【作者】 成庆利；

【导师】 贾新成； 马向东；

【作者基本信息】 河南农业大学 ， 微生物学， 2002， 硕士

【摘要】 α-淀粉酶是工业上应用的重要的酶类之一，并且也是酶学研究中最活跃的领域。本实验构建了野油菜黄单胞菌（Xanthomonas campestris pv. campestris 8004）的基因组文库，克隆到了α-淀粉酶基因，并对其进行了分析。 黄单胞菌的总DNA和载体pUC18质粒用不同的方法分别抽提。采用0.5U MobI限制内切酶对抽提的黄单胞菌总DNA进行部分酶切，酶切时间要控制好。时间过长或过短，都会影响酶切效果。本实验用琼脂糖凝胶电泳来来检测酶切效果，并控制酶切时间，最终选定为20分钟。回收集中在2-7kb之间的片段。pUC18质粒用BamHI进行单酶切，酶切一定要完全。用胶回收试剂盒回收。为防止质粒本身自连，回收后用CIAP去磷酶去磷，反应一定要完全。回收的总DNA与pUC18质粒按一定比例混和，加入T₄DNA连接酶、连接酶缓冲液、去离子水使反应体积为20μl，在14-16℃条件下反应16-18小时。因为酶连效果较低，扩大反应体系为50μl，再继续反应16-18小时。用高效感受态细胞DH5α进行常规转化，得到了20000多个转化子。采用LBSP平板初筛，进一步用I₂-淀粉法鉴定，筛选到了α-淀粉酶的阳性克隆。经质粒大小验证、酶切验证、以及功能验证，得到了大小约为2Kb的α-淀粉酶基因片段，并绘制了其限制酶图谱。 经上海生工生物工程技术有限公司测序部测序，外源片段为1899bp。经序列分析，找到了α-淀粉酶基因的开放阅读框架，这个框架编码了一个有475个氨基酸残基的蛋白质。推测的氨基酸序列与Pseudomonas sp. KFCC10818的α-淀粉酶氨基酸序列有45％同源性。它同时含有在其他组织的α-淀粉酶中4个同 源序列。 以 pUC为载体的基因在大肠杆菌 DHS a 中的表达量非常 小，并且一部分表达产物被分泌到胞外。因此提取总蛋白时需 破碎细胞，浓缩发酵液，用三氯乙酸和丙酮抽提。经SDSPAGE 凝胶电泳门2洲／V)分析，a－淀粉酶的分子量约为45KDa，与 从核苦酸序列推测的结果相同。更多还原

【Abstract】 Alpha-amylase was one of important enzymes in industry field, and the activist fields in enzyme studying .Genomic library of Xanthomonas campestris pv. campestris was constructed, and alpha- amylase gene was cloned> sequenced and analysed.The total DNA of Xanthomonas campestris pv. campestris and pUCIS plasmid were extracted respectively by different methods. The genome DNA was partially digested by 0.5U Mobl restriction enzyme . The time of digestion must be controlled. The resμlt of digestion woμld be effected whether longer or shorter. Agarose gel electrophorase coμld be used to detect the digestion resμlt and control the time .20 minutes were selected. Pieces of 2-7 Kb DNA was extracted. pUCIS plasmid was totally digested with BamHl enzyme and extracted by gel purification. To prevent the self ligation , CLAP was used to dephosphorylate totally . 2-7 Kb DNA and pUCIS plasmid were mixed.. T4 DNA ligase , ligase buffer and deioned water were added to 20 μl reaction volume . Because the ligation efficiency was very low, after ligation for 16-18h at 14℃ to 16℃,the reaction volume was enlarged to 50μl in the same condition for 16 to 18 h again. The ligation product was transformed with high efficiency competent cells, and more than 20000 transformants were gotten. LBSP plate screening was first used , then Idione- starch method was taken, and positive cloning of alpha- amylase gene were identified. After checking the length of positive cloning plasmid , digesting it with the restriction enzymes and testing of its ruction ,alpha- amylase gene of about 2Kb was cloned andits restriction enzyme map was made.The piece of DNA sequenced by Sangon was an 1899 bp fragment. Sequence analysis revealed a potential ORF encoding a protein of 475 amino acid residues. The deduced amino acid sequence had 45% homology to that of the alpha-amylase of Pseudomonas sp. KFCC10818. It also contained all four homology sequences highly conserved in the alpha-amylase from a wide range of organisms.The expression of the Amy gene in Escherichia coli was poor, and some of the expression were secreted. To extract the total protein, the cells were broken, the germination liguid was enriched, and TCA and Acetone A.R. were used. SDS-PAGE (12%W/V) showed that the molecular weight of alpha-amylase was 45 KD, which was the same as the result deduced from the nucleotide sequence.更多还原