家蚕30K蛋白的鉴定和功能研究
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摘要
家蚕是鳞翅目的模式昆虫,具有开放式的血液循环系统。血液在家蚕的生命活动中承担着重要的生理功能,它是体内各种营养物质和激素等的交换场所。家蚕的血液重量占整个体重的21%-25%,而血液蛋白质是血液的主要成分。家蚕30K蛋白是末龄幼虫和蛹期血液中含量最大的一群蛋白质,也是胚胎中主要的卵黄蛋白。30K蛋白属于鳞翅目昆虫低分子量脂蛋白家族。到目前为止,已在家蚕、烟草天蛾和粟夜蛾三个物种中鉴定到了该家族的同源蛋白。
家蚕9倍基因组的组装成功,以及各种鳞翅目昆虫EST数据的公布,为系统性鉴定鳞翅目昆虫低分子量脂蛋白家族的成员奠定了良好的基础。本研究在此基础上,对鳞翅目昆虫的低分子量脂蛋白进行了鉴定,并对它们进行了结构分析和进化分析。此外,利用已公布的家蚕基因组和芯片数据,我们还对家蚕的低分子量脂蛋白基因进行了染色体定位分析、组织表达谱分析和发育时期表达谱分析。
在获得了以上数据的基础上,本研究重点对家蚕低分子量脂蛋白家族中的典型30K蛋白亚家族进行了功能研究。利用已经制备获得的BmLP1和BmLP7的抗体,我们对30K蛋白基因的合成、分泌、转运和利用等进行了系统性的研究。另外,本研究还从家蚕血液中纯化获得了几种30K蛋白,利用氯仿和甲醇法提取了30K蛋白所结合的脂类,并进一步利用LC-MS/MS对30K蛋白所结合的脂类进行了质谱鉴定。
本研究获得的主要结果如下：
1.鳞翅目昆虫低分子量脂蛋白基因的鉴定
(1)鳞翅目昆虫低分子量脂蛋白基因的鉴定：在家蚕基因组精细图序列数据和各种鳞翅目昆虫EST数据的基础上,本研究对鳞翅目昆虫的低分子量脂蛋白家族进行了鉴定。在蚕蛾总科、夜蛾总科和螟蛾总科的12个物种中,共鉴定到73个低分子量脂蛋白基因,它们的分布情况如下：家蚕46个、蓖麻蚕5个、琥珀蚕6个、印度柞蚕4个、烟草天蛾3个、草地贪夜蛾1个、粟夜蛾1个、西方豆类夜蛾1个、棉铃虫1个、甘蓝夜蛾1个、粉纹夜蛾1个和豆荚野螟蛾1个。在鉴定获得的73个基因中,有56个基因为首次鉴定。
(2)低分子量脂蛋白的结构分类：对鉴定到的基因进行序列比对,发现它们根据结构可以分为三个亚家族：典型30K蛋白(Typical30KP)、富含丝氨酸和苏氨酸的30K蛋白(S/T-rich30KP)和ENF肽结合蛋白(ENF-BP)。这三个亚家族有着相似的C端序列,都由一个NTD和一个CTD组成,而它们的N端序列差别很大。典型30K蛋白的N端由信号肽和一段很短的不保守区域组成；S/T-rich30KP的N端则由信号肽和一个富含丝氨酸和苏氨酸的结构域(STD)组成；ENF肽结合蛋白的N端由一个恶臭假单胞菌同源区(PPD)组成。
(3)低分子量脂蛋白的进化分析：在蚕蛾总科中,同时存在典型30K蛋白、富含丝氨酸和苏氨酸的30K蛋白和ENF肽结合蛋白亚家族。但在夜蛾总科和螟蛾总科中,我们只鉴定到了ENF肽结合蛋白亚家族。并且,在全基因组已被测序的棉铃虫中,也只能鉴定到ENF肽结合蛋白亚家族。这些结果表明,ENF肽结合蛋白亚家族是三个亚家族中起源最早的一个亚家族。富含丝氨酸和苏氨酸的30K蛋白只在家蚕中鉴定到,可能为家蚕特有的一群低分子量脂蛋白。
2.家蚕低分子量脂蛋白基因的特征分析
(1)家蚕低分子量脂蛋白基因的染色体定位：家蚕中共鉴定到46个低分子量脂蛋白基因,其中的43个基因能定位到染色体上。Bmlpl-Bmlp32(24个典型30K蛋白基因和8个富含丝氨酸和苏氨酸的30K蛋白基因)全部定位于第20号染色体的nscaf2795重叠群上的970Kbp(1380K-2350K)区域内,它们在染色体上呈串联重复分布。家蚕的10个ENF肽结合蛋白基因中,有8个基因构成了4个基因对(Bmlp37/38、Bmlp40/39、Bmlp41/42和Bmlp44/45),定位于第7号、22号和24号染色体上,每个基因对内的2个基因的转录方向相反。
(2)家蚕低分子量脂蛋白基因的表达特征分析：家蚕中的三群低分子量脂蛋白基因的组织和时期表达特征各不相同,暗示它们存在功能的分化。典型30K蛋白基因主要在脂肪体和体壁中表达,部分30K蛋白基因在一龄到四龄幼虫期表达,部分在五龄幼虫到蛾期表达。ENF肽结合蛋白基因主要在血细胞中表达,可能参与了血细胞的免疫调控过程。富含丝氨酸和苏氨酸的30K蛋白基因主要在羽化前一天和化蛾第一天有表达,在精巢中的表达量很高,而在卵巢中几乎不表达。根据其富含丝氨酸和苏氨酸的特征,推测可能参与受精或者精子的形成过程。
3.家蚕30K蛋白的合成、分泌、转运和利用
(1)家蚕30K蛋白的合成和分泌：通过RT-PCR技术分析不同发育时期脂肪体中30K蛋白基因的合成模式。同时,通过western blotting技术对不同发育时期血液中30K蛋白的变化情况进行分析。综合以上两方面结果,表明30K蛋白是由脂肪体合成,然后分泌入血液。
