单 位：
新疆农业大学农学院;中国农业科学院植物保护研究所/农业部作物有害生物综合治理重点实验室
关键词：
链孢粘帚霉;内切葡聚糖酶;核盘菌;基因敲除转化子;荧光定量PCR;菌寄生
摘 要：
为研究链孢粘帚霉Gliocladium catenulatum HL-1-1寄生核盘菌菌核的相关基因,从实验室前期构建的差异表达cDNA文库中随机挑选504个阳性克隆进行测序和比对分析,获得内切葡聚糖酶基因,并命名为eg8-20。该基因与木霉Trichoderma sp.SSL内切葡聚糖酶基因的同源性高达94%。采用RACE技术获得了eg8-20全长cDNA,该基因长度为1479 bp(GenBank登录号JQ728996),开放阅读框(ORF)长1269 bp,编码423个氨基酸。对HL-1-1菌株在菌核粉培养基中内切葡聚糖酶基因表达水平的定量监测结果表明,eg8-20表达水平随菌核诱导培养时间的延长而提高,96 h后表达量升高9倍。为进一步验证其功能,通过同源重组将G418抗性标记基因定点插入到链孢粘帚霉HL-1-1中,获得eg8-20缺失转化子ED-3。表型研究发现,基因敲除转化子的生长速度、产孢量与野生型无显著差异。室内生物测定表明,转化子能够抑制核盘菌菌核萌发,但其侵染能力明显减弱,寄生级别由野生型菌株的四级降为二级,证明内切葡聚糖酶在链孢粘帚霉寄生核盘菌菌核过程中发挥着重要的作用。
译 名：
Cloning and Functional Analysis of Endoglucanase Gene from Gliocladium catenulatum HL-1-1
作 者：
ZHANG Ye1,2,SUN Manhong1,LI Shidong1,LUO Ming2(1.Key Laboratory of Integrated Pest Management in Crops,Ministry of Agriculture/Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing,100081;2.Xinjiang Agricultural University,Wulumuqi,8300521.)
关键词：
Gliocladium catenulatum;endoglucanase;Sclerotinia sclerotiorum;gene-deficient transformant; real-time PCR;mycoparasitism
摘 要：
A subtractive cDNA library of Gliocladium catenulatum HL-1-1 was constructed previously to investigate genes associated with mycoparasitism on sclerotia of Sclerotinia sclerotiorum in authors' lab.Authors in this paper selected 504 positive clones randomly.After sequencing,we obtained a fragment of endoglucanase gene named eg8-20.Blastx analysis indicated that its deduced amino acid sequence was of 94% homology to endoglucanase from Trichoderma sp.SSL.Its cDNA sequence was completed by RACE,showing 1479 bp in length(GenBank accession number JQ728996),including 1269 bp of open reading frame which encoded 423 amino acids. Expression of eg8-20 was quantitatively monitored by real-time PCR when G.catenulatum HL-1-1 was cultured in sclerotia powder broth.Results showed that the expression level of eg8-20 increased as the incubation continued. And the expression level was 9 times higher at 96 h than that at 0 h.An eg8-20 gene-deficient mutant,ED-3,was constructed by inserting resistant gene G418 into HL-1-1 genome using homologous recombination technique. Determination of fungal characteristics indicated that the wild and the gene-deleted strains were same in growth rate and sporulation.However,parasitism of the mutant to S.sclerotiorum was greatly decreased to 2nd degree from 4th by wild type.The results demonstrated that endoglucanase secreted by G.catenulatum plays an important role in mycoparasitism to S.sclerotiorum,in addition to other components involved in the parasitic processing.
