摘要
【目的】本研究旨在阐明长足大竹象Cyrtotrachelus buqueti内切葡聚糖酶最适反应条件,并挖掘长足大竹象消化道内切葡聚糖酶关键基因。【方法】采用3,5-二硝基水杨酸(DNS)法,设置以羧甲基纤维素钠(MC)为反应底物的单因素和正交优化试验测定内切葡聚糖酶反应的最适条件。通过对长足大竹象发育转录组内切葡聚糖酶编码基因进行生物信息学分析,并将基因表达量与酶活性数据进行关联分析,筛选出发育时期中关键内切葡聚糖酶基因,采用实时荧光定量PCR对不同发育时期长足大竹象消化道内切葡聚糖酶关键基因表达量进行验证确定。【结果】研究表明,长足大竹象成虫内切葡聚糖酶的最适反应条件为:温度45℃,pH 5.6,底物浓度2%,酶比活力59.85 U/mg(雌)和52.87 U/mg(雄);幼虫内切葡聚糖酶的最适反应条件为:温度35℃,pH 4.8,底物浓度2%,酶比活力38.34 U/mg。筛选出长足大竹象消化道内切葡聚糖酶关键基因c64192_g1和c57057_g1。实时荧光定量PCR结果表明c64192_g1和c57507_g1基因在成虫时期表达量高于幼虫。【结论】长足大竹象雌雄成虫的内切葡聚糖酶比活力均高于幼虫,存在两个影响内切葡聚糖酶活性的关键基因c64192_g1和c57507_g1。这些研究成果丰富了内切葡聚糖酶来源,并为长足大竹象内切葡聚糖酶异源表达提供数据参考,进而为木质纤维素预处理和生物质能源的开发利用奠定理论基础。
【Aim】 This study aims to investigate the optimal reaction conditions of endoglucanases from Cyrtotrachelus buqueti, and to explore the key genes of endoglucanases in the gut of C. buqueti. 【Methods】 Single factor and orthogonal optimization experiments were carried out. The optimal reaction conditions for endoglucanases were determined by using 3,5-dinitrosalicylic acid (DNS) method with sodium carboxymethyl cellulose (MC) as the substrate. The endoglucanase-encoding gene sequences in the transcriptomes of different developmental stages of C. buqueti were subjected to bioinformatics analysis. Through correlation analysis between gene expression level and enzyme activity, key endoglucanase genes in different developmental stages of C. buqueti were screened. And the expression levels of key genes of endoglucanases in the gut of C. buqueti of different developmental stages were validated by using qRT-PCR. 【Results】 The results showed that the optimum reaction conditions for endoglucanases in C. buqueti adults were 45℃, pH 5.6, and 2% substrate concentration, with the enzyme activity of 59.85 and 52.87 U/mg in female and male, respectively, while the optimum reaction conditions for endoglucanases in C. buqueti larvae were 35℃, pH 4.8, and 2% substrate concentration, with the enzyme activity of 38.34 U/mg. Moreover, two key endoglucanase genes, c64192_g1 and c57057_g1, were identified, and they exhibited higher expression level in adults than in larvae. 【Conclusion】 The endoglucanase activities in female and male adults of C. buqueti are higher than that in larvae, and two key genes, c64192_g1 and c57057_g1, affecting the endoglucanase activity in C. buqueti were screened. These foundings enrich the endoglucanase source and provide data reference for the heterologous expression of endoglucanase gene in C. buqueti, laying a foundation for the pretreatment of lignocellulose and the development and utilization of biomass energy.
作者
王明珺
王昌吉
陈晓雅
李沅秋
梁娇娇
罗朝兵
WANG Ming-Jun;WANG Chang-Ji;CHEN Xiao-Ya;LI Yuan-Qiu;LIANG Jiao-Jiao;LUO Chao-Bing(Bamboo Diseases and Pests Control and Resources Development Key Laboratory of Sichuan Province, Leshan Normal University, Leshan, Sichuan 614000, China)
出处
《昆虫学报》
CAS
CSCD
北大核心
2019年第10期1150-1161,共12页
Acta Entomologica Sinica
基金
四川省科技计划项目(2019YFG0139)
关键词
长足大竹象
内切葡聚糖酶
基因表达
酶活性
正交优化
Cyrtotrachelus buqueti
endoglucanase
gene expression
enzyme activity
orthogonal optimization