(2)家蚕30K蛋白的转运：在蛹期和蛾期时,血液中的30K蛋白逐渐减少,而卵巢中的30K蛋白却逐渐增加。RT-PCR结果表明,卵巢中的30K蛋白并非自身合成而来。因此,卵巢中的30K蛋白可能是从血液中转运而来。通过制备30K蛋白亲和柱,对卵巢膜蛋白进行pull-down实验,发现卵黄原蛋白受体能与30K蛋白结合,因此推测卵黄原蛋白受体可能是介导30K蛋白转运的受体。免疫组织化学定位结果表明,30K蛋白主要转运进卵巢的卵母细胞内储存起来。
(3)家蚕30K蛋白的利用：对不同发育时期的胚胎蛋白质进行western blotting分析,发现30K蛋白主要在胚胎的最后两天减少。对不同发育时期的胚胎进行酶活分析,发现酶活从胚胎第5天后升高,到蚁蚕时达到最大值。提取蚁蚕粗提物,并与30K蛋白进行体外孵育,发现蚁蚕粗提物能够降解30K蛋白。对第6天和第10天的胚胎进行免疫组织化学定位,结果表明30K蛋白在胚胎早期主要定位于胚外的卵黄颗粒中,在胚胎后期定位于胚子的肠腔内。综合以上结果,发现30K蛋白在反转期前定位于胚外卵黄颗粒中,反转期后由胚子吞入肠腔内,再由肠腔内的蛋白酶对30K蛋白进行降解,为蚁蚕的孵化提供能量来源。
4.家蚕30K蛋白的纯化及其结合脂类的鉴定
(1)30K蛋白的纯化：通过硫酸铵沉淀、疏水作用层析和阳离子交换层析成功地从家蚕五龄第7天血液中纯化到BmLP1、BmLP7、含2种30K蛋白的组分和含4种30K蛋白的组分。
(2)30K蛋白载脂功能的验证：对纯化的30K蛋白进行SDS-PAGE电泳,并用油红O染色法染色,结果表明纯化得到的30K蛋白含有脂类,表明30K蛋白具有载脂功能。
(3)30K蛋白所结合脂类的鉴定：采用氯仿和甲醇法提取30K蛋白所结合的脂类,并用LC-MS/MS进行鉴定。共鉴定到12种含量较高的脂类,其中分子式为C22H35O6、C25H31O8和C12H13O4的脂类较多,尤以分子式为C25H31Os的脂类最多,且该脂类含有多个同分异构体。但是,由于这些脂类分子结构较稳定,质谱碎裂时无法得到它们的结构信息,因而目前无法推测它们的结构式,需要进一步实验对其结构式进行鉴定。
(4)胚胎中脂类的利用：利用薄层层析方法对不同发育时期胚胎中的脂类进行分离,结果显示脂类主要在胚胎后期被消化吸收,而30K蛋白也是在胚胎后期被利用。因此,我们认为在胚胎利用30K蛋白的同时,30K蛋白所结合的脂类也一起被胚胎利用。
综合第3和第4点的结果,我们对家蚕30K蛋白的功能有了进一步的认识。从五龄期到上蔟期,几个30K蛋白基因(Bmlpl-Bmlp4,Bmlp7)在家蚕脂肪体中高量表达,之后分泌入家蚕血液中。在五龄末期的血液中30K蛋白含量最大,之后可能由卵黄原蛋白受体介导转运进卵巢的卵母细胞内。储存于卵巢内的30K蛋白直到下一个世代的胚胎期才被利用。30K蛋白最初位于胚外的卵黄颗粒中,到反转期后由胚子吞入肠腔内,肠腔内合成的降解酶对30K蛋白进行降解而被利用。同时,30K蛋白所结合的脂类也一同被利用,为蚁蚕孵化提供能量
Bombyx mori, the domesticated silkworm, is a lepidopteran model insect. Similar to most lepidoptera, the silkworm has an open vessel system. Hemolymph is involved in many physiological function processes, such as nutrient and hormone transportation. Silkworm hemolymph comprises21%～25%of the body weight, whereas proteins account for6%of the hemolymph.30K proteins (30KP) are the most abundant protein components at the late fifth instar larval and pupal stages, and30K proteins are also plentiful in silkworm eggs.30K proteins are classified into lepidopteran low molecular weight lipoproteins family. To date,30K proteins homologs have only been identified in Bombyx mori, Manduca sexta and Pseudaletia separata.
