松材线虫Bx-Daf-22基因鉴定及表达
|
摘要
为探究松材线虫抗逆态幼虫DL3形成的分子机理,对其鸟苷酸环化酶路径中的抗逆态基因BxDaf-22进行了鉴定和表达研究。笔者应用BLAST比对技术在松材线虫基因组数据中鉴定得到固醇载体蛋白SCP2同源基因,命名为Bx-Daf-22。根据松材线虫基因组数据设计引物,应用PCR技术扩增BxDaf-22完整蛋白编码区。进一步对Bx-Daf-22基因编码的氨基酸序列进行同源序列系统进化、系统发育分析、保守结构域分析和蛋白质三维结构比对等生物信息学分析。应用荧光定量Q-PCR对不同虫龄Bx-Daf-22表达丰度进行分析。结果表明:Bx-Daf-22由4个α螺旋,1个310螺旋位于碳(C)端,在1号和2号α螺旋中间包围着5个β折叠。Bx-Daf-22具有固醇载体蛋白SCP2构域,可将起始信号传递到c GMP路径的膜结合受体上,表明该蛋白可以完成cGMP路径中受体蛋白的功能。以二龄幼虫作为标准,抗逆态幼虫DL3的Bx-Daf-22表达量显著上调,雄成虫Bx-Daf-22表达量下调显著。Bx-Daf-22蛋白质结构及不同虫龄Bx-Daf-22表达量表明,该基因参与与cGMP路径且与DL3形成直接相关。
To explore the molecular mechanism of Bursaphelenchus xylophilus dauer larvae 3 formation, thecGMP pathway gene Bx-Daf-22 was identified. Based on the B. xylophilus genome, a SCP2 homologous genewas identified by BLAST, and named as Bx-Daf-22. Bx-Daf-22 was cloned by the method of PCR. And somebioinformatics analysis, such as phylogenetic and conserved domain analysis were conducted based on theamino acid sequence of Bx-Daf-22. Then, the orthologous alignment and 3 D structure of the Bx-Daf-22 wereconstructed. The expression levels of Bx-Daf-22 in different larvae and adults were tasted by Q-PCR. Theresults indicated that: the Bx-Daf-22 consisted of 4 α helices, 1 310 helix at the C-terminal, and 5 β-pleatedsheets among the middle of the α helix. Compared with the expression level in L2, the expression of DL3 weresignificantly up-regulated, the expression of Bx-Daf-22 in male adult was significantly suppressed. Thestructure of Bx-Daf-22 and the expression of Bx-Daf-22 indicate that the gene is involved in cGMP pathwayand directly related to DL3 formation.
To explore the molecular mechanism of Bursaphelenchus xylophilus dauer larvae 3 formation, thecGMP pathway gene Bx-Daf-22 was identified. Based on the B. xylophilus genome, a SCP2 homologous genewas identified by BLAST, and named as Bx-Daf-22. Bx-Daf-22 was cloned by the method of PCR. And somebioinformatics analysis, such as phylogenetic and conserved domain analysis were conducted based on theamino acid sequence of Bx-Daf-22. Then, the orthologous alignment and 3 D structure of the Bx-Daf-22 wereconstructed. The expression levels of Bx-Daf-22 in different larvae and adults were tasted by Q-PCR. Theresults indicated that: the Bx-Daf-22 consisted of 4 α helices, 1 310 helix at the C-terminal, and 5 β-pleatedsheets among the middle of the α helix. Compared with the expression level in L2, the expression of DL3 weresignificantly up-regulated, the expression of Bx-Daf-22 in male adult was significantly suppressed. Thestructure of Bx-Daf-22 and the expression of Bx-Daf-22 indicate that the gene is involved in cGMP pathwayand directly related to DL3 formation.
引文
[1]田浩楷,张帅,柳小龙,等.扩散型与繁殖型松材线虫数字基因表达谱对比分析[J].生物安全学报,2017,26(2):111-121.
[2]张星耀.我国松材线虫病防控基础研究进展[C].第三届全国生物入侵大会论文集,2010:4-5.
[3] Mamiya Y. Pathology of the pine wilt disease caused by Bursaphelenchus xylophilus[J].Annual Review of Phytopathology,1983,21(21):201-220.
[4] Wang B, Ma L, Wang F, et al. Low temperature extends the lifespan of Bursaphelenchus xylophilus through the cGMP Pathway[J].International Journal of Molecular Sciences,2017,18(11):2320.
