Abstract：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 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 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.
陈观水, 周以飞, 林生，等. 2006. 甘薯抗病基因NBS类似物的分离与序列分析[J]. 热带亚热带植物学报, 14(5): 359-365. (Chen G S, Zhou Y F, Lin S, et al. 2006. Isolation and Sequence analysis of NBS-type resistance gene analogues in sweet potato(Ipomoea batatas(L.)[J]. Journal of Tropical and Subtropical Botany，14(5): 359-365.)
丁国华, 秦智伟, 刘宏宇, 等. 2005. 黄瓜NBS类型抗病基因同源序列的克隆与分析[J]. 园艺学报, (32)4: 638-642. (Ding G H, Qin Z W，Liu H Y, et al. 2005. Analysis and cloning of NBS class disease resistant gene analog in cucumber[J]. Acta Horticulturae Sinica. (32)4: 638-642.)
高丽华，周以飞，郑伟文，等. 2007. 南瓜NBS类抗病基因同源序列的克隆与分析[J]. 长江蔬菜, 8: 40-42. (Gao L H, Zhou Y F, Zhen W W, et al. 2007. Cloning and analysis of NBS class disease resistant gene analogous in squash(Cucurbita moschata Duch.)[J]. Journal of Changjiang Vegetables, 8: 40-42.)
郭绍贵. 2005. 西瓜枯萎病R基因同源序列的克隆与分析[D]. 硕士学位论文, 扬州大学,导师：陈学好，许勇. 4: pp. 793-800. (Guo S G. 2005. Analysis and cloning of R gene analogous in water melon(Citrullus lanatus)[D]. Thesis for M.S., Yangzhou University, Supervisor: Chen X H, Xu Y, 4: pp. 793-800.)
李德葆. 1996. 基因工程操作技术[M]. 科学技术出版社,上海. pp. 64-98. (Li D B. 1996. Technology of Gene Engineering[M]. Science Technology Press, Shanghai. pp. 64-98.)
卢圣栋. 1998. 现代分子生物学实验技术[M]. 高等教育出版社,北京. pp.123-165. (Lu S D. 1998. Experimental Technology of Modern Molecular Biology[M]. High Education Press,Beijing. pp.123-165.)
阙友雄，许莉萍，张木清，等. 2009. 甘蔗中一个 NBS-LRR 类基因的全长克隆与表达分析[J]. 作物学报, 35(6):1161?1166. (Que Y X, Xu L P, Zhang M Q, et al. 2009. Cloning and expression analysis of an NBS-LRR type gene from sugarcane[J]. Acta Agronominca Sinica, 35(6):1161?1166.)
孙涌栋, 罗未蓉, 李新峥, 等. 2014. NaHS对NaHCO3胁迫下黑籽南瓜种子萌发及生理特性的影响[J]. 植物学报, 49(1): 98-104. (Sun Y D, Luo W R, Li X Z, et al. 2014. Effects of exogenous NaHS on seed germination and physiological characteristics of Cucurbita ficifolia under NaHCO3 stress[J]. Chinese Bulletin of Botany[J], 49(1): 98-104.)
王贤磊，高兴旺，张铁钢，等. 2011. 甜瓜抗病基因同源序列的克隆与分析[J]. 新疆大学学报(自然科学版) 28(2): 136-144. (Wang X L, Gao X W, Zhang T G, et al. 2011. Cloning and analysis of melon resistant gene analog[J]. Journal of Xinjiang University(Natural Science Edition), 28(2): 136-144.)
杨明挚，陈小兰，尹梅, 等. 2005. 黑子南瓜中STK类抗病基因同源序列的克隆及序列分析[J].云南大学学报(自然科学版), 27(2): 176-179. (Yang M Z, Chen X L, Yin M, et al. 2005. Clone and sequence analysis of STK type disease-resistance gene analogous from figleaf gourd[J]. Journal of Yunnan University(Natural Science Edition), 27(2): 176-179.)
赵芹，谢大森，何晓明，等. 2015. 基于NBS-LRR类R基因保守结构域克隆瓠瓜抗病基因同源序列[J]. 华南农业大学学报, 36(5):, 92-98. (Zhao Q, Xie D S, He X M,et al. 2015. Cloning of resistance gene analogs from Lagenaria siceraria based on conserved domains of NBS-LRR type R gene[J]. Journal of South China Agricultural University, 36(5):, 92-98.)