The9x coverage silkworm genome data has been assembled, as well as many publicly lepidopteran EST datasets are available. In our study, we identified lepidopteran low molecular weight lipoprotein genes based on these datasets, and then analyzed their structure and evolution. By using the published silkworm genome and microarray data, we analyzed the genome locations and the temporal-spatial expression patterns of lepidopteran low molecular weight lipoprotein genes.
On this basis, the function of silkworm typical30K proteins of lepidopteran low molecular weight lipoproteins family was characterized. In our study, we performed systematic studies of30K proteins and revealed their synthesis, transportation and degradation by using the BmLP1and BmLP7antibody. The results provide valuable preferences for other30K proteins. We also purified several30K proteins, and then extracted lipids which bind to30K proteins, and identified them by LC-MS/MS.
The main results are as follows:
1. Identification of lepidopteran low molecular weight lipoprotein genes
(1) Identification of lepidopteran low molecular weight lipoprotein genes:We performed an extensive survey of lepidopteran-derived genome and EST datasets. We identified7330KP homologous genes in12lepidopteran species (belong to Bombycoidea, Noctuoidea, and Pyraloidea), including B. mori (46), Samia cynthia ricini (5), Antheraea assama (6), Antheraea mylitta (4), M. sexta (3), Spodoptera frugiperda (1), Pseudaletia separate (1), Striacosta albicosta (1), Heliothis virescens (1), Mamestra brassicae (1), Trichoplusia ni (1), and Maruca vitrata (1). Among these identified genes,5630KP genes are novel members.
(2) Classification of lepidopteran low molecular weight lipoproteins:The multiple sequence alignment results revealed that the identified30KP homologous genes could be classified into three groups:ENF-BP genes, typical30KP genes, and serine/threonine-rich30KP (S/T-rich30KP) genes. The C-terminal regions are common to all the three subfamilies (NTD and CTD), but the N-termini are highly variable. The N-terminal region of typical30KP is composed of the signal peptides and an unconserved region. S/T-rich30KP have an exclusive S/T-rich domain between signal peptides and NTD in the N terminus. ENF-BP was found to contain a special domain in the N terminus, which is homologous to Pp-0912of Pseudomonas putida.
(3) Phylogenetic analysis of lepidopteran low molecular weight lipoprotein genes: In Bombycoidea insects, we found typical30KP genes, S/T-rich30KP and ENF-BP genes. However, in Noctuidea and Pyralidea, we only found ENF-BP genes. By searching against the whole genome data of H.virescens (unpublished data), we identify a total of four30KP genes, all of which belong to ENF-BP subfamily. Therefore, ENF-BP subfamily may be the origin of lepidopteran low molecular weight lipoprotein family. S/T-rich30KPs were only found in B. mori, and may be a Bombyx-specific low molecular weight lipoprotein subfamily.
2. Characterization of silkworm low molecular weight lipoproteins
(1) Genome locations of silkworm low molecular weight lipoprotein genes:46low molecular weight lipoprotein genes were identified in the silkworm, and43genes could be located on the chromosomes.24typical30KP genes (Bmlpl-Bmlp24) and8S/T-rich30KP genes (BmIp24-Bmlp32) exist in nscaf2795of Chr.20. The32genes are confined within970Kbp (1380K-2350K) and display a tandem pattern. Four sense and antisense transcnptional pairs (Bmlp37/38and Bmlp40/39and Bmlp41/42and Bmlp44/45) of ENF-BPs exist on chromosome7,22, and24in B. mori.
