黑翅土白蚁体内纤维素酶编码基因的克隆与表达
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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。
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 genes
With 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 coli
The 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 cells
The 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 castes
We 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.
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。
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 genes
With 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 coli
The 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 cells
The 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 castes
We 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.
引文
蔡邦华,陈宁生,陈安国等.长江大堤上黑翅土白蚁的地面活动与其巢位的关系[J].昆虫学报,1965,14：128-139.
崔琰,寇叙.纤维素酶的作用及在养殖业中的应用[J].养殖技术顾问,2008,6：145-146.
黄复生,朱世模,平正明等.中国动物志-昆虫纲(第一七卷)等翅目[M].北京：科学出版社,2000：146-158.
姜淑荣.浅谈纤维素酶在酒类生产中的应用[J].中国酿造,2008,9：12-15.
李珺,张贞华,李桃生.低等白蚁肠道内原生动物的分布及其进化学意义[J].白蚁科技,1999,16：1-7.
李朝丽,郭建生.生物酶在纺织上的应用[J].现代纺织技术,2009,2：58-61.
李明华,张大伟,楚杰等.饲料纤维素酶的研究与应用进展[J].新饲料,2006,7：65-68.
刘刚,余少文,孔舒等.碱性纤维素酶及其应用的研究进展[J].生物加工过程,2005,3：9-14.
牟晓红.生物质转化燃料乙醇纤维素酶的研究[J].石油化工应用,2009,28：16-19.
全国白蚁防治中心编.中国白蚁防治专业培训教程[M].中国物价出版社,2004.
邵学良,刘志伟.纤维素酶的性质及其在食品工业中的应用[J].中国食物与营养,2009,8：34-36.
邵震宇,谢益民.纤维素酶在制浆造纸工业中的应用[J].江苏造纸,2008,2：38-42.
史龙君.纤维素酶在酱油酿造上的应用研究[J].中国调味品,2008,9：61-64.
魏亚琴,李红玉.纤维素酶高产菌选育研究进展及未来趋势[J].兰州大学学报(自然科学版),2008,44：107-114.
杨友坤,朱凤香,王卫平等.纤维素酶及其应用现状[J].安徽农学通报,2009,15：59-62.
Aanen D K, Eggleton P, Rouland-Lefevre C, et al. The evolution of fungus-growing termites and their mutualistic fungal symbionts [J]. Proceedings of the National Academy of Sciences,2002, 99:14887-14892.
Abe T, Bignell D E, Higashi M. Termites:Evolution, Sociality, Synbiosis, Ecology [M]. Netherlands:Kluwer Academic Publishers,2000,209-231.
Bhat M K. Cellulases and related enzymes in biotechnology [J]. Biotechnology Advances,2000, 18:355-383.
Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding [J]. Analytical Biochemistry,1976,72: 248-254.
Breznak J A, Brune A. Role of micoorganisms in the digestion of lignocellulose by termites [J]. Annual Review of Entomology,1994,39:453-487.
Breznak J A, Pankratz H S. In situ morphology of the gut microbiota of wood-eating termites [Reticulitermes flavipes (Kollar) and Coptotermes formosanus Shiraki] [J]. Applied and Environmental Microbiology,1977,33:406-426.
Collins N M. Consumption of wood by artificially isolated colonies of the fungus-growing termite Macroterm.es bellicosus [J]. Entomologia Experimentalis et Applicata,1981,29:313-320.
Czolij R, Slaytor M, O'Brien R W. Bacterial flora of the mixed segment and the hindgut of the higher termite Nasutitermes exitiosus Hill (Termitidae. Nasutitermitinae) [J]. Applied and Environmental Microbiology,1985,49:1226-1236.
Dolan M F. Speciation of termite gut protests:the role of bacterial symbionts [J]. International Microbioloy,2001,4:203-208.
Enari T M, Paavola M L. Enzymic hydrolysis of cellulose [J]. Critical Reviews in Biotechnology, 1987,51:67-87.
