嗜卷书虱P450基因的分子生物学特性及其异源表达研究
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摘要
书虱(psocids)属于虱啮目Psopotera、书虱科Liposcelididae、书虱属Liposcelis,是一类重要的储藏物害虫,大量发生时可造成严重的经济损失,且对化学药剂的抗性发展很快,已经引起了全世界储藏物工作者的高度重视。然而与其它农业害虫相比,以往对该类害虫包括其它储藏物害虫的研究主要集中在应用研究层面,较少涉及基础或应用基础研究领域。昆虫细胞色素P450是昆虫体内一种重要的代谢酶,在昆虫的生命活动中具有重要的生理功能。其中细胞色素P450对杀虫剂的代谢作用增强是大多数昆虫产生抗药性的主要机制之一,这一机制也常被认为是最为重要的一个抗性机制,而且由于其催化底物的多样性,细胞色素P450极有可能介导昆虫的交互抗性。本学位论文在国家自然科学基金(30871631)、教育部新世纪优秀人才支持计划(NCET-04-0854)以及西南大学研究生创新基金优秀博士生项目(kb2008001)的资助下,以目前世界范围内危害严重的嗜卷书虱Liposcelis bostrychophila Badonnel为对象,瞄准昆虫P450功能研究的这一热点问题,率先开展了嗜卷书虱P450基因分子生物学特性及异源表达方面的研究,旨在认识嗜卷书虱细胞色素P450的生理功能、了解P450与书虱抗性形成与发展的相互关系、鉴定书虱抗性相关P450基因以及揭示其介导书虱抗药性的分子机制。通过近4年的研究,取得了如下研究结果：
1嗜卷书虱Housekeeping基因的克隆及内参基因的筛选
1.1 Housekeeping基因的克隆及序列分析
结合反转录PCR(reverse transcriptase-PCR,RT-PCR)与cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)等技术,首次成功地从嗜卷书虱体内分离克隆获得了4个不同的Housekeeping基因Lbp-Actin1、Lbβ-Actin2、Lba-Tubulin及LbGapdh的cDNA全序列,GenBank登录号分别为FJ196622、FJ447483、FJ595242及FJ595241.通过序列分析,明确了4个Housekeeping基因的开放阅读框,并推导了其编码的氨基酸序列。进一步利用Protparam、Scanprosite、PSORT、TMHMM、SignalP 3.0以及ProtFun等生物信息学软件分析了上述Housekeeping基因编码蛋白质的理化性质、保守基序、细胞内的定位、跨膜结构、信号肽序列以及潜在的生理功能。使用Mega 4.01软件应用Neighbor Joining方法构建了其相应的系统发育树并明确了与其它物种相应Housekeeping基因的遗传距离。此外,应用蛋白质三维结构同源模拟工具SWISS-MODEL,成功地构建了各自的三维结构模型。研究结果丰富了嗜卷书虱的遗传信息,并且为将来深入探讨啮目昆虫的进化关系提供了可能的分子标记。1.2内参基因的筛选
研究表明,在实时定量PCR(qPCR)中选用不同的内参基因可能会对基因表达转录分析的结果产生重要影响。然而在昆虫学的相关研究中,内参基因筛选这个重要的问题却常被忽视。本研究在成功建立嗜卷书虱5个候选内参基因(Lbβ-Actin1、Lbβ-Actin2、Lba-Tubulin、LbGapdh及18S rRNA)qPCR反应体系的基础上,利用NormFinder和geNorm分析软件综合评估了这5个候选内参基因在嗜卷书虱不同发育阶段、不同品系以及溴氰菊酯诱导条件下mRNA表达的稳定性,筛选出了适用于不同试验条件的嗜卷书虱基因表达转录分析的内参基因Lbp-Actin1。进一步利用绝对定量方法分析了嗜卷书虱经药剂诱导其体内Lbβ-Actin1 mRNA表达量的时间动态(8、12、24、36及48 h),验证了其作为嗜卷书虱基因表达转录分析中内参基因的稳定性。同时以最稳定的Lbβ-Actinl及最不稳定的18S rRNA分别作为内参对嗜卷书虱CYP6CE2在不同发育阶段、药剂诱导前后的相对表达量进行比较分析,结果发现无论是在不同的发育阶段,还是在药剂诱导的情况下以18S rRNA作为内参时CYP6CE2的表达模式均与以Lbp-Actin1作为内参基因时的表达模式差异极大,进一步证实了前人关于核糖体基因不适宜用作内参基因的推测,充分证明了基因表达转录分析中内参基因的选用对定量分析的结果具有重要的影响。
2嗜卷书虱P450基因的克隆及其表达模式解析
2.1 P450基因的克隆及序列分析
结合RT-PCR与RACE等技术,首次成功地从嗜卷书虱体内分离克隆了5个全新的P450基因CYP6CE、CYP6CE2、CYP4CB1、CYP4CC1及CYP4CD1的cDNA全序列,GenBank登录号分别为EF421245、EF421246、EU979550、EU979549和EU979551。通过序列分析明确了上述5个P450基因的开放阅读框,并推导了其编码的氨基酸序列。进一步利用Protparam、Scanprosite、PSORT、TMHMM、SignalP3.0以及ProtFun等生物信息学软件分析了推导蛋白质的理化性质、保守基序、细胞内的定位、跨膜结构、信号肽序列以及潜在的生理功能。序列分析结果表明克隆获得的5个细胞色素P450基因均为细胞微粒体型P450,极有可能在嗜卷书虱体内参与外源化合物的代谢。在GenBank中选取了近40条已发布且功能己知的P450基因(主要为第四家族和第六家族成员)编码的氨基酸序列,使用Mega 4.01软件应用Neighbor Joining方法构建了系统发育树并明确了与其它物种P450基因的遗传距离。结果表明CYP6CE1、CYP6CE2与多个抗性相关的CYP6家族成员具有较高的同源性,据此推测CYP6CE1、CYP6CE2可能参与嗜卷书虱抗药性的形成。此外,应用蛋白质三维结构同源模拟工具SWISS-MODEL成功地构建了CYP6CE1及CYP6CE2的三维结构模型。然而,由于在蛋白质晶体结构数据库中还未有与CYP4CB1、CYP4CC1及CYP4CD1同源性达到20%以上的蛋白,因此无法对嗜卷书虱这3个细胞色素P450蛋白的三维结构进行模拟。
2.2 P450在不同发育阶段的表达模式
利用嗜卷书虱在27.5℃的条件下完成各个不同发育阶段所需要不同时间的这一生物学特性,通过时间上的控制,获得了处于不同发育阶段(卵、一龄、二龄、三龄、四龄若虫以及成虫)的书虱用于总RNA的提取。进而通过制作标准曲线、计算引物的扩增效率以及分析扩增产物的熔解曲线,成功建立了嗜卷书虱CYP6CE1、CYP6CE2、CYP4CB1、CYP4CC1和CYP4CD1等5个P450基因的qPCR反应体系。以Lbp-Actin1基因作为内参,对5个P450基因在嗜卷书虱不同发育阶段的mRNA表达水平进行了相对定量分析。结果表明,嗜卷书虱3个CYP4家族的P450基因均在成虫期表达量最高。其中,CYP4CB1在嗜卷书虱二龄若虫体内的相对表达量显著低于其它龄期,在成虫期表达量最高,其余4个龄期内的表达均无显著差异。CYP4CC1的相对表达量在成虫体内最高,在卵和二龄若虫中较低。其中成虫期表达量约为卵期的7倍,二龄若虫期表达量约为卵期的0.5倍,而其余3个龄期的相对表达量波动幅度不大均在2左右。CYP4CD1在卵期的表达量最低,成虫期的表达量约为卵期的7倍,其余各龄期间的相对表达量近乎恒定,维持在卵期表达量的2倍左右,且无显著差异。
2.3 P450在药剂诱导后的表达模式
在生物测定明确了嗜卷书虱对溴氰菊酯、甲基对氧磷和涕灭威毒力的基础上,建立了两种不同的诱导体系：即低剂量(LC10)长时间持续诱导和较高剂量(LC50)短时间诱导体系。利用qPCR技术,以Lbβ-Actin1为内参基因,分析了嗜卷书虱5个P450基因在低剂量(LC10)溴氰菊酯诱导前后其mRNA表达量的变化,明确了嗜卷书虱5个P450基因分别对低剂量溴氰菊酯的诱导反应。结果表明,低剂量(LC10)溴氰菊酯诱导可显著提高嗜卷书虱体内CYP6CE1、CYP6CE2、CYP4CB1、CYP4CC1基因的相对表达量。其中,CYP6CE1表达量上升最高大约为对照组的2.8倍；CYP6CE2、CYP4CB1、CYP4CC1相对表达量上升了2倍左右；然而,CYP4CD1的相对表达量经低剂量溴氰菊酯诱导后却显著地下降。
此外,利用较高剂量(LC50)的溴氰菊酯、甲基对氧磷、涕灭威处理嗜卷书虱30 min后,采用qPCR技术分析了上述P450基因经药剂诱导后其mRNA表达的时间动态(8、12、24、36及48 h)。qPCR结果表明CYP6CE1、CYP6CE2、CYP4CB1及CYP4CC1可被溴氰菊酯、甲基对氧磷显著的诱导。其中,溴氰菊酯诱导后36 h CYP6CE1、CYP6CE2、CYP4CB1及CYP4CC1的相对表达量达到最高分别为对照的2.l、1.5、3.0以及1.8倍；甲基对氧磷诱导后24 h这4个P450基因的相对表达量达到最高峰分别为对照的3.9、1.5、6.6及2.6倍。涕灭威对嗜卷书虱4个可被溴氰菊酯、甲基对氧磷诱导的P450基因均无显著的诱导作用,而CYP4CD1的表达量在涕灭威的诱导后36 h达到高峰为对照的4.7倍。由此可见,CYP4CD1可能与其它4个P450基因具有不同的生理功能：CYP6CE1、CYP6CE2、CYP4CB1及CYP4CC1极有可能参与了溴氰菊酯、甲基对氧磷在嗜卷书虱体内的代谢过程,而CYP4CD1可能参与了涕灭威在嗜卷书虱体内的代谢过程。
2.4 P450在不同品系中的表达模式
利用qPCR技术,以Lbp-Actin1为内参基因,分析了嗜卷书虱5个P450基因在实验室选育并保存的嗜卷书虱DDVP、PH3抗性品系及敏感品系中的表达模式。结果表明,除CYP4CC1外其它4个P450基因在2个抗性品系中均有不同程度的过量表达,但上调表达的幅度不高(均未超过2倍),这一现象暗示这4个P450基因极有可能共同参与嗜卷书虱抗性的形成。由此可见,书虱抗性形成并非由单一的基因或酶类介导,而是其体内多种酶类协同作用的结果。
3嗜卷书虱P450基因的原核表达
基因异源表达技术是基因工程技术的核心,是功能基因组学研究中明确基因功能的一个基础研究。本研究采用Gateway(?)支术成功构建了嗜卷书虱CYP6CE1和Lbβ-Actin1基于pDestl7的原核表达载体,经Western Blot检测证实大肠杆菌中表达的重组蛋白就是CYP6CE1-pDestl7和Lbβ-Actinl-pDestl 7的重组蛋白,从而实现了这2个基因在大肠杆菌体内的异源表达。同时利用BamHI和Xhol的双酶切以及DNA重组技术构建了嗜卷书虱CYP6CE1、CYP4CB1、CYP4CC1及CYP4CD1基于pET4317.1a(+)的原核表达载体。为了提高P450基因异源表达产物的产量和产物的分离纯化等,通过对PCR上游引物5，的改造实现了对细胞色素P450蛋白的N端修饰。包括去除信号肽序列,将起始密码子ATG之后的第二个密码子替换为GCT,在该密码子之后引入4个连续的CAT编码组氨酸的标签等。最后,利用BamHI和XbaI的双酶切以及DNA重组技术,成功构建了嗜卷书虱CYP6CE1、CYP4CB1、CYP4CC1及CYP4CD1基于pCW的原核表达载体。异源表达的实现为将来深入研究表达产物的生化及毒理学特性奠定了坚实的基础。
4嗜卷书虱CYP6CE1等位基因多态性