(2) Temporal-spatial expression patterns of silkworm low molecular weight lipoprotein genes:The three groups have their respective temporal-spatial expression patterns, which revealed the functional divergence of three subfamilies. ENF-BP genes are mainly expressed in the hemocyte. Typical30KP genes are expressed mainly in the fat body and integument. The cluster analysis on30KP genes suggested that they show five different temporal expression profiles in the whole life cycle. S/T-rich30KP genes are expressed highly in testis, whereas lowly in ovary during the metamorphosis in which a pupa turns into an adult. S/T-rich30KPs may be involved in the process of spermatogenesis or fertilization because they are expressed highly in testis.
3. The synthesis, transportation, degradation and utilization of30K proteins
(1) The synthesis and secretion of30K proteins:RT-PCR was used to analyze the developmental expression profile of30K protein genes in the fat body. The protein level of30K protein in the hemolymph was detected by western blotting using the antibody. Comparative analysis revealed that30K proteins were synthesized in fat body and then secreted into hemolymph.
(2) The transportation of30K proteins:In the pupal and adult stage, the protein level of30K proteins decreases gradually in the hemolymph, whereas increasing in the ovary. Our results revealed that the30K proteins in the ovary were transported mostly from the hemolymph and not the result of mRNA translation in the ovary. We made a30K proteins affinity chromatography column. The result of pull-down assays of membrane proteins of ovary showed that30K proteins could interact with vitellogenin receptor. We speculated that vitellogenin receptor is the receptor of30K proteins, but further investigation is needed before we address a definite conclusion. We used immunohistochemistry to confirm the location of30K proteins in the ovary and found that most of30K proteins were located in the oocyte of eggs.
(3) The utilization of30K proteins:Western blotting analysis showed that30K proteins were present at only very low levels at days9and10during embryogenesis. The proteolytic activity of the crude extracts from eggs or newly hatched larvae was measured throughout embryogenesis of the silkworm, and suggested that protease activity increase from day5eggs and attained a maximum in the newly hatched larvae. The densitometry showed that30K proteins were degraded after being incubated with the crude extracts of the newly hatched larvae. Immunohistochemistry revealed that strong signals for30K proteins were detected in the extraembryonic yolk granules at day6eggs. At day10eggs,30K proteins filled the whole gut lumen. Integrated these data suggest that3OK proteins first loacted in the extraembryonic yolk granules at day6eggs, when the embryonic reverse (blastokinesis) is completed,30K proteins are then ingested into the gut lumen. Our data clearly shows that degradation of30K proteins takes place in the gut lumen. We considered that degraded30K proteins may provide essential nutrients for hatching newly larvae.
4. Purification of30K proteins and identification of the lipids which bind to30K proteins
(1) Purification of30K proteins:We purified several30K proteins from day7fifth instar larvae by using ammonium sulfate precipitation, hydrophobic chromatography and cation exchange chromatography. The30K proteins including fraction of2kinds of30K proteins, fraction of4kinds of30K proteins, BmLPl and BmLP7.
(2)30K proteins can bind to lipids:The purified30K proteins were separated by SDS-PAGE. The result of oil red-O staining suggested that all purified30K proteins could bind to lipids.
(3) Identification of the lipids of30K proteins:The lipids of30K proteins were extracted by using chloroform/methanol (2/1) mixture and then analyzed by LC-MS/MS. In total, we identified12lipids, and three lipids (molecular formulas are C22H35O6, C25H31O8, and C12H13O4) are abundant component. The lipid whose molecular formula is C25H31O8is the most abundant, and it has various isomerides. However, these lipids are too stable to obtain more information about their structure formula. So, we can not detecte their structure formula and we will detect the structure formula in future.
(4) The utilization of lipids during embryogenesis:Thin-layer chromatography was performed to analyze the change pattern of lipids during embryogenesis. We found that the lipids decrease in the anaphase of embryogenesis. Therefore, we suggested that30K proteins together with the lipids were utilized in the anaphase of embryogenesis to provide essential nutrients for hatching newly larvae.
Taken together with3and4, we found that30K protein genes(Bmlp1-Bmlp4, Bmlp7) were synthesized in fat body and then secreted into hemolymph from the fifth instar larval stage to wandering stage.30K proteins attained the maximum in the anaphase of the fifth instar, and then transported to the oocyte of eggs of ovary. Vitellogenin receptor is the possible receptor of30K proteins.30K proteins did not be utilized until the next generation.30K proteins were first loacted in the extraembryonic yolk granules, when the embryonic reverse is completed,30K proteins could be easily ingested into the gut lumen.30K proteins were degraded by de novo proteases in the gut lumen.30K proteins together with their binding lipids were utilized in the anaphase of embryogenesis to provide essential nutrients for embryogenesis.
