摘要
昆虫铁蛋白在铁转运和贮藏、抗氧化胁迫及病菌侵染等诱导的免疫反应等过程中发挥重要作用。为探索烟粉虱Bemisia tabaci铁蛋白功能,本研究以烟粉虱MED隐种Bemisia tabaci MED为研究对象,采用反转录PCR(reverse transcription PCR,RT-PCR)克隆铁蛋白基因,采用荧光定量PCR检测了该基因在烟粉虱不同发育时期及在吡虫啉胁迫条件下成虫体内mRNA的表达量,并利用原核表达载体pMAL-BtFer1对该蛋白进行体外表达和纯化。克隆获得烟粉虱铁蛋白基因的cDNA序列,并命名为BtFer1(GenBank登录号:JX865415)。该基因cDNA长度为1 043 bp,编码224个氨基酸残基,序列中含有铁蛋白典型的铁结合功能域,以及由19个氨基酸残基构成的信号肽。荧光定量分析显示,BtFer1在不同发育时期均有表达,以成虫和3龄若虫期的表达量较高,并且该基因的表达受吡虫啉胁迫处理的强烈诱导。体外表达、纯化和电泳检测显示,在0.3 mmol/L浓度IPTG诱导下融合蛋白在上清和沉淀中均能表达,电泳检测到一条约68 kD的外源蛋白,与预测融合蛋白分子量大小相符。本研究明确了该铁蛋白基因在烟粉虱MED隐种不同发育阶段及在低浓度吡虫啉胁迫下成虫体内的表达水平,为深入研究其功能提供理论依据。
Insect ferritins play key roles in iron transport, response to oxidative stress and the immune response to pathogen infection and others. In this study, cDNA identification and expression of recombinant ferritin from Bemisia tabaci MED were done in order to elucidate the function of ferritin in this insect. The cDNA was amplified by RT-PCR and named BtFer1 （GenBank accession number： JX865415）. The partial sequence of BtFer1 is 1 043 bp in length, encoding 224 amino acids. Sequence analysis indicated that the protein has the characteristic features of typical ferritin ironbinding region signature and a 19residue signal peptide. q-PCR assay displayed that BtFer1 was expressed in various developmental stages of B. tabaci and the expression levels in B. tabaci at the adult and 3rd instar nymphal stages were much higher than those at other stages. The expression profiling also showed that BtFer1 was up-regulated in adults treated with 1 mg/L imidacloprid. Furthermore, the BtFer1 gene was constructed into the expression vector pMAL-c2x for protein expression in prokaryotic cells and purified by amylose affinity. The results showed that the fused MBP-BtFer1 proteins could be effectively induced with 0.3 mmol/L IPTG, and the fusion protein was about 68 kDa, which was consistent with the predicted result. This study makes clear the expression levels of BtFer1 in various developmental stages and in adults of B. tabaci MED treated with lower concentration of imidacloprid, and provides a basis for further functional study of this gene.
出处
《昆虫学报》
CAS
CSCD
北大核心
2013年第7期738-746,共9页
Acta Entomologica Sinica
基金
国家自然科学基金项目(30571219)
国家转基因生物新品种培育重大专项(2009ZX08012-007B)
关键词
烟粉虱MED隐种
铁蛋白
基因克隆
表达分析
原核表达
吡虫啉
Bemisia tabaci MED ferritin gene cloning expression profiling prokaryotic expression imidacloprid
