家蚕围食膜的蛋白质组及几丁质去乙酰化酶的功能研究
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摘要
几丁质(Chitin)是由N-乙酰葡萄糖胺(N-acetyl glucosamine, GlcNAc)经β-1,4糖苷键连接而成的寡聚物,是自然界中广泛存在的天然有机化合物之一,其数量仅次于纤维素。几丁质广泛存在于真菌及藻类的细胞壁、无脊椎动物的外骨骼及表皮。前人的研究证明,几丁质是昆虫表皮和围食膜(Peritrophic membrane,PM)的重要组成成分之一,昆虫生长、发育的各个时期都需要一定量的几丁质,几丁质代谢随着昆虫不同生长发育阶段而变化,因此,几丁质在昆虫的正常生长发育过程中有着至关重要的。家蚕(Bombyx mori)既是重要的经济昆虫,也是鳞翅目昆虫的典型代表。家蚕围食膜已知的主要组分有几丁质、蛋白质,围食膜蛋白质对维持着围食膜的结构的完整性,蛋白质多糖的疏水性则有助于围食膜几丁质微纤维网状结构的形成,并维持其强度。围食膜作为家蚕体内防御病原微生物入侵的第一道防线,通过降解合成的几丁质或破坏蛋白与几丁质的结合,即可以抑制围食膜的形成以及昆虫的生长发育等,达到防治害虫的目的,具有极大的发展潜力,同时也是国内外研究的热点。
在昆虫中,几丁质去乙酰化酶(Chitin Deacetylase, CDA)不仅是围食膜重要结构蛋白质之一,也可以对几丁质进行修饰,因此,可作为破坏围食膜结构的新靶标。本文通过蛋白质组学技术对家蚕围食膜进行全蛋白质组分析,筛选家蚕BmCDA7蛋白质,对家蚕CDAs家族成员进行系统分析。利用原核表达的重组蛋白质制备多克隆抗体,分析其在组织和时期中的表达方式。通过真核表达得到具有活性的家蚕BmCDA7蛋白,首次比较准确的测定昆虫CDAs的活性,然后对家蚕中肠特异BmCDA7基因进行转基因增量表达和干涉,检测中肠围食膜的变化,研究家蚕CDA基因与几丁质的关系,进一步阐述家蚕BmCDA7基因的功能。主要研究结果如下：
1.家蚕围食膜的结构及其蛋白质组成
家蚕围食膜包裹着食物,为无色、透明的管状结构,类似于I型围食膜。解剖过程中发现围食膜几乎布满整个中肠,并且富有弹性。从家蚕横切面可以清楚的看到有围食膜位于食物和中肠之间。通过几丁质染色,证明家蚕围食膜和其他昆虫的结构也是一样,都是由几丁质丝组成的网状结构,围食膜的这种结构保证了其对中肠的防护功能。
对家蚕围食膜进行扫描电镜观察,从家蚕围食膜的外表面可以发现家蚕围食膜至少有两层,内表面绝大部分是光滑致密,具有明显的褶皱,该结构不仅有利于食物的吸收,而且对中肠的保护作用。
我们对家蚕五龄第3天围食膜的总蛋白质进行shotgun分析,最后成功鉴定305个可信度较高的蛋白质。这些蛋白质的分子量大部分分布在8.02kDa和788.52kDa之间,除了两个蛋白质例外：BGIBMGA010471-PA的分子量大小为1538.87kDa, BGIBMGA006856-PA的分子量甚至达到2002.29kDa；但是小于100kDa的蛋白质还是占大多数的,占79.34%。家蚕围食膜蛋白质极酸蛋白质和极碱蛋白质的等电点分别为3.39和12.91。
我们同时构建了围食膜的双向电泳图谱,检测到较为明显的蛋白点在60多个,这些蛋白质点主要分布在分子量10-60kDa,等电点pI4-9的范围内。通过MALDI-TOF MS鉴定,有12个蛋白质点得到了鉴定(均包含于shotgun数据中)。通过信息分析和功能注释,我们找到了两个较为重要的围食膜结构蛋白质：Chitin deacetylase(几丁质去乙酰化酶)、Peritrophic membrane chitin binding protein2(围食膜几丁质结合蛋白质2)。
利用GO分类对鉴定到的蛋白质进行功能分析：305个蛋白质中有216个蛋白质获得至少一个GO号,这些蛋白质被分为三大类,主要参与细胞成分、分子功能和生物过程。对这些蛋白质进行KEGG pathway分析,他们主要分为代谢(metabolism),遗传信息处理(genetic information processing),环境信息处理(environmental information processing),细胞过程(cellular processes),有机体系统(organismal systems)和人类疾病(human diseases)五大类。它们具体主要参与蛋白吸收和消化、碳水化合物代谢、解毒代谢及免疫反应过程等。其中消化系统(digestive system)在有机体系统中占有比例最高,而参与免疫系统(immune system)的有5个pathway。这正好解释围食膜不仅帮助中肠进行酶解食物等消化吸收作用,还可以参与了家蚕的免疫防御过程,对中肠起保护作用。我们比较感兴趣的正是这些与免疫相关的蛋白质,比如,Cadherin membrane protein、Aminopeptidase N、Alkaline phosphatase、Serine protease、Lipase等。通过芯片数据分析,这些蛋白质都是在中肠高量特异性的表达。通过RT-PCR对具有几丁质结合蛋白质和丝氨酸蛋白酶抑制剂对应的基因进行表达检测,进一步确认它们都是中肠特异的基因,由此,我们推测这些蛋白质应该都是围食膜的蛋白质。
2.家蚕BmCDA7基因的全长克隆及序列分析
结合家蚕基因9×数据,利用Race技术我们获得了BmCDA7基因全长cDNA序列1357bp(含ployA)大小,该序列具有一个1140bp大小的编码区,27bp大小的5'UTR和190bp大小的3'UTR。该基因定位于28号染色体,编码379个氨基酸,前面16个氨基酸为信号肽,分子量为41.26kDa,等电点为5.12,富含15个半胱氨酸,具有一个几丁质去乙酰化酶结构域。在第168个氨基酸可能发生N连接-糖基化,在第209,215个氨基酸可能发生O连接-糖基化。
从家蚕基因组中鉴别出了8个家蚕CDAs基因,并把它们命名为BmCDA1～BmCDA8,它们都具有一个几丁质去乙酰化酶功能域及五个典型的motif。所有CDAs成员都具有信号肽和糖基化位点,其中BmCDA2和BmCDA5都具有两种拼接形式,其中只有BmCDA3和BmCDA4没有EST证据。系统发生分析结果表明,昆虫CDAs分为5类群,BmCDA7属于V群,仅仅只含有几丁质去乙酰化酶结构域。
结合家蚕全基因组的表达谱芯片和RT-PCR的结果,发现BmCDA7基因在胚胎第9天后开始表达,一直持续到五龄第7天,以后基本不表达。这个时期正好是家蚕中肠从形成到凋亡的过程。另外BmCDA7基因在每龄起蚕都比眠蚕表达量有所提高,这个正是家蚕围食膜更替的时间点,暗示着该基因可能跟围食膜的更新有关。
3.家蚕BmCDA7基因的原核表达及组织定位