Bent A F, Kankel B N, Dahlbeck D, et al. 1994. RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes[J]. Science, 265(5180): 1856–1860.
Bent AF. 1996. Plant disease resistance genes function meets structure[J]. Plant Cell, 8(10): 1757 -1771.
Bourne H R, Sanders D A, Mc Cormick F. 1991. The GTPase superfamily: conserved structure and molecular mechanism[J]. Nature, 349: 117-127.
Brody J D, Roger W I. 2006. Plant NBS-LRR proteins in pathogen sensing and host defense[J]. Nature Immunology, 7(12): 1243–1249.
Chen Ru-gang, Zhang Li-ying, Zhang Jun-hong. 2007. CaMi, a root-knot nematode resistance gene from hot pepper(Capsium annuum L.) confers nematode resistance in tomato[J]. Plant Cell Reports, 26(7): 895-905.
Dixon M S, Hatzixanthis K, Jones D A, et al. 1998. The tomato Cf-5 disease resistance gene and six homologous show pronounced allelic variation in leucine-rich repeat copy number[J]. Plant Cell, 10(11): 915–1925.
Johal G S, Briggs S I. 1992. Reductase activity encoded by the HM1 disease resistance gene in maize[J]. Science, 258(5084): 985-987.
Liu X, Lin F, Wang L, Pan Q. 2007. The in silico map-based cloning of Pi36, a rice coiled-coil
nucleotide-binding site leucine-rich repeat gene that confers race-specific resistance to the blast
fungus[J]. Genetics, 176(4): 2541–2549.
Meyers B C, Dickerman A W, Michelmore R W, et al. 1999. Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding super family[J]. Plant Journal, 20(3): 317–332.
Meyers B C, Kozik A, Griego A, et al. 2003. Genome-wide analysis of NBS-LRR-encoding genes in Aarabidopsis[J]. Plant Cell, 15(4): 809-834.
Saraste M, Sibbald P R, Wittinghofer A. 1990. The P-loop a common motif in ATP-and GTP binding proteins[J]. Trends Biochemical Sciences, 15(11): 430-434.
Staskawicz B J, Ausubel F M, Baker B J, et al. 1995. Molecular genetics of plant disease resistance[J]. Science, 268(5211): 661-667., 陈观水, 周以飞, 林生，等. 2006. 甘薯抗病基因NBS类似物的分离与序列分析[J]. 热带亚热带植物学报, 14(5): 359-365. (Chen G S, Zhou Y F, Lin S, et al. 2006. Isolation and Sequence analysis of NBS-type resistance gene analogues in sweet potato(Ipomoea batatas(L.)[J]. Journal of Tropical and Subtropical Botany，14(5): 359-365.)
丁国华, 秦智伟, 刘宏宇, 等. 2005. 黄瓜NBS类型抗病基因同源序列的克隆与分析[J]. 园艺学报, (32)4: 638-642. (Ding G H, Qin Z W，Liu H Y, et al. 2005. Analysis and cloning of NBS class disease resistant gene analog in cucumber[J]. Acta Horticulturae Sinica. (32)4: 638-642.)
高丽华，周以飞，郑伟文，等. 2007. 南瓜NBS类抗病基因同源序列的克隆与分析[J]. 长江蔬菜, 8: 40-42. (Gao L H, Zhou Y F, Zhen W W, et al. 2007. Cloning and analysis of NBS class disease resistant gene analogous in squash(Cucurbita moschata Duch.)[J]. Journal of Changjiang Vegetables, 8: 40-42.)
郭绍贵. 2005. 西瓜枯萎病R基因同源序列的克隆与分析[D]. 硕士学位论文, 扬州大学,导师：陈学好，许勇. 4: pp. 793-800. (Guo S G. 2005. Analysis and cloning of R gene analogous in water melon(Citrullus lanatus)[D]. Thesis for M.S., Yangzhou University, Supervisor: Chen X H, Xu Y, 4: pp. 793-800.)
李德葆. 1996. 基因工程操作技术[M]. 科学技术出版社,上海. pp. 64-98. (Li D B. 1996. Technology of Gene Engineering[M]. Science Technology Press, Shanghai. pp. 64-98.)
卢圣栋. 1998. 现代分子生物学实验技术[M]. 高等教育出版社,北京. pp.123-165. (Lu S D. 1998. Experimental Technology of Modern Molecular Biology[M]. High Education Press,Beijing. pp.123-165.)