P450等位基因的点突变对其蛋白结构、底物识别、催化活性及功能具有重要影响。本研究采用RT-PCR技术从嗜卷书虱敏感品系和2个抗性品系体内分离克隆了3个CYP6CE1的等位基因CYP6CE1v1、CYP6CE1v2和CYP6CE1v3(GenBank登录号分别为EF421245、EU266572及EU266573)。序列比对发现CYP6CE1v2与CYP6CE1v3核苷酸序列中有15个多态性位点及其所在的位置。序列分析明确了CYP6CE1v2的15处多态性位点仅导致了其编码的蛋白质1处氨基酸替换,而CYP6CE1v3编码的蛋白质却发生了5处氨基酸替换。利用Protparam软件对CYP6CE1v1-3所编码蛋白质的基本参数进行预测,发现CYP6CE1v2与CYP6CE1v1编码的蛋白质在性质上差异不大,但与CYP6CE1v3编码的蛋白质在性质上存在明显差异。进一步使用蛋白质三维结构同源模拟工具SWISS-MODEL对上述3个等位基因编码蛋白质的三维结构进行模拟,在理论上证实了CYP6CE1v3编码蛋白的3处氨基酸替换均对其三维结构产生一定的影响。
综上所述,本研究克隆获得了嗜卷书虱体内的4个持家基因cDNA全序列,丰富了嗜卷书虱的遗传信息,并且为将来深入探讨啮目昆虫的进化关系提供了可能的分子标记；分离获得了5个细胞色素P450新基因的cDNA全序列,为深入研究嗜卷书虱P450酶系统的功能奠定了坚实的基础；利用分子生物学最新研究成果筛选出了基因表达转录分析中稳定表达的内参基因,搭建了书虱基因表达转录分析的研究平台；并在此基础上,利用实时定量PCR技术全面解析了这5个细胞色素P450基因在嗜卷书虱不同发育阶段、不同品系以及药剂诱导后的表达模式；此外,还进行了细胞色素P450的原核表达和重组蛋白离体研究。研究成果将促进对嗜卷书虱细胞色素P450基因生理功能的认识,为嗜卷书虱抗性相关P450基因的鉴定提供理论依据,并对阐释细胞色素P450介导嗜卷书虱代谢抗性的分子生物学机制具有重要的理论意义。
The psocid, Liposcelis bostrychophila Badonnel (Psocoptera:Liposcelidae), is an important stored-product insect. It is distributed worldwide and is commonly found in various processed and unprocessed dry foods in households, granaries, and warehouses. Outbreaks can lead to significant economic losses and rapid resistance to insecticides resulting in control failure have made L. bostrychophila a pest of interest to researchers globally. However, compared to other agricultural insect pests, stored products pest research (including psocids) was mainly focused on control aspects, while basic research was lacking. P450 enzymes (cytochrome P450 monooxygenases), an important enzyme system in insecticide detoxification, are a complex family of heme-binding enzymes found in most organisms. In insects, the diverse functions of P450 enzymes range from the synthesis and degradation of ecdysteroids and juvenile hormones to the metabolism of xenobiotics. P450 enzymes also play important roles in the adaptation of insects to toxic compounds in their host plants, and are involved in metabolism of almost all commonly used insecticides. Because of the diversity of their substrates, P450-mediated detoxification has the potential to confer cross-resistance to toxins independent of their target site, and thus P450-mediated resistance is now regarded as the most frequent type of metabolism-based insecticide resistance. Therefore, we initiated the study on the molecular characterizations and heterologous expression of P450s in L. bostrychophila. The purpose of the current study was to understand the physiological roles of psocid P450s; to explore the relationship between P450s and insecticide resistance in psocids; to identify P450 genes linked to resistance; and to reveal the molecular mechanisms of insecticide resistance in psocids. The research was supported by the National Natural Sciences Foundation (30871631),Program for New Century Excellent Talents in University (NCET-04-0854) and the Science and Technology Innovation Foundation for Graduate Students (kb2008001).The main results are as follows:
1 Housekeeping genes cloning and reference selection
1.1 Molecular cloning and sequences analysis of housekeeping genes
Four housekeeping genes, Lbβ-Actinl,Lbβ-Actin2, Lba-Tubulin, and LbGapdh, were cloned from L. bostrychophila using the combined techniques of reverse transcriptase-PCR (RT-PCR) with rapid amplification of cDNA ends (RACE). The GenBank accession numbers were FJ196622, FJ447483,FJ595242, and FJ595241,respectively. Molecular characterizations of the putative proteins have been predicted by Protparam.The full-length cDNA of Lbβ-Actinl was a 1772 bp sequence with an open reading frame (ORF) encoding 376 amino acids, while Lbβ-Actin2 was 1350 bp in length containing an ORF encoding 376 amino acids. In addition, the 1565 bp cDNA of Lba-Tubulin had an ORF of 1350 bp encoding 450 amino acids and LbGapdh had an ORF of 333 amino acids. Furthermore, the bioinformatics software such as Scanprosite, PSORT, TMHMM, SignalP 3.0 and ProtFun were used to predict the conserved motif, membrane location, transmembrane structure, signal peptide and potential functions of the putative protein. Phylogenetic trees generated from the nucleotide sequences of their coding regions revealed a relationship that was closer to other insects than to mammals. In addition, their 3-D structure models were constructed by SWISS-MODEL. The housekeeping gene sequences obtained will be helpful to future studies of the psocid L. bostrychophila and related species.
1.2 Evaluation and validation of reference genes for gene expression profiling
