摘要
实验经限制性酶切和双向Ｓｏｕｔｈｅｒｎ杂交将重组质粒ｐＣＢＨＩＩ－１４的１４ｋｂ外源片段上的绿色木霉纤维素酶ＣＢＨＩＩ基因定位于４．４ｋｂ的ＥｃｏＲＩ片段上，将该片段克隆于质粒ｐＵＣ１９，获得重组质粒ｐＣＢＨＩＩ。通过限制性分析构建了ｐＣＢＨＩＩ上４．４ｋｂＥｃｏＲＩ片段的物理图谱。在此基础上用质粒制作该片段的亚克隆，采用Ｓａｎｇｅｒ双脱氧链终止法测定了含绿色木霉纤维素酶ＣＢＨＩＩ基因的４０７４ｂｐ的ＤＮＡ序列，由ＧＴ－ＡＧ规则和ｃＤＮＡ序列推知该结构基因由４个外显子和３个内含子组成，总长１６１３ｂｐ，５′端存在与一般真核基因类似的两个特征性调控序列，但３′端不存在真核基因通常具有的特征序列而存在功能未明的短重复序列和嘧啶富集区。并对纤维素酶基因间的同源性和可能的功能作了初步的探讨。
Restriction analysis and bi-directional southern blotting were conducted to localize the CBHII gene of Trichoderma viride in 4kb SolI insert cloned in plasmid pUC19.A 4.4kb EcoRI fragment containing T. viride CBHII gene was verified and subcloned into plasmid pUC19. The physical map of the 4.4kb EcoRI fragment in recombinant pCBHⅡ was then established. Based on the map 18 restriction fragments were subcloned into appropriate plasmids. A11 the short fragments were sequenced by the method of Sanger′s dideoxy chain termination,and 4074bp of DNA sequence was determined which revealed that the CBHII gene of T. viride had a length of 1613bp,flanked by 744bp at 5′end and 1718bp at 3'end. It was deduced that the CBH II gene was composed of 4 exons and 3 introns. The cis-regulatory elements,similar to those in most eukaryotic genes, were found upstream of the CBHII structural gene,but the downstream region of the gene lacked of the sequences associated with the function of transcriptional termination and polyadenylation of mRNA in most eukaryotes, whereas,this region had short repeated sequences and pyrimidine-rich sequences with unknown function. The homology between cellulase genes and their possible function were also discussed.
