桑椹缩小型菌核病菌丝裂原活化蛋白激酶基因Mmk1的克隆与分析
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摘要
根据核盘菌菌核形成相关丝裂原活化蛋白激酶Smk1的氨基酸序列设计出简并引物进行RT-PCR,克隆获得长度为856 bp的桑椹缩小型菌核病菌丝裂原活化蛋白激酶基因Mmk1的cDNA片段。将测序结果与GenBank数据库进行Blastx比对后,发现克隆片段所编码的氨基酸序列与其他真菌的MAPK氨基酸序列具有较高的同源性。实时荧光定量PCR分析表明,该基因的表达量随着菌株的生长而减少。Mmk1基因的分子克隆为进一步研究其在菌核形成中的作用奠定了基础。
The degenerate primers were designed according to the mitogen-activated protein kinases Smk1 from Sclerotinia sclerotiorum. Using the degenerate primers to carry out RT-PCR, length of 856 bp cDNA fragments of mitogen-activated protein kinases Mmk1 of S. shiraiana were cloned. Sequencing results were queried for similarity against the GenBank using Blastx, and found that the amino acids sequences encoded by cloned fragments had high homology with MAPK amino acid sequences of other fungi. Real time quantitative PCR analysis revealed that expression of the gene decreased with the growth of the strain. Molecular characterization of Mmk1 will be useful for the further functional determination of the gene involving in the development of sclerotium.
The degenerate primers were designed according to the mitogen-activated protein kinases Smk1 from Sclerotinia sclerotiorum. Using the degenerate primers to carry out RT-PCR, length of 856 bp cDNA fragments of mitogen-activated protein kinases Mmk1 of S. shiraiana were cloned. Sequencing results were queried for similarity against the GenBank using Blastx, and found that the amino acids sequences encoded by cloned fragments had high homology with MAPK amino acid sequences of other fungi. Real time quantitative PCR analysis revealed that expression of the gene decreased with the growth of the strain. Molecular characterization of Mmk1 will be useful for the further functional determination of the gene involving in the development of sclerotium.
引文
[1]操红缨.桑椹研究进展[J].时珍国医国药,1999,10(8):626-628.
[2]KIM S B,CHANG B Y,JO Y H,et al.Macrophage activating activity of pyrrole alkaloids from Morus alba fruits[J].Journal of Ethnopharmacology,2013,145(1):393-396.
[3]李玉峰.湖州市桑果产业的现状与发展对策研究[D].杭州:浙江大学,2012.
[4]邹宇晓,廖森泰,肖更生,等.蚕桑资源多元化开发利用新技术研究进展[J].蚕业科学,2016(4):561-569.
[5]蒯元璋,吴福安.桑椹菌核病病原及病害防治技术综述[J].蚕业科学,2012,38(6):1099-1104.
[6]HONG S K,WAN G K,SUNG G B,et al.Identification and distribution of two fungal species causing sclerotial disease on mulberry fruits in korea[J].Mycobiology,2007,35(2):87-90.
[7]吕蕊花,赵爱春,余建,等.桑椹肥大性菌核病病原菌生物学特性及流行性[J].微生物学报,2017,57(3):388-398.
[8]CHEN C B,HAREL A R,YARDEN O,et al.MAPK regulation of sclerotial development in Sclerotinia sclerotiorum is linked with pH and cAMP sensing[J].Mol Plant Microbe Interact,2004,17(4):404.
[9]刘蒙蒙,宋超,邢咏梅,等.猪苓菌丝形成菌核的MAPK基因克隆及表达分析[J].微生物学通报,2015,42(12):2345-2350.
[10]GOUGH N R.Focus issue:Recruiting players for a game of ERK[J].Science Signaling,2011,4(196):eg9.
[11]苏正川.桑葚缩小型菌核病病原学及其分子检测体系[D].重庆:西南大学,2015.
[12]郑章云,杨义,任杰群,等.2种木霉菌制剂对桑椹菌核病的田间防治试验[J].蚕业科学,2016(1):168-170.