Hongoh Y, Ohkuma M, Kudo T. Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera:Rhinotermitidae) [J]. FEMS Microbiology Ecology, 2003,44:231-242.
Hyodo F, Inone T', Azuma J-I et al. Role of the mutualistic fungus in lignin degradation in the fungus-growing termite Macrotermes gilyus (Isoptera:Macrotermitinae) [J]. Soil Biology and Biochemistry,2000,32:653-658.
Kouame L P, Niamke S, Diopoh J, et al. Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus [J]. Biotechnology Letters,2001,23:1575-1581.
Konig H, Frohlich J, Hertel H. Diversity and lignocellulolytic activities of cultured microorganisms [M]. In Intestinal Microorganisms of termites and other invertebrates (Edited by Konig H and Varma A), Berlin:Springer,2006:271-301.
Martin M M, Martin J S. Cellulose digestion in the midgut of the fungus-growing termites Macrotermes natalensis:the role of acquired digestive enzymes [J]. Science,1978,199: 1453-1455.
Martin M M. The evolution of cellulose digestion in insects [J]. Philosophical Transactions of the Royal Society of London B:Biological Sciences,1991,333:281-288.
Nakashima K, Azuma J. Distribution and properties of endo-β-1,4-glucanase from a lower termite, Coptotermes formosanus (Shiraki) [J]. Bioscience Biotechnology Biochemistry,2000,64: 1500-1506.
Nakashima K, Watanabe H, Saitoh H, et al. Dual cellulose-digesting system of the wood-feeding termite, Coptotermes formosanus Shiraki [J]. Insect Biochemistry and Molecular Biology,2002, 32:777-784.
Nelson N, A photometric adaptation of the Somogyi method for the determination of glucose [J]. Journal of Biological Chemistry,1951,193:375-380.
Odelson D A, Breznak J A. Volatile fatty acid production by the hindgut microbiota of xylopltagous termites [J]. Applied and Environmental Microbiology,45:1602-1613,
Ohkuma M, Noda S, Hongoh Y, et al. Coevolution of symbiotic systems of termites and their gut microorganisms [J]. RIKENReview,2001,41:73-74.
Radek R. Flagellates, bacteria, and fungi associated with termites:diversity and function in nutrition-a review [J]. Ecotropica,1999,5:183-196.
Reese E T. Polysaccharase and the hydrolysis of insoluble substrates [J]. Process,1976,6:9-12.
Retief L W, Hewitt P H. Digestive carbohydrases of the harvester termite Hodotermes mossambicus:a-glycosidases [J]. Journal of Insect Physiology,1973,19:105-113.
Rouland-Lefievre C, Diouf M N, Brauman A et al. Phylogenetic relationships in Termitomyces (family Agaricaceae) based on the nucleotide sequence of ITS:first approach to elucidate the evolutionary history of the symbiosis between fungus-growing termites and their fungi [J]. Molecular Phylogenetics and Evolution,2002,22:423-429.
Sands W A. The association of termites and fungi. In Biology of Termites, (Krishna K and Weesner F M, Eds.), Academic Press, New York and London,1969, pp.495-524.
Scharf M E, Wu-Scharf D, Pittendrigh B R et al. Caste-and development-associated gene expression in a lower termite [J].Genome Biology,2003,4:1-11.
Slaytor M. Cellulose digestion in termites and cockroaches:what role do symbionts play? [J]. Comparative Biochemistry and Physiology,1992,103:775-784.
Smant G, Stokkemans J P, Yan Y T, et al. Endogenous cellulases in animals:isolation of beta-1,4-endoglucanase genes from two species of plant-par-asitic cyst nematodes [J]. Proceedings of the National Academy of Sciences,1998,95:4906-4911.
Sun J Z, Zhou X G. Utilization of Lignocellulose-feeding insects for viable biofuels:an emerging and promising area of entomological science [M]. In Recent Advances in Entomological Research:from Molecular Biology to Pest management (Edited by Liu T X and Kang L, published by China High Education Press, In English),2009:261-300.
