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橡胶树(Hevea brasiliensis Muell.Arg).的传统种植区主要分布于高温多雨的热带地区,抗旱基因或DNA标记的筛选鉴定对于橡胶耐旱品系的选育及橡胶树向温光和雨量相对不足的非传统植胶区的拓展具有十分重要的意义。RAPD分析是目前遗传多态性研究及特异性基因片段鉴定最有效的分子学工具之一。本研究借助聚合酶链式反应(PCR)对来自37个橡胶无性系的基因组DNAs进行了RAPD分析。结果显示,多态性及特异性扩增条带的数量随无性系及引物的不同而有所差异,有些扩增带为一些无性系共有,而另一些则仅存在于个别或少数无性系之中。根据引物OPB-12的两个特异性扩增条带(1.2和1.4kb)在各无性系中单独或同时存在与否,我们鉴定出了两个相应的特异性RAPD标记,其中1.4kb扩增带尤其引人注目。克隆及测序结果表明,该RAPD片段与酿酒酵母(Saccharomycescerevisiae)脯氨酸特异性通透酶基因存在一定程度同源性。针对该片段设计一对特异性引物并再次对基因组DNA进行更严格条件下的PCR扩增,结果从14个参试无性系中获得了该1.4kb条带。这种由RAPD标记通过特异性引物设计转化得到的SCAR(序列特异性扩增区)标记可用于橡胶树优良或特异性基因的筛选及鉴定。
The traditional planting areas of Hevea brasiliensis Muell.Arg are mainly distributed in hot and rainy tropical regions. The selection of drought-resistant genes or DNA markers is of great significance to the breeding of drought-resistant rubber strains and the non-traditional Rubber zone expansion has very important significance. RAPD analysis is currently one of the most effective molecular tools for genetic polymorphism and specific gene fragment identification. In this study, RAPD analysis of genomic DNA from 37 rubber clones was performed by polymerase chain reaction (PCR). The results showed that the number of polymorphic and specific amplified bands varied with clones and primers. Some amplified bands were shared by some clones while others were only present in some or a few clones in. Based on the presence of two specific amplification bands (1.2 and 1.4 kb) of primer OPB-12, individually or simultaneously in each clonal line, we identified two corresponding specific RAPD markers, of which the 1.4 kb amplification The band is particularly noticeable. The cloning and sequencing results showed that there was some homology between the RAPD fragment and the proline-specific permease gene of Saccharomyces cerevisiae. A pair of specific primers was designed for this fragment and again genomic DNA was subjected to PCR amplification under more stringent conditions. As a result, the 1.4 kb band was obtained from 14 tested clones. This SCAR (sequence-specific amplification region) marker obtained by RAPD-specific design and transformation of primers can be used for screening and identification of excellent or specific genes in rubber trees.