家蚕9倍基因组的组装成功,以及各种鳞翅目昆虫EST数据的公布,为系统性鉴定鳞翅目昆虫低分子量脂蛋白家族的成员奠定了良好的基础。本研究在此基础上,对鳞翅目昆虫的低分子量脂蛋白进行了鉴定,并对它们进行了结构分析和进化分析。此外,利用已公布的家蚕基因组和芯片数据,我们还对家蚕的低分子量脂蛋白基因进行了染色体定位分析、组织表达谱分析和发育时期表达谱分析。
在获得了以上数据的基础上,本研究重点对家蚕低分子量脂蛋白家族中的典型30K蛋白亚家族进行了功能研究。利用已经制备获得的BmLP1和BmLP7的抗体,我们对30K蛋白基因的合成、分泌、转运和利用等进行了系统性的研究。另外,本研究还从家蚕血液中纯化获得了几种30K蛋白,利用氯仿和甲醇法提取了30K蛋白所结合的脂类,并进一步利用LC-MS/MS对30K蛋白所结合的脂类进行了质谱鉴定。
本研究获得的主要结果如下：
1.鳞翅目昆虫低分子量脂蛋白基因的鉴定
(1)鳞翅目昆虫低分子量脂蛋白基因的鉴定：在家蚕基因组精细图序列数据和各种鳞翅目昆虫EST数据的基础上,本研究对鳞翅目昆虫的低分子量脂蛋白家族进行了鉴定。在蚕蛾总科、夜蛾总科和螟蛾总科的12个物种中,共鉴定到73个低分子量脂蛋白基因,它们的分布情况如下：家蚕46个、蓖麻蚕5个、琥珀蚕6个、印度柞蚕4个、烟草天蛾3个、草地贪夜蛾1个、粟夜蛾1个、西方豆类夜蛾1个、棉铃虫1个、甘蓝夜蛾1个、粉纹夜蛾1个和豆荚野螟蛾1个。在鉴定获得的73个基因中,有56个基因为首次鉴定。
(2)低分子量脂蛋白的结构分类：对鉴定到的基因进行序列比对,发现它们根据结构可以分为三个亚家族：典型30K蛋白(Typical30KP)、富含丝氨酸和苏氨酸的30K蛋白(S/T-rich30KP)和ENF肽结合蛋白(ENF-BP)。这三个亚家族有着相似的C端序列,都由一个NTD和一个CTD组成,而它们的N端序列差别很大。典型30K蛋白的N端由信号肽和一段很短的不保守区域组成；S/T-rich30KP的N端则由信号肽和一个富含丝氨酸和苏氨酸的结构域(STD)组成；ENF肽结合蛋白的N端由一个恶臭假单胞菌同源区(PPD)组成。
(3)低分子量脂蛋白的进化分析：在蚕蛾总科中,同时存在典型30K蛋白、富含丝氨酸和苏氨酸的30K蛋白和ENF肽结合蛋白亚家族。但在夜蛾总科和螟蛾总科中,我们只鉴定到了ENF肽结合蛋白亚家族。并且,在全基因组已被测序的棉铃虫中,也只能鉴定到ENF肽结合蛋白亚家族。这些结果表明,ENF肽结合蛋白亚家族是三个亚家族中起源最早的一个亚家族。富含丝氨酸和苏氨酸的30K蛋白只在家蚕中鉴定到,可能为家蚕特有的一群低分子量脂蛋白。
2.家蚕低分子量脂蛋白基因的特征分析
(1)家蚕低分子量脂蛋白基因的染色体定位：家蚕中共鉴定到46个低分子量脂蛋白基因,其中的43个基因能定位到染色体上。Bmlpl-Bmlp32(24个典型30K蛋白基因和8个富含丝氨酸和苏氨酸的30K蛋白基因)全部定位于第20号染色体的nscaf2795重叠群上的970Kbp(1380K-2350K)区域内,它们在染色体上呈串联重复分布。家蚕的10个ENF肽结合蛋白基因中,有8个基因构成了4个基因对(Bmlp37/38、Bmlp40/39、Bmlp41/42和Bmlp44/45),定位于第7号、22号和24号染色体上,每个基因对内的2个基因的转录方向相反。
(2)家蚕低分子量脂蛋白基因的表达特征分析：家蚕中的三群低分子量脂蛋白基因的组织和时期表达特征各不相同,暗示它们存在功能的分化。典型30K蛋白基因主要在脂肪体和体壁中表达,部分30K蛋白基因在一龄到四龄幼虫期表达,部分在五龄幼虫到蛾期表达。ENF肽结合蛋白基因主要在血细胞中表达,可能参与了血细胞的免疫调控过程。富含丝氨酸和苏氨酸的30K蛋白基因主要在羽化前一天和化蛾第一天有表达,在精巢中的表达量很高,而在卵巢中几乎不表达。根据其富含丝氨酸和苏氨酸的特征,推测可能参与受精或者精子的形成过程。
3.家蚕30K蛋白的合成、分泌、转运和利用
(1)家蚕30K蛋白的合成和分泌：通过RT-PCR技术分析不同发育时期脂肪体中30K蛋白基因的合成模式。同时,通过western blotting技术对不同发育时期血液中30K蛋白的变化情况进行分析。综合以上两方面结果,表明30K蛋白是由脂肪体合成,然后分泌入血液。
