家蚕血细胞特异标记BmintegrinaPS3的鉴定及BmUsh基因的造血分化调控研究
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摘要
家蚕为鳞翅目的模式昆虫,其整个体腔都处在循环血淋巴中。根据形态的不同,家蚕血细胞分为五种类型：原血细胞,颗粒细胞,浆细胞,拟绛色细胞和小球细胞,但各种血细胞在体内的增殖分化及发育形成过程仍不清楚。
本研究利用细胞增殖分子标记对家蚕幼虫时期血细胞的增殖进行系统分析。基于家蚕基因组数据库筛选并鉴定得到家蚕颗粒细胞的一个特异性分子标记BmintegrinaPS3,利用其表达特性追踪家蚕颗粒细胞的来源,并探讨颗粒细胞在免疫反应中的参与过程。克隆鉴定家蚕造血作用调控因子BmUsh基因,并分析其对造血作用的调控功能。主要研究结果如下：
1.家蚕幼虫血细胞的增殖
(1)家蚕循环血细胞的增殖：利用BrdU、PHH3及Tubulin抗体进行免疫荧光实验,发现家蚕5龄幼虫的循环血淋巴中不断有血细胞发生DNA复制,同时进入到细胞分裂。除了小球细胞,其他4种循环血细胞：原血细胞、浆细胞、颗粒细胞、拟绛色细胞均能被BrdU标记。血细胞增殖率从5龄起蚕到5龄盛食期逐步增高；在5龄5天时,为了给变态时期准备足够数量的血细胞,增殖率达到5龄时期的最大值；从5龄6天到上蔟一天,增殖率一直维持在一个较高的水平。使用灭活的大肠杆菌诱导家蚕5龄幼虫进行细胞免疫,总体细胞的增殖率增高,进行吞噬反应中的血细胞也能发生增殖。
(2)家蚕造血器官的结构、形态变化：造血器官位于翅原基中央,周围由脂肪体组织包裹。以大造为实验材料,利用DAPI染料对L5D1到L5D7的造血器官进行全组织染色,观察发现造血器官体积从5龄1天到5龄6天逐渐增大,而到5龄7天时,造血器官体积急剧减小,通过细胞凋亡检测及体外培养发现,5龄末期造血器官的减小主要是由于造血器官内的血细胞大量释放到循环血淋巴中,而非细胞凋亡。
(3)家蚕造血干细胞及造血前体细胞的鉴定：利用哺乳动物中干细胞鉴定的方法,延长BrdU体内标记时间进行BrdU滞留标记实验,发现家蚕循环血细胞中存在两种能被BrdU标记上的细胞：一种是能被BrdU长期标记上的血细胞,该细胞中的BrdU信号饱满,细胞周期缓慢,为潜在的造血干细胞；另一种是能被BrdU短期标记上的血细胞,它们能够不断地分裂导致BrdU信号被稀释,这一类细胞为造血前体细胞。对造血器官内的血细胞进行BrdU滞留标记实验,发现造血器官内血细胞分裂十分活跃,且其中增殖的血细胞不断排入到循环血细胞中,造血器官中的血细胞作为造血前体细胞不断地参与到幼虫的造血过程中。
2.家蚕颗粒细胞的标记物鉴定及其免疫功能研究
(1)BmintegrinαPS3的克隆及序列分析：通过对家蚕基因组芯片数据库进行分析,筛选发现预测基因BG1BMGA006624在血液组织中特异性地高量表达。通过预测序列及RACE技术,克隆得到包括其完整ORF的cDNA序列。该基因位于10号染色体的nscaf2855上,DNA全长为25988bp,含有25个外显子和24个内含子。ORF框长为2895bp,编码965aa,预测蛋白分子量为107.1kDa,等电点为5.16,含有整合素家族保守的integrina结构域,N端含有一个20aa的信号肽,在C端有典型的跨膜结构域。3D结构预测显示,该蛋白从24到596氨基酸的胞外域能形成一个屈膝结构。对各物种中的integrina同源序列进行多序列比对,构建系统发生树,结果显示该蛋白与组织特异表达性的PS3亚家族蛋白聚为一类,所以命名为BmintegrinaPS3。(2) BmintegrinaPS3基因的表达分析：利用RT-PCR及qRT-PCR检测Bm integrinaPS3基因在家蚕5龄3天各组织中的表达情况,发现其除了在头部微弱表达以外,在血细胞特异并高量表达。进一步检测Bm integrin aPS3基因在5龄各时期血细胞中的表达,发现其表达量从4龄眠期开始逐渐增高,在5龄6天时达到最高值,L5D7至变态期,都维持在一个较高的表达水平。
(3)BmintegrinaPS3的血细胞标记：原核诱导表达并纯化制得BmintegrinoaPS3重组蛋白,通过免疫新西兰兔后制得免疫血清。利用免疫血清进行Western blotting检测原核诱导产物,结果显示免疫血清能够特异识别重组表达的BmintegrinaPS3蛋白。Western blotting检测家蚕循环血细胞,发现免疫血清能够识别两个大小不同的蛋白亚型,并且分子量较小的一条带定位于细胞膜上。免疫荧光检测家蚕循环血细胞,发现免疫血清在体内能够特异识别家蚕循环血液中的颗粒细胞。通过与Tubulin抗体共染,显示BmintegrinaPS3的信号主要位于家蚕体内颗粒细胞细胞膜上,呈点状分布,推测该蛋白在体内执行功能时主要位于细胞膜上。
(4)BmintegrinαPS3蛋白的亚细胞定位变化：为了进一步鉴定BmintegrinαPS3蛋白的表达特性,构建家蚕BmintegrinαPS3全长表达载体转染进入家蚕胚胎细胞系,发现BmintegrinαPS3-EGFP融合蛋白均匀分布于细胞质中。将3个Bmintegrinp因子全长表达载体分别与BmintegrinαPS3全长表达载体共同转染时,发现有β亚基存在时,BmintegrinαPS3能够聚集到β亚基所在之处。尤其是6150β亚基能够完全招募所有的BmintegrinαPS3分子,构成αPS3β36150二聚体。以上结果显示,αPS3亚基的定位在体内可能存在两种表达形式,一种是在没有β亚基等因子的情况下,其表达定位于细胞质中,另外一种则是在有β亚基等因子的情况下有,αPS3亚基能够被活化形成一个较小分子量的蛋白,定位于细胞膜上。
(5)家蚕颗粒细胞的来源：利用RT-PCR及qRT-PCR检测Bm integrin αPS3基因在家蚕各时期胚胎的表达情况,发现胚胎第7天,BmintegrinαPS3开始表达,直到胚胎末期都维持一个较高的表达水平,说明颗粒细胞在胚胎第7天逐渐开始产生,之后一直存在于循环血淋巴中。通过造血器官的体外培养及剥离实验,发现造血器官中的血细胞在排到循环血淋巴后,一部分细胞能分化形成颗粒细胞。
(6)家蚕颗粒细胞的免疫功能：对家蚕幼虫注射凝胶珠后发现Bm integrinαPS3的表达量在注射3h时就已经上调表达,在12h-24h时,维持在一个显著性增高的表达水平；在48h时,其表达量降低到接近正常的水平。对家蚕幼虫注射灭活后的大肠杆菌后发现BmintegrinαPS3的表达量在6h时,就已经大幅度地增加,而到12h时微弱地降低,在24h时,已经接近于正常的水平。以上实验说明颗粒细胞作为细胞免疫的主要成员能参与到包囊反应及吞噬反应中,在进行包囊反应时,过程相对较长,而在进行吞噬反应时,能迅速地参与并完成吞噬反应。
3.家蚕造血作用因子的克隆及功能鉴定