将BmCDA7基因的编码区亚克隆到p28表达载体,构建成p28-BmCDA7原核表达重组质粒转化大肠杆菌BL21(DE3)表达菌株,以加入终浓度为0.2mM的IPTG,37℃培养4h为诱导条件,与对照相比,在35～45kDa之间位置处有特异条带,与目的蛋白预测分子质量大小相符合,说明均BmCDA7重组蛋白获得了表达。经蛋白组分鉴定发现,BmCDA7重组蛋白以包涵体的形式在大肠杆菌表达菌株表达。
利用Ni柱结合切胶的方法纯化原核表达的BmCDA7重组蛋白,并成功制备BmCDA7重组蛋白的多克隆抗体。Western blotting检测家蚕组织中BmCDA7蛋白质的分布情况,结果表明BmCDA7蛋白质在家蚕5龄3天的中肠和围食膜中都能够检测到,在前肠、后肠和除去消化道的整蚕都未检测到,表明BmCDA7蛋白质在mRNA水平和蛋白质水平上都有表达,并且在·mRNA水平和蛋白质水平的表达情况相一致。证实BmCDA7蛋白质在中肠和围食膜是特异的蛋白质。
4.家蚕BmCDA7基因的真核表达及其蛋白酶活测定
将BmCDA7基因亚克隆到pPIC9K真核表达载体,线性化质粒DNA后电转化GS115菌株上,通过转化子筛选获得整合入酵母染色体上的重组酵母重组质粒,在毕赤酵母中进行真核表达。Western blotting的结果进一步确实目的蛋白质得到了表达。诱导96h的上清液经过硫酸钱粗纯化后,再用PEG20,000进行浓缩。参考前人对CDA酶活的测定方法对粗纯化BmCDA7重组蛋白质样品进行酶活测定。我们测得粗纯化BmCDA7重组蛋白质样品的酶活力为1.85U/mL。
5.家蚕BmCDA7基因的功能研究
为了获得可遗传的转录后基因增量表达或基因沉默,我们构建了两个转基因载体：转基因增量表达载体pBac[P2-BmCDA7-SV40,3×P3-EGFP-SV40],转基因干涉表达载体pBac[P2-BmCDA7S-I-BmCDA7A-SV40,3×P3-EGFP]。显微注射非制育蚕卵,并在G1代进行荧光检测,均获得了转基因阳性个体。利用反向PCR技术,对转基因系统的插入位点进行鉴定,结果显示：转基因增量和干涉表达系统的插入位点分别位于第10号染色体的nscaf2859上以及第15号染色体的nscaf2888上。
分析两个转基因系统中的围食膜通透性,结果发现在这两个转基因品系的围食膜内腔中蓝色葡聚糖的含量都要比对照多,说明了这两个转基因品系的围食膜通透性都比对照有所提高。通过围食膜超微结构的观察,我们发现这两个转基因品系中的围食膜上的几丁质都发生了明显的变化,都有所减少或变小,其排列也不同于对照。可能是由于在增量表达转基因系中BmCDA7蛋白提高了,几丁质被过度修饰了,从而影响了通透性；而干涉了该基因后可能是由几丁质合成后没有该酶的修饰,发生了几丁质畸形。进一步对围食膜的几丁质含量进行了测定,发现这两个转基因品系的围食膜几丁质的含量都比降低。这些变化可能就是引起这两个转基因品系的围食膜通透性提高的原因。综上,我们推测BmCDA7对几丁质的修饰作用在围食膜几丁质的形成排列起着重要的作用。
Chitin is a linear polymer of (3-(1-4)-linked N-acetylglucosamines (GlcNAc), that most common natural amino polysaccharide in nature.It is is second only to cellulose in abundances and mainly synthesized by fungi, nematodes and arthropods. In insects, chitin is a constituent part of the cuticle and peritrophic membrane (PM)in insects. The chitin metabolism have been reported to change at different growth stages of insects. So the chitin is crucial for insect growth and development. The domesticated silkworm, Bombyx mori, is not only a very important economic insect that contributes to the national economy of many countries, but is also an excellent model organism of Lepidoteran insects for basic research. Chitin and protein are the major constituent of PM. The structural integrity of the PM is maintained by the protein component present in the PM. The hydrophobic of protein polysaccharides have the function to help maintain the chitin micro fiber reticular formation and its strength of PM. the PM is first line of defense against invading microorganisms. Degrading chitin or disturbing the combination of protein and chitin can inhibit peritrophic membrane formation and insect growth and development. It is become the domestic and foreign scholars study hotspots, and has great to control pests.
Chitin deacetylase (CDA) is the structural peritrophic matrix proteins and could modify chitin. So, it can be an attractive target to disturb the PM. Our study use proteomic methods to identify comprehensively the proteins from the PM of silkworm. Meanwhile we perform further study on the BmCDA7gene by the molecular techniques of gene cloning, reverse-transcription PCR, western blotting and prokaryotic and eukaryotic expression. Then we used the transgene technology to analyze the function of BmCDA7gene. The main results are as follows:
