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时间:2020-08-11
《中性内切-β-葡聚糖酶基因在里氏木霉中的重组与表达.pdf》由会员上传分享,免费在线阅读,更多相关内容在行业资料-天天文库。
1、第27卷第1期高校化学工程学报No.1、b1.272013年2月JournalofChemicalEngineeringofChineseUniversitiesFeb2Ol3文章编号:1003—9015(2013)01—0108-05网络出版地址:http://wwwcnki.net/kcms/detail/33.1141.TQ.20121229.I718.004-htmI中性内切一p-葡聚糖酶基因在里氏木霉中的重组与表达顾斌涛,江守坤,夏黎明(浙江大学化学工程与生物工程学系,浙江杭州31002
2、7)摘要:中性内切一p一葡聚糖酶在棉织物生物整理方面具有重要的应用价值,研究首先从特异腐质霉(Humicolains0le菌株中克隆到一个中性内切-p一葡聚糖酶基因,将该基因置于里氏木霉(TrichodermareeseO纤维二糖水解酶I强启动子Pcbhl(及其信号肽)和终止子Tcbhl之间,并以pCAMBIA1300为载体骨架构建重组质粒pCB-PHT。采用根瘤农杆菌介导转化技术将重组质粒pCB-PHT导入里氏木霉的分生孢子中,进一步筛选得到八个重组里氏木霉转化子。摇瓶发酵培养72h时,发酵液的
3、中性内切.p·葡聚糖酶活力最高可达到98.8IU.mL左右,是出发菌株的5.1倍。研究成功地实现了外源中性内切.p.葡聚糖酶在丝状真菌里氏木霉中的重组与胞外表达,有关研究结果将在牛仔布水洗工业中发挥出重要作用。关键词:中性内切.B-葡聚糖酶;里氏木霉;基因重组;表达;牛仔布;生物整理中图分类号:Q556.9文献标识码:ADOh10.3969/j.issn.1003.9015.2013.01.0l8RecombinationandExpressionofNeutralEndo——GlucanaseG
4、enefromHumico~InsolensinTrichodermaReeseiGUBin-tao,JIANGShou-kun,XIALi·ming(DepartmentofChemicalEngineeringandBioengineering,ZhejiangUniversity,Hangzhou310027,China)Abstract:Neutralendo-p-glucanaseiswidelyusedforthebiofinishingapplicationinthetextilei
5、ndustry.Inordertoimprovetheneutralendo-p—glucanaseactivityofTrichodermreesei,theendo—p-glucanasegenefromHumicolainsolenswasclonedbvmereversetranscriptionPCRmethod,andwasinsertedbetweenreeseistrongpromoterPcbhl(inc1udingsecretingsignalpeptidesequence)a
6、ndterminator,furtherligatedtopCAMBIA1300vectortoconstructplasmidpCB-PHT.TheplasmidpCB-PHTWastransformedintoreeseiZU.02viaanoptimizedAgrobacteriumtumefaciensmediatedtransformation。andtheextracellularendo一6一glucanaseWasexpressedunderthecontrolofreeseist
7、rongpromoterPcbh1.Eightreeseitransformantswithsuccessfulintegrationoftheendo--glucanasegeneexpressioncassettewereobtained.After72hshaking。flaskfermentation,theendo.D—glucanaseactivityofthereeseitransformantinthefermentationsolutionreaches98.8IU.mL_。.w
8、hichiS5.1.foldashighasthatofthehoststrain.Thisresultshowsthattheendo.13.glucanasegeneiSsuccessfullytransformedandexpressedinreesei.TheTreeseitransformantsareparticularlysuitableforusesindenimwashingindustry.Keywords:neutralendo一13-glucanase;Tr
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