(1)BmUsh的克隆及序列分析：通过家蚕基因组数据库预测序列及RACE技术,克隆得到家蚕BmUsh基因完整的cDNA序列。该基因位于4号染色体上,含有3个外显子和2个内含子。ORF框长为2409bp,编码803aa,预测分子量为85.89kDa,等电点为8.3。BmUsh含有8个典型的C2H2型锌指蛋白结构域,属于FOG蛋白家族成员。对各物种FOG同源序列进行多序列比对构建系统发生树,结果显示,FOG从无脊椎动物到脊椎动物都是保守的,各种昆虫中的FOG成员都只有Ush,而由于长期进化的原因,在哺乳动物的形成过程中,FOG基因也发生了扩增,存在两个FOG同源基因FOG-1,FOG-2。
(2) BmUsh基因的表达分析：BmUsh基因从胚胎第1天开始至胚胎第4天表达量逐渐降低,胚胎第5天至胚胎第9天几乎不表达。在家蚕5龄3天各组织中均有表达,但在血细胞中的表达量显著性地高于其他组织。检测BmUsh基因在5龄各时期血细胞中的表达情况,结果显示BmUsh基因在眠期、盛时期、上蔟时期都上调表达。
(3) BmUsh核定位信号鉴定：Ush作为转录因子定位到细胞核内,通过序列分析预测,发现BmUsh蛋白中450-459这10个位点的氨基酸中有4个精氨酸的串联,为典型的核定位信号基序。将这一基序与DsRed共同构建到瞬时表达载体中,转染家蚕细胞系后,发现DsRed能够完全定位到细胞核中。同时将4个精氨酸逐一进行点突变,发现第1位和第3位精氨酸突变后核定位信号能力完全丢失,DsRed定位在了细胞质中。以上结果说明450-459这10个位点的氨基酸为BmUsh的核定位信号,其中456和458位精氨酸起着不可缺少的作用。
(4) BmUsh的相互作用因子：为了进一步寻找到BmUsh蛋白的相互作用因子,构建了家蚕BmUsh全长表达载体,BmUsh-DsRed融合蛋白完全表达定位于细胞核中。同时将BmUsh全长蛋白中456和458位的精氨酸进行点突变,发现突变蛋白都不能正常进入到细胞核内。说明该位点精氨酸在全长蛋白中确定决定着核定位信号。将另外一个造血作用因子BmLz全长表达载体与Bm Ush全长表达载体共同转染时,发现BmLz蛋白能与正常的BmUsh蛋白共同定位表达于细胞核内；同时将BmLz全长表达载体分别与BmUsh突变载体共同转染时,发现BmLz蛋白能将BmUsh突变蛋白由细胞质内迁移到细胞核内,但也有非常少数的情况,Lz(?)能被BmUsh点突变蛋白召集到细胞质中。利用昆虫杆状病毒表达载体同时高量表达BmUsh和BmLz的融合蛋白,免疫共沉淀实验直接证明了BmUsh和BmLz能够相互作用。综上,推测BmLz蛋白为BmUsh蛋白的相互作用因子,能够在细胞核内发挥重要的功能。
(5) BmUsh的造血作用调控功能：在原代培养的循环血细胞中转染BmUsh全长表达载体,发现BmUsh融合蛋白在家蚕血细胞中表达于细胞核内。观察对照质粒转染的结果,发现转染成功的细胞100%为浆细胞,而与之相比BmUsh融合蛋白所表达的血细胞形态发生了形态上的变化,类似于体外培养的拟绛色细胞。推测BmUsh蛋白在浆细胞细胞核内高表达之后,促进浆细胞分化成为拟绛色细胞。
合成双链RNA后注射5龄5天家蚕幼虫,mRNA表达水平检测显示BmUsh被成功下调表达,同时黑化效应因子PPO1,PPO2及其上游的激酶PPAE及BAEE在血细胞中的表达量同样也被下调。在表型上,发现注射BmUsh干涉组的蚕体血淋巴不能正常进行黑化反应。以上结果与前面的高表达实验结果共同说明了BmUsh基因作为细胞核内转录因子调控浆细胞分化为拟绛色细胞。
(6) BmUsh参与血细胞的先天免疫：对家蚕幼虫注射灭活后的大肠杆菌后发现BmUsh的表达量在6h时增加了20倍,而在12h-24h时已经接近于正常的水平。黑化途径效应因子PPO1和PPO2的表达变化与BmUsh的表达量变化趋势一致。推测BmUsh通过调控拟绛色细胞的产生,参与到细胞免疫黑化反应中。
Silkworm is the typical model insects, and all the tissues are surrounded by the circulating hemolymph. According to their different morphology, five types of hemocytes have been identified in silkworm, Bombyx mori:prohemocytes, plasmatocytes, granulocytes, spherulocytes, and oenocytoids. The study of hemocytes in circulatory system and HPO in vitro has been well investigated, but precise lineage relationships among different hemocyte types remain unclear.
In the present study, we used molecular marker to monitor the proliferation of hemocytes in vivo. Based on the silkworm database, a graunulocyte specific marker BmintegrinαPS3were found and characterized. By the expression of BmintegrinαPS3, the sources and immune function of granulocytes were investigated. Through the homology search, we cloned and functional analysed the hematopoiesis regulator gene Bm Ush in silkworm. The results are as follows:
1. The proliferation of larval hemocytes
