic DNAs of four subtropical fruit tree species (lichi, longan, orange and pomelo) and an ancient fruit tree species Ginkgo via polymerase chain reaction (PCR). Among fifteen different sequences obtained, twelve from the four subtropical fruit tree species shared various degree of amino-acid identity with several plant R-gene sequences and many RGAs cloned from other plant species, while the rest three sequences from Ginkgo had no similarity to known R genes. The results suggest that the PCR-based approach using degenerate primers based on the conserved NBS domains of cloned R genes can provide an attractive strategy for amplifying RGAs in fruit trees, but the R genes in the ancient species Ginkgo might be much different from those of modern fruit tree species.
出处
《福建果树》
2002年第z1期1-4,共4页
Fujian Fruits