1. Peritrophic membrane of silkworm structure and analysis of its protein components
The silkworm PM that almost cover the entire midgut wrap with food bolus, and is a colorless, transparent, flexible tubular structure, which is similar to the type I PM. The PM clearly locates between the food and the midgut from cross-section of the silkworm, Which indicates that the silkworm has PM as most of the Lepidoptera. The silkworm PM has the chitin network that protect midgut from invading microorganisms.
There are at least two layers can be found on the PM. And the inner surface of PM is smooth and compact, but still has an obvious fold. This structure suggests the PM not only plays important roles in facilitating food digestion and providing protection to the gut epithelium.
To study the protein profiles of silkworm PM, the total proteins extracted from day3of the5th instar larvae of silkworm PM were separated by shotgun techonology, and a total of305proteins were identified. The molecular weights mostly ranged between8.02kDa and788.52kDa, except for BGIBMGA006856-PA (2002.29kDa) and BGIBMGA010471-PA (1538.87kDa). A total of79.34%of the identified proteins were smaller than100kDa. The most acidic and basic proteins identified by LC-MS/MS had pIs of3.39and12.91, respectively.
The protein extracts from the PM were aslo separated by2-DE. More than60protein spots were detected. Most of the resolved protein spots had pI values between pH5and9with molecular weight of10-66kDa. We detected more spots in silkworm PM than other insects using2-DE. And then30spots from PM were excised and further investigated by MALDI-TOF MS. In addition, there were12proteins were successfully identified. This investigation revealed that these proteins were components of the PM. There were two structural peritrophic matrix proteins, chitin deacetylase and peritrophic membrane chitin binding protein2.
Gene Ontology tools were used to analyze the PM proteome to present an overall view on the functional categories of PM proteins. The results indicate that216of the305identified proteins show at least one matched GO annotation. The proteins were classified into cellular component, molecular function, and biological process according to the GO hierarchy using WEGO. Majority of the proteins were assigned to the cell and cell part in terms of "cellular component." The highest distribution was associated with binding and catalytic activity in terms of "molecular function." The remaining proteins were linked to different activities, such as antioxidation, electron transport, and enzyme regulation, and among others. The proteins were classified according to different categories based on "biological process."The highest number of proteins was mapped to proteins involved in metabolic process, followed by proteins associated with cellular process.