(1) The proliferation of larval hemocytes in circulatory system:During the first four days of the final instar, hemocyte proliferation gradually increased, while BrdU incorporation attained its highest level on L5D5. Subsequently, the BrdU labelling indices were maintained from L5D6to wandering stage. The double immunofluorescence analysis showed that the circulating hemocytes could symmetrically divide into two new daughter cells.All types of hemocytes in circulatory system, excluding spheruloeytes,could be labelled by BrdU. By the stimulus of heat-inactivated E.coli, the percentage of BrdU positive hemocytes significantly increased. The hemocytes could keep proliferating to produce more new cells to meet the requirement for cellular defence.
(2) The structures of HPOs during the final instar:HPOs are surrounded by some fat bodies and are located on the wing discs of larvae. By DAPI staining, the developments of HPOs during5th instar larvae were investigated. The data showed that the sizes of HPOs were gradually increased from L5D1to L5D6, while the size on L5D7significantly decreased because of the hemocytes'discharge into hemolymph, not cell apoptosis.
(3) The hematopoietic stem cells and hematopoietic progenitors:To locate the hemocyte precursor cells, the label time was extended to carry the BrdU label retention assay.A small numbers of hemocytes retained intense BrdU signals and were termed long-term label-retention cells which were be putative stem cells, while there were many short-term BrdU label hemocytes in circulatory hemolymph which would be hematopoietic progenitors. At the same time, we did BrdU label retention analysis in HPO and found that the hemocytes in HPO actively divided and continually discharge the proliferating hemocytes into hemolymph. So there were short-term BrdU label hemocytes in HPO which would be hematopoietic progenitors.
2. The molecular marker and cell immune function of granulocytes
(1) Granulocyte-specific genes cloning and sequence analysis:By searching the silkworm genome database, we find BmintegrinαcPS3was highly and specifically expressed in hemocytes. Through the known sequence data and RACE, the BmintegrinαPS3cDNA including the complete ORF sequence was cloned. BmintegrinaPS3locates on chromosome10, the DNA total length is25988bp, containing25exons and24introns. The ORF length is2895bp, encoding965aa, molecular weight is106.5kDa, and isoelectric point is5.16. BmintegrinocPS3contains5conserved integrin alpha domains, has a length of20amino acid signal peptide at the N-terminus, and a typical transmembrane domain at the C terminus. The3D structure of BmintegrinαPS3was predicted to be bent that is very similar to the resolution structure of integrins in mammal.The phylogenetic tree of integrin homologues from different species showed that BmintegrinaPS3was closer to the PS3family.
(2) The mRNA expression pattern of BmintegrinαPS3in silkworm: BmintegrinαPS3was expressed at a high level in hemocytes and at a very low level in head. During each period of the fifth instar, from the4th instar molting stage expression level of BmintegrinαPS3began gradually increasing, until L5D6its expression levels reached the highest, and from L5D7to W2, its expression maintained high levels.