Several studies have shown that the reference genes used for the quantification of mRNA expression may be affected by the experimental design or cell type, however, this important aspect is often neglected in gene expression studies of insects. In the current study, the stabilities of five reference gene candidates (Lbβ-Actinl,Lbβ-Actin2, Lba-Tubulin, LbGapdh, and 18S rRNA) were systematically analyzed among the developmental stages and between different strains after deltamethrin induction. The aim of this study was to evaluate housekeeping genes of L. bostrychophila for their suitability as reference genes in quantitative real-time PCR (qPCR) using geNorm and NormFinder, thus provide appropriate reference genes to explore the gene expression patterns of the detoxifying and target enzymes in psocids to clarify the resistance mechanisms at a molecular level. According to the analysis by geNorm, we ranked the stability of the five psocid housekeeping genes after insecticide induction in the following order:Lbβ-Actinl>LbGapdh> Lba-Tubulin>Lbβ-Actin2>Lb18S rRNA. Furthermore, the mRNA expression of selected psocid genes at different life stages was ranked in the following order:Lbβ-Actinl>Lba-Tubulin> Lbβ-Actin2>LbGapdh>Lb18S rRNA.Additionally, the ranking for different strains was Lbβ-Actinl>LbGapdh>Lba-Tubulin>Lbβ-Actin2>18s rRNA. Although there were differences between NormFinder and geNorm, most the results were the same. Therefore, combined the analysis of NormFinder and geNorm, Lbβ-Actinl was selected as the most appropriate reference gene for accurate normalization, while the 18S rRNA is the least stable gene.
Two representative reference genes (Lbβ-Actinl,the most stable one; Lb18S rRNA, the least stable one) were applied to determine the expression pattern of a P450 gene encoding CYP6CE2. For deltamethrin induction, the CYP6CE2 transcripts increased significantly normalized to Lbβ-Actinl and Lb18S rRNA, respectively. However, the increase was falsely amplified from 1.9 to 3.4 fold when normalized to Lb18S rRNA.Furthermore, the developmental expression profile of CYP6CE2 normalized to Lbβ-Actinl was different when normalized to Lb18S rRNA, suggesting that the stability of the reference gene had an effect on gene profiling. In addition, the expression stability of Lbβ-Actinl was further investigated in psocids after insecticide induction. These results found that there was no significant difference between the induced and control groups, suggesting that Libβ-actinl was an appropriate internal control for the gene expression profiling in this insect. Our study demonstrated that each candidate reference gene should be validated before use to make sure it is stably expressed under the given experimental manipulation to ensure accurate normalization.
2 P450 gene cloning and gene expression profiling
2.1 Molecular cloning and sequences analysis of P450 genes
Five novel P450 genes, CYP6CE1,CYP6CE2, CYP4CB1,CYP4CC1 and CYP4CD1,were cloned from L. bostrychophila using the combined techniques of reverse transcriptase-PCR (RT-PCR) with rapid amplification of cDNA ends (RACE). GenBank accession numbers were EF421245, EF421246, EU979550, EU979549 and EU979551,respectively. Molecular characterizations of the putative proteins have been predicted by Protparam. The full-length cDNA of CYP6CE1 was a 2025 bp sequence with an open reading frame (ORF) encoded 527 amino acids, while CYP6CE2 was 1822 bp in length containing an ORF encoded 521 amino acids. Furthermore, the 1772 bp cDNA of CYP4CB1 had an ORF encoding 511 amino acids, while CYP4CCl.was 1853 bp with an ORF encoding 504 amino acids. Moreover, CYP4CD1 possessed an ORF of 512 amino acids. Furthermore, the bioinformatics software such as Scanprosite, PSORT, TMHMM, SignalP 3.0 and ProtFun were used to predict the conserved motif, membrane location, transmembrane structure, signal peptide and potential functions of the putative proteins. In addition, the 3-D structure models of CYP6CE1 and CYP6CE2 were constructed by SWISS-MODEL using CYP3A4 as template; however, we were unable to construct structure models for other P450s because the protein database does not currently have a sufficient number of template proteins.
2.2 Developmental expression profiles of the five P450 genes
In the current study, we analyzed the developmental expression patterns of the cloned P450 genes from L. bostrychophila to provide insights as to the protein physiological functions. We obtained L.bostrychophila in different, but uniform developmental stages including egg,1st stadium nymph,2nd stadium nymph,3rd stadium nymph,4th stadium nymph, and adult to extract total RNA for gene expression profiling. According to the results of qPCR, transcripts of the five P450 genes were detected at all the life stages tested and none were found to be stage specific. Higher expression levels of CYP6CE2 were observed at nymphal stages than at adulthood. The CYP6CE2 transcripts decrease progressively from early to late developmental stages. The relative quantity of CYP6CE2 was highest in 1st nymph and gradually decreased to the lowest in adults. However, there was no significant difference in expression among the 3rd,4th stadium and the adults (P>0.05). Additionally, as indicated by qPCR, the relative quantity for all life stages of CYP6CE1 was between 1 and 2 folds. The three CYP4 members (CYP4CB1,CYP4CC1 and CYP4CD1)were relatively stable. Similar to CYP6CE1,the relative quantity for all life stages was between 1 and 2 folds. Interestingly, all of them were more highly expressed at adulthood. These results imply that CYP6CE1,CYP4CB1,CYP4CC1 and CYP4CD1 may have important roles in adult psocids, while CYP6CE2 may be more crucial to nymphal psocids.