The PM proteins were mapped to the KEGG ortholog level for the KEGG analysis. Two hundred and thirty-four different pathways were linked to the PM. These pathways were classified into metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and human diseases. Among the organismal systems, the digestive system was the most active. Moreover, five pathways were related to the immune system. This phenomenon is likely because the PM not only could help the midgut in coordinating the movement of enzymes and nutrient uptake, but also plays an important role in insect defense strategy.
2. Full-length cDNA cloning and sequence analysis of silkworm BmCDA7gene
Based on the assembled9X coverage genome sequence, we cloned and sequenced the BmCDA7gene. The putative BmCDA7cDNA contains an open reading frame (ORF) of1,140bp. RACE experiments were performed to obtain the5'and3'ends of BmCDA7,27bp upstream-untranslated region and190bp down stream-untranslated region. So, the full length of the cDNA of BmCDA7is1357bp followed by an AT rich region with two typical polyadenylation signal signal sequence, AATAAA. The putative BmCDA7cDNA encoded for a379-amino acids protein consisting of a16-amino acid signal peptide by the software SignalP and a mature polypeptide of363amino acids. After removal of the signal peptide, the deduced protein is predicted to have a molecular weight of41.26kDa and the theoretical pI of5.12by the ExPASy server. Prediction of potential glycosylation sites using the NetNglyc1.0and NetOglyc3.1server shown that the protein contains one putative N-glycosylation site at Asn168, one putative O-glycosylation site at Thr209.,215. BmCDA7has a putative polysaccharide deacetylase-like domain (residues46-182) and15cysteine residues which maight be a basic module that combined with other protein sequences to generate new function or modify existing function by SMART analysis.
8putative silkworm CD As were identified. Based on the EST, there are two genes (BmCDA3and BmCDA4)have expression evidence. BmCDA2and BmCDA5have alternative splicing. Phylogenetic analysis shows that all of the CDA-like proteins from insects originated from one root. The silkworm CDAs also grouped the proteins into five major classes, groups I through V, and BmCDA7was divided into group V.
The expression of BmCDA7was also studied by RT-PCR. It show this gene has expression from9d after oviposition to day7of5th instar larva and has high expression at the molting silkworm. It is just period from the formation to the process of apoptosis of silkworm midgut. The foregut, midgut, hindgut and remaining carcasses were dissected from Day-3of the fifth instar larvae to use in this study. The signal was only detected in midgut, but no signals were found in the foregut, hindgut and remaining carcasses. This suggested that the gene should be updated with PM.
3. Prokaryotic expression and tissue localization of silkworm BmCDA7gene
The complete CDS of BmCDA7gene was subcloned into the p28prokaryotic expression vector, the BmCDA7-p28plasmids were introduced into Escherichia coli BL21(DE3), and the recombinant protein was abundantly expressed as insoluble inclusion body after induction by0.2mM IPTG,37℃,4h. There was one specific band corresponding to molecular weights of BmCDA7and purified using the Ni2+-NTA affinity column. Then the purified proteins were injected into rabbit to generate polyclonal antibodies. In addition to the PM, the tissue distribution of BmCDA7in silkworm larvae was detected by western blot using the antibody to the recombinant protein. BmCDA7was detected in midgut and PM tissue, but was not detectable from the larval foregut, hindgut and remaining carcasses. This result was also confirmed by the translational levels analysis of BmCDA7, and suggested the BmCDA7was specific expression protein in the midgut and PM.
4. Eukaryotic expression of silkworm BmCDA7gene and activity determination
The recombinant expression vector pPIC9K-BmCDA7plasmid and pPIC9K plasmid were transformed into P. pastoris by electroporation. After transformation and primary screening by histidine-deficient medium and G418, The positive yeast cells containing plasmid was selected for cultivation in in BMMY medium after induction by1%methanol at30℃. SDS-PAGE analysis of the crude supernatants at the induction period from96h appeared no significant additional band corresponding to the molecular masses of BmCDA7in the induced yeast containing recombinant pPIC9K. But we detected specific band from crude supernatants of pPIC9K-BmCDA7using the BmCDA7antibodies. This result indicated that BmCDA7was successfully expressed in the yeast. Using the differences method as previous described, we demonstrated that recombinant BmCDA7is active, and the CDA activity of BmCDA7is1.85U/mL.
5. Function analysis of silkworm BmCDA7gene
We have successfully constructed two transgenic vectors, the transgenic overexpression vector ([P2-BmCDA7-SV40,3×P3-EGFP-SV40]) and the transgenic RNAi vector (pBac[P2-BmCDA7S-I-BmCDA7A-SV40,3xP3-EGFP]). Comicro-inject the transgenic vectors and helper vector into the silkworm eggs which have been broken embryonic diapause. Scan the G1by fluorescence microscope and positive individuals were obtained. The inverse PCR results show that insertion site of transgenic overexpression and transgenic RNAi system are localed on nscaf2859of chromosome10and nscaf2888of chromosome15, respectively.