阙友雄，许莉萍，张木清，等. 2009. 甘蔗中一个 NBS-LRR 类基因的全长克隆与表达分析[J]. 作物学报, 35(6):1161?1166. (Que Y X, Xu L P, Zhang M Q, et al. 2009. Cloning and expression analysis of an NBS-LRR type gene from sugarcane[J]. Acta Agronominca Sinica, 35(6):1161?1166.)
孙涌栋, 罗未蓉, 李新峥, 等. 2014. NaHS对NaHCO3胁迫下黑籽南瓜种子萌发及生理特性的影响[J]. 植物学报, 49(1): 98-104. (Sun Y D, Luo W R, Li X Z, et al. 2014. Effects of exogenous NaHS on seed germination and physiological characteristics of Cucurbita ficifolia under NaHCO3 stress[J]. Chinese Bulletin of Botany[J], 49(1): 98-104.)
王贤磊，高兴旺，张铁钢，等. 2011. 甜瓜抗病基因同源序列的克隆与分析[J]. 新疆大学学报(自然科学版) 28(2): 136-144. (Wang X L, Gao X W, Zhang T G, et al. 2011. Cloning and analysis of melon resistant gene analog[J]. Journal of Xinjiang University(Natural Science Edition), 28(2): 136-144.)
杨明挚，陈小兰，尹梅, 等. 2005. 黑子南瓜中STK类抗病基因同源序列的克隆及序列分析[J].云南大学学报(自然科学版), 27(2): 176-179. (Yang M Z, Chen X L, Yin M, et al. 2005. Clone and sequence analysis of STK type disease-resistance gene analogous from figleaf gourd[J]. Journal of Yunnan University(Natural Science Edition), 27(2): 176-179.)
赵芹，谢大森，何晓明，等. 2015. 基于NBS-LRR类R基因保守结构域克隆瓠瓜抗病基因同源序列[J]. 华南农业大学学报, 36(5):, 92-98. (Zhao Q, Xie D S, He X M,et al. 2015. Cloning of resistance gene analogs from Lagenaria siceraria based on conserved domains of NBS-LRR type R gene[J]. Journal of South China Agricultural University, 36(5):, 92-98.)
Bent A F, Kankel B N, Dahlbeck D, et al. 1994. RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes[J]. Science, 265(5180): 1856–1860.
Bent AF. 1996. Plant disease resistance genes function meets structure[J]. Plant Cell, 8(10): 1757 -1771.
Bourne H R, Sanders D A, Mc Cormick F. 1991. The GTPase superfamily: conserved structure and molecular mechanism[J]. Nature, 349: 117-127.
Brody J D, Roger W I. 2006. Plant NBS-LRR proteins in pathogen sensing and host defense[J]. Nature Immunology, 7(12): 1243–1249.
Chen Ru-gang, Zhang Li-ying, Zhang Jun-hong. 2007. CaMi, a root-knot nematode resistance gene from hot pepper(Capsium annuum L.) confers nematode resistance in tomato[J]. Plant Cell Reports, 26(7): 895-905.
Dixon M S, Hatzixanthis K, Jones D A, et al. 1998. The tomato Cf-5 disease resistance gene and six homologous show pronounced allelic variation in leucine-rich repeat copy number[J]. Plant Cell, 10(11): 915–1925.
Johal G S, Briggs S I. 1992. Reductase activity encoded by the HM1 disease resistance gene in maize[J]. Science, 258(5084): 985-987.
Liu X, Lin F, Wang L, Pan Q. 2007. The in silico map-based cloning of Pi36, a rice coiled-coil
nucleotide-binding site leucine-rich repeat gene that confers race-specific resistance to the blast
fungus[J]. Genetics, 176(4): 2541–2549.
Meyers B C, Dickerman A W, Michelmore R W, et al. 1999. Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding super family[J]. Plant Journal, 20(3): 317–332.
Meyers B C, Kozik A, Griego A, et al. 2003. Genome-wide analysis of NBS-LRR-encoding genes in Aarabidopsis[J]. Plant Cell, 15(4): 809-834.
Saraste M, Sibbald P R, Wittinghofer A. 1990. The P-loop a common motif in ATP-and GTP binding proteins[J]. Trends Biochemical Sciences, 15(11): 430-434.
Staskawicz B J, Ausubel F M, Baker B J, et al. 1995. Molecular genetics of plant disease resistance[J]. Science, 268(5211): 661-667.