(2)家蚕30K蛋白的转运：在蛹期和蛾期时,血液中的30K蛋白逐渐减少,而卵巢中的30K蛋白却逐渐增加。RT-PCR结果表明,卵巢中的30K蛋白并非自身合成而来。因此,卵巢中的30K蛋白可能是从血液中转运而来。通过制备30K蛋白亲和柱,对卵巢膜蛋白进行pull-down实验,发现卵黄原蛋白受体能与30K蛋白结合,因此推测卵黄原蛋白受体可能是介导30K蛋白转运的受体。免疫组织化学定位结果表明,30K蛋白主要转运进卵巢的卵母细胞内储存起来。
(3)家蚕30K蛋白的利用：对不同发育时期的胚胎蛋白质进行western blotting分析,发现30K蛋白主要在胚胎的最后两天减少。对不同发育时期的胚胎进行酶活分析,发现酶活从胚胎第5天后升高,到蚁蚕时达到最大值。提取蚁蚕粗提物,并与30K蛋白进行体外孵育,发现蚁蚕粗提物能够降解30K蛋白。对第6天和第10天的胚胎进行免疫组织化学定位,结果表明30K蛋白在胚胎早期主要定位于胚外的卵黄颗粒中,在胚胎后期定位于胚子的肠腔内。综合以上结果,发现30K蛋白在反转期前定位于胚外卵黄颗粒中,反转期后由胚子吞入肠腔内,再由肠腔内的蛋白酶对30K蛋白进行降解,为蚁蚕的孵化提供能量来源。
4.家蚕30K蛋白的纯化及其结合脂类的鉴定
(1)30K蛋白的纯化：通过硫酸铵沉淀、疏水作用层析和阳离子交换层析成功地从家蚕五龄第7天血液中纯化到BmLP1、BmLP7、含2种30K蛋白的组分和含4种30K蛋白的组分。
(2)30K蛋白载脂功能的验证：对纯化的30K蛋白进行SDS-PAGE电泳,并用油红O染色法染色,结果表明纯化得到的30K蛋白含有脂类,表明30K蛋白具有载脂功能。
(3)30K蛋白所结合脂类的鉴定：采用氯仿和甲醇法提取30K蛋白所结合的脂类,并用LC-MS/MS进行鉴定。共鉴定到12种含量较高的脂类,其中分子式为C22H35O6、C25H31O8和C12H13O4的脂类较多,尤以分子式为C25H31Os的脂类最多,且该脂类含有多个同分异构体。但是,由于这些脂类分子结构较稳定,质谱碎裂时无法得到它们的结构信息,因而目前无法推测它们的结构式,需要进一步实验对其结构式进行鉴定。
(4)胚胎中脂类的利用：利用薄层层析方法对不同发育时期胚胎中的脂类进行分离,结果显示脂类主要在胚胎后期被消化吸收,而30K蛋白也是在胚胎后期被利用。因此,我们认为在胚胎利用30K蛋白的同时,30K蛋白所结合的脂类也一起被胚胎利用。
综合第3和第4点的结果,我们对家蚕30K蛋白的功能有了进一步的认识。从五龄期到上蔟期,几个30K蛋白基因(Bmlpl-Bmlp4,Bmlp7)在家蚕脂肪体中高量表达,之后分泌入家蚕血液中。在五龄末期的血液中30K蛋白含量最大,之后可能由卵黄原蛋白受体介导转运进卵巢的卵母细胞内。储存于卵巢内的30K蛋白直到下一个世代的胚胎期才被利用。30K蛋白最初位于胚外的卵黄颗粒中,到反转期后由胚子吞入肠腔内,肠腔内合成的降解酶对30K蛋白进行降解而被利用。同时,30K蛋白所结合的脂类也一同被利用,为蚁蚕孵化提供能量
Bombyx mori, the domesticated silkworm, is a lepidopteran model insect. Similar to most lepidoptera, the silkworm has an open vessel system. Hemolymph is involved in many physiological function processes, such as nutrient and hormone transportation. Silkworm hemolymph comprises21%～25%of the body weight, whereas proteins account for6%of the hemolymph.30K proteins (30KP) are the most abundant protein components at the late fifth instar larval and pupal stages, and30K proteins are also plentiful in silkworm eggs.30K proteins are classified into lepidopteran low molecular weight lipoproteins family. To date,30K proteins homologs have only been identified in Bombyx mori, Manduca sexta and Pseudaletia separata.
