| 摘要: |
| 【目的】研究陕南地区桑天牛内切β-1,4-葡聚糖酶基因，为构建高效分解纤维素的工程菌奠定基础。【方法】用质量分数为1%的羟甲基纤维素钠(CMC Na)测定桑天牛幼虫内切β-1,4-葡聚糖酶活性，Trizol法提取桑天牛肠道总mRNA，扩增其内切β-1,4-葡聚糖酶基因，用DNAStar和NCBI上的BLAST进行序列分析。根据测序得到的基因序列设计1对引物，扩增桑天牛内切β-1,4-葡聚糖酶基因CDs区，构建原核表达载体pET-APEG，并在大肠杆菌中表达。【结果】桑天牛内切β-1,4-葡聚糖酶最适pH为4.4～5.6，pH值为5.0时酶活性最高；最适温度为30～50 ℃，50 ℃时酶活最高；酶液在37 ℃稳定性好，而在60 ℃的水浴中处理30 min，酶活性急剧下降。桑天牛内切β-1,4-葡聚糖酶基因CDs区长度为978 bp，编码325个氨基酸残基；BLAST结果显示，该基因序列与黄星天牛属于GHF5的纤维素酶基因序列相似性达85%，氨基酸序列相似性达90%，与已报道的桑天牛Ag-EaseⅢ的序列相似性达98%，氨基酸序列相似性达99%；构建原核表达载体pET-APEG在大肠杆菌BL21(DE3)中进行表达，SDS-PAGE检测结果表明，诱导表达沉淀在约55 ku处有1条特异蛋白条带，与预测蛋白的分子量相符。【结论】陕南地区桑天牛β-1,4-葡聚糖酶为内切葡聚糖酶，属于GHF5家族，其可以在大肠杆菌中表达。 |
| 关键词: 桑天牛 内切β-1,4-葡聚糖酶 内切β-1,4-葡聚糖酶基因 克隆 表达 |
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| 基金项目:农业部现代农业产业技术体系建设专项（nycytx-40） |
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| Enzyme analysis,gene CDs cloning and expression in E.coli of Mulberry Borer (Apriona germari hope) endogen β-1,4-endoglucanase |
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| Abstract: |
| 【Objective】This objective focused on endogen β-1,4-endoglucanase gene of mulberrying borer,with the purpose of supplying the theory basis for constructing engineering bacteria which can decompose cellulose effectively. 【Method】The activities of endogen β-1,4-endoglucanase were measured by 1% of CMC-Na,total RNA was isolated from Mucberry Borer gut by trizol, the first cDNA chian was used as template,a pair of primers was designed based on the gene whose accession number is AY771358.1,for PCR endogen β-1,4-endoglucanase gene of Mulberry Borer,DNASTAR and BLAST of NCBI were used for sequence analysis;another pair of primers was designed based on the sequence of this study,which was to add restriction enzyme cutting site BamHⅠfor up site and SacⅠfor down site,PCR endogen β-1,4-endoglucanase gene of Mulberry Borer,and structure pET-APEG for prokaryotic expression in Escherichia coli BL21(DE3).【Result】 The most suitable pH range is from 4.4 to 5.6,with peak at 5.0;the most suitable range temperature is from 30 to 50 ℃,with peak at 50 ℃.If solution of endoglucanase is treated in water bath of 37 ℃ for 30 min,the activities hardly change;However,after treated in water bath of 60 ℃ for 30 min,the activities plummet.The length of CDs of β-1 and 4-endoglucanase is 978 bp,ecoding 325 amino acid residues;The result of BLAST showed that,the sequence identity of this gene is 85% compared with the cellulase gene of yellow spotted longicorn beetle,Psacothea hilaris which belongs to the GHF5,and the sequence identity of protein reaches 90%,the sequence identity of this gene is 98% compared with the sequence of Ag EaseⅢ which has been reported in mulberry borer,the sequence identity of protein reaches 99%.Structed pET-APEG for expression in E.coil BL21(DE3) and SDS-PAGE result showed,pET-APEG vector was expressed as about 55 ku polypeptide in E.coil BL21 (DE3).【Conclusion】 The mulberry borer of Shannan area belongs to GHF5,and has multi-domains of endogen β-1,4-endoglucanase,and it’s gene can be expressed in E.coil BL21(DE3). |
| Key words: Mulberry Borer endogen β-1,4-endoglucanase endogen β-1,4-endoglucanase gene cloning expression |
