花生RGA片段的克隆及初步分析

Cloning and Primary Analysis for RGA Fragments in Peanut

目的:通过同源克隆获得了花生闽花6号的RGA片段,为其抗性的研究及抗性育种提供了参考资料。方法:试验分为两组:其一通过利用抗性基因的NBS保守区所设计的简并引物对花生品种闽花六号进行了RGA片段扩增,其二结合已登录的花生RGA片段序列经过多元比对后设计简并引物进行RGA片段的扩增及序列分析;分析比较两组克隆方法的效果。结果:测序分析表明:前者20条随机测序序列中没有一条与已知RGA片段序列相似;后者20条随机测序序列中有18条为RGA片段序列,其登录号为GenBankEU639668-EU639685。结论:前一种方法克隆扩增RGA基因片段的效率很低,而后一种方法克隆扩增效果更好,这为闽花6号花生的遗传改良提供了理论基础。

Objective： RGA fragments were cloned to investigate resistance and provide theoretical guidance for breeding in peanut. Methods： In this study, degenerate PCR primers firstly designed to bind to DNA regions encoding conserved motifs within NBS （nucleotide binding site） were used to amplify NBS-encoding regions from A. hypogaea L. Minhua Number 6. At the same time a pair of degenerate primers was designed according to the result of multiple sequence alignment from known RGA sequences in peanut. RGA fragments were amplified using homology cloning on the peanut variety Minhua number 6. Results： It was showed that for former there was no sequence obtained in the study homologous to known sequences of RGA according to the sequencing results. While for later there are 18 RGA sequences in 20 clones selected randomly manifested by BLASTN sequence analysis, GenBank numbers of which are EU639668-EU639685. Conclusion： It shows that it is more efficiently to amplify corresponding RGA fragments by means of the RGA sequence known to this species, which provides a shortcut for following RGA study and use. For that matter it provides theory basis for genetic improvement in Minhua Number 6.