【作者】 蓝希钳；

【导师】 周泽扬； 下坂诚；

【作者基本信息】 西南农业大学 ， 微生物学， 2002， 硕士

【摘要】 气单孢菌Aeromonas sp. SUWA-9菌株是从日本国长野县Suwa Lake的湖水中分离到的细菌。该菌株可产生多种活性很强的胞内几丁质酶。利用限制性核酸内切酶Sau 3AI将该菌的基因组DNA部分酶切成各种大小的DNA片段，经蔗糖密度梯度离心后收集2-10Kb的DNA片段，再与经BamHI/BAP处理的载体质粒pUC118连接，转入大肠杆菌中表达，得到了8株阳性重组菌，编号clone-1，2，3，4，5，6，8，9。经底物反应和限制性内切酶图谱分析，确定其中的clone-1，4，5，6，8含有相同的几丁质酶基因；而clone-2，3，5，9四株重组菌则含有不同的几丁质酶基因。SDS-PAGE活性染色显示这些酶所作用的底物有所不同，既有内切型酶，也有外切型酶，分子量从64KD-83KD不等。用不同的限制性核酸内切酶对clone-5重组质粒进行酶切，构建了它的酶切图谱。根据clone-5质粒的酶切图谱，利用多种核酸内切酶进行消化，再用连接酶进行连接，构建了一系列的重组亚克隆质粒。通过测定这些亚克隆质粒的两端核苷酸序列，再设计引物，分别从两个方向测定了clone-5质粒的核苷酸序列，共3，205bp。利用基因分析软件genetyx对clone-5质粒的核苷酸序列进行分析，并搜索日本DNA数据库(DNA Data Bank of Japan，简称DDBJ)，推断clone-5质粒的几丁质酶基因含有一个2，261bp的开放阅读框(ORF)，编码一条887个氨基酸的几丁质酶蛋白；ORF上游有一个启动子。经检索，与DDBJ中登陆的一个来源于Aeromonas的几丁质酶基因有90％的同源性。 另一方面，在生物遗传转化的研究中，理想报告基因的探索一直是重要研究内容之一。自然界的微生物中约99％是目前还不能培养的，这些未能培养的微生物蕴藏着极其巨大的基因资源，是筛选有用基因的重要来源。在研究和开发未能培养的微生物的过程中，从土壤细菌中克隆到一条全长4.7kb的DNA片段，与质粒pUC118连接后转入大肠杆菌JM109菌株中，其编码的产物可以使重组菌体在紫外线照射下呈现红色荧光。将基因称为红色基因(red gene)，含该基因的重组菌称为Red-clone。利用多种限制性核酸内切酶对Red-clone的重组质粒插入片段进行消化，构建了该插入片段的限制性内切酶图谱；并以其图谱为指导，构建了一系列亚克隆重组质粒。其中有一个亚克隆(称为Red-SacⅠ-SalⅠ)尽管仅含1.5kb左右原插入片段，仍然能在紫外线下发射红色荧光。再用各种限制性核酸内切酶对 Red－rl－Sail进行消化、连接和转化，继续亚克隆。测定各亚克隆的核昔酸序列，确定Red伽 I－Sa的插入片段为 1，54fop。利用软件分析，并搜索DDBJ，推断red gene的开放阅读框为77fop，编码257个氨基酸。在开放阅读框的上游没有找到启动子，但在开放阅读框的内部却发现了一个启动子，估计Red－gene的启动子是基因内启动子，或者是利用质粒pUC的 lac启动子。在 DDBJ中搜索到多种细菌来源的 cobA基因（编码uroporph仰nogen h methyltransferase）与 red sene有 40－50％的同源性，并据报道，其中一个来源于 PmpboibaclerilllAslldelll切Chit的 CObA的基因，转人大肠杆菌、酵母菌及动物细胞后能使表达载体在紫外线下发射红色荧光。由此推断Reddeene可能也是cobA gene中的一种，具有类似的功能。 为验证red ghe能否用来作为分子生物学研究的报告基因，将克隆在clon。5的几丁质酶基因与Red唱ene连接后再插入质粒pUC 18中，转入大肠杆菌M109菌株，得到含有几丁质酶基因和红色基因的重组菌株。该重组菌株既能产生几丁质酶，又能使菌体在紫外线下呈现红色荧光。可见，所克隆到的红色基因用作以大肠杆菌为表达载体的报告基因具有一定的潜力。更多还原

【Abstract】 Aeromonas sp. SUWA-9 is a bacterium isolated from the Suwa Lake of Japan as a producer of several chitin-degrading enzymes. To isolating genes coding for chitin-degrading enzymes, genomic DNA was prepared from SUWA-9, partially degested with Sau 3AI, and size-fractionated by sucrose density gradient gentrifugation. The resulting 2-20 kb DNA fragments were ligated with plasmid pUC118 previously digested with Bam HI and dephosphorylated by bacterial alkaline phosphatase (BAP). After transformation into Ecoli. JM109, 8 out total 30,000 recombmant colonies were selected as producing chitinase activity on M9 selection medium cotaining substrates, methylumberyferrol (MU)-(N-acetylglucosamine)n=(1-3). By comparing restriction maps of plasmids possessed in these clones, 5 clones (clones 1, 4, 5, 6, and 8) were found to contain the same chitinase gene, while the other three clones (clones 2, 3 and 9) contain different chitinase genes one another. SDS-PAGE and activity staining analysis revealed that all of clones 2, 5 and 9 exhibited a single active band with 83-kD in size, whereas clone 3 exhibited four different bands with 64 kD, 70kD, 76kD and 83 kD After construcing a series of deleted subclones from the plasmid prepared from clone-5, a complete nucleotide sequences comprising of 3,205 bps wes determined. One open reading frame (ORF) was found in this sequence, which was comprised of 2,661 bps and could encode a polypeptide with 887 amino acid residues. A search for database in the DNA Data Bank of Japan (DDBJ) revealed that the amino acid sequence of the above ORF shows 90% identity with that of one putative chitinase from Aeromonas hydrophilu in which only a primary sequence has been reported to date.Reporter gene is very important for molecular biology. Until now more than 99% microorganisms in the nature have not been cultured. It has been wildly accepted that it is a good idea to search new genes from these uncultured microorganisms During the course of study to isolate new genes from total DNAs prepared from uncultured bacteria in soil, we obtained a 4.7-kb DNA fragment that made E. coli host cells produce a redfluorescence light under exposure of ultraviolet (UV). The minimum region of DNA fragment required for an expression of red fluorescent light could be reduced to 1.5 kb. The nucleotide sequence of this 1.5 kb DNA fragment was determined, and one ORF was found and named as red gene, which was comprised of 771 bp and coded for a polypeptide of 257 amino acid residues in size. A database search revealed that the putative sequence of the red gene shows 40-50% identity with those of uroporphyrinogen III methyltransferase (encoded by cobA gene) from various kinds of bacteria. An over-expression of the cobA gene in E. coli was reported to lead to an accumulation of trimethylated derivative of porphyrin termed trimethylpyrrocorphin and Factor II , which emit strong red fluorescence under UV. Therefore, the red gene isolated from uncultured microorganisms must be a novel cobA gene.Our interesting is to use the red gene as a reporter gene. The red gene and chitinase gene from clone-5 were ligated together with pUC118(2fcwwH I/BAP), and then transformed into Ecoli. JM109. The recombinant cells exhibited chitinase activity and shew red fluorescence under UV. So we can say that the red gene may also be used as a reporter gene.更多还原