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为选育抗根结线虫的李属砧木,以新疆野生樱桃李(Prunus sogdiana)扦插苗为试验材料,对其接种南方根结线虫(Meloidogyne incognita),从中筛选出12株抗病植株。根据已知的抗根结线虫基因的NBS-LRR保守结构域设计简并引物,并以抗病植株的DNA和cDNA为模板对抗根结线虫同源基因序列进行扩增。获得6个抗根结线虫同源基因片段,依次命名为psoRPM1、psoRPM2、psoRPM3、psoRPM4、psoRPM5和psoRPM6,GenBank登录号为:HM593974、HM593975、HM593970、HM593971、HM593972和HM593973。其中前4个基因片段与桃、甜樱桃、酸樱桃以及樱花中的抗病基因的同源性高达90%以上。基因序列分析表明,这6个基因片段均具有典型的NBS保守结构域。根据其氨基酸序列的特点,推断这6个基因片段均属于non TIR-NBS-LRR型抗病基因。RT-PCR表达分析结果显示,psoRPM1和psoRPM6在接种南方根结线虫12和24 h后表达量显著增加,预示着这2个基因片段可能参与了樱桃李对根结线虫的识别和防御过程。本研究在樱桃李中克隆出抗根结线虫相关的NBS-LRR型基因片段,为进一步克隆樱桃李中的抗根结线虫基因全长序列及转基因育种奠定了基础。
Is the rootstock of Prunus mongolica which is resistant to Meloidogyne incognita, and 12 resistant plants were selected from Prunus sogdiana cuttings in Xinjiang. Degenerate primers were designed according to the known NBS-LRR conserved domain of root knot nematode gene. The root knot nematode homologous gene sequences were amplified by using the DNA and cDNA of the resistant plant as a template. Six root knot nematode homologous gene fragments were obtained and named as psoRPM1, psoRPM2, psoRPM3, psoRPM4, psoRPM5 and psoRPM6 in turn, GenBank accession numbers were: HM593974, HM593975, HM593970, HM593971, HM593972 and HM593973. Among them, the first four genes shared more than 90% homology with resistance genes in peach, sweet cherry, sour cherry and cherry. Sequence analysis showed that these six gene fragments all have typical NBS conserved domains. According to the characteristics of its amino acid sequence, it is inferred that these six gene fragments belong to the non TIR-NBS-LRR type disease resistance gene. The results of RT-PCR showed that the expression of psoRPM1 and psoRPM6 significantly increased after inoculated with M. root-knot nematode 12 and 24 h, indicating that these two genes may be involved in the identification and defense of root-knot nematode. In this study, we cloned the NBS-LRR gene fragment associated with root-knot nematode in cherry plum, which laid the foundation for further cloning the full-length gene and transgenic breeding of root knot nematode in cherry plum.