中国生物防治学报
中國生物防治學報
중국생물방치학보
CHINESE JOURNAL OF BIOLOGICAL CONTROL
2013年
1期
74-82
,共9页
张晔%孙漫红%李世东%罗明
張曄%孫漫紅%李世東%囉明
장엽%손만홍%리세동%라명
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
鏈孢粘帚黴%內切葡聚糖酶%覈盤菌%基因敲除轉化子%熒光定量PCR%菌寄生
련포점추매%내절포취당매%핵반균%기인고제전화자%형광정량PCR%균기생
Gliocladium catenulatum%endoglucanase%Sclerotinia sclerotiorum%gene-deficient transformant%real-time PCR%mycoparasitism
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。
為研究鏈孢粘帚黴Gliocladium catenulatum HL-1-1寄生覈盤菌菌覈的相關基因,從實驗室前期構建的差異錶達cDNA文庫中隨機挑選504箇暘性剋隆進行測序和比對分析,穫得內切葡聚糖酶基因,併命名為eg8-20。該基因與木黴Trichoderma sp. SSL內切葡聚糖酶基因的同源性高達94%。採用RACE技術穫得瞭eg8-20全長cDNA,該基因長度為1479 bp(GenBank登錄號JQ728996),開放閱讀框(ORF)長1269 bp,編碼423箇氨基痠。對HL-1-1菌株在菌覈粉培養基中內切葡聚糖酶基因錶達水平的定量鑑測結果錶明, eg8-20錶達水平隨菌覈誘導培養時間的延長而提高,96 h後錶達量升高9倍。為進一步驗證其功能,通過同源重組將G418抗性標記基因定點插入到鏈孢粘帚黴HL-1-1中,穫得eg8-20缺失轉化子ED-3。錶型研究髮現,基因敲除轉化子的生長速度、產孢量與野生型無顯著差異。室內生物測定錶明,轉化子能夠抑製覈盤菌菌覈萌髮,但其侵染能力明顯減弱,寄生級彆由野生型菌株的四級降為二級,證明內切葡聚糖酶在鏈孢粘帚黴寄生覈盤菌菌覈過程中髮揮著重要的作用。
위연구련포점추매Gliocladium catenulatum HL-1-1기생핵반균균핵적상관기인,종실험실전기구건적차이표체cDNA문고중수궤도선504개양성극륭진행측서화비대분석,획득내절포취당매기인,병명명위eg8-20。해기인여목매Trichoderma sp. SSL내절포취당매기인적동원성고체94%。채용RACE기술획득료eg8-20전장cDNA,해기인장도위1479 bp(GenBank등록호JQ728996),개방열독광(ORF)장1269 bp,편마423개안기산。대HL-1-1균주재균핵분배양기중내절포취당매기인표체수평적정량감측결과표명, eg8-20표체수평수균핵유도배양시간적연장이제고,96 h후표체량승고9배。위진일보험증기공능,통과동원중조장G418항성표기기인정점삽입도련포점추매HL-1-1중,획득eg8-20결실전화자ED-3。표형연구발현,기인고제전화자적생장속도、산포량여야생형무현저차이。실내생물측정표명,전화자능구억제핵반균균핵맹발,단기침염능력명현감약,기생급별유야생형균주적사급강위이급,증명내절포취당매재련포점추매기생핵반균균핵과정중발휘착중요적작용。
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 cDNAsequence 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.