克隆渐狭蜡蚧菌几丁质酶基因，为进一步明确该菌产生的几丁质酶对定居性根结类和孢囊类线虫卵孵化抑制作用提供理论依据。采用分离自黑龙江大豆孢囊线虫孢囊寄生真菌CGMCC5328，通过形态学特征及ITS序列比较分析，鉴定该菌株为渐狭蜡蚧菌。通过简并引物设计和RACE技术，克隆渐狭蜡蚧菌中几丁质酶基因，利用生物学软件分析该基因序列及其编码的氨基酸序列。首次从渐狭蜡蚧菌中克隆得到一个几丁质酶基因 LACHI1，该基因DNA序列长1 743 bp，含3个内含子，包含1 272 bp开放阅读框，编码423个氨基酸，理论分子量45.9 kDa，等电点5.90。 LACHI1 基因编码的几丁质酶，属于糖基水解酶18家族几丁质酶。同源性比对表明该菌产生的几丁质酶与昆虫寄生真菌和食线虫真菌产生的几丁质酶有很高的同源性。
To clone chitinolytic genes from Lecanicillium attenuatum and provide theoretical basis for the inhibition of the chitinases on egg-hatching of root knot and cyst nematode.In this study,an nematode egg-parasitic fungus CGMCC5328 was isolated from Heterodera glycines infecting soybean in Heilongjiang Province.Based on morphological characters and molecular analysis of ITS-rDNA,the strain was identified as L.attenuatum.The chitinase gene LACHI1 from L.attenuatum was cloned using the degenerate PCR primers and RACE techniques.The analysis of the gene LACHI1 and its amino acid sequence was by biology software.We have cloned a chitinase gene LACHI1 from L.attenuatum for the first time.The gene is 1 743 bp in length and contains three putative introns.The ORF of LACHI1 is 1 272 bp in size with encoding protein of 423 aa,molecular mass of 45.9 kDa and pI of 5.90.The chitinase deduced by LACHI1 belongs to glycosyl hydrolase family 18 chitinase.Comparison of the chitinase amino acid sequence with other chitinases from entomopathogenic fungi and nematophagous fungi revealed that the enzymes were highly similar.
[1] Van Nguyen N,Kim Y J,Oh K T,et al.The role of chitinase from Lecanicillium antillanum B-3 in parasitism to root-knot nematode Meloidogyne incognita eggs [J].Biocontrol Science and Technology,2007,17(9/10):1047-1058.
[2] Kerry B R.Rhizosphere interactions and the exploitation of microbial agents for the biological control of plant-parasitic nematodes [J].Annual Review of Phytopathology,2000,38(1):423-441.
[3] Bartnick-Garcia S.Cell wall chemistry,morphogenesis,and taxonomy of fungi [J].Annual Review of Microbiology,1968,22:87-108.
[4] Merzendorfer H,Zimoch L.Chitin metabolism in insects:structure,function and regulation of chitin synthases and chitinases [J].Journal of Experimental Biology,2003,206(24):4393-4412.
[5] Wharton D A.Nematode egg-shells [J].Parasitology,1980,81(2):447-463.
[6] Warren R A J.Microbial hydrolysis of polysaccharides [J].Annual Review of Microbiology,1996,50:183-212.
[7] Shaikh S A,Deshpande M V.Chitinolytic enzymes:their contribution to basic and applied research [J].World Journal of Microbiology and Biotechnology,1993,9(4):468-475.
[8] Herrera-Estrella A,Chet I.Chitinases in biological control [J].EXS,1999,87(1):171-184.
[9] Henrissat B.A classification of glycosyl hydrolases based on amino acid sequence similarities [J].Biochemical Journal,1991,280(2):309-316.
[10] Henrissat B,Bairoch A.New families in the classification of glycosyl hydrolases based on amino acid sequence similarities [J].The Biochemical Journal,1993,293(Pt3):781-788.
[11] Charnley A K.Fungal pathogens of insects cuticle:degrading enzymes and toxins [J].Advances in Botanical Research,2003,40:241-321.
[12] Tian H L,Riggs R D,Crippen D L.Control of soybean cyst nematode by chitinolytic bacteria with chitin substrate[J]. Journal of Nematology,2000,32(4):370-376.