(3) The subcellular location of BmintegrinocPS3in hemocyte:The BmintegrinocPS3fusion protein was prokaryotic expressed in E.coli, then was affinity purified. Antiserum was produced by immunizing rabbits and was used to detect induced fusion protein. The results proved that antiserum specifically recognized the BmintegrinαPS3fusion protein. Western blotting analysis was carried out in hemocytes with antiserum, and the results showed that BmintegrinocPS3had two different forms:a larger form expressed in cell cytoplasm and a smaller form expressed on cell membrane. Using antiserum for immunofluorescence, we found that the BmintegrinaPS3can specifically recognize granulocytes and mainly distributed on the cell membrane.
(4) The activation of BmintegrinaPS3:To further characterize the BmintegrinaPS3protein, the full-length expression vector was constructed and transfected into BmE-SWU3cell line. The results showed that BmintegrinαPS3protein uniformly distributed in cell cytoplasm of BmE-SWU3cells. When three Bmintegrinp full-length expression vectors were respectively cotransfected with BmintegrinocPS3full-length expression vector, BmintegrinαPS3could be gathered to where β subunit existed. Especially, the6150β subunit could gather all the BmintegrinαPS3molecule that one cell expressed. That suggested that the interaction between BmintegrinαPS3and6150β subunit was closer than other two β subunits. BmintegrinocPS3and6150β subunit could form dimmer on cell membrane. All the above, BmintegrinαPS3could exist in two forms:one form expressed in cell cytoplasm when no β subunit and other factors; the other form cutted by some factors and gathered on cell membrane by β subunit.
(5) The source of silkworm granulocytes:The RT-PCR and Real-time PCR showed that BmintegrinαPS3expressed from7th day of embryo stage and maintained a high expression level untill the end of embryo stage. It suggests that the granulocytes begin to be produced from precursor cells on7th day of embryo stage and exist in circulatory hemolymph. Through the culture of HPO in vitro and loss of HPO in vivo, it shows that a small part of hemocytes from HPO were differentiated into granulocytes in hemolymph.
(6) The immune function of silkworm granulocytes:Injection of bead could induce the encapsulation response by the hemocytes. During the immune response, the expression of BmintegrinαPS3was upregulated at3h, maintained a significantly higher expression levels at12h, and back to the normal level at24h. With the same method, injection of heat-inactivated E.coli could induce the phagocytosis response by the hemocytes. The expression of BmintegrinαPS3upregulated a lot at6h, down regulated weekly at12h, and already close to normal level at24h. All these results indicated that granulocytes as the executor of cell immune response, participate into encapsulation and phagocytosis in different speed.
3. Cloning and functional analysis of the hematopoiesis regulation factor BmUsh in silkworm
(1) Cloning and sequence analysis of BmUsh:BmUsh cDNA including the complete ORF sequence was cloned. BmUsh locates on chromosome4, containing3exons and2introns. The ORF length is2409bp, encoding803aa, molecular weight is85.89kDa, and isoelectric point is8.3. BmUsh contains8conserved C2H2zinc domains.The phylogenetic tree of FOG homologues from different species showed that FOGs were conserved from invertebrates to vertebrate. There is one FOG in every insect species and during molecular evolution there are two FOGs in every mammal species. BmUsh was closer to the insect Ush family.
(2) The mRNA expression pattern of BmUsh in silkworm:The Real-time PCR with cDNA of different stage of embryo showed the expression of BmUsh decreased gradually from lth day to9th day. The Real-time PCR with cDNA of different tissues on L5D3showed that BmUsh expressed in several tissues, but significantly higher in hemocytes than other tissues. The Real-time PCR with cDNA of different stage hemocyte in5th showed that BmUsh were upregulated at the molting stage, the middle day of larve, and the wandering stage.
(3) Identification of Nuclear Localization Signal (NLS) in BmUsh: Ush as transcription factors must be targeted to the nucleus. By sequence analysis,10aa at the450-459amino acids site with4arginine series was predicted to be typical NLS motif. This motif with DsRed was together cloned into transient expression vector, and expressed in BmE-SWU3, while each of the four arginine were site mutated respectively and constructed into vector in the same method. The results showed that the predicted NLS motif was able to target all DsRed molecules to nucleus, but the NLS motif with lth and3th arginine mutation could not target any DsRed molecules to nucleus.
(4) The interaction factor of BmUsh:In order to find the interaction factor of BmUsh, the full-length expression vector of BmUsh was constructed and transfected into BmE-SWU3. The BmUsh-DsRed were highly expressed and clustered in nucleus. Accoring to the known NLS site, we mutated the456and458arginines of full-length BmUsh and found that the mutant BmUsh could not enter into nucleus. Direct evidence showed the locus indeed played the role of the nuclear localization signal in the full-length protein of BmUsh. Another hematopoiesis regulation factor BmLz was clone into full-length expression vector and cotransfected with BmUsh and mutant BmUsh. The results showed that BmLz was co-located with BmUsh in same site of nucleus, and the BmLz could gather the mutant BmUsh into nucleus or enter into cell cytoplasma to co-locate with mutant BmUsh. BmUsh and BmLz were simultaneously overexpressed by Bac-to-Bac/BmNPV expression system. The immunoprecipitation assay by recombinant bacmid illustrates that BmUsh and BmLz as a protein-protein interaction performs functions in nucleus.