Analysis of the PM permeability of the two transgenic system, we found the blue dextran in the transgenic system more than the control. This in the PM permeability of transgenic system has improved. In the transgenic overexpression system, the over-expression of BmCDA7maight excessive modification the chitin. In the transgenic RNAi system, the suppression expression of BmCDA7maight made chitin deformity. The chitin in PM of the two transgenic system have obvious change(reduced or smaller), and disorder arrangement. Ang the chitin content of two transgenic system have reduce. So, we verify the BmCDA7'modification of chitin play an important role in formation and arrangement of chitin.
在昆虫中,几丁质去乙酰化酶(Chitin Deacetylase, CDA)不仅是围食膜重要结构蛋白质之一,也可以对几丁质进行修饰,因此,可作为破坏围食膜结构的新靶标。本文通过蛋白质组学技术对家蚕围食膜进行全蛋白质组分析,筛选家蚕BmCDA7蛋白质,对家蚕CDAs家族成员进行系统分析。利用原核表达的重组蛋白质制备多克隆抗体,分析其在组织和时期中的表达方式。通过真核表达得到具有活性的家蚕BmCDA7蛋白,首次比较准确的测定昆虫CDAs的活性,然后对家蚕中肠特异BmCDA7基因进行转基因增量表达和干涉,检测中肠围食膜的变化,研究家蚕CDA基因与几丁质的关系,进一步阐述家蚕BmCDA7基因的功能。主要研究结果如下：
1.家蚕围食膜的结构及其蛋白质组成
家蚕围食膜包裹着食物,为无色、透明的管状结构,类似于I型围食膜。解剖过程中发现围食膜几乎布满整个中肠,并且富有弹性。从家蚕横切面可以清楚的看到有围食膜位于食物和中肠之间。通过几丁质染色,证明家蚕围食膜和其他昆虫的结构也是一样,都是由几丁质丝组成的网状结构,围食膜的这种结构保证了其对中肠的防护功能。
对家蚕围食膜进行扫描电镜观察,从家蚕围食膜的外表面可以发现家蚕围食膜至少有两层,内表面绝大部分是光滑致密,具有明显的褶皱,该结构不仅有利于食物的吸收,而且对中肠的保护作用。
我们对家蚕五龄第3天围食膜的总蛋白质进行shotgun分析,最后成功鉴定305个可信度较高的蛋白质。这些蛋白质的分子量大部分分布在8.02kDa和788.52kDa之间,除了两个蛋白质例外：BGIBMGA010471-PA的分子量大小为1538.87kDa, BGIBMGA006856-PA的分子量甚至达到2002.29kDa；但是小于100kDa的蛋白质还是占大多数的,占79.34%。家蚕围食膜蛋白质极酸蛋白质和极碱蛋白质的等电点分别为3.39和12.91。
我们同时构建了围食膜的双向电泳图谱,检测到较为明显的蛋白点在60多个,这些蛋白质点主要分布在分子量10-60kDa,等电点pI4-9的范围内。通过MALDI-TOF MS鉴定,有12个蛋白质点得到了鉴定(均包含于shotgun数据中)。通过信息分析和功能注释,我们找到了两个较为重要的围食膜结构蛋白质：Chitin deacetylase(几丁质去乙酰化酶)、Peritrophic membrane chitin binding protein2(围食膜几丁质结合蛋白质2)。
利用GO分类对鉴定到的蛋白质进行功能分析：305个蛋白质中有216个蛋白质获得至少一个GO号,这些蛋白质被分为三大类,主要参与细胞成分、分子功能和生物过程。对这些蛋白质进行KEGG pathway分析,他们主要分为代谢(metabolism),遗传信息处理(genetic information processing),环境信息处理(environmental information processing),细胞过程(cellular processes),有机体系统(organismal systems)和人类疾病(human diseases)五大类。它们具体主要参与蛋白吸收和消化、碳水化合物代谢、解毒代谢及免疫反应过程等。其中消化系统(digestive system)在有机体系统中占有比例最高,而参与免疫系统(immune system)的有5个pathway。这正好解释围食膜不仅帮助中肠进行酶解食物等消化吸收作用,还可以参与了家蚕的免疫防御过程,对中肠起保护作用。我们比较感兴趣的正是这些与免疫相关的蛋白质,比如,Cadherin membrane protein、Aminopeptidase N、Alkaline phosphatase、Serine protease、Lipase等。通过芯片数据分析,这些蛋白质都是在中肠高量特异性的表达。通过RT-PCR对具有几丁质结合蛋白质和丝氨酸蛋白酶抑制剂对应的基因进行表达检测,进一步确认它们都是中肠特异的基因,由此,我们推测这些蛋白质应该都是围食膜的蛋白质。
2.家蚕BmCDA7基因的全长克隆及序列分析
结合家蚕基因9×数据,利用Race技术我们获得了BmCDA7基因全长cDNA序列1357bp(含ployA)大小,该序列具有一个1140bp大小的编码区,27bp大小的5'UTR和190bp大小的3'UTR。该基因定位于28号染色体,编码379个氨基酸,前面16个氨基酸为信号肽,分子量为41.26kDa,等电点为5.12,富含15个半胱氨酸,具有一个几丁质去乙酰化酶结构域。在第168个氨基酸可能发生N连接-糖基化,在第209,215个氨基酸可能发生O连接-糖基化。
从家蚕基因组中鉴别出了8个家蚕CDAs基因,并把它们命名为BmCDA1～BmCDA8,它们都具有一个几丁质去乙酰化酶功能域及五个典型的motif。所有CDAs成员都具有信号肽和糖基化位点,其中BmCDA2和BmCDA5都具有两种拼接形式,其中只有BmCDA3和BmCDA4没有EST证据。系统发生分析结果表明,昆虫CDAs分为5类群,BmCDA7属于V群,仅仅只含有几丁质去乙酰化酶结构域。
