华北农学报
華北農學報
화북농학보
ACTA AGRICULTURAE BOREALI-SINICA
2009年
5期
73-76
,共4页
白爱枝%闫祖威%唐国敏%梁运章
白愛枝%閆祖威%唐國敏%樑運章
백애지%염조위%당국민%량운장
黑曲霉%木聚糖酶%基因克隆%序列分析
黑麯黴%木聚糖酶%基因剋隆%序列分析
흑곡매%목취당매%기인극륭%서렬분석
Aspergillus niger%Xylanase%Gene cloning%Sequence analysis
以黑曲霉(Aspergillus niger)A3的基因组DNA为模板,根据已报道的xynB基因序列设计简并引物,在合适的PCR反应条件下有效地扩增出了xynB的结构基因及其5'调控区序列片段,并克隆到pBS-T载体上,序列测定表明,该目的片段全长1 611 bp.通过序列分析:结构基因部分长744 bp,其中含有一个66 bp的内含子和18个氨基酸的信号肽序列;xynB基因的cDNA序列大小为678 bp,编码225个氨基酸,与GenBank上检索的木聚糖酶基因的核苷酸序列同源性最高达98%,氨基酸序列同源性达99%.在翻译起始点上游92 bp和118 bp处找到转录起始位点和类"TATA"box的核心启动子区特征元件以及"CAAT"box,表明所克隆的调控区序列具有真核生物启动子特点,这为构建木聚糖酶高效表达载体,用于工业育种奠定了基础.
以黑麯黴(Aspergillus niger)A3的基因組DNA為模闆,根據已報道的xynB基因序列設計簡併引物,在閤適的PCR反應條件下有效地擴增齣瞭xynB的結構基因及其5'調控區序列片段,併剋隆到pBS-T載體上,序列測定錶明,該目的片段全長1 611 bp.通過序列分析:結構基因部分長744 bp,其中含有一箇66 bp的內含子和18箇氨基痠的信號肽序列;xynB基因的cDNA序列大小為678 bp,編碼225箇氨基痠,與GenBank上檢索的木聚糖酶基因的覈苷痠序列同源性最高達98%,氨基痠序列同源性達99%.在翻譯起始點上遊92 bp和118 bp處找到轉錄起始位點和類"TATA"box的覈心啟動子區特徵元件以及"CAAT"box,錶明所剋隆的調控區序列具有真覈生物啟動子特點,這為構建木聚糖酶高效錶達載體,用于工業育種奠定瞭基礎.
이흑곡매(Aspergillus niger)A3적기인조DNA위모판,근거이보도적xynB기인서렬설계간병인물,재합괄적PCR반응조건하유효지확증출료xynB적결구기인급기5'조공구서렬편단,병극륭도pBS-T재체상,서렬측정표명,해목적편단전장1 611 bp.통과서렬분석:결구기인부분장744 bp,기중함유일개66 bp적내함자화18개안기산적신호태서렬;xynB기인적cDNA서렬대소위678 bp,편마225개안기산,여GenBank상검색적목취당매기인적핵감산서렬동원성최고체98%,안기산서렬동원성체99%.재번역기시점상유92 bp화118 bp처조도전록기시위점화류"TATA"box적핵심계동자구특정원건이급"CAAT"box,표명소극륭적조공구서렬구유진핵생물계동자특점,저위구건목취당매고효표체재체,용우공업육충전정료기출.
In this paper,the cloning of a β-xylanase structural gene and its 5'flanking regions gene from Aspergillus niger was studied. On the base of nucleic acid DNA sequence of β-xylanase gene, a pair of degenerate primers was de-signed . Using the genomic DNA of Aspergillus niger as template, a 1.6 kb fragment was amplified by PCR and cloned into the vector pBS-T. Sequence analysis showed that the structural gene of β-xylanase is 744 bp length including a 66 bp in-tron and a 678 bp encoding region, which encoded a polypeptide of 225 amino acids and included the signal sequence of 18 amino acids.The homology analysis of the xynB cDNA sequence shares 98% in base sequence with the β-xylanase gene in GeneBank and the amino acid sequence shares 99 % . Upstream of the tranlation start site, there were classic core element of Eukaryotic Gene Promoter,such as transcription start site,TATA box and CAAT box elements.The work was beneficial in construction of homologus expression for xylanase efficient expression vector.