(5) The hematopoiesis regulation function of BmUsh:BmUsh was overexpressed in primary culture of circulating hemocytes with the full-length expression vector of BmUsh. BmUsh protein was distributed in nucleus of hemocytes. Comparision between the control plasmid and BmUsh plasmid, the BmUsh could promote the plasmatocyte differentiating into the oenocytoid. Larvae on L5D5were injected with synthetic double-stranded RNA, and the expression of BmUsh was successful knock down. Meanwhile, the expressions of PPO1, PPO2, PPAE, and BAEE in hemocytes were also down. The melanization response of RNAi larvae was not processed. The overexpression and knowndown assay show that BmUsh regulate the differentiation of oenocytoid.
(6) BmUsh involve the immune response:Injection L5D3larvae with heat-inactivated E.coli could induce the phagocytosis response of the hemocytes. The results showed that at6h, the expression level of BmUsh increased20-fold; at12h-24h, the expression level of BmUsh already were close to normal level. All these indicate that BmUsh is involved in the melanization response by regulating the differentiation of oenocytoid.
本研究利用细胞增殖分子标记对家蚕幼虫时期血细胞的增殖进行系统分析。基于家蚕基因组数据库筛选并鉴定得到家蚕颗粒细胞的一个特异性分子标记BmintegrinaPS3,利用其表达特性追踪家蚕颗粒细胞的来源,并探讨颗粒细胞在免疫反应中的参与过程。克隆鉴定家蚕造血作用调控因子BmUsh基因,并分析其对造血作用的调控功能。主要研究结果如下：
1.家蚕幼虫血细胞的增殖
(1)家蚕循环血细胞的增殖：利用BrdU、PHH3及Tubulin抗体进行免疫荧光实验,发现家蚕5龄幼虫的循环血淋巴中不断有血细胞发生DNA复制,同时进入到细胞分裂。除了小球细胞,其他4种循环血细胞：原血细胞、浆细胞、颗粒细胞、拟绛色细胞均能被BrdU标记。血细胞增殖率从5龄起蚕到5龄盛食期逐步增高；在5龄5天时,为了给变态时期准备足够数量的血细胞,增殖率达到5龄时期的最大值；从5龄6天到上蔟一天,增殖率一直维持在一个较高的水平。使用灭活的大肠杆菌诱导家蚕5龄幼虫进行细胞免疫,总体细胞的增殖率增高,进行吞噬反应中的血细胞也能发生增殖。
(2)家蚕造血器官的结构、形态变化：造血器官位于翅原基中央,周围由脂肪体组织包裹。以大造为实验材料,利用DAPI染料对L5D1到L5D7的造血器官进行全组织染色,观察发现造血器官体积从5龄1天到5龄6天逐渐增大,而到5龄7天时,造血器官体积急剧减小,通过细胞凋亡检测及体外培养发现,5龄末期造血器官的减小主要是由于造血器官内的血细胞大量释放到循环血淋巴中,而非细胞凋亡。
(3)家蚕造血干细胞及造血前体细胞的鉴定：利用哺乳动物中干细胞鉴定的方法,延长BrdU体内标记时间进行BrdU滞留标记实验,发现家蚕循环血细胞中存在两种能被BrdU标记上的细胞：一种是能被BrdU长期标记上的血细胞,该细胞中的BrdU信号饱满,细胞周期缓慢,为潜在的造血干细胞；另一种是能被BrdU短期标记上的血细胞,它们能够不断地分裂导致BrdU信号被稀释,这一类细胞为造血前体细胞。对造血器官内的血细胞进行BrdU滞留标记实验,发现造血器官内血细胞分裂十分活跃,且其中增殖的血细胞不断排入到循环血细胞中,造血器官中的血细胞作为造血前体细胞不断地参与到幼虫的造血过程中。
2.家蚕颗粒细胞的标记物鉴定及其免疫功能研究
(1)BmintegrinαPS3的克隆及序列分析：通过对家蚕基因组芯片数据库进行分析,筛选发现预测基因BG1BMGA006624在血液组织中特异性地高量表达。通过预测序列及RACE技术,克隆得到包括其完整ORF的cDNA序列。该基因位于10号染色体的nscaf2855上,DNA全长为25988bp,含有25个外显子和24个内含子。ORF框长为2895bp,编码965aa,预测蛋白分子量为107.1kDa,等电点为5.16,含有整合素家族保守的integrina结构域,N端含有一个20aa的信号肽,在C端有典型的跨膜结构域。3D结构预测显示,该蛋白从24到596氨基酸的胞外域能形成一个屈膝结构。对各物种中的integrina同源序列进行多序列比对,构建系统发生树,结果显示该蛋白与组织特异表达性的PS3亚家族蛋白聚为一类,所以命名为BmintegrinaPS3。(2) BmintegrinaPS3基因的表达分析：利用RT-PCR及qRT-PCR检测Bm integrinaPS3基因在家蚕5龄3天各组织中的表达情况,发现其除了在头部微弱表达以外,在血细胞特异并高量表达。进一步检测Bm integrin aPS3基因在5龄各时期血细胞中的表达,发现其表达量从4龄眠期开始逐渐增高,在5龄6天时达到最高值,L5D7至变态期,都维持在一个较高的表达水平。