2.3 Expression profiles of the five P450 genes induced by insecticides
We determined the susceptibility of L. bostrychophila to deltamethrin, paraoxon-methyl and aldicarb using a survivorship bioassay. To determine the induction responses of P450s of the cloned P450s, three insecticides were used to induce the psocids. Based on the bioassay, the psocids were induced by the insecticides in two different regimes:LL, low dose (LC10)+long time (24 h) and HS, high dose (LC50)+short time (0.5 h). Real-time PCR with Lbβ-Actinl as the reference gene was used to determine the relative expression quantity of the P450 genes. As indicated by real-time RT-PCR, four P450 genes (CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1)transcripts increased significantly after exposure to 2 mg/L deltamethrin for 24 h (P<0.05), while CYP4CD1 decreased a little. Compared to the control group, CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1 transcripts increased 2.8,1.9,1.8 and 2.3 folds, respectively. However, the relative quantity of CYP4CD1 decreased significantly (P<0.05).
In addition, we also investigated the time course effects of insecticides (deltamethrin, paraoxon-methyl and aldicarb) on the expression patterns of the five P450 genes following HS regime. With the exception of CYP4CD1,the other P450s expression was highest at 36 h after deltamethrin induction. Compared to the control, CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1 transcripts increased 2.5,1.5,3.0 and 1.8 folds. Similar to deltamethrin, paraoxon-methyl showed good induction to CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1,but had no obvious effect on CYP4CD1.Twenty four hours after the paraoxon-methyl induction, the relative quantity of CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1 reached the 3.9,1.5,6.6 and 2.6 folds, respectively. CYP6CE1 transcripts increased 1.5 fold after 36 h of aldicarb induction, while CYP6CE2 and CYP4CB1 had no significant response. Additionally, the expression of CYP4CC1 was suppressed during the treatment. Interestingly CYP4CD1 transcripts, which had no response to deltamethrin and paraoxon-methyl, increased quickly 8 h after aldicarb induction and reached the peak 36 h later. These results suggest that CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1 are inducible to deltamethrin and paraoxon-methyl, while CYP4CD1 was induced by aldicarb. Furthermore, CYP6CE2, CYP4CB1 and CYP4CC1 may be involved in the metabolism of deltamethrin and paraoxon-methyl in L.bostrychophila. Meanwhile, CYP4CD1 was related to aldicarb metabolism.
However, we do not know which pathway is involved in controlling P450s transcriptional response to insecticides in psocids. We also do not know what triggers it and how. Further studies including the cloning and analyzing the nuclear receptors and the upstream regions of these P450 genes may provide some information.
2.4 Expression profiles of the five P450 genes among different strains
To verify whether P450s are over-expressed in resistant psocids, real-time PCR with the Lbβ-Actin1 as the reference gene was employed to determine the relative expression quantity of the P450 genes in three different strains of the psocids. Three strains, including susceptible strain (SS), DDVP resistant strain (DDVP-R) and PH3 resistant strain (PH3-R), were started at Southwest University Key Laboratory of Entomology and Pest Control Engineering and subjected to artificial selection to applied pesticides since the 1990s. As indicated by qPCR, four P450 genes (CYP6CE1, CYP6CE2, CYP4CB1 and CYP4CC1)transcripts in DDVP-R and PH3-R strains were significantly higher than SS strain (P<0.05), while the expression of CYP4CD1 was not significantly different (P>0.05), which implied that the overexpressed P450s were possibly involved in psocids resistance. However, the relative expression levels of the overexpressed P450s were relatively low (less than 2 fold). Moreover, as has been demonstrated by our previous studies several other key enzymes such as GSTs and AChE were related to pesticide resistance. Therefore, we assumed that the psocids resistance was a result of the cooperation of the enzymes mentioned above. However, we currently lack sufficient evidence to show that CYP6CE1 and CYP6CE2 are involved in deltamethrin metabolism in psocids. Future studies on substrate specificity of the recombinant proteins will provide direct evidence.
3 Heterologous expression of psocids P450 in Escherichia coli
Heterologous expression of foreign genes is the core field in functional genomics research, which will provide great insights to the gene function. Thus, biochemical and toxicological properties determination of the recombinant proteins based on the heterologous expression will provide direct evidence to the P450s function study. In the present study, on the basis of Gateway(?) technology, we constructed prokaryotic expression vectors (ligated to pDest 17) for CYP6CE1 and Lb(3-Actinl.Moreover, CYP6CE1 and Lbβ-Actin1 were successfully expressed in E. coli and the recombinant proteins were further confirmed by Western Blot analysis. Additionally, applying the double digestion of BamHI and Xhol restriction enzymes with the DNA recombination technology, we constructed expression vectors for CYP6CE1,CYP4CB1,CYP4CC1 and CYP4CD1 based on pET4317.1a (+)vector. In order to improve the heterologous expression efficiency and convenience to recombinant protein purification, N-terminus modification as suggested by previous researcher was performed for expression vector construction. Signal peptide sequences of the putative protein sequences were discarded, the second codon behind the start codon ATG was changed to GCT. If necessary, four continuous codons of CAT could be added to form a 4×His tag for recombinant protein purification. According to the N-terminus modification, we constructed expression vectors for CYP6CE1,CYP4CB1,CYP4CC1 and CYP4CD1 based on pCW plasmid. Our results formed a solid basis to express psocid P450s effectively in E. coli.
4 Polymorphism of CYP6CE1 alleles
It has been reported that mutations of P450 alleles could result in changes in protein structure, substrate recognition and catalytic activity. In this study, three alleles (GenBank accession numbers were EF421245, EU266572 and EU266573) of CYP6CE1 were cloned from 3 strains (DDVP, PH3 resistant strains and susceptible strain) of L.bostrychophila using the combined techniques of reverse transcriptase-PCR and sequenced. According to the sequences alignment, fifteen polymorphism sites were found in the nucleotide sequences of CYP6CElv2 and CYP6CElv3, respectively. We also determined their locations in the sequences. Based on the sequence analysis, the 15 nucleotide polymorphism sites of CYP6CE1v2 resulted in only one amino acid substitution, while five amino acid alterations were found in CYP6CElv3.Protein parameters of CYP6CElvl-3 were predicted by Proparam.As indicated by the prediction, there was no significant difference in properties of CYP6CE1v2 and CYP6CE1v1 protein, which were different from CYP6CE1v3 protein, especially in theoretical pⅠ.Furthermore,3-D structure homology modeling of these three proteins by SWISS-MODEL demonstrated that three amino acid substitutions in CYP6CE1v3 resulted in its structure alteration.
