基于PCR技术的香栓菌和毛栓菌快速检测
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摘要
木材腐朽在多数的情况下是由对木材具有腐蚀能力的相关真菌的生长繁殖所造成的。为了鉴定造成木材腐蚀的菌种,本文首先从腐朽的木材上分离获得木材腐朽菌,对其进行培养并进行形态学的初步鉴定,再通过分子生物学领域PCR法对其进行有效性鉴定。分别采用蜗牛酶法、CTAB法、SDS法和蛋白酶K对木材腐朽真菌的全基因进行提取,且对提取的DNA进行紫外吸收光谱和琼脂糖凝胶电泳(AGE)检测,然后进行PCR扩增并测序,构建系统发育树,最后对其进行综合的比对分析得知:木材腐朽真菌1为香栓菌,2为毛栓菌。
In most cases,wood decay is caused by the growth and reproduction of related fungi that have the ability to corrode the wood itself. To identify the species that caused the wood corrosion,in this paper,wood decay fungi were firstly isolated from decaying wood,cultured and morphologically identified,and then identified by PCR in the field of molecular biology. The whole genes of wood decay fungi were extracted by using snail enzyme method,CTAB method,SDS method,and proteinase K respectively,and the extracted DNA was subjected to ultraviolet absorption spectrum and agarose gel electrophoresis( AGE) detection. Then PCR amplification along with sequencing was processed and a phylogenetic tree was constructed. At last,a comprehensive comparative analysis was made,and the results were: the decaying wood fungi 1 was Trametes suavelones and decaying wood fungi 2 was Trametes hirstuta.
In most cases,wood decay is caused by the growth and reproduction of related fungi that have the ability to corrode the wood itself. To identify the species that caused the wood corrosion,in this paper,wood decay fungi were firstly isolated from decaying wood,cultured and morphologically identified,and then identified by PCR in the field of molecular biology. The whole genes of wood decay fungi were extracted by using snail enzyme method,CTAB method,SDS method,and proteinase K respectively,and the extracted DNA was subjected to ultraviolet absorption spectrum and agarose gel electrophoresis( AGE) detection. Then PCR amplification along with sequencing was processed and a phylogenetic tree was constructed. At last,a comprehensive comparative analysis was made,and the results were: the decaying wood fungi 1 was Trametes suavelones and decaying wood fungi 2 was Trametes hirstuta.
引文
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[14]张晋彦.春兰‘宋梅’和杂交兰组培苗的菌根化研究[D].杭州:浙江农林大学,2016.ZHANG J Y. Study on mycorrhizalization of spring orchid’Songmei’and hybrid orchid culture seedlings[D]. Hangzhou:Zhejiang A&F University,2016.
[15] Jaszek M,Grzywnowicz K,Malarczyk E,et al. Enhanced extracellular laccase activity as a part of the response system of white fot fungi:Trametes versicolor and abortiporus beinnis to paraquatcaused oxidative stress conditions[J]. Pesticide Biochemistry and Physiology,2006,85(3):147-154.
[16]冯璐,戚大伟.基于PCR技术的木材腐朽菌鉴定方法的研究[J].森林工程,2016,32(5):35-39.FENG L,QI D W. Research on the identification method of wood decay bacteria based on PCR[J]. Forest Engineering,2016,32(5):35-39.
[17]孙婧,戚大伟,池玉杰,等.两种木腐真菌的分离及鉴定[J].森林工程,2017,33(5):45-49.SUN J,QI D W,CHI Y J,et al. Isolation and identification of two kinds of wood rot fungi[J]. Forest Engineering,2017,33(5):45-49.
[2]周贤武,高玉磊,苏明垒,等.基因工程改良木材性质研究进展[J].林业科学,2018,54(3):152-160.ZHOU X W,GAO Y L,SU M L,et al. Research progress on genetic engineering improvement of wood properties[J]. Forestry Science,2018,54(3):152-160.
[3] MAHMOUD M G,RIFAAT H M,EI SAYED O H,et al. Effect of inducers and process parameters on laccase production by locally isolated marine streptomyces lydicus from red sea,Egypt[J]. International Journal of Chem Tech Research,2013,5(1):15-23.