(3)BmintegrinaPS3的血细胞标记：原核诱导表达并纯化制得BmintegrinoaPS3重组蛋白,通过免疫新西兰兔后制得免疫血清。利用免疫血清进行Western blotting检测原核诱导产物,结果显示免疫血清能够特异识别重组表达的BmintegrinaPS3蛋白。Western blotting检测家蚕循环血细胞,发现免疫血清能够识别两个大小不同的蛋白亚型,并且分子量较小的一条带定位于细胞膜上。免疫荧光检测家蚕循环血细胞,发现免疫血清在体内能够特异识别家蚕循环血液中的颗粒细胞。通过与Tubulin抗体共染,显示BmintegrinaPS3的信号主要位于家蚕体内颗粒细胞细胞膜上,呈点状分布,推测该蛋白在体内执行功能时主要位于细胞膜上。
(4)BmintegrinαPS3蛋白的亚细胞定位变化：为了进一步鉴定BmintegrinαPS3蛋白的表达特性,构建家蚕BmintegrinαPS3全长表达载体转染进入家蚕胚胎细胞系,发现BmintegrinαPS3-EGFP融合蛋白均匀分布于细胞质中。将3个Bmintegrinp因子全长表达载体分别与BmintegrinαPS3全长表达载体共同转染时,发现有β亚基存在时,BmintegrinαPS3能够聚集到β亚基所在之处。尤其是6150β亚基能够完全招募所有的BmintegrinαPS3分子,构成αPS3β36150二聚体。以上结果显示,αPS3亚基的定位在体内可能存在两种表达形式,一种是在没有β亚基等因子的情况下,其表达定位于细胞质中,另外一种则是在有β亚基等因子的情况下有,αPS3亚基能够被活化形成一个较小分子量的蛋白,定位于细胞膜上。
(5)家蚕颗粒细胞的来源：利用RT-PCR及qRT-PCR检测Bm integrin αPS3基因在家蚕各时期胚胎的表达情况,发现胚胎第7天,BmintegrinαPS3开始表达,直到胚胎末期都维持一个较高的表达水平,说明颗粒细胞在胚胎第7天逐渐开始产生,之后一直存在于循环血淋巴中。通过造血器官的体外培养及剥离实验,发现造血器官中的血细胞在排到循环血淋巴后,一部分细胞能分化形成颗粒细胞。
(6)家蚕颗粒细胞的免疫功能：对家蚕幼虫注射凝胶珠后发现Bm integrinαPS3的表达量在注射3h时就已经上调表达,在12h-24h时,维持在一个显著性增高的表达水平；在48h时,其表达量降低到接近正常的水平。对家蚕幼虫注射灭活后的大肠杆菌后发现BmintegrinαPS3的表达量在6h时,就已经大幅度地增加,而到12h时微弱地降低,在24h时,已经接近于正常的水平。以上实验说明颗粒细胞作为细胞免疫的主要成员能参与到包囊反应及吞噬反应中,在进行包囊反应时,过程相对较长,而在进行吞噬反应时,能迅速地参与并完成吞噬反应。
3.家蚕造血作用因子的克隆及功能鉴定
(1)BmUsh的克隆及序列分析：通过家蚕基因组数据库预测序列及RACE技术,克隆得到家蚕BmUsh基因完整的cDNA序列。该基因位于4号染色体上,含有3个外显子和2个内含子。ORF框长为2409bp,编码803aa,预测分子量为85.89kDa,等电点为8.3。BmUsh含有8个典型的C2H2型锌指蛋白结构域,属于FOG蛋白家族成员。对各物种FOG同源序列进行多序列比对构建系统发生树,结果显示,FOG从无脊椎动物到脊椎动物都是保守的,各种昆虫中的FOG成员都只有Ush,而由于长期进化的原因,在哺乳动物的形成过程中,FOG基因也发生了扩增,存在两个FOG同源基因FOG-1,FOG-2。
(2) BmUsh基因的表达分析：BmUsh基因从胚胎第1天开始至胚胎第4天表达量逐渐降低,胚胎第5天至胚胎第9天几乎不表达。在家蚕5龄3天各组织中均有表达,但在血细胞中的表达量显著性地高于其他组织。检测BmUsh基因在5龄各时期血细胞中的表达情况,结果显示BmUsh基因在眠期、盛时期、上蔟时期都上调表达。
(3) BmUsh核定位信号鉴定：Ush作为转录因子定位到细胞核内,通过序列分析预测,发现BmUsh蛋白中450-459这10个位点的氨基酸中有4个精氨酸的串联,为典型的核定位信号基序。将这一基序与DsRed共同构建到瞬时表达载体中,转染家蚕细胞系后,发现DsRed能够完全定位到细胞核中。同时将4个精氨酸逐一进行点突变,发现第1位和第3位精氨酸突变后核定位信号能力完全丢失,DsRed定位在了细胞质中。以上结果说明450-459这10个位点的氨基酸为BmUsh的核定位信号,其中456和458位精氨酸起着不可缺少的作用。
(4) BmUsh的相互作用因子：为了进一步寻找到BmUsh蛋白的相互作用因子,构建了家蚕BmUsh全长表达载体,BmUsh-DsRed融合蛋白完全表达定位于细胞核中。同时将BmUsh全长蛋白中456和458位的精氨酸进行点突变,发现突变蛋白都不能正常进入到细胞核内。说明该位点精氨酸在全长蛋白中确定决定着核定位信号。将另外一个造血作用因子BmLz全长表达载体与Bm Ush全长表达载体共同转染时,发现BmLz蛋白能与正常的BmUsh蛋白共同定位表达于细胞核内；同时将BmLz全长表达载体分别与BmUsh突变载体共同转染时,发现BmLz蛋白能将BmUsh突变蛋白由细胞质内迁移到细胞核内,但也有非常少数的情况,Lz(?)能被BmUsh点突变蛋白召集到细胞质中。利用昆虫杆状病毒表达载体同时高量表达BmUsh和BmLz的融合蛋白,免疫共沉淀实验直接证明了BmUsh和BmLz能够相互作用。综上,推测BmLz蛋白为BmUsh蛋白的相互作用因子,能够在细胞核内发挥重要的功能。
(5) BmUsh的造血作用调控功能：在原代培养的循环血细胞中转染BmUsh全长表达载体,发现BmUsh融合蛋白在家蚕血细胞中表达于细胞核内。观察对照质粒转染的结果,发现转染成功的细胞100%为浆细胞,而与之相比BmUsh融合蛋白所表达的血细胞形态发生了形态上的变化,类似于体外培养的拟绛色细胞。推测BmUsh蛋白在浆细胞细胞核内高表达之后,促进浆细胞分化成为拟绛色细胞。
合成双链RNA后注射5龄5天家蚕幼虫,mRNA表达水平检测显示BmUsh被成功下调表达,同时黑化效应因子PPO1,PPO2及其上游的激酶PPAE及BAEE在血细胞中的表达量同样也被下调。在表型上,发现注射BmUsh干涉组的蚕体血淋巴不能正常进行黑化反应。以上结果与前面的高表达实验结果共同说明了BmUsh基因作为细胞核内转录因子调控浆细胞分化为拟绛色细胞。
(6) BmUsh参与血细胞的先天免疫：对家蚕幼虫注射灭活后的大肠杆菌后发现BmUsh的表达量在6h时增加了20倍,而在12h-24h时已经接近于正常的水平。黑化途径效应因子PPO1和PPO2的表达变化与BmUsh的表达量变化趋势一致。推测BmUsh通过调控拟绛色细胞的产生,参与到细胞免疫黑化反应中。