In summary, in the present study we cloned the full-length cDNA of four housekeeping genes from L. bostrychophila, which extended sequence information for Psocoptera and provided potential molecular markers to investigate the evolutionary relationship between the Psocoptera and other insects. Five novel P450 genes were isolated form L. bostrychophila, which formed a solid basis to explore the physiological functions undertaken by P450 enzyme system.We systematically evaluated and validated the expression stability of housekeeping genes, and selected only those stably expressed as reference. This constructed a platform for the gene expression profiling in L. bostrychophila using real-time PCR. Based on the reference selection, we determined the developmental expression patterns of these P450s and their expression profiles in the adults after exposure to several insecticides, as well as the relative expression quantity in different strains showing different resistance to DDVP and PH3.Furthermore, we constructed different expression vectors for heterologous expression of psocid P450 in E. coli, in which CYP6CE1 and Lbβ-Actinl were successfully expressed. Our results will not only provide great insights into exploring the functions of the psocid P450s system in development and physiology, but also provide evidence for clarifying the adaptive mechanisms mechanism of psocids to environment. In the meantime, we may enrich and develop the scientific theoretical study system for the resistance mechanism of stored-product insect pests.
1嗜卷书虱Housekeeping基因的克隆及内参基因的筛选
1.1 Housekeeping基因的克隆及序列分析
结合反转录PCR(reverse transcriptase-PCR,RT-PCR)与cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)等技术,首次成功地从嗜卷书虱体内分离克隆获得了4个不同的Housekeeping基因Lbp-Actin1、Lbβ-Actin2、Lba-Tubulin及LbGapdh的cDNA全序列,GenBank登录号分别为FJ196622、FJ447483、FJ595242及FJ595241.通过序列分析,明确了4个Housekeeping基因的开放阅读框,并推导了其编码的氨基酸序列。进一步利用Protparam、Scanprosite、PSORT、TMHMM、SignalP 3.0以及ProtFun等生物信息学软件分析了上述Housekeeping基因编码蛋白质的理化性质、保守基序、细胞内的定位、跨膜结构、信号肽序列以及潜在的生理功能。使用Mega 4.01软件应用Neighbor Joining方法构建了其相应的系统发育树并明确了与其它物种相应Housekeeping基因的遗传距离。此外,应用蛋白质三维结构同源模拟工具SWISS-MODEL,成功地构建了各自的三维结构模型。研究结果丰富了嗜卷书虱的遗传信息,并且为将来深入探讨啮目昆虫的进化关系提供了可能的分子标记。1.2内参基因的筛选
研究表明,在实时定量PCR(qPCR)中选用不同的内参基因可能会对基因表达转录分析的结果产生重要影响。然而在昆虫学的相关研究中,内参基因筛选这个重要的问题却常被忽视。本研究在成功建立嗜卷书虱5个候选内参基因(Lbβ-Actin1、Lbβ-Actin2、Lba-Tubulin、LbGapdh及18S rRNA)qPCR反应体系的基础上,利用NormFinder和geNorm分析软件综合评估了这5个候选内参基因在嗜卷书虱不同发育阶段、不同品系以及溴氰菊酯诱导条件下mRNA表达的稳定性,筛选出了适用于不同试验条件的嗜卷书虱基因表达转录分析的内参基因Lbp-Actin1。进一步利用绝对定量方法分析了嗜卷书虱经药剂诱导其体内Lbβ-Actin1 mRNA表达量的时间动态(8、12、24、36及48 h),验证了其作为嗜卷书虱基因表达转录分析中内参基因的稳定性。同时以最稳定的Lbβ-Actinl及最不稳定的18S rRNA分别作为内参对嗜卷书虱CYP6CE2在不同发育阶段、药剂诱导前后的相对表达量进行比较分析,结果发现无论是在不同的发育阶段,还是在药剂诱导的情况下以18S rRNA作为内参时CYP6CE2的表达模式均与以Lbp-Actin1作为内参基因时的表达模式差异极大,进一步证实了前人关于核糖体基因不适宜用作内参基因的推测,充分证明了基因表达转录分析中内参基因的选用对定量分析的结果具有重要的影响。
2嗜卷书虱P450基因的克隆及其表达模式解析
2.1 P450基因的克隆及序列分析
结合RT-PCR与RACE等技术,首次成功地从嗜卷书虱体内分离克隆了5个全新的P450基因CYP6CE、CYP6CE2、CYP4CB1、CYP4CC1及CYP4CD1的cDNA全序列,GenBank登录号分别为EF421245、EF421246、EU979550、EU979549和EU979551。通过序列分析明确了上述5个P450基因的开放阅读框,并推导了其编码的氨基酸序列。进一步利用Protparam、Scanprosite、PSORT、TMHMM、SignalP3.0以及ProtFun等生物信息学软件分析了推导蛋白质的理化性质、保守基序、细胞内的定位、跨膜结构、信号肽序列以及潜在的生理功能。序列分析结果表明克隆获得的5个细胞色素P450基因均为细胞微粒体型P450,极有可能在嗜卷书虱体内参与外源化合物的代谢。在GenBank中选取了近40条已发布且功能己知的P450基因(主要为第四家族和第六家族成员)编码的氨基酸序列,使用Mega 4.01软件应用Neighbor Joining方法构建了系统发育树并明确了与其它物种P450基因的遗传距离。结果表明CYP6CE1、CYP6CE2与多个抗性相关的CYP6家族成员具有较高的同源性,据此推测CYP6CE1、CYP6CE2可能参与嗜卷书虱抗药性的形成。此外,应用蛋白质三维结构同源模拟工具SWISS-MODEL成功地构建了CYP6CE1及CYP6CE2的三维结构模型。然而,由于在蛋白质晶体结构数据库中还未有与CYP4CB1、CYP4CC1及CYP4CD1同源性达到20%以上的蛋白,因此无法对嗜卷书虱这3个细胞色素P450蛋白的三维结构进行模拟。
2.2 P450在不同发育阶段的表达模式
利用嗜卷书虱在27.5℃的条件下完成各个不同发育阶段所需要不同时间的这一生物学特性,通过时间上的控制,获得了处于不同发育阶段(卵、一龄、二龄、三龄、四龄若虫以及成虫)的书虱用于总RNA的提取。进而通过制作标准曲线、计算引物的扩增效率以及分析扩增产物的熔解曲线,成功建立了嗜卷书虱CYP6CE1、CYP6CE2、CYP4CB1、CYP4CC1和CYP4CD1等5个P450基因的qPCR反应体系。以Lbp-Actin1基因作为内参,对5个P450基因在嗜卷书虱不同发育阶段的mRNA表达水平进行了相对定量分析。结果表明,嗜卷书虱3个CYP4家族的P450基因均在成虫期表达量最高。其中,CYP4CB1在嗜卷书虱二龄若虫体内的相对表达量显著低于其它龄期,在成虫期表达量最高,其余4个龄期内的表达均无显著差异。CYP4CC1的相对表达量在成虫体内最高,在卵和二龄若虫中较低。其中成虫期表达量约为卵期的7倍,二龄若虫期表达量约为卵期的0.5倍,而其余3个龄期的相对表达量波动幅度不大均在2左右。CYP4CD1在卵期的表达量最低,成虫期的表达量约为卵期的7倍,其余各龄期间的相对表达量近乎恒定,维持在卵期表达量的2倍左右,且无显著差异。
2.3 P450在药剂诱导后的表达模式
在生物测定明确了嗜卷书虱对溴氰菊酯、甲基对氧磷和涕灭威毒力的基础上,建立了两种不同的诱导体系：即低剂量(LC10)长时间持续诱导和较高剂量(LC50)短时间诱导体系。利用qPCR技术,以Lbβ-Actin1为内参基因,分析了嗜卷书虱5个P450基因在低剂量(LC10)溴氰菊酯诱导前后其mRNA表达量的变化,明确了嗜卷书虱5个P450基因分别对低剂量溴氰菊酯的诱导反应。结果表明,低剂量(LC10)溴氰菊酯诱导可显著提高嗜卷书虱体内CYP6CE1、CYP6CE2、CYP4CB1、CYP4CC1基因的相对表达量。其中,CYP6CE1表达量上升最高大约为对照组的2.8倍；CYP6CE2、CYP4CB1、CYP4CC1相对表达量上升了2倍左右；然而,CYP4CD1的相对表达量经低剂量溴氰菊酯诱导后却显著地下降。
此外,利用较高剂量(LC50)的溴氰菊酯、甲基对氧磷、涕灭威处理嗜卷书虱30 min后,采用qPCR技术分析了上述P450基因经药剂诱导后其mRNA表达的时间动态(8、12、24、36及48 h)。qPCR结果表明CYP6CE1、CYP6CE2、CYP4CB1及CYP4CC1可被溴氰菊酯、甲基对氧磷显著的诱导。其中,溴氰菊酯诱导后36 h CYP6CE1、CYP6CE2、CYP4CB1及CYP4CC1的相对表达量达到最高分别为对照的2.l、1.5、3.0以及1.8倍；甲基对氧磷诱导后24 h这4个P450基因的相对表达量达到最高峰分别为对照的3.9、1.5、6.6及2.6倍。涕灭威对嗜卷书虱4个可被溴氰菊酯、甲基对氧磷诱导的P450基因均无显著的诱导作用,而CYP4CD1的表达量在涕灭威的诱导后36 h达到高峰为对照的4.7倍。由此可见,CYP4CD1可能与其它4个P450基因具有不同的生理功能：CYP6CE1、CYP6CE2、CYP4CB1及CYP4CC1极有可能参与了溴氰菊酯、甲基对氧磷在嗜卷书虱体内的代谢过程,而CYP4CD1可能参与了涕灭威在嗜卷书虱体内的代谢过程。
2.4 P450在不同品系中的表达模式
利用qPCR技术,以Lbp-Actin1为内参基因,分析了嗜卷书虱5个P450基因在实验室选育并保存的嗜卷书虱DDVP、PH3抗性品系及敏感品系中的表达模式。结果表明,除CYP4CC1外其它4个P450基因在2个抗性品系中均有不同程度的过量表达,但上调表达的幅度不高(均未超过2倍),这一现象暗示这4个P450基因极有可能共同参与嗜卷书虱抗性的形成。由此可见,书虱抗性形成并非由单一的基因或酶类介导,而是其体内多种酶类协同作用的结果。