Sun Y, Cheng J Y. Hydrolysis of lignocellulosic materials for ethanol production:a review [J]. Bioresource Technology,2002,831:1-11.
Thayer D W. Facultative wood-digesting bacteria from the hind-gut of the termite Reticulitermes hesperus [J]. Journal of General Microbiology,1976,95:287-296.
Thayer D W. Carboxymethylcellulase produced by facultative bacteria from the hind-gut of the termite Reticulitermes hesperus [J]. Journal of General Microbiology,1978,106:13-18.
Tokuda G, Saito H, Watanabe H. A digestive-glucosidase from the salivary glands of the termite, Neotermes koshunesis (Shiraki):distribution, characterization and isolation of its precursor cDNA by 5'-and 3'-RACE amplifications with degenerate primers [J]. Insect Biochemistry and molecular Biology,2002,32:1681-1689.
Tokuda G, Lo N, Watanabe H, et al. Metazoan cellulase genes from termites:intron/exon structures and sites of expression [J]. Biochimica et Biophysica Acta,1999,1447:146-159.
Tokuda G, Lo N, Watanabe H, et al. Major alteration of the expression site of endogenous cellulases in members of an apical termite lineage [J]. Molecular Ecology,2004,13:3219-3228.
Trager W. A cellulase from the symbiotic intestinal flagellates of termite and of the roach, Cryptocercus punctulatus [J]. Biochemical Journal,1932,26:1762-1771.
Varma A, Kolli B K, Paul J, et al. Lignocellulose degradation by microorganisms from termite hills and termite guts:a survey on the present state of art [J]. FEMS Microbiology Reviews, 1994,15:9-28.
Watanabe H, Noda H, Tokuda G, et al. A cellulase gene of termite origin [J]. Nature,1998,394: 330-331.
Yamin MA. Flagellates of the orders Trichomonadida Kirby, Oxymonadida Grasse, and Hypermastigida Grassi and Foa reported from lower termites (Isoptera farallies Mastotermitidae, Kaloterm itidae, Hodotermitidae, Termnpsidae, Rhinotermitidae, and Serritermitidae) and from the wood-feeding roach Cryptocercus (Dictyoptera:Cryptocercidae) [J]. Sociobiology,1979,4:1-120.
崔琰,寇叙.纤维素酶的作用及在养殖业中的应用[J].养殖技术顾问,2008,6：145-146.
黄复生,朱世模,平正明等.中国动物志-昆虫纲(第一七卷)等翅目[M].北京：科学出版社,2000：146-158.
姜淑荣.浅谈纤维素酶在酒类生产中的应用[J].中国酿造,2008,9：12-15.
李珺,张贞华,李桃生.低等白蚁肠道内原生动物的分布及其进化学意义[J].白蚁科技,1999,16：1-7.
李朝丽,郭建生.生物酶在纺织上的应用[J].现代纺织技术,2009,2：58-61.
李明华,张大伟,楚杰等.饲料纤维素酶的研究与应用进展[J].新饲料,2006,7：65-68.
刘刚,余少文,孔舒等.碱性纤维素酶及其应用的研究进展[J].生物加工过程,2005,3：9-14.
牟晓红.生物质转化燃料乙醇纤维素酶的研究[J].石油化工应用,2009,28：16-19.
全国白蚁防治中心编.中国白蚁防治专业培训教程[M].中国物价出版社,2004.
邵学良,刘志伟.纤维素酶的性质及其在食品工业中的应用[J].中国食物与营养,2009,8：34-36.
邵震宇,谢益民.纤维素酶在制浆造纸工业中的应用[J].江苏造纸,2008,2：38-42.
史龙君.纤维素酶在酱油酿造上的应用研究[J].中国调味品,2008,9：61-64.
魏亚琴,李红玉.纤维素酶高产菌选育研究进展及未来趋势[J].兰州大学学报(自然科学版),2008,44：107-114.
杨友坤,朱凤香,王卫平等.纤维素酶及其应用现状[J].安徽农学通报,2009,15：59-62.
