PmACRE基因的克隆及遗传转化拟南芥
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摘要
Avr9/Cf-9 rapidly elicited(ACRE)gene是富含亮氨酸重复单元,参与蛋白与蛋白互作,特异性识别病原物激发子的关键基因,在植物抗病过程中有重要意义。利用RT-PCR从马尾松中克隆出Avr9/Cf-9 rapidly elicited gene,命名为PmACRE(Gen Bank登录号:MF630966)。测序结果显示,PmACRE c DNA序列全长862 bp,其中完整编码区长度624 bp,编码207个氨基酸;以PFGFP Bar 137质粒为基本载体,构建了由组成型CaMV35S启动子驱动PmACRE基因的植物表达载体PFGFP Bar 137-PmACRE;采用冻融法转入农杆菌AGL1菌株,通过花序浸染法对拟南芥进行了遗传转化,并获得了4株阳性苗,经目的基因ACRE和GFP基因双重PCR检测,证实目的基因已整合到拟南芥基因组中。研究结果为进一步分析马尾松ACRE基因的功能和深入揭示R基因调控马尾松响应松材线虫侵染的作用机制奠定基础。
The Avr9/Cf-9 rapidly elicited( ACRE) gene belongs to leucine rich repeats( LRR),which is the key gene involved in the process of protein-protein interaction and specific identification of pathogen elicitors and it plays a significant role in disease risistance. The Avr9/Cf-9 rapidly elicited gene from Pinus massoniana( Gen Bank accession number: MF630966) was cloned through RT-PCR and named as Pm ACRE. The ACRE of P. massoniana,whose c DNA length is 862 bp,contained an open reading frame of 624 bp which encoded a polypeptide of 207 amino acids. The plant expression vector PFGFP Bar 137 was constructed,in which the Pm ACRE gene was driven by the constitutive promoter Ca MV35 S. The constructed expression vector was transformed into Agrobacterium tumefaciens AGL1 strain by the floral dip method. Leaf segments of Arabidopsis thaliana were inoculated with A. tumefaciens AGL1 containing plasmid PFGFP Bar 137-Pm ACRE. The transgenic lines were selected by ACRE and GFP gene PCR detection and the result showed that the target gene had been integrated into the genome of A. thaliana and the 4 positive plants have been obtained. The results of this study gave a foundation for the further analysis of the function of ACRE and revealing the mechanism on the P. massoniana in response to Bursaphelenchus xylophilus infestation.
The Avr9/Cf-9 rapidly elicited( ACRE) gene belongs to leucine rich repeats( LRR),which is the key gene involved in the process of protein-protein interaction and specific identification of pathogen elicitors and it plays a significant role in disease risistance. The Avr9/Cf-9 rapidly elicited gene from Pinus massoniana( Gen Bank accession number: MF630966) was cloned through RT-PCR and named as Pm ACRE. The ACRE of P. massoniana,whose c DNA length is 862 bp,contained an open reading frame of 624 bp which encoded a polypeptide of 207 amino acids. The plant expression vector PFGFP Bar 137 was constructed,in which the Pm ACRE gene was driven by the constitutive promoter Ca MV35 S. The constructed expression vector was transformed into Agrobacterium tumefaciens AGL1 strain by the floral dip method. Leaf segments of Arabidopsis thaliana were inoculated with A. tumefaciens AGL1 containing plasmid PFGFP Bar 137-Pm ACRE. The transgenic lines were selected by ACRE and GFP gene PCR detection and the result showed that the target gene had been integrated into the genome of A. thaliana and the 4 positive plants have been obtained. The results of this study gave a foundation for the further analysis of the function of ACRE and revealing the mechanism on the P. massoniana in response to Bursaphelenchus xylophilus infestation.
引文
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[2]PANWAR P,JHA A K,PANDEY P K,et al.Functional markers based molecular characterization and cloning of resistance gene analogs encoding NBS-LRR disease resistance proteins in finger millet(Eleusine coracana)[J].Molecular Biology Reports,2011,38(5):3 427-3 436.
[3]李少锋,苏晓华,张冰玉.林木基因克隆研究进展[J].植物学报,2011,46(1):79-107.
[4]尹佟明.林木基因组及功能基因克隆研究概述[J].遗传,2010,32(7):677-684.
[5]张星耀.中国松材线虫病危险性评估及对策[M].北京:科学出版社,2011.
[6]潘沧桑.松材线虫病研究进展[J].厦门大学学报(自然科学版),2011,50(2):476-483.
[7]张锴,梁军,严冬辉,等.中国松材线虫病研究[J].世界林业研究,2010,23(3):59-63.
[8]ZHENG H,XU M,XU F,et al.A comparative proteomics analysis of Pinus massoniana inoculated with Bursaphelenchus xylophilus[J].Pakistan Journal of Botany,2015,47(4):1 271-1 280.
[9]XIE W,HUANG A,LI H,et al.Identification and comparative analysis of microRNAs in Pinus massoniana infected by Bursaphelenchus xylophilus[J].Plant Growth Regulation,2017,83(2):223-232.
[10]LI G S,ASIEGBU F O.Induction of Pinus sylvestris Ps ACRE,a homology of Avr9/Cf-9 rapidly elicited defense-related gene following infection with root rot fungus Heterobasidion annosum[J].Plant Science,2004,167(3):535-540.
[11]ROWLAND O,LUDWIG A A,MERRICK C J,et al.Functional analysis of Avr9/Cf-9 rapidly elicited genes identifies a protein kinase,ACIK1,that is essential for full Cf-9-dependent disease resistance in tomato[J].Plant Cell,2005,17(1):295-310.
[12]丁丽娜,杨国兴.植物抗病机制及信号转导的研究进展[J].生物技术通报,2016,32(10):109-117.
[13]WU L T,DU C F,ZENG Z,et al.Study on the signal transduction pathway of plant defense to pathogens[J].Agricultural Science&Technology,2014,15(4):517-519.
[14]张艳.植物抗病防卫相关基因的克隆及其转化烟草的研究[D].福州:福建农林大学,2011.
[15]戴小枫,陈捷胤,田李,等.植物抗病基因和提高植物抗病性的方法[P].2012-09-05.
[16]王友红,张鹏飞,陈建群.植物抗病基因及其作用机理[J].植物学通报,2005,22(1):92-99.
[17]袁亮,张伟彬.植物抗病基因作用机理及克隆研究进展[J].安徽农业科学,2009,37(4):1 513-1 515.
[18]JONES J D G,DANGL J L.The plant immune system[J].Nature,2006,444(7 117):323-329.
[19]JIA R Z,MING R,ZHU Y J.Genome-wide analysis of nucleotide-binding site(NBS)disease resistance(R)genes in sacred lotus(Nelumbo nucifera Gaertn.)reveals their transition role during early evolution of land plants[J].Tropical Plant Biology,2013,6(2):98-116.
[20]HEESE A,LUDWIG A A,JONES J.Rapid phosphorylation of a syntaxin during the Avr9/Cf-9-race-specific signaling pathway[J].Plant Physiology,2005,138(4):2 406-2 416.
[21]骆有庆,张星耀.中国森林重大生物灾害[M].北京:中国林业出版社,2003.
[22]GHEYSEN G,FENOLL C.Gene expression in nematode feeding sites[J].Annual Review of Phytopathology,2002,40(4):191-219.
[23]袁广胜.玉米抗穗粒腐病差异表达基因的分离及其功能分析[D].雅安:四川农业大学,2011.
[24]茆振川,谢丙炎,杨之为,等.根结线虫与植物的分子互作[J].园艺学报,2006,33(4):901-907.