The9x coverage silkworm genome data has been assembled, as well as many publicly lepidopteran EST datasets are available. In our study, we identified lepidopteran low molecular weight lipoprotein genes based on these datasets, and then analyzed their structure and evolution. By using the published silkworm genome and microarray data, we analyzed the genome locations and the temporal-spatial expression patterns of lepidopteran low molecular weight lipoprotein genes.
On this basis, the function of silkworm typical30K proteins of lepidopteran low molecular weight lipoproteins family was characterized. In our study, we performed systematic studies of30K proteins and revealed their synthesis, transportation and degradation by using the BmLP1and BmLP7antibody. The results provide valuable preferences for other30K proteins. We also purified several30K proteins, and then extracted lipids which bind to30K proteins, and identified them by LC-MS/MS.
The main results are as follows:
1. Identification of lepidopteran low molecular weight lipoprotein genes
(1) Identification of lepidopteran low molecular weight lipoprotein genes:We performed an extensive survey of lepidopteran-derived genome and EST datasets. We identified7330KP homologous genes in12lepidopteran species (belong to Bombycoidea, Noctuoidea, and Pyraloidea), including B. mori (46), Samia cynthia ricini (5), Antheraea assama (6), Antheraea mylitta (4), M. sexta (3), Spodoptera frugiperda (1), Pseudaletia separate (1), Striacosta albicosta (1), Heliothis virescens (1), Mamestra brassicae (1), Trichoplusia ni (1), and Maruca vitrata (1). Among these identified genes,5630KP genes are novel members.
(2) Classification of lepidopteran low molecular weight lipoproteins:The multiple sequence alignment results revealed that the identified30KP homologous genes could be classified into three groups:ENF-BP genes, typical30KP genes, and serine/threonine-rich30KP (S/T-rich30KP) genes. The C-terminal regions are common to all the three subfamilies (NTD and CTD), but the N-termini are highly variable. The N-terminal region of typical30KP is composed of the signal peptides and an unconserved region. S/T-rich30KP have an exclusive S/T-rich domain between signal peptides and NTD in the N terminus. ENF-BP was found to contain a special domain in the N terminus, which is homologous to Pp-0912of Pseudomonas putida.
(3) Phylogenetic analysis of lepidopteran low molecular weight lipoprotein genes: In Bombycoidea insects, we found typical30KP genes, S/T-rich30KP and ENF-BP genes. However, in Noctuidea and Pyralidea, we only found ENF-BP genes. By searching against the whole genome data of H.virescens (unpublished data), we identify a total of four30KP genes, all of which belong to ENF-BP subfamily. Therefore, ENF-BP subfamily may be the origin of lepidopteran low molecular weight lipoprotein family. S/T-rich30KPs were only found in B. mori, and may be a Bombyx-specific low molecular weight lipoprotein subfamily.
2. Characterization of silkworm low molecular weight lipoproteins
(1) Genome locations of silkworm low molecular weight lipoprotein genes:46low molecular weight lipoprotein genes were identified in the silkworm, and43genes could be located on the chromosomes.24typical30KP genes (Bmlpl-Bmlp24) and8S/T-rich30KP genes (BmIp24-Bmlp32) exist in nscaf2795of Chr.20. The32genes are confined within970Kbp (1380K-2350K) and display a tandem pattern. Four sense and antisense transcnptional pairs (Bmlp37/38and Bmlp40/39and Bmlp41/42and Bmlp44/45) of ENF-BPs exist on chromosome7,22, and24in B. mori.