[13] Tikhonov V E,Lopez-Llorca L V,Salinas J,et al.Purification and characterization of chitinases from the nematophagous fungi Verticillium chlamydosporium and V.suchlasporium [J].Fungal Genetics and Biology,2002,35(1):67-78.
[14] Kobayashi D Y,Reedy R M,Bick J A,et al.Characterization of a chitinase gene from Stenotrophomonas maltophilia strain 34S1 and its involvement in biological control [J].Applied and Environmental Microbiology,2002,68(3):1047-1054.
[15] Moosavi M R,Zare R,Zamanizaden H R,et al.Pathogenicity of Verticillium epiphytum isolates against Meloidogyne javanica [J].International Journal of Pest Management,2011,57(4):291-297.
[16] Khan A,Williams K,Molloy M P,et al.Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels [J].Protein Expression and Purification,2003,32(2):210-220.
[17] 张成敏,武侠,才秀华.厚垣普奇尼亚菌 Pochonia chlamydosporia 产生的几丁质酶对南方根结线虫卵孵化的影响[J].中国农业科学,2009,42(10):3509-3515.
[18] 林森,武侠,曹君正,等.产生几丁质酶的交枝顶孢(Acremonium implicatum)对南方根结线虫生防潜力[J].植物病理学报,2013,43(5):509-517.
[19] Khan A,Williams K L,Nevalainen H M.Effects of Paecilomyces lilacinus protease and chitinase on the eggshell structures and hatching of Meloidogyne javanica juveniles [J].Biological Control,2004,31(3):346-352.
[20] Duo-Chuan L.Review of fungal chitinases [J].Mycopathologia,2006,161(6):345-360.
[21] Zare R,Gams W.A revision of Verticillium section Prostrata.IV.The genera Lecanicillium and Simplicillium.gen.nov[J].Nova Hedwigia,2001,73(1):1-50.
[22] Kim J J,Goettel M S,Gillespie D R.Potential of Lecanicillium species for dual microbial control of aphids and the cucumber powdery mildew fungus, Sphaerotheca fuliginea [J].Biological Control,2007,40(3):327-332.
[23] Zare R,Gams W,Culham A.A revision of Verticillium sect. Prostrata.I.Phylogenetic studies using ITS sequences [J].Nova Hedwigia,2000,71(3-4):465-480.
[24] Kim H Y,Lee H B,Kim Y C,et al.Laboratory and field evaluations of entomopathogenic Lecanicillium attenuatum CNU-23 for control of green peach aphid(Myzus persicae)[J].Journal of Microbiology Biotechnology,2008,18(12):1915-1918.
[25] Kim J J,Kim K C.Selection of a highly virulent isolate of Lecanicillium attenuatum against cotton aphid [J].Journal of Asia-Pacific Entomology,2008,11(1):1-4.
[26] Gan Z W,Yang J K,Tao N,et al.Cloning of the gene Lecanicillium psalliotae chitinase Lpchi1 and identification of its potential role in the biocontrol of root-knot nematode Meloidogyne incognita [J].Applied Microbiology and Biotechnology,2007,76(6):1309 1317.
[27] Lu Z X,Laroche A,Huang H C.Isolation and characterization of chitinases from Verticillium lecanii [J].Canadian Journal of Microbiology,2005,51(12):1045-1055.
[28] Zhu Y P,Pan J R,Qiu J Z,et al.Isolation and characterization of a chitinase gene from entomopathogenic fungus Verticillium lecanii [J].Brazilian Journal of Microbiology,2008,39(2):314-320.
[29] Milner R J.Prospects for biopesticides for aphid control [J].BioControl,1997,42(1-2):227-239.
[30] Faria M R,Wraight S P.Mycoinsecticides and Mycoacaricides:a comprehensive list with worldwide coverage and international classification of formulation types [J].Biological Control,2007,43(3):237-256.
[31] 叶向勇,孙建国,马超,等.渐狭蜡蚧菌产生的几丁质酶对芦笋茎枯病菌的抑制作用[J].生物技术通讯,2012,23(5):698-700.
[32] Dong L Q,Yang J K,Zhang K Q.Cloning and phylogenetic analysis of the chitinase gene from the facultative pathogen Paecilomyces lilacinus [J].Journal of Applied Microbiology,2007,103(6):2476-2488.