Silkworm is the typical model insects, and all the tissues are surrounded by the circulating hemolymph. According to their different morphology, five types of hemocytes have been identified in silkworm, Bombyx mori:prohemocytes, plasmatocytes, granulocytes, spherulocytes, and oenocytoids. The study of hemocytes in circulatory system and HPO in vitro has been well investigated, but precise lineage relationships among different hemocyte types remain unclear.
In the present study, we used molecular marker to monitor the proliferation of hemocytes in vivo. Based on the silkworm database, a graunulocyte specific marker BmintegrinαPS3were found and characterized. By the expression of BmintegrinαPS3, the sources and immune function of granulocytes were investigated. Through the homology search, we cloned and functional analysed the hematopoiesis regulator gene Bm Ush in silkworm. The results are as follows:
1. The proliferation of larval hemocytes
(1) The proliferation of larval hemocytes in circulatory system:During the first four days of the final instar, hemocyte proliferation gradually increased, while BrdU incorporation attained its highest level on L5D5. Subsequently, the BrdU labelling indices were maintained from L5D6to wandering stage. The double immunofluorescence analysis showed that the circulating hemocytes could symmetrically divide into two new daughter cells.All types of hemocytes in circulatory system, excluding spheruloeytes,could be labelled by BrdU. By the stimulus of heat-inactivated E.coli, the percentage of BrdU positive hemocytes significantly increased. The hemocytes could keep proliferating to produce more new cells to meet the requirement for cellular defence.
(2) The structures of HPOs during the final instar:HPOs are surrounded by some fat bodies and are located on the wing discs of larvae. By DAPI staining, the developments of HPOs during5th instar larvae were investigated. The data showed that the sizes of HPOs were gradually increased from L5D1to L5D6, while the size on L5D7significantly decreased because of the hemocytes'discharge into hemolymph, not cell apoptosis.
(3) The hematopoietic stem cells and hematopoietic progenitors:To locate the hemocyte precursor cells, the label time was extended to carry the BrdU label retention assay.A small numbers of hemocytes retained intense BrdU signals and were termed long-term label-retention cells which were be putative stem cells, while there were many short-term BrdU label hemocytes in circulatory hemolymph which would be hematopoietic progenitors. At the same time, we did BrdU label retention analysis in HPO and found that the hemocytes in HPO actively divided and continually discharge the proliferating hemocytes into hemolymph. So there were short-term BrdU label hemocytes in HPO which would be hematopoietic progenitors.