[13]SULTANA R,KIM K.Bacillus thuringiensis C25 suppresses popcorn disease caused by Ciboria shiraiana in mulberry(Morus australis L.)[J].Biocontrol Science and Technology,2016,26(2):145-162.
[14]THOMPSON J D,GIBSON T J,PLEWNIAK F,et al.The CLUSTAL_X windows interface:Flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Research,1997,25(25):4876-4882.
[15]夏瑞,陆旺金,李建国,等.简并引物的程序化设计与荔枝HMGR基因片段的克隆[J].果树学报,2006,23(6):903-906.
[16]刘荣.核盘菌两个新型RNA病毒基因组的分子生物学特性研究[D].武汉:华中农业大学,2015.
[17]李运合,孙光明.利用CODEHOP和iCODEHOP设计简并引物克隆芒果LAX基因家族片段[J].热带作物学报,2011,32(12):2278-2282.
[18]魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979.
[19]王爱印.桑椹菌核病病原菌的分离、鉴定及其拮抗性桑树内生菌的研究[D].重庆:西南大学,2016.
[2]KIM S B,CHANG B Y,JO Y H,et al.Macrophage activating activity of pyrrole alkaloids from Morus alba fruits[J].Journal of Ethnopharmacology,2013,145(1):393-396.
[3]李玉峰.湖州市桑果产业的现状与发展对策研究[D].杭州:浙江大学,2012.
[4]邹宇晓,廖森泰,肖更生,等.蚕桑资源多元化开发利用新技术研究进展[J].蚕业科学,2016(4):561-569.
[5]蒯元璋,吴福安.桑椹菌核病病原及病害防治技术综述[J].蚕业科学,2012,38(6):1099-1104.
[6]HONG S K,WAN G K,SUNG G B,et al.Identification and distribution of two fungal species causing sclerotial disease on mulberry fruits in korea[J].Mycobiology,2007,35(2):87-90.
[7]吕蕊花,赵爱春,余建,等.桑椹肥大性菌核病病原菌生物学特性及流行性[J].微生物学报,2017,57(3):388-398.
[8]CHEN C B,HAREL A R,YARDEN O,et al.MAPK regulation of sclerotial development in Sclerotinia sclerotiorum is linked with pH and cAMP sensing[J].Mol Plant Microbe Interact,2004,17(4):404.
[9]刘蒙蒙,宋超,邢咏梅,等.猪苓菌丝形成菌核的MAPK基因克隆及表达分析[J].微生物学通报,2015,42(12):2345-2350.
[10]GOUGH N R.Focus issue:Recruiting players for a game of ERK[J].Science Signaling,2011,4(196):eg9.
[11]苏正川.桑葚缩小型菌核病病原学及其分子检测体系[D].重庆:西南大学,2015.
[12]郑章云,杨义,任杰群,等.2种木霉菌制剂对桑椹菌核病的田间防治试验[J].蚕业科学,2016(1):168-170.
[13]SULTANA R,KIM K.Bacillus thuringiensis C25 suppresses popcorn disease caused by Ciboria shiraiana in mulberry(Morus australis L.)[J].Biocontrol Science and Technology,2016,26(2):145-162.
[14]THOMPSON J D,GIBSON T J,PLEWNIAK F,et al.The CLUSTAL_X windows interface:Flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Research,1997,25(25):4876-4882.
[15]夏瑞,陆旺金,李建国,等.简并引物的程序化设计与荔枝HMGR基因片段的克隆[J].果树学报,2006,23(6):903-906.
[16]刘荣.核盘菌两个新型RNA病毒基因组的分子生物学特性研究[D].武汉:华中农业大学,2015.
[17]李运合,孙光明.利用CODEHOP和iCODEHOP设计简并引物克隆芒果LAX基因家族片段[J].热带作物学报,2011,32(12):2278-2282.
[18]魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979.
[19]王爱印.桑椹菌核病病原菌的分离、鉴定及其拮抗性桑树内生菌的研究[D].重庆:西南大学,2016.