(2) Temporal-spatial expression patterns of silkworm low molecular weight lipoprotein genes:The three groups have their respective temporal-spatial expression patterns, which revealed the functional divergence of three subfamilies. ENF-BP genes are mainly expressed in the hemocyte. Typical30KP genes are expressed mainly in the fat body and integument. The cluster analysis on30KP genes suggested that they show five different temporal expression profiles in the whole life cycle. S/T-rich30KP genes are expressed highly in testis, whereas lowly in ovary during the metamorphosis in which a pupa turns into an adult. S/T-rich30KPs may be involved in the process of spermatogenesis or fertilization because they are expressed highly in testis.
3. The synthesis, transportation, degradation and utilization of30K proteins
(1) The synthesis and secretion of30K proteins:RT-PCR was used to analyze the developmental expression profile of30K protein genes in the fat body. The protein level of30K protein in the hemolymph was detected by western blotting using the antibody. Comparative analysis revealed that30K proteins were synthesized in fat body and then secreted into hemolymph.
(2) The transportation of30K proteins:In the pupal and adult stage, the protein level of30K proteins decreases gradually in the hemolymph, whereas increasing in the ovary. Our results revealed that the30K proteins in the ovary were transported mostly from the hemolymph and not the result of mRNA translation in the ovary. We made a30K proteins affinity chromatography column. The result of pull-down assays of membrane proteins of ovary showed that30K proteins could interact with vitellogenin receptor. We speculated that vitellogenin receptor is the receptor of30K proteins, but further investigation is needed before we address a definite conclusion. We used immunohistochemistry to confirm the location of30K proteins in the ovary and found that most of30K proteins were located in the oocyte of eggs.
(3) The utilization of30K proteins:Western blotting analysis showed that30K proteins were present at only very low levels at days9and10during embryogenesis. The proteolytic activity of the crude extracts from eggs or newly hatched larvae was measured throughout embryogenesis of the silkworm, and suggested that protease activity increase from day5eggs and attained a maximum in the newly hatched larvae. The densitometry showed that30K proteins were degraded after being incubated with the crude extracts of the newly hatched larvae. Immunohistochemistry revealed that strong signals for30K proteins were detected in the extraembryonic yolk granules at day6eggs. At day10eggs,30K proteins filled the whole gut lumen. Integrated these data suggest that3OK proteins first loacted in the extraembryonic yolk granules at day6eggs, when the embryonic reverse (blastokinesis) is completed,30K proteins are then ingested into the gut lumen. Our data clearly shows that degradation of30K proteins takes place in the gut lumen. We considered that degraded30K proteins may provide essential nutrients for hatching newly larvae.
4. Purification of30K proteins and identification of the lipids which bind to30K proteins
(1) Purification of30K proteins:We purified several30K proteins from day7fifth instar larvae by using ammonium sulfate precipitation, hydrophobic chromatography and cation exchange chromatography. The30K proteins including fraction of2kinds of30K proteins, fraction of4kinds of30K proteins, BmLPl and BmLP7.
(2)30K proteins can bind to lipids:The purified30K proteins were separated by SDS-PAGE. The result of oil red-O staining suggested that all purified30K proteins could bind to lipids.
(3) Identification of the lipids of30K proteins:The lipids of30K proteins were extracted by using chloroform/methanol (2/1) mixture and then analyzed by LC-MS/MS. In total, we identified12lipids, and three lipids (molecular formulas are C22H35O6, C25H31O8, and C12H13O4) are abundant component. The lipid whose molecular formula is C25H31O8is the most abundant, and it has various isomerides. However, these lipids are too stable to obtain more information about their structure formula. So, we can not detecte their structure formula and we will detect the structure formula in future.
(4) The utilization of lipids during embryogenesis:Thin-layer chromatography was performed to analyze the change pattern of lipids during embryogenesis. We found that the lipids decrease in the anaphase of embryogenesis. Therefore, we suggested that30K proteins together with the lipids were utilized in the anaphase of embryogenesis to provide essential nutrients for hatching newly larvae.
Taken together with3and4, we found that30K protein genes(Bmlp1-Bmlp4, Bmlp7) were synthesized in fat body and then secreted into hemolymph from the fifth instar larval stage to wandering stage.30K proteins attained the maximum in the anaphase of the fifth instar, and then transported to the oocyte of eggs of ovary. Vitellogenin receptor is the possible receptor of30K proteins.30K proteins did not be utilized until the next generation.30K proteins were first loacted in the extraembryonic yolk granules, when the embryonic reverse is completed,30K proteins could be easily ingested into the gut lumen.30K proteins were degraded by de novo proteases in the gut lumen.30K proteins together with their binding lipids were utilized in the anaphase of embryogenesis to provide essential nutrients for embryogenesis.
引文
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