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本研究以抗镰刀菌枯萎病香蕉种质“金手指”(AAAB)为材料,根据已克隆的抗枯萎病基因的 NBS保守结构域设计简并引物,通过同源序列克隆获得了 20 条来自基因组 DNA 的 RGA 片段,大小为 530 bp 左右。根据其推断的氨基酸序列,经保守结构域分析,其结构域均为 NB-ARC,属于 non-TIR-NBS 类候选抗病基因类序列。它们均具有 P-loop (GMGGVGKTT),Kinase-2 (LLVLDDIW),RNBS-B (CKVLFTTRS)以及疏水氨基酸结构域 GLPL (GLPLALKVL)等 4 个保守氨基酸基元。20 个 RGA 之间核苷酸序列的相似性在 41.1%～99.3%之间,氨基酸序列的相似性在 33.2%～96.3%之间。同时,对分离得到的 20 条 RGAs 进行系统发育树分析,发现它们分布在 5 个不同的区域。并且所编码的氨基酸序列与已知抗枯萎病基因 Fom-2、I2C-1、I2C-2和 I2 等编码的氨基酸序列表现出 28%～54%的同源性,证明了抗病基因在进化上具有一定的保守性。因此,这些抗病基因同源片段(RGA)的分离将为进一步从香蕉中分离抗枯萎病基因打下基础,也可作为分子标记筛选香蕉抗枯萎病的候选基因。
In this study, we designed degenerate primers based on the NBS conserved domain of the Fusarium wilt resistance gene Fusarium wilt “Golden Finger” (AAAB), cloned 20 homologous sequences RGA fragments from genomic DNA, about 530 bp in size. According to its deduced amino acid sequence, its domain was NB-ARC based on conserved domain analysis, belonging to non-TIR-NBS candidate resistance gene sequence. They all have four conserved amino acid motifs, P-loop (GMGGVGKTT), Kinase-2 (LLVLDDIW), RNBS-B (CKVLFTTRS), and hydrophobic amino acid domain GLPL (GLPLALKVL). The similarity of the 20 RGA nucleotide sequences was between 41.1% and 99.3%, and the amino acid sequences were similar between 33.2% and 96.3%. Meanwhile, phylogenetic tree analysis of the 20 isolated RGAs showed that they distributed in five different regions. The deduced amino acid sequence showed 28% ~ 54% homology with amino acid sequence encoded by Fom-2, I2C-1, I2C-2 and I2, On a certain degree of conservation. Therefore, the segregation of these resistance gene homologous fragments (RGA) will lay a foundation for further isolation of Fusarium wilt resistance genes from banana, and may also be used as a molecular marker to screen candidate genes for Fusarium wilt resistance in banana.