结合家蚕全基因组的表达谱芯片和RT-PCR的结果,发现BmCDA7基因在胚胎第9天后开始表达,一直持续到五龄第7天,以后基本不表达。这个时期正好是家蚕中肠从形成到凋亡的过程。另外BmCDA7基因在每龄起蚕都比眠蚕表达量有所提高,这个正是家蚕围食膜更替的时间点,暗示着该基因可能跟围食膜的更新有关。
3.家蚕BmCDA7基因的原核表达及组织定位
将BmCDA7基因的编码区亚克隆到p28表达载体,构建成p28-BmCDA7原核表达重组质粒转化大肠杆菌BL21(DE3)表达菌株,以加入终浓度为0.2mM的IPTG,37℃培养4h为诱导条件,与对照相比,在35～45kDa之间位置处有特异条带,与目的蛋白预测分子质量大小相符合,说明均BmCDA7重组蛋白获得了表达。经蛋白组分鉴定发现,BmCDA7重组蛋白以包涵体的形式在大肠杆菌表达菌株表达。
利用Ni柱结合切胶的方法纯化原核表达的BmCDA7重组蛋白,并成功制备BmCDA7重组蛋白的多克隆抗体。Western blotting检测家蚕组织中BmCDA7蛋白质的分布情况,结果表明BmCDA7蛋白质在家蚕5龄3天的中肠和围食膜中都能够检测到,在前肠、后肠和除去消化道的整蚕都未检测到,表明BmCDA7蛋白质在mRNA水平和蛋白质水平上都有表达,并且在·mRNA水平和蛋白质水平的表达情况相一致。证实BmCDA7蛋白质在中肠和围食膜是特异的蛋白质。
4.家蚕BmCDA7基因的真核表达及其蛋白酶活测定
将BmCDA7基因亚克隆到pPIC9K真核表达载体,线性化质粒DNA后电转化GS115菌株上,通过转化子筛选获得整合入酵母染色体上的重组酵母重组质粒,在毕赤酵母中进行真核表达。Western blotting的结果进一步确实目的蛋白质得到了表达。诱导96h的上清液经过硫酸钱粗纯化后,再用PEG20,000进行浓缩。参考前人对CDA酶活的测定方法对粗纯化BmCDA7重组蛋白质样品进行酶活测定。我们测得粗纯化BmCDA7重组蛋白质样品的酶活力为1.85U/mL。
5.家蚕BmCDA7基因的功能研究
为了获得可遗传的转录后基因增量表达或基因沉默,我们构建了两个转基因载体：转基因增量表达载体pBac[P2-BmCDA7-SV40,3×P3-EGFP-SV40],转基因干涉表达载体pBac[P2-BmCDA7S-I-BmCDA7A-SV40,3×P3-EGFP]。显微注射非制育蚕卵,并在G1代进行荧光检测,均获得了转基因阳性个体。利用反向PCR技术,对转基因系统的插入位点进行鉴定,结果显示：转基因增量和干涉表达系统的插入位点分别位于第10号染色体的nscaf2859上以及第15号染色体的nscaf2888上。
分析两个转基因系统中的围食膜通透性,结果发现在这两个转基因品系的围食膜内腔中蓝色葡聚糖的含量都要比对照多,说明了这两个转基因品系的围食膜通透性都比对照有所提高。通过围食膜超微结构的观察,我们发现这两个转基因品系中的围食膜上的几丁质都发生了明显的变化,都有所减少或变小,其排列也不同于对照。可能是由于在增量表达转基因系中BmCDA7蛋白提高了,几丁质被过度修饰了,从而影响了通透性；而干涉了该基因后可能是由几丁质合成后没有该酶的修饰,发生了几丁质畸形。进一步对围食膜的几丁质含量进行了测定,发现这两个转基因品系的围食膜几丁质的含量都比降低。这些变化可能就是引起这两个转基因品系的围食膜通透性提高的原因。综上,我们推测BmCDA7对几丁质的修饰作用在围食膜几丁质的形成排列起着重要的作用。
Chitin is a linear polymer of (3-(1-4)-linked N-acetylglucosamines (GlcNAc), that most common natural amino polysaccharide in nature.It is is second only to cellulose in abundances and mainly synthesized by fungi, nematodes and arthropods. In insects, chitin is a constituent part of the cuticle and peritrophic membrane (PM)in insects. The chitin metabolism have been reported to change at different growth stages of insects. So the chitin is crucial for insect growth and development. The domesticated silkworm, Bombyx mori, is not only a very important economic insect that contributes to the national economy of many countries, but is also an excellent model organism of Lepidoteran insects for basic research. Chitin and protein are the major constituent of PM. The structural integrity of the PM is maintained by the protein component present in the PM. The hydrophobic of protein polysaccharides have the function to help maintain the chitin micro fiber reticular formation and its strength of PM. the PM is first line of defense against invading microorganisms. Degrading chitin or disturbing the combination of protein and chitin can inhibit peritrophic membrane formation and insect growth and development. It is become the domestic and foreign scholars study hotspots, and has great to control pests.
