黑籽南瓜中NBS类抗病基因同源序列的克隆与表达分析  

Cloning and Expression Analysis of NBS Type Disease-resistance Genes Homologues from Cucurbita ficifotia

作　　者：丁玉梅[1,2,3] 高婷[4] 仲莎[4] 姚春馨[2,3] 周晓罡[2,3] 杨正安[4] 张兴国[1] DING Yu-Mei;GAO Ting;ZHONG Sha;YAO Chun-Xin;ZHOU Xiao-Gang;YANG Zheng-An;ZHANG Xing-Guo(School of Horticulture and Landscape Architecture,Southwest University,Chongqing 400715,China;Biotechnology and Germplasm Institute,Yunnan Academy of Agricultural Science,Kunming 650223,China;Key Laboratory of Agricultural Biotechnology of Yunnan,Kunming 650223,China;College of Horticulture and Landscape,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]西南大学园艺园林学院,重庆400715 [2]云南省农业科学院生物技术与种质资源研究所,昆明650223 [3]云南省农业生物技术重点实验室,昆明650223 [4]云南农业大学园林园艺学院,昆明650201

出　　处：《农业生物技术学报》2018年第8期1305-1317,共13页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31460516);云南省高水平大学园艺学创新人才培养基地(云教高[2015]57)

摘　　要：黑籽南瓜(Cucurbita ficifolia)是云南特有的对病害有较强抗性的一种瓜类作物。为发掘利用其蕴含的优异抗病基因资源,本研究根据已克隆到的植物核苷酸结合位点(nucleotide binding site,NBS)类抗病基因的保守氨基酸结构域基因设计简并引物,通过PCR扩增从黑籽南瓜基因组中分离了8条黑籽南瓜抗病基因同源序列(resistance gene analogous,RGAs),聚类分析和相似性比较结果显示,黑籽南瓜抗病基因同源序列2(black seed squash resistance gene analogous 2,HQRGA2(Gen Bank No.:MG946756)单独为一类,且具有核苷酸结合衔接子(nucleotide-binding adaptor shared by APAF-1,R proteins and CED-4;NBARC)结构,其余7条序列均不具有NBS保守结构域。核苷酸相似性分析表明,HQRGA2与部分抗病基因序列的相似性达到87%~99%,其中与南瓜(Cucurbita maschata)NBS类抗病蛋白基因13(squash resistance gene analogous,SQRGA-13)和SQRGA-8的相似性分别达到98.0%和97.0%。对HQRGA2编码的氨基酸保守结构域分析表明,其含有NBS类抗病基因的4个保守模块:P-loop、Kinase-2、Kinase-3a和GLPL区,且属于无Toll蛋白或白介素1受体类似的NBS富亮氨酸重复(non toll/interleukin-1 receptor like of NBS plus leucine-rich region,non-TIR-NBS-LRR)类抗病基因同源序列。HQRGA2编码氨基酸的同源性分析结果表明,其与南瓜抗病蛋白基因SQRGA-13氨基酸同源性较高,达到96.6%,与其余抗病基因同源性在16.6%~43.8%之间,NBS区域氨基酸系统进化树分析结果表明,HQRGA2可能是一个新的抗病基因片段。q RTPCR分析表明,黑籽南瓜HQRGA2片段的表达受尖孢镰刀菌(Fusarium oxysporum)的胁迫诱导,表现出先扬后抑的表达模式,说明HQRGA2可能为抗病相关基因片段。黑籽南瓜中NBS类抗病基因同源序列HQRGA2的获得为分离克隆其完整抗病基因提供了新线索。Cucurbita ficifolia is one of characteristic germplasm crops that has strong resistance abilities to many disease pathogens stress, especially resistance to Fusarium oxysporum. Based on the degenerated primers being designed according to the conserved regions of NBS type genes in other plants, eight RGA sequences were cloned and sequenced from Cucurbita ficifolia genome by PCR amplification. Among them, the sequence of HQRGA2 had got NB-ARC structure domain and would be classified an individual category, yet the other seven sequences hadn’t got the NBS domains by multi alignment sequence contrast. Furthermore, the results of nucleotide homology analysis indicated that 98% and 97% of nucleotides of HQRGA2 were identity to the resistant genes SQRGA-13 and SQRGA-8 from Cucurbita moschata respectively. Furthermore, the amino acid sequence analysis results showed that the sequence of HQRGA2 belonged to the class of Non-TIR-NBS-LRR disease-resistance genes analogues, which contains the conserved regions of P-loop, Kinase-2, Kinase-3, and GLPL domain. We also obtained that 96.6% amino acids were homology to RGAs of Cucurbita moschata by comparison analysis of amino acid sequences. The results of qRT- PCR showed that the expression of HQRGA2 from C. ficifolia was influenced and induced by the fungi of Fusarium oxysporum, with the expression pattern of ‘up-down’, which indicated its relation to resistance gene in the aspect. The obtaining of NBS type of R-gene analogues provided us a clue for the further cloning of disease- resistance genes from Cucurbita ficifolia., Abstract Cucurbita ficifolia is one of characteristic germplasm crops that has strong resistance abilities to many disease pathogens stress, especially resistance to Fusarium oxysporum. Based on the degenerated primers being designed according to the conserved regions of NBS type genes in other plants, eight RGA sequences were cloned and sequenced from Cucurbita ficifolia genome by PCR amplification. Among them, the sequence of HQRGA2 had got NB-ARC str

关 键 词：黑籽南瓜 核苷酸结合位点(NBS)类基因 保守结构域 qRT-PCR 

分 类 号：S642[农业科学—蔬菜学]