3嗜卷书虱P450基因的原核表达
基因异源表达技术是基因工程技术的核心,是功能基因组学研究中明确基因功能的一个基础研究。本研究采用Gateway(?)支术成功构建了嗜卷书虱CYP6CE1和Lbβ-Actin1基于pDestl7的原核表达载体,经Western Blot检测证实大肠杆菌中表达的重组蛋白就是CYP6CE1-pDestl7和Lbβ-Actinl-pDestl 7的重组蛋白,从而实现了这2个基因在大肠杆菌体内的异源表达。同时利用BamHI和Xhol的双酶切以及DNA重组技术构建了嗜卷书虱CYP6CE1、CYP4CB1、CYP4CC1及CYP4CD1基于pET4317.1a(+)的原核表达载体。为了提高P450基因异源表达产物的产量和产物的分离纯化等,通过对PCR上游引物5，的改造实现了对细胞色素P450蛋白的N端修饰。包括去除信号肽序列,将起始密码子ATG之后的第二个密码子替换为GCT,在该密码子之后引入4个连续的CAT编码组氨酸的标签等。最后,利用BamHI和XbaI的双酶切以及DNA重组技术,成功构建了嗜卷书虱CYP6CE1、CYP4CB1、CYP4CC1及CYP4CD1基于pCW的原核表达载体。异源表达的实现为将来深入研究表达产物的生化及毒理学特性奠定了坚实的基础。
4嗜卷书虱CYP6CE1等位基因多态性
P450等位基因的点突变对其蛋白结构、底物识别、催化活性及功能具有重要影响。本研究采用RT-PCR技术从嗜卷书虱敏感品系和2个抗性品系体内分离克隆了3个CYP6CE1的等位基因CYP6CE1v1、CYP6CE1v2和CYP6CE1v3(GenBank登录号分别为EF421245、EU266572及EU266573)。序列比对发现CYP6CE1v2与CYP6CE1v3核苷酸序列中有15个多态性位点及其所在的位置。序列分析明确了CYP6CE1v2的15处多态性位点仅导致了其编码的蛋白质1处氨基酸替换,而CYP6CE1v3编码的蛋白质却发生了5处氨基酸替换。利用Protparam软件对CYP6CE1v1-3所编码蛋白质的基本参数进行预测,发现CYP6CE1v2与CYP6CE1v1编码的蛋白质在性质上差异不大,但与CYP6CE1v3编码的蛋白质在性质上存在明显差异。进一步使用蛋白质三维结构同源模拟工具SWISS-MODEL对上述3个等位基因编码蛋白质的三维结构进行模拟,在理论上证实了CYP6CE1v3编码蛋白的3处氨基酸替换均对其三维结构产生一定的影响。
综上所述,本研究克隆获得了嗜卷书虱体内的4个持家基因cDNA全序列,丰富了嗜卷书虱的遗传信息,并且为将来深入探讨啮目昆虫的进化关系提供了可能的分子标记；分离获得了5个细胞色素P450新基因的cDNA全序列,为深入研究嗜卷书虱P450酶系统的功能奠定了坚实的基础；利用分子生物学最新研究成果筛选出了基因表达转录分析中稳定表达的内参基因,搭建了书虱基因表达转录分析的研究平台；并在此基础上,利用实时定量PCR技术全面解析了这5个细胞色素P450基因在嗜卷书虱不同发育阶段、不同品系以及药剂诱导后的表达模式；此外,还进行了细胞色素P450的原核表达和重组蛋白离体研究。研究成果将促进对嗜卷书虱细胞色素P450基因生理功能的认识,为嗜卷书虱抗性相关P450基因的鉴定提供理论依据,并对阐释细胞色素P450介导嗜卷书虱代谢抗性的分子生物学机制具有重要的理论意义。
The psocid, Liposcelis bostrychophila Badonnel (Psocoptera:Liposcelidae), is an important stored-product insect. It is distributed worldwide and is commonly found in various processed and unprocessed dry foods in households, granaries, and warehouses. Outbreaks can lead to significant economic losses and rapid resistance to insecticides resulting in control failure have made L. bostrychophila a pest of interest to researchers globally. However, compared to other agricultural insect pests, stored products pest research (including psocids) was mainly focused on control aspects, while basic research was lacking. P450 enzymes (cytochrome P450 monooxygenases), an important enzyme system in insecticide detoxification, are a complex family of heme-binding enzymes found in most organisms. In insects, the diverse functions of P450 enzymes range from the synthesis and degradation of ecdysteroids and juvenile hormones to the metabolism of xenobiotics. P450 enzymes also play important roles in the adaptation of insects to toxic compounds in their host plants, and are involved in metabolism of almost all commonly used insecticides. Because of the diversity of their substrates, P450-mediated detoxification has the potential to confer cross-resistance to toxins independent of their target site, and thus P450-mediated resistance is now regarded as the most frequent type of metabolism-based insecticide resistance. Therefore, we initiated the study on the molecular characterizations and heterologous expression of P450s in L. bostrychophila. The purpose of the current study was to understand the physiological roles of psocid P450s; to explore the relationship between P450s and insecticide resistance in psocids; to identify P450 genes linked to resistance; and to reveal the molecular mechanisms of insecticide resistance in psocids. The research was supported by the National Natural Sciences Foundation (30871631),Program for New Century Excellent Talents in University (NCET-04-0854) and the Science and Technology Innovation Foundation for Graduate Students (kb2008001).The main results are as follows:
1 Housekeeping genes cloning and reference selection
1.1 Molecular cloning and sequences analysis of housekeeping genes
Four housekeeping genes, Lbβ-Actinl,Lbβ-Actin2, Lba-Tubulin, and LbGapdh, were cloned from L. bostrychophila using the combined techniques of reverse transcriptase-PCR (RT-PCR) with rapid amplification of cDNA ends (RACE). The GenBank accession numbers were FJ196622, FJ447483,FJ595242, and FJ595241,respectively. Molecular characterizations of the putative proteins have been predicted by Protparam.The full-length cDNA of Lbβ-Actinl was a 1772 bp sequence with an open reading frame (ORF) encoding 376 amino acids, while Lbβ-Actin2 was 1350 bp in length containing an ORF encoding 376 amino acids. In addition, the 1565 bp cDNA of Lba-Tubulin had an ORF of 1350 bp encoding 450 amino acids and LbGapdh had an ORF of 333 amino acids. Furthermore, the bioinformatics software such as Scanprosite, PSORT, TMHMM, SignalP 3.0 and ProtFun were used to predict the conserved motif, membrane location, transmembrane structure, signal peptide and potential functions of the putative protein. Phylogenetic trees generated from the nucleotide sequences of their coding regions revealed a relationship that was closer to other insects than to mammals. In addition, their 3-D structure models were constructed by SWISS-MODEL. The housekeeping gene sequences obtained will be helpful to future studies of the psocid L. bostrychophila and related species.