[4]池玉杰.东北林区64种木材腐朽菌木材分解能力的研究[J].林业科学,2001,37(5):107-112.CHI Y J. Study on wood decomposition ability of 64 species of wood decay bacteria in northeast forest area[J]. Forestry Science,2001,37(5):107-112.
[5] MORIN N,VALLAEYS T,HENDRICKX L,et al. An efficient DNA isolation protocol for filamentous cyanobacteria of the genus Arthrospira[J]. Journal of Microbiological Methods,2010,80(2):148-154.
[6]池玉杰.木材腐朽菌培养特性的研究综述[J].菌物学报,2004,23(1):158-164.CHI Y J. Study on culture characteristics of wood decay bacteria[J]. Journal of Bacteria,2004,23(1):158-164.
[7]刘素玲,冉玉平,曾蔚,等.一种适用于PCR反应的酵母菌、无绿藻及丝状真菌DNA提取方法[J].中国真菌学杂志,2006,1(6):340-342.LIU S L,RAN Y P,ZENG W,et al. A DNA extraction method for yeast,green algae and filamentous fungi suitable for PCR reaction[J]. Chinese Mycology Journal,2006,1(6):340-342.
[8]李晓琴,徐煜.软腐—一种特殊的木材腐朽[J].林业科技,2003,28(3):46-46.LI X Q,XU Y. Soft rot-a special kind of wood decay[J]. Forestry Technology,2003,28(3):46-46.
[9]张文超.聚合酶链反应(PCR)技术与基因扩增分析仪器(PCR仪)[J].生命科学仪器,2005,3(3):13-19.ZHANG W C. Polymerase chain reaction(PCR)and gene amplification analysis instrument(PCR)[J]. Life Science Instruments,2005,3(3):13-19.
[10] LEE S Y,KIM M,KIM S H,et al. Transcriptomic analysis of the white rot fungus Polyporus brumalis provides insight into sesquiterpene biosynthesis[J]. Microbiological Research,2016,182(8):141-149.
[11]张伟,高国平,祁金玉,等. 3种林木病原腐朽菌生长特性研究[J].西北林学院学报,2010,25(3):122-125.ZHANG W,GAO G P,QI J Y,et al. Study on growth characteristics of 3 forest pathogenic decay bacteria[J]. Journal of Northwest Forestry College,2010,25(3):122-125.
[12]吴发红,黄东益,黄小龙,等.几种真菌DNA提取方法的比较[J].中国农学通报,2009,25(8):62-64.WU F H,HUANG D Y,HUANG X L,et al. Comparison of several methods for extracting fungal DNA[J]. Chinese Agricultural Bulletin,2009,25(8):62-64.
[13]戴玉成.中国木本植物病原木材腐朽菌研究[J].菌物学报,2012,31(4):493-509.DAI Y C. Study on rot bacteria of Chinese woody plant pathogen[J]. Journal of Bacteria,2012,31(4):493-509.
[14]张晋彦.春兰‘宋梅’和杂交兰组培苗的菌根化研究[D].杭州:浙江农林大学,2016.ZHANG J Y. Study on mycorrhizalization of spring orchid’Songmei’and hybrid orchid culture seedlings[D]. Hangzhou:Zhejiang A&F University,2016.
[15] Jaszek M,Grzywnowicz K,Malarczyk E,et al. Enhanced extracellular laccase activity as a part of the response system of white fot fungi:Trametes versicolor and abortiporus beinnis to paraquatcaused oxidative stress conditions[J]. Pesticide Biochemistry and Physiology,2006,85(3):147-154.
[16]冯璐,戚大伟.基于PCR技术的木材腐朽菌鉴定方法的研究[J].森林工程,2016,32(5):35-39.FENG L,QI D W. Research on the identification method of wood decay bacteria based on PCR[J]. Forest Engineering,2016,32(5):35-39.
[17]孙婧,戚大伟,池玉杰,等.两种木腐真菌的分离及鉴定[J].森林工程,2017,33(5):45-49.SUN J,QI D W,CHI Y J,et al. Isolation and identification of two kinds of wood rot fungi[J]. Forest Engineering,2017,33(5):45-49.
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