Aanen D K, Eggleton P, Rouland-Lefevre C, et al. The evolution of fungus-growing termites and their mutualistic fungal symbionts [J]. Proceedings of the National Academy of Sciences,2002, 99:14887-14892.
Abe T, Bignell D E, Higashi M. Termites:Evolution, Sociality, Synbiosis, Ecology [M]. Netherlands:Kluwer Academic Publishers,2000,209-231.
Bhat M K. Cellulases and related enzymes in biotechnology [J]. Biotechnology Advances,2000, 18:355-383.
Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding [J]. Analytical Biochemistry,1976,72: 248-254.
Breznak J A, Brune A. Role of micoorganisms in the digestion of lignocellulose by termites [J]. Annual Review of Entomology,1994,39:453-487.
Breznak J A, Pankratz H S. In situ morphology of the gut microbiota of wood-eating termites [Reticulitermes flavipes (Kollar) and Coptotermes formosanus Shiraki] [J]. Applied and Environmental Microbiology,1977,33:406-426.
Collins N M. Consumption of wood by artificially isolated colonies of the fungus-growing termite Macroterm.es bellicosus [J]. Entomologia Experimentalis et Applicata,1981,29:313-320.
Czolij R, Slaytor M, O'Brien R W. Bacterial flora of the mixed segment and the hindgut of the higher termite Nasutitermes exitiosus Hill (Termitidae. Nasutitermitinae) [J]. Applied and Environmental Microbiology,1985,49:1226-1236.
Dolan M F. Speciation of termite gut protests:the role of bacterial symbionts [J]. International Microbioloy,2001,4:203-208.
Enari T M, Paavola M L. Enzymic hydrolysis of cellulose [J]. Critical Reviews in Biotechnology, 1987,51:67-87.
Hongoh Y, Ohkuma M, Kudo T. Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera:Rhinotermitidae) [J]. FEMS Microbiology Ecology, 2003,44:231-242.
Hyodo F, Inone T', Azuma J-I et al. Role of the mutualistic fungus in lignin degradation in the fungus-growing termite Macrotermes gilyus (Isoptera:Macrotermitinae) [J]. Soil Biology and Biochemistry,2000,32:653-658.
Kouame L P, Niamke S, Diopoh J, et al. Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus [J]. Biotechnology Letters,2001,23:1575-1581.
Konig H, Frohlich J, Hertel H. Diversity and lignocellulolytic activities of cultured microorganisms [M]. In Intestinal Microorganisms of termites and other invertebrates (Edited by Konig H and Varma A), Berlin:Springer,2006:271-301.
Martin M M, Martin J S. Cellulose digestion in the midgut of the fungus-growing termites Macrotermes natalensis:the role of acquired digestive enzymes [J]. Science,1978,199: 1453-1455.
Martin M M. The evolution of cellulose digestion in insects [J]. Philosophical Transactions of the Royal Society of London B:Biological Sciences,1991,333:281-288.
Nakashima K, Azuma J. Distribution and properties of endo-β-1,4-glucanase from a lower termite, Coptotermes formosanus (Shiraki) [J]. Bioscience Biotechnology Biochemistry,2000,64: 1500-1506.
Nakashima K, Watanabe H, Saitoh H, et al. Dual cellulose-digesting system of the wood-feeding termite, Coptotermes formosanus Shiraki [J]. Insect Biochemistry and Molecular Biology,2002, 32:777-784.
Nelson N, A photometric adaptation of the Somogyi method for the determination of glucose [J]. Journal of Biological Chemistry,1951,193:375-380.
Odelson D A, Breznak J A. Volatile fatty acid production by the hindgut microbiota of xylopltagous termites [J]. Applied and Environmental Microbiology,45:1602-1613,
Ohkuma M, Noda S, Hongoh Y, et al. Coevolution of symbiotic systems of termites and their gut microorganisms [J]. RIKENReview,2001,41:73-74.
Radek R. Flagellates, bacteria, and fungi associated with termites:diversity and function in nutrition-a review [J]. Ecotropica,1999,5:183-196.