[5] Mamiya Y. The life history of the pine wood nematode,Bursaphelenchus lignicolus[J].Japanese Journal of Nematology,1975,5:16-25.
[6]王峰,李丹蕾,马玲,等.松材线虫抗逆态基因Bx-DAF6鉴定及基因沉默[J].北京林业大学学报,2016,38(1):21-27.
[7] Wang D D, Cheng X Y, Wang Y S, et al. Characterization and expression of daf-9, and daf-12, genes in the pinewood nematode,Bursaphelenchus xylophilus[J].Forest Pathology,2013,43(2):144-152.
[8] Goldejn W, Riddle D L. A pheromone influences land development in the nematode Caenorhabditis elegans[J].Science,1982,218(4572):578-580.
[9]杨振德,赵博光,郭建.松材线虫行为学研究进展[J].南京林业大学学报:自然科学版,2003,27(1):87-92.
[10] Goldejn W, Riddle D L. The Caenorhabditis elegans dauer larva:developmental effects of food, pheromone and temperature[J].Dev.Biol.,1984,102:368-378.
[11] Taub J, Lau J F, Ma C, et al. A cytosolic catalase is needed to extend adult lifespan in C. elegans daf-C and clk-1 mutants[J].Nature,2003,421(6924):764.
[12]韩京华.秀丽隐杆线虫对苯甲醛嗅觉适应信号途径中Gα的鉴定及功能的研究[D].昆明:云南大学,2016:4-6.
[13] Thomajs H, Birnby D A, Vow J J. Evidence for parallel processing of sensory information controlling dauer formation in C. eleguns[J].Genetics,1993,134:1105-1117.
[14] Inoue T, Thomas J H. Targets of TGF-βsignaling in Caenorhabditis elegans dauer formation[J].Developmental Biology,2000,217(1):192-204.
[15] Kikuchi T, Cotton J A, Dalzell J, et al. Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus[J].PLoS Pathogens,2011,7(9):e1002219.
[16] Garsin D, Villanueva J, Begun J, et al. Long-lived C. elegans daf-2mutants are resistant to bacterial pathogens[J].Science,2003,300(5627):1921.
[17] Feng Wang, Danlei Li, Qiaoli Chen, et al. Genome-wide survey and characterization of the small heat shock protein gene family in Bursaphelenchus xylophilus[J].Gene,2016,579(2):153-161.
[18] Feng Wang, Zhiying Wang, Danlei Li, et al. Identification and characterization of a Bursaphelenchus xylophilus Aphelenchida:(Aphelenchoididae)thermotolerance-related gene:Bx-HSP90[J].Int J Mol Sci,2012,13(7):8819-33.
[19]王峰,马玲,陈俏丽,等.松材线虫热激转录因子Bx-HSF-1基因的克隆及表达分析[J].林业科学,2016,52(12):92-98.
[20]刘立宏,王峰,王步勇,等.松材线虫Bx-sHSP16A基因克隆及蛋白质结构研究[J].中国农学通报,2013,29(22):40-45.
[21] Ishibashi N, Kondo E. Occurrence and survival of the dispersal forms of pine wood nematode, Bursaphelenchus lignicolus MAMIYA and KIYOHARA:ecological significance of dormancy in plant parasitic nematodes[J].Applied Entomology and Zoology,1977,12(4):293-302.
[22]刘立宏,王峰,马玲,等.松材线虫乙酰胆碱酯酶1基因沉默及蛋白抑制剂[J].林业科学,2015,51(1):66-73.
[23]零雅茗,贾岩岩,李丹蕾,等.落叶松-杨栅锈菌效应因子Mlp-AvrM基因克隆[J].森林工程,2017,33(2):41-44.
[24] Golden J W, Riddle D L. A gene affecting production of the Caenorhabditis elegans dauer-inducing pheromone[J].Mol. Gen.Genet,1985(3):534-6.
[25] White J Q, Nicholas T J, Gritton J, et al. The sensory circuitry for sexual attraction in C. elegans males[J].Curr. Biol,2007(21):1847-1857.
[26] Guo X, Zhang H, Zheng X, et al. Structural and functional characterization of a novel gene, Hc-daf-22, from the strongylid nematode Haemonchus contortus[J].Parasites&Vectors,2016,9(1):422-422.
[2]张星耀.我国松材线虫病防控基础研究进展[C].第三届全国生物入侵大会论文集,2010:4-5.
[3] Mamiya Y. Pathology of the pine wilt disease caused by Bursaphelenchus xylophilus[J].Annual Review of Phytopathology,1983,21(21):201-220.