Chitin deacetylase (CDA) is the structural peritrophic matrix proteins and could modify chitin. So, it can be an attractive target to disturb the PM. Our study use proteomic methods to identify comprehensively the proteins from the PM of silkworm. Meanwhile we perform further study on the BmCDA7gene by the molecular techniques of gene cloning, reverse-transcription PCR, western blotting and prokaryotic and eukaryotic expression. Then we used the transgene technology to analyze the function of BmCDA7gene. The main results are as follows:
1. Peritrophic membrane of silkworm structure and analysis of its protein components
The silkworm PM that almost cover the entire midgut wrap with food bolus, and is a colorless, transparent, flexible tubular structure, which is similar to the type I PM. The PM clearly locates between the food and the midgut from cross-section of the silkworm, Which indicates that the silkworm has PM as most of the Lepidoptera. The silkworm PM has the chitin network that protect midgut from invading microorganisms.
There are at least two layers can be found on the PM. And the inner surface of PM is smooth and compact, but still has an obvious fold. This structure suggests the PM not only plays important roles in facilitating food digestion and providing protection to the gut epithelium.
To study the protein profiles of silkworm PM, the total proteins extracted from day3of the5th instar larvae of silkworm PM were separated by shotgun techonology, and a total of305proteins were identified. The molecular weights mostly ranged between8.02kDa and788.52kDa, except for BGIBMGA006856-PA (2002.29kDa) and BGIBMGA010471-PA (1538.87kDa). A total of79.34%of the identified proteins were smaller than100kDa. The most acidic and basic proteins identified by LC-MS/MS had pIs of3.39and12.91, respectively.
The protein extracts from the PM were aslo separated by2-DE. More than60protein spots were detected. Most of the resolved protein spots had pI values between pH5and9with molecular weight of10-66kDa. We detected more spots in silkworm PM than other insects using2-DE. And then30spots from PM were excised and further investigated by MALDI-TOF MS. In addition, there were12proteins were successfully identified. This investigation revealed that these proteins were components of the PM. There were two structural peritrophic matrix proteins, chitin deacetylase and peritrophic membrane chitin binding protein2.
Gene Ontology tools were used to analyze the PM proteome to present an overall view on the functional categories of PM proteins. The results indicate that216of the305identified proteins show at least one matched GO annotation. The proteins were classified into cellular component, molecular function, and biological process according to the GO hierarchy using WEGO. Majority of the proteins were assigned to the cell and cell part in terms of "cellular component." The highest distribution was associated with binding and catalytic activity in terms of "molecular function." The remaining proteins were linked to different activities, such as antioxidation, electron transport, and enzyme regulation, and among others. The proteins were classified according to different categories based on "biological process."The highest number of proteins was mapped to proteins involved in metabolic process, followed by proteins associated with cellular process.
The PM proteins were mapped to the KEGG ortholog level for the KEGG analysis. Two hundred and thirty-four different pathways were linked to the PM. These pathways were classified into metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and human diseases. Among the organismal systems, the digestive system was the most active. Moreover, five pathways were related to the immune system. This phenomenon is likely because the PM not only could help the midgut in coordinating the movement of enzymes and nutrient uptake, but also plays an important role in insect defense strategy.