Reese E T. Polysaccharase and the hydrolysis of insoluble substrates [J]. Process,1976,6:9-12.
Retief L W, Hewitt P H. Digestive carbohydrases of the harvester termite Hodotermes mossambicus:a-glycosidases [J]. Journal of Insect Physiology,1973,19:105-113.
Rouland-Lefievre C, Diouf M N, Brauman A et al. Phylogenetic relationships in Termitomyces (family Agaricaceae) based on the nucleotide sequence of ITS:first approach to elucidate the evolutionary history of the symbiosis between fungus-growing termites and their fungi [J]. Molecular Phylogenetics and Evolution,2002,22:423-429.
Sands W A. The association of termites and fungi. In Biology of Termites, (Krishna K and Weesner F M, Eds.), Academic Press, New York and London,1969, pp.495-524.
Scharf M E, Wu-Scharf D, Pittendrigh B R et al. Caste-and development-associated gene expression in a lower termite [J].Genome Biology,2003,4:1-11.
Slaytor M. Cellulose digestion in termites and cockroaches:what role do symbionts play? [J]. Comparative Biochemistry and Physiology,1992,103:775-784.
Smant G, Stokkemans J P, Yan Y T, et al. Endogenous cellulases in animals:isolation of beta-1,4-endoglucanase genes from two species of plant-par-asitic cyst nematodes [J]. Proceedings of the National Academy of Sciences,1998,95:4906-4911.
Sun J Z, Zhou X G. Utilization of Lignocellulose-feeding insects for viable biofuels:an emerging and promising area of entomological science [M]. In Recent Advances in Entomological Research:from Molecular Biology to Pest management (Edited by Liu T X and Kang L, published by China High Education Press, In English),2009:261-300.
Sun Y, Cheng J Y. Hydrolysis of lignocellulosic materials for ethanol production:a review [J]. Bioresource Technology,2002,831:1-11.
Thayer D W. Facultative wood-digesting bacteria from the hind-gut of the termite Reticulitermes hesperus [J]. Journal of General Microbiology,1976,95:287-296.
Thayer D W. Carboxymethylcellulase produced by facultative bacteria from the hind-gut of the termite Reticulitermes hesperus [J]. Journal of General Microbiology,1978,106:13-18.
Tokuda G, Saito H, Watanabe H. A digestive-glucosidase from the salivary glands of the termite, Neotermes koshunesis (Shiraki):distribution, characterization and isolation of its precursor cDNA by 5'-and 3'-RACE amplifications with degenerate primers [J]. Insect Biochemistry and molecular Biology,2002,32:1681-1689.
Tokuda G, Lo N, Watanabe H, et al. Metazoan cellulase genes from termites:intron/exon structures and sites of expression [J]. Biochimica et Biophysica Acta,1999,1447:146-159.
Tokuda G, Lo N, Watanabe H, et al. Major alteration of the expression site of endogenous cellulases in members of an apical termite lineage [J]. Molecular Ecology,2004,13:3219-3228.
Trager W. A cellulase from the symbiotic intestinal flagellates of termite and of the roach, Cryptocercus punctulatus [J]. Biochemical Journal,1932,26:1762-1771.
Varma A, Kolli B K, Paul J, et al. Lignocellulose degradation by microorganisms from termite hills and termite guts:a survey on the present state of art [J]. FEMS Microbiology Reviews, 1994,15:9-28.
Watanabe H, Noda H, Tokuda G, et al. A cellulase gene of termite origin [J]. Nature,1998,394: 330-331.
Yamin MA. Flagellates of the orders Trichomonadida Kirby, Oxymonadida Grasse, and Hypermastigida Grassi and Foa reported from lower termites (Isoptera farallies Mastotermitidae, Kaloterm itidae, Hodotermitidae, Termnpsidae, Rhinotermitidae, and Serritermitidae) and from the wood-feeding roach Cryptocercus (Dictyoptera:Cryptocercidae) [J]. Sociobiology,1979,4:1-120.