[4] Wang B, Ma L, Wang F, et al. Low temperature extends the lifespan of Bursaphelenchus xylophilus through the cGMP Pathway[J].International Journal of Molecular Sciences,2017,18(11):2320.
[5] Mamiya Y. The life history of the pine wood nematode,Bursaphelenchus lignicolus[J].Japanese Journal of Nematology,1975,5:16-25.
[6]王峰,李丹蕾,马玲,等.松材线虫抗逆态基因Bx-DAF6鉴定及基因沉默[J].北京林业大学学报,2016,38(1):21-27.
[7] Wang D D, Cheng X Y, Wang Y S, et al. Characterization and expression of daf-9, and daf-12, genes in the pinewood nematode,Bursaphelenchus xylophilus[J].Forest Pathology,2013,43(2):144-152.
[8] Goldejn W, Riddle D L. A pheromone influences land development in the nematode Caenorhabditis elegans[J].Science,1982,218(4572):578-580.
[9]杨振德,赵博光,郭建.松材线虫行为学研究进展[J].南京林业大学学报:自然科学版,2003,27(1):87-92.
[10] Goldejn W, Riddle D L. The Caenorhabditis elegans dauer larva:developmental effects of food, pheromone and temperature[J].Dev.Biol.,1984,102:368-378.
[11] Taub J, Lau J F, Ma C, et al. A cytosolic catalase is needed to extend adult lifespan in C. elegans daf-C and clk-1 mutants[J].Nature,2003,421(6924):764.
[12]韩京华.秀丽隐杆线虫对苯甲醛嗅觉适应信号途径中Gα的鉴定及功能的研究[D].昆明:云南大学,2016:4-6.
[13] Thomajs H, Birnby D A, Vow J J. Evidence for parallel processing of sensory information controlling dauer formation in C. eleguns[J].Genetics,1993,134:1105-1117.
[14] Inoue T, Thomas J H. Targets of TGF-βsignaling in Caenorhabditis elegans dauer formation[J].Developmental Biology,2000,217(1):192-204.
[15] Kikuchi T, Cotton J A, Dalzell J, et al. Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus[J].PLoS Pathogens,2011,7(9):e1002219.
[16] Garsin D, Villanueva J, Begun J, et al. Long-lived C. elegans daf-2mutants are resistant to bacterial pathogens[J].Science,2003,300(5627):1921.
[17] Feng Wang, Danlei Li, Qiaoli Chen, et al. Genome-wide survey and characterization of the small heat shock protein gene family in Bursaphelenchus xylophilus[J].Gene,2016,579(2):153-161.
[18] Feng Wang, Zhiying Wang, Danlei Li, et al. Identification and characterization of a Bursaphelenchus xylophilus Aphelenchida:(Aphelenchoididae)thermotolerance-related gene:Bx-HSP90[J].Int J Mol Sci,2012,13(7):8819-33.
[19]王峰,马玲,陈俏丽,等.松材线虫热激转录因子Bx-HSF-1基因的克隆及表达分析[J].林业科学,2016,52(12):92-98.
[20]刘立宏,王峰,王步勇,等.松材线虫Bx-sHSP16A基因克隆及蛋白质结构研究[J].中国农学通报,2013,29(22):40-45.
[21] Ishibashi N, Kondo E. Occurrence and survival of the dispersal forms of pine wood nematode, Bursaphelenchus lignicolus MAMIYA and KIYOHARA:ecological significance of dormancy in plant parasitic nematodes[J].Applied Entomology and Zoology,1977,12(4):293-302.
[22]刘立宏,王峰,马玲,等.松材线虫乙酰胆碱酯酶1基因沉默及蛋白抑制剂[J].林业科学,2015,51(1):66-73.
[23]零雅茗,贾岩岩,李丹蕾,等.落叶松-杨栅锈菌效应因子Mlp-AvrM基因克隆[J].森林工程,2017,33(2):41-44.
[24] Golden J W, Riddle D L. A gene affecting production of the Caenorhabditis elegans dauer-inducing pheromone[J].Mol. Gen.Genet,1985(3):534-6.
[25] White J Q, Nicholas T J, Gritton J, et al. The sensory circuitry for sexual attraction in C. elegans males[J].Curr. Biol,2007(21):1847-1857.
[26] Guo X, Zhang H, Zheng X, et al. Structural and functional characterization of a novel gene, Hc-daf-22, from the strongylid nematode Haemonchus contortus[J].Parasites&Vectors,2016,9(1):422-422.