【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。更多还原

【Abstract】 Cucurbita ficifolia is one of characteristic germplasm crops in Yunnan that has strong resistant abilities to wilt caused by Fusarium oxysporum,besides it has tolerant abilities to many abiotic stresses,such as coldness,drought and barren soil.The exploration and utilization of its advantageous gene resources is of great significance in disease resistant breeding of Cucurbita crops.In view of the situations,six degenerated primers were designed according to the conserved regions of nucleotide binding site(NBS) type genes in other plants,and consequently 8 resistance gene analogous(RGAs) were isolated and sequenced from C.ficifolia genome by using PCR amplification.The cluster analysis showed that the 8 RGAs would be classified3 categories,among them the sequence named black seed squash resistance gene analogous 2(HQRGA2(Gen Bank No.:MG946756) had got nucleotide-binding adaptor shared by APAF-1,R proteins and CED-4(NBARC) conservative structure domain which belongs to NBS class and would be classified an individual category,yet the other 7 sequences hadn’t got the NBS domains,moreover there was higher nucleotide identity in each category and lower identity among different categories,the nucleotide diversities of those RGAs was rich by multi alignment sequence contrast.Furthermore,the results of nucleotide similarity analysis indicated the identity of disease resistance genes from Cucurbitaceae crops varied from 87.0% to 99.0% when compared with the sequence of HQRGA2,yet 98.0% and 97.0% of nucleotides of HQRGA2 were shown to have similarity comparable to that of resistant genes SQRGA-13 and SQRGA-8 from C.moschata respectively.By amino acid sequences contrast of HQRGA2 with the NBS region of the previously known disease resistant genes,the results showed that HQRGA2 contained the designated regions,that is P-loop,Kinase-2,Kinase-3,and GLPL domain,and it belonged to the class of non toll/interleukin-1 receptor like of NBS plus leucine-rich region(non-TIR-NBS-LRR) disease-resistance genes analogues for the last amino acid of kinase-2 was tryptophan.However,more RGAs should be isolated from C.ficifolia to ensure whether its genome contains anther class of toll/interleukin-1 receptor like of NBS plus leucine-rich region(TIR-NBS-LRR) gene analogues in the further investigations.The results of amino acid homology analysis in this paper that the homologous percentages were from 16.6% to 96.6% when contrasted with the known disease resistance gene,among them,the gene SQRGA-13 from C.moschata has got the highest percentage of 96.6% and the gene Mi-1.1 fromLycopersicon esculentum was 43.8% respectively.The amino acid homology analysis results illustrated that C.ficifolia had a close genetic relationship with C.moschata,yet the neighbor-jioning tree based on aligment of amino acid squences indicated that HQRGA2 would possibly be a new type of disease resistant gene fragment.To testify whether HQRGA2 induced by pathogen,The relative expression of HQRGA2 were measured in the seedlings after infected with the fungi of F.oxysporum on different inoculation stages,the results of q RT-PCR showed that the relative expression of HQRGA2 was gradually increasing during 72 h inoculation,and the treatments were significantly higher than uninoculated control at stages of 4,12,24,48 and 72 h.The expression of HQRGA2 reached to a peak at 72 h which had 3.71 times of control.In succession,the expression was decreasing since then till 120 h(significantly lower than control).The process diagram presented the expression pattern of up-down,which indicated HQRGA2 was involved in the response to the pathogen and was related to disease resistant gene in the aspect.Anyhow in this investigation,The NBS type disease-resistance gene analogue of HQRGA2 were obtained and thus a new clue for the cloning of the complete gene from C.ficifolia was provided,at the same time,this would be the basis for the creations of new varieties resistant to wilt in further breeding research of Cucurbita crops.更多还原