【作者】 陈春润；

【导师】 莫建初；

【作者基本信息】 浙江大学 ， 环境生物学， 2010， 博士

【摘要】 在纤维素降解过程中,至少需要内切-β-1,4-葡聚糖酶(endo-P-1,4-glucanase, EG, EC 3.2.1.4)、外切-β-1,4-葡聚糖酶(exo-β-1,4-glucanase, CBH, EC 3.2.1.91)和β-葡萄糖苷酶(β-glucosidase, BG, EC 3.2.1.21)三种成分的共同作用,但白蚁的内源性纤维素酶只包括内切-β-1,4-葡聚糖酶和β-葡萄糖苷酶两种成分,这两种成分由唾液腺和中肠上皮细胞分泌产生。低等白蚁后肠富含细菌、原生动物等共生微生物,它们分泌产生的纤维素酶,与白蚁内源性纤维素酶一道共同完成食物中纤维素类物质的降解。高等白蚁后肠缺乏原生动物,但其巢体上的共生真菌具丰富的纤维素酶,因此白蚁通常通过取食巢体上的共生真菌来获得外源性纤维素酶,协助完成食物中纤维素的消化与吸收。本文利用分子生物学手段对黑翅土白蚁内源性纤维素酶进行了初步的研究,从其体内克隆到了两个纤维素酶基因,利用异源表达系统对其进行了表达,并对其性质进行了初步研究,为今后进一步研究白蚁分解纤维素机理及更好的利用白蚁纤维素酶奠定了基础。1纤维素酶基因的克隆利用5’和3’RACE方法从黑翅土白蚁体内克隆到了两个纤维素酶基因的全长cDNA序列,并将其推导氨基酸序列与NCBI数据库中的其它白蚁的纤维素酶氨基酸序列进行了比对,发现内切-β-1,4-葡聚糖酶的同源性在75%以上,β-葡萄糖苷酶的同源性在52%以上。2纤维素酶基因在大肠杆菌中的表达利用pET-28a载体构建原核表达载体,将纤维素酶基因在大肠杆菌中进行诱导表达。结果发现在45-66.2kDa之间出现特异性条带,利用抗His标签单克隆抗体进行Western blot分析,证实成功地表达了目的蛋白。但是,对表达产物进行酶活测定后发现,原核表达产物没有纤维素酶活性。3纤维素酶基因在昆虫细胞中的表达利用Bac-to-Bac昆虫杆状病毒表达系统将目的基因在昆虫细胞中进行了表达,并利用制备的多克隆抗体进行Western blot分析,证实目的蛋白在昆虫细胞中成功表达。对表达产物的粗酶液进行酶活测定,结果显示重组内切-β-1,4-葡聚糖酶的酶活为0.190U,重组p-葡萄糖苷酶的酶活为0.117U。4黑翅土白蚁体内纤维素酶的性质分析通过半定量RT-PCR和Western blot的方法检测了纤维素酶在不同品级白蚁和不同组织间的表达情况和分布情况,并对不同品级白蚁和不同组织的纤维素酶活性进行了初步测定。结果显示：两种纤维素酶由唾液腺和中肠上皮细胞分泌产生,且这两个部位的纤维素酶活性最高。更多还原

【Abstract】 In nature, degradation of cellulose is performed by mixtures of hydrolytic enzymes collectively known as cellulases. The cellulases are divided into three major groups: endoglucanases (EG, EC 3.2.1.4), cellobiohydrolases (exoglucanases, CBH, EC 3.2.1.91), andβ-glucosidases (BG, EC 3.2.1.21). The study of cellulose digestion in insects is most advanced in termites, which are classified into lower termites, which possess large populations of cellulolytic protozoa in the hindgut, and higher termites, which do not have cellulolytic symbiotes in the gut. The endogenous cellulolytic enzymes of termites primarily consist of endo-P-1,4-glucanase andβ-glucosidase.In this study, we report the cloning, expression and analysis of the distribution and enzyme activity of the cellulases from the termite, Odontotermes formosanus.1 Cloning of cellulase genesWith 5’-and 3’-RACE method, we obtain two complete cellulase cDNAs from 0. formosanus. Blasting on the NCBI with the deduced proteins, we found many cellusase sequences from other termites. The endo-β-1,4-glucanase andβ-glucosidase sequence presented more than 75% and 52% identity with other termite cellulases, respectively.2 Recombinant expression of cellulase genes in Escherichia coliThe cDNAs of two cellulases were transformed into the expression vector of pET-28a, and the recombinant vector was transformed into E. coli of BL21.Two distinctive bands was observed between 45.0 and 66.2 kDa, and western blot analysis using anti His-tag antibody confirmed that the inserted two cellulase cDNAs was overexpressed in E. coli. Unfortunately, no cellulase activity was detected in the recombinant proteins. 3 Recombinant expression of cellulase genes in insect cellsThe two cellulase genes were expressed in insect cells using Baculovirus Expression Vector System (BEVS). The recombinant proteins were present as a single band by SDS-PAGE, and the consistent result of Western blot using polyclonal antiserum confirmed that the inserted cellulase cDNAs were expressed in insect cells. The results of enzyme activity assay showed that, the activities of endo-β-1, 4-glucanase andβ-glucosidase were 0.190 and 0.117U, respectively.4 Variations of the transcripts and expression levels of cellulases in tissues and castesWe conducted RT-PCR experiments using cDNAs prepared from different castes and worker tissues. And by the analysis of SDS-PAGE using total protein extracts from different castes and worker tissues and Western blot using polyclonal antiserum, we investigated the transcript variations of two cellulases. At last, we examined the enzyme activity of two cellulases in different casts and worker tissues. We found that the two endogenous cellulases were excreted by salivary glands and midgut epithelia.更多还原