金針菇漆酶基因的克隆及其在畢赤酵母中的表達(dá)研究

Cloning of A Laccase Gene from Flammulina velutipes and Study on Its Expression in Pichia pastoris

張銀波　姜瓊　江木蘭　馬立新　

摘　要：綜合運(yùn)用cDNA末端快速擴(kuò)增(Rapid Amplification of cDNA Ends,RACE )和基因組步行等技術(shù)克隆到一個(gè)金針菇(Flammulina velutipes)的漆酶結(jié)構(gòu)基因和其對(duì)應(yīng)的全長(zhǎng)cDNA,經(jīng)測(cè)序和BLAST比對(duì)分析表明該基因?qū)儆诙嚆~氧化酶基因家族,與已發(fā)表的漆酶基因(AF176230) 的同源性最高,在氨基酸水平為72%.該結(jié)構(gòu)基因命名為glccFv,cDNA命名為lccFv,其序列提交GenBank,登錄號(hào)分別為AY485826和AY450406.將lccFv的開(kāi)放閱讀框克隆到畢赤酵母表達(dá)載體pHBM906,轉(zhuǎn)化畢赤酵母GS115且實(shí)現(xiàn)了分泌表達(dá).將重組畢赤酵母GS115 (pHBM565)誘導(dǎo)產(chǎn)酶,在培養(yǎng)溫度20℃、甲醇流加量為1.0%(V/V)的情況下,其分泌表達(dá)的LCCFv的最高酶活為0.1070 U/mL,最適反應(yīng)溫度為45℃,最適反應(yīng)pH值為3.9,在最適反應(yīng)條件下其熱穩(wěn)定性和pH值穩(wěn)定性均較好.

關(guān)鍵詞：cDNA末端快速擴(kuò)增, 基因組步行, 漆酶基因, 畢赤酵母, 分泌表達(dá)

分類號(hào)：Q939 　文獻(xiàn)標(biāo)識(shí)碼：A

文章編號(hào)：0001-6209(2004)06-0775-05

基金項(xiàng)目：國(guó)家"863計(jì)劃"(2001AA214161;2002AA227011)

作者簡(jiǎn)介：張銀波(1974-),男,湖北應(yīng)城人,助理研究員,碩士研究生,從事微生物基因工程方面的研究.E-mail:ybzhang04@yahoo.com.馬立新,通訊作者. Tel/Fax: 86-27-88666349;E-mail: malixin9@hotmail.com

作者單位：張銀波（湖北大學(xué),分子微生物學(xué)與基因工程研究室,武漢,430062;中國(guó)農(nóng)業(yè)科學(xué)院油料作物研究所,農(nóng)業(yè)部油料作物遺傳改良重點(diǎn)開(kāi)放實(shí)驗(yàn)室,武漢,430062）　

姜瓊（湖北大學(xué),分子微生物學(xué)與基因工程研究室,武漢,430062）　

江木蘭（中國(guó)農(nóng)業(yè)科學(xué)院油料作物研究所,農(nóng)業(yè)部油料作物遺傳改良重點(diǎn)開(kāi)放實(shí)驗(yàn)室,武漢,430062）　

馬立新（湖北大學(xué),分子微生物學(xué)與基因工程研究室,武漢,430062）　

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收稿日期：2004年4月23日

修稿日期：2004年6月4日

出版日期：2004年12月1日

微生物學(xué)報(bào)

ACTA MICROBIOLOGICA SINICA

2004 Vol.44 No.6 P.775-779