2. Full-length cDNA cloning and sequence analysis of silkworm BmCDA7gene
Based on the assembled9X coverage genome sequence, we cloned and sequenced the BmCDA7gene. The putative BmCDA7cDNA contains an open reading frame (ORF) of1,140bp. RACE experiments were performed to obtain the5'and3'ends of BmCDA7,27bp upstream-untranslated region and190bp down stream-untranslated region. So, the full length of the cDNA of BmCDA7is1357bp followed by an AT rich region with two typical polyadenylation signal signal sequence, AATAAA. The putative BmCDA7cDNA encoded for a379-amino acids protein consisting of a16-amino acid signal peptide by the software SignalP and a mature polypeptide of363amino acids. After removal of the signal peptide, the deduced protein is predicted to have a molecular weight of41.26kDa and the theoretical pI of5.12by the ExPASy server. Prediction of potential glycosylation sites using the NetNglyc1.0and NetOglyc3.1server shown that the protein contains one putative N-glycosylation site at Asn168, one putative O-glycosylation site at Thr209.,215. BmCDA7has a putative polysaccharide deacetylase-like domain (residues46-182) and15cysteine residues which maight be a basic module that combined with other protein sequences to generate new function or modify existing function by SMART analysis.
8putative silkworm CD As were identified. Based on the EST, there are two genes (BmCDA3and BmCDA4)have expression evidence. BmCDA2and BmCDA5have alternative splicing. Phylogenetic analysis shows that all of the CDA-like proteins from insects originated from one root. The silkworm CDAs also grouped the proteins into five major classes, groups I through V, and BmCDA7was divided into group V.
The expression of BmCDA7was also studied by RT-PCR. It show this gene has expression from9d after oviposition to day7of5th instar larva and has high expression at the molting silkworm. It is just period from the formation to the process of apoptosis of silkworm midgut. The foregut, midgut, hindgut and remaining carcasses were dissected from Day-3of the fifth instar larvae to use in this study. The signal was only detected in midgut, but no signals were found in the foregut, hindgut and remaining carcasses. This suggested that the gene should be updated with PM.
3. Prokaryotic expression and tissue localization of silkworm BmCDA7gene
The complete CDS of BmCDA7gene was subcloned into the p28prokaryotic expression vector, the BmCDA7-p28plasmids were introduced into Escherichia coli BL21(DE3), and the recombinant protein was abundantly expressed as insoluble inclusion body after induction by0.2mM IPTG,37℃,4h. There was one specific band corresponding to molecular weights of BmCDA7and purified using the Ni2+-NTA affinity column. Then the purified proteins were injected into rabbit to generate polyclonal antibodies. In addition to the PM, the tissue distribution of BmCDA7in silkworm larvae was detected by western blot using the antibody to the recombinant protein. BmCDA7was detected in midgut and PM tissue, but was not detectable from the larval foregut, hindgut and remaining carcasses. This result was also confirmed by the translational levels analysis of BmCDA7, and suggested the BmCDA7was specific expression protein in the midgut and PM.
4. Eukaryotic expression of silkworm BmCDA7gene and activity determination
The recombinant expression vector pPIC9K-BmCDA7plasmid and pPIC9K plasmid were transformed into P. pastoris by electroporation. After transformation and primary screening by histidine-deficient medium and G418, The positive yeast cells containing plasmid was selected for cultivation in in BMMY medium after induction by1%methanol at30℃. SDS-PAGE analysis of the crude supernatants at the induction period from96h appeared no significant additional band corresponding to the molecular masses of BmCDA7in the induced yeast containing recombinant pPIC9K. But we detected specific band from crude supernatants of pPIC9K-BmCDA7using the BmCDA7antibodies. This result indicated that BmCDA7was successfully expressed in the yeast. Using the differences method as previous described, we demonstrated that recombinant BmCDA7is active, and the CDA activity of BmCDA7is1.85U/mL.
5. Function analysis of silkworm BmCDA7gene
We have successfully constructed two transgenic vectors, the transgenic overexpression vector ([P2-BmCDA7-SV40,3×P3-EGFP-SV40]) and the transgenic RNAi vector (pBac[P2-BmCDA7S-I-BmCDA7A-SV40,3xP3-EGFP]). Comicro-inject the transgenic vectors and helper vector into the silkworm eggs which have been broken embryonic diapause. Scan the G1by fluorescence microscope and positive individuals were obtained. The inverse PCR results show that insertion site of transgenic overexpression and transgenic RNAi system are localed on nscaf2859of chromosome10and nscaf2888of chromosome15, respectively.
Analysis of the PM permeability of the two transgenic system, we found the blue dextran in the transgenic system more than the control. This in the PM permeability of transgenic system has improved. In the transgenic overexpression system, the over-expression of BmCDA7maight excessive modification the chitin. In the transgenic RNAi system, the suppression expression of BmCDA7maight made chitin deformity. The chitin in PM of the two transgenic system have obvious change(reduced or smaller), and disorder arrangement. Ang the chitin content of two transgenic system have reduce. So, we verify the BmCDA7'modification of chitin play an important role in formation and arrangement of chitin.
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