1.2 Evaluation and validation of reference genes for gene expression profiling
Several studies have shown that the reference genes used for the quantification of mRNA expression may be affected by the experimental design or cell type, however, this important aspect is often neglected in gene expression studies of insects. In the current study, the stabilities of five reference gene candidates (Lbβ-Actinl,Lbβ-Actin2, Lba-Tubulin, LbGapdh, and 18S rRNA) were systematically analyzed among the developmental stages and between different strains after deltamethrin induction. The aim of this study was to evaluate housekeeping genes of L. bostrychophila for their suitability as reference genes in quantitative real-time PCR (qPCR) using geNorm and NormFinder, thus provide appropriate reference genes to explore the gene expression patterns of the detoxifying and target enzymes in psocids to clarify the resistance mechanisms at a molecular level. According to the analysis by geNorm, we ranked the stability of the five psocid housekeeping genes after insecticide induction in the following order:Lbβ-Actinl>LbGapdh> Lba-Tubulin>Lbβ-Actin2>Lb18S rRNA. Furthermore, the mRNA expression of selected psocid genes at different life stages was ranked in the following order:Lbβ-Actinl>Lba-Tubulin> Lbβ-Actin2>LbGapdh>Lb18S rRNA.Additionally, the ranking for different strains was Lbβ-Actinl>LbGapdh>Lba-Tubulin>Lbβ-Actin2>18s rRNA. Although there were differences between NormFinder and geNorm, most the results were the same. Therefore, combined the analysis of NormFinder and geNorm, Lbβ-Actinl was selected as the most appropriate reference gene for accurate normalization, while the 18S rRNA is the least stable gene.
Two representative reference genes (Lbβ-Actinl,the most stable one; Lb18S rRNA, the least stable one) were applied to determine the expression pattern of a P450 gene encoding CYP6CE2. For deltamethrin induction, the CYP6CE2 transcripts increased significantly normalized to Lbβ-Actinl and Lb18S rRNA, respectively. However, the increase was falsely amplified from 1.9 to 3.4 fold when normalized to Lb18S rRNA.Furthermore, the developmental expression profile of CYP6CE2 normalized to Lbβ-Actinl was different when normalized to Lb18S rRNA, suggesting that the stability of the reference gene had an effect on gene profiling. In addition, the expression stability of Lbβ-Actinl was further investigated in psocids after insecticide induction. These results found that there was no significant difference between the induced and control groups, suggesting that Libβ-actinl was an appropriate internal control for the gene expression profiling in this insect. Our study demonstrated that each candidate reference gene should be validated before use to make sure it is stably expressed under the given experimental manipulation to ensure accurate normalization.
2 P450 gene cloning and gene expression profiling
2.1 Molecular cloning and sequences analysis of P450 genes
Five novel P450 genes, CYP6CE1,CYP6CE2, CYP4CB1,CYP4CC1 and CYP4CD1,were cloned from L. bostrychophila using the combined techniques of reverse transcriptase-PCR (RT-PCR) with rapid amplification of cDNA ends (RACE). GenBank accession numbers were EF421245, EF421246, EU979550, EU979549 and EU979551,respectively. Molecular characterizations of the putative proteins have been predicted by Protparam. The full-length cDNA of CYP6CE1 was a 2025 bp sequence with an open reading frame (ORF) encoded 527 amino acids, while CYP6CE2 was 1822 bp in length containing an ORF encoded 521 amino acids. Furthermore, the 1772 bp cDNA of CYP4CB1 had an ORF encoding 511 amino acids, while CYP4CCl.was 1853 bp with an ORF encoding 504 amino acids. Moreover, CYP4CD1 possessed an ORF of 512 amino acids. Furthermore, the bioinformatics software such as Scanprosite, PSORT, TMHMM, SignalP 3.0 and ProtFun were used to predict the conserved motif, membrane location, transmembrane structure, signal peptide and potential functions of the putative proteins. In addition, the 3-D structure models of CYP6CE1 and CYP6CE2 were constructed by SWISS-MODEL using CYP3A4 as template; however, we were unable to construct structure models for other P450s because the protein database does not currently have a sufficient number of template proteins.
2.2 Developmental expression profiles of the five P450 genes
In the current study, we analyzed the developmental expression patterns of the cloned P450 genes from L. bostrychophila to provide insights as to the protein physiological functions. We obtained L.bostrychophila in different, but uniform developmental stages including egg,1st stadium nymph,2nd stadium nymph,3rd stadium nymph,4th stadium nymph, and adult to extract total RNA for gene expression profiling. According to the results of qPCR, transcripts of the five P450 genes were detected at all the life stages tested and none were found to be stage specific. Higher expression levels of CYP6CE2 were observed at nymphal stages than at adulthood. The CYP6CE2 transcripts decrease progressively from early to late developmental stages. The relative quantity of CYP6CE2 was highest in 1st nymph and gradually decreased to the lowest in adults. However, there was no significant difference in expression among the 3rd,4th stadium and the adults (P>0.05). Additionally, as indicated by qPCR, the relative quantity for all life stages of CYP6CE1 was between 1 and 2 folds. The three CYP4 members (CYP4CB1,CYP4CC1 and CYP4CD1)were relatively stable. Similar to CYP6CE1,the relative quantity for all life stages was between 1 and 2 folds. Interestingly, all of them were more highly expressed at adulthood. These results imply that CYP6CE1,CYP4CB1,CYP4CC1 and CYP4CD1 may have important roles in adult psocids, while CYP6CE2 may be more crucial to nymphal psocids.
2.3 Expression profiles of the five P450 genes induced by insecticides
We determined the susceptibility of L. bostrychophila to deltamethrin, paraoxon-methyl and aldicarb using a survivorship bioassay. To determine the induction responses of P450s of the cloned P450s, three insecticides were used to induce the psocids. Based on the bioassay, the psocids were induced by the insecticides in two different regimes:LL, low dose (LC10)+long time (24 h) and HS, high dose (LC50)+short time (0.5 h). Real-time PCR with Lbβ-Actinl as the reference gene was used to determine the relative expression quantity of the P450 genes. As indicated by real-time RT-PCR, four P450 genes (CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1)transcripts increased significantly after exposure to 2 mg/L deltamethrin for 24 h (P<0.05), while CYP4CD1 decreased a little. Compared to the control group, CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1 transcripts increased 2.8,1.9,1.8 and 2.3 folds, respectively. However, the relative quantity of CYP4CD1 decreased significantly (P<0.05).
In addition, we also investigated the time course effects of insecticides (deltamethrin, paraoxon-methyl and aldicarb) on the expression patterns of the five P450 genes following HS regime. With the exception of CYP4CD1,the other P450s expression was highest at 36 h after deltamethrin induction. Compared to the control, CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1 transcripts increased 2.5,1.5,3.0 and 1.8 folds. Similar to deltamethrin, paraoxon-methyl showed good induction to CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1,but had no obvious effect on CYP4CD1.Twenty four hours after the paraoxon-methyl induction, the relative quantity of CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1 reached the 3.9,1.5,6.6 and 2.6 folds, respectively. CYP6CE1 transcripts increased 1.5 fold after 36 h of aldicarb induction, while CYP6CE2 and CYP4CB1 had no significant response. Additionally, the expression of CYP4CC1 was suppressed during the treatment. Interestingly CYP4CD1 transcripts, which had no response to deltamethrin and paraoxon-methyl, increased quickly 8 h after aldicarb induction and reached the peak 36 h later. These results suggest that CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1 are inducible to deltamethrin and paraoxon-methyl, while CYP4CD1 was induced by aldicarb. Furthermore, CYP6CE2, CYP4CB1 and CYP4CC1 may be involved in the metabolism of deltamethrin and paraoxon-methyl in L.bostrychophila. Meanwhile, CYP4CD1 was related to aldicarb metabolism.
However, we do not know which pathway is involved in controlling P450s transcriptional response to insecticides in psocids. We also do not know what triggers it and how. Further studies including the cloning and analyzing the nuclear receptors and the upstream regions of these P450 genes may provide some information.
2.4 Expression profiles of the five P450 genes among different strains
To verify whether P450s are over-expressed in resistant psocids, real-time PCR with the Lbβ-Actin1 as the reference gene was employed to determine the relative expression quantity of the P450 genes in three different strains of the psocids. Three strains, including susceptible strain (SS), DDVP resistant strain (DDVP-R) and PH3 resistant strain (PH3-R), were started at Southwest University Key Laboratory of Entomology and Pest Control Engineering and subjected to artificial selection to applied pesticides since the 1990s. As indicated by qPCR, four P450 genes (CYP6CE1, CYP6CE2, CYP4CB1 and CYP4CC1)transcripts in DDVP-R and PH3-R strains were significantly higher than SS strain (P<0.05), while the expression of CYP4CD1 was not significantly different (P>0.05), which implied that the overexpressed P450s were possibly involved in psocids resistance. However, the relative expression levels of the overexpressed P450s were relatively low (less than 2 fold). Moreover, as has been demonstrated by our previous studies several other key enzymes such as GSTs and AChE were related to pesticide resistance. Therefore, we assumed that the psocids resistance was a result of the cooperation of the enzymes mentioned above. However, we currently lack sufficient evidence to show that CYP6CE1 and CYP6CE2 are involved in deltamethrin metabolism in psocids. Future studies on substrate specificity of the recombinant proteins will provide direct evidence.
3 Heterologous expression of psocids P450 in Escherichia coli
Heterologous expression of foreign genes is the core field in functional genomics research, which will provide great insights to the gene function. Thus, biochemical and toxicological properties determination of the recombinant proteins based on the heterologous expression will provide direct evidence to the P450s function study. In the present study, on the basis of Gateway(?) technology, we constructed prokaryotic expression vectors (ligated to pDest 17) for CYP6CE1 and Lb(3-Actinl.Moreover, CYP6CE1 and Lbβ-Actin1 were successfully expressed in E. coli and the recombinant proteins were further confirmed by Western Blot analysis. Additionally, applying the double digestion of BamHI and Xhol restriction enzymes with the DNA recombination technology, we constructed expression vectors for CYP6CE1,CYP4CB1,CYP4CC1 and CYP4CD1 based on pET4317.1a (+)vector. In order to improve the heterologous expression efficiency and convenience to recombinant protein purification, N-terminus modification as suggested by previous researcher was performed for expression vector construction. Signal peptide sequences of the putative protein sequences were discarded, the second codon behind the start codon ATG was changed to GCT. If necessary, four continuous codons of CAT could be added to form a 4×His tag for recombinant protein purification. According to the N-terminus modification, we constructed expression vectors for CYP6CE1,CYP4CB1,CYP4CC1 and CYP4CD1 based on pCW plasmid. Our results formed a solid basis to express psocid P450s effectively in E. coli.
4 Polymorphism of CYP6CE1 alleles
It has been reported that mutations of P450 alleles could result in changes in protein structure, substrate recognition and catalytic activity. In this study, three alleles (GenBank accession numbers were EF421245, EU266572 and EU266573) of CYP6CE1 were cloned from 3 strains (DDVP, PH3 resistant strains and susceptible strain) of L.bostrychophila using the combined techniques of reverse transcriptase-PCR and sequenced. According to the sequences alignment, fifteen polymorphism sites were found in the nucleotide sequences of CYP6CElv2 and CYP6CElv3, respectively. We also determined their locations in the sequences. Based on the sequence analysis, the 15 nucleotide polymorphism sites of CYP6CE1v2 resulted in only one amino acid substitution, while five amino acid alterations were found in CYP6CElv3.Protein parameters of CYP6CElvl-3 were predicted by Proparam.As indicated by the prediction, there was no significant difference in properties of CYP6CE1v2 and CYP6CE1v1 protein, which were different from CYP6CE1v3 protein, especially in theoretical pⅠ.Furthermore,3-D structure homology modeling of these three proteins by SWISS-MODEL demonstrated that three amino acid substitutions in CYP6CE1v3 resulted in its structure alteration.
In summary, in the present study we cloned the full-length cDNA of four housekeeping genes from L. bostrychophila, which extended sequence information for Psocoptera and provided potential molecular markers to investigate the evolutionary relationship between the Psocoptera and other insects. Five novel P450 genes were isolated form L. bostrychophila, which formed a solid basis to explore the physiological functions undertaken by P450 enzyme system.We systematically evaluated and validated the expression stability of housekeeping genes, and selected only those stably expressed as reference. This constructed a platform for the gene expression profiling in L. bostrychophila using real-time PCR. Based on the reference selection, we determined the developmental expression patterns of these P450s and their expression profiles in the adults after exposure to several insecticides, as well as the relative expression quantity in different strains showing different resistance to DDVP and PH3.Furthermore, we constructed different expression vectors for heterologous expression of psocid P450 in E. coli, in which CYP6CE1 and Lbβ-Actinl were successfully expressed. Our results will not only provide great insights into exploring the functions of the psocid P450s system in development and physiology, but also provide evidence for clarifying the adaptive mechanisms mechanism of psocids to environment. In the meantime, we may enrich and develop the scientific theoretical study system for the resistance mechanism of stored-product insect pests.
引文
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