2. The molecular marker and cell immune function of granulocytes
(1) Granulocyte-specific genes cloning and sequence analysis:By searching the silkworm genome database, we find BmintegrinαcPS3was highly and specifically expressed in hemocytes. Through the known sequence data and RACE, the BmintegrinαPS3cDNA including the complete ORF sequence was cloned. BmintegrinaPS3locates on chromosome10, the DNA total length is25988bp, containing25exons and24introns. The ORF length is2895bp, encoding965aa, molecular weight is106.5kDa, and isoelectric point is5.16. BmintegrinocPS3contains5conserved integrin alpha domains, has a length of20amino acid signal peptide at the N-terminus, and a typical transmembrane domain at the C terminus. The3D structure of BmintegrinαPS3was predicted to be bent that is very similar to the resolution structure of integrins in mammal.The phylogenetic tree of integrin homologues from different species showed that BmintegrinaPS3was closer to the PS3family.
(2) The mRNA expression pattern of BmintegrinαPS3in silkworm: BmintegrinαPS3was expressed at a high level in hemocytes and at a very low level in head. During each period of the fifth instar, from the4th instar molting stage expression level of BmintegrinαPS3began gradually increasing, until L5D6its expression levels reached the highest, and from L5D7to W2, its expression maintained high levels.
(3) The subcellular location of BmintegrinocPS3in hemocyte:The BmintegrinocPS3fusion protein was prokaryotic expressed in E.coli, then was affinity purified. Antiserum was produced by immunizing rabbits and was used to detect induced fusion protein. The results proved that antiserum specifically recognized the BmintegrinαPS3fusion protein. Western blotting analysis was carried out in hemocytes with antiserum, and the results showed that BmintegrinocPS3had two different forms:a larger form expressed in cell cytoplasm and a smaller form expressed on cell membrane. Using antiserum for immunofluorescence, we found that the BmintegrinaPS3can specifically recognize granulocytes and mainly distributed on the cell membrane.
(4) The activation of BmintegrinaPS3:To further characterize the BmintegrinaPS3protein, the full-length expression vector was constructed and transfected into BmE-SWU3cell line. The results showed that BmintegrinαPS3protein uniformly distributed in cell cytoplasm of BmE-SWU3cells. When three Bmintegrinp full-length expression vectors were respectively cotransfected with BmintegrinocPS3full-length expression vector, BmintegrinαPS3could be gathered to where β subunit existed. Especially, the6150β subunit could gather all the BmintegrinαPS3molecule that one cell expressed. That suggested that the interaction between BmintegrinαPS3and6150β subunit was closer than other two β subunits. BmintegrinocPS3and6150β subunit could form dimmer on cell membrane. All the above, BmintegrinαPS3could exist in two forms:one form expressed in cell cytoplasm when no β subunit and other factors; the other form cutted by some factors and gathered on cell membrane by β subunit.
(5) The source of silkworm granulocytes:The RT-PCR and Real-time PCR showed that BmintegrinαPS3expressed from7th day of embryo stage and maintained a high expression level untill the end of embryo stage. It suggests that the granulocytes begin to be produced from precursor cells on7th day of embryo stage and exist in circulatory hemolymph. Through the culture of HPO in vitro and loss of HPO in vivo, it shows that a small part of hemocytes from HPO were differentiated into granulocytes in hemolymph.
(6) The immune function of silkworm granulocytes:Injection of bead could induce the encapsulation response by the hemocytes. During the immune response, the expression of BmintegrinαPS3was upregulated at3h, maintained a significantly higher expression levels at12h, and back to the normal level at24h. With the same method, injection of heat-inactivated E.coli could induce the phagocytosis response by the hemocytes. The expression of BmintegrinαPS3upregulated a lot at6h, down regulated weekly at12h, and already close to normal level at24h. All these results indicated that granulocytes as the executor of cell immune response, participate into encapsulation and phagocytosis in different speed.
3. Cloning and functional analysis of the hematopoiesis regulation factor BmUsh in silkworm
(1) Cloning and sequence analysis of BmUsh:BmUsh cDNA including the complete ORF sequence was cloned. BmUsh locates on chromosome4, containing3exons and2introns. The ORF length is2409bp, encoding803aa, molecular weight is85.89kDa, and isoelectric point is8.3. BmUsh contains8conserved C2H2zinc domains.The phylogenetic tree of FOG homologues from different species showed that FOGs were conserved from invertebrates to vertebrate. There is one FOG in every insect species and during molecular evolution there are two FOGs in every mammal species. BmUsh was closer to the insect Ush family.
(2) The mRNA expression pattern of BmUsh in silkworm:The Real-time PCR with cDNA of different stage of embryo showed the expression of BmUsh decreased gradually from lth day to9th day. The Real-time PCR with cDNA of different tissues on L5D3showed that BmUsh expressed in several tissues, but significantly higher in hemocytes than other tissues. The Real-time PCR with cDNA of different stage hemocyte in5th showed that BmUsh were upregulated at the molting stage, the middle day of larve, and the wandering stage.
(3) Identification of Nuclear Localization Signal (NLS) in BmUsh: Ush as transcription factors must be targeted to the nucleus. By sequence analysis,10aa at the450-459amino acids site with4arginine series was predicted to be typical NLS motif. This motif with DsRed was together cloned into transient expression vector, and expressed in BmE-SWU3, while each of the four arginine were site mutated respectively and constructed into vector in the same method. The results showed that the predicted NLS motif was able to target all DsRed molecules to nucleus, but the NLS motif with lth and3th arginine mutation could not target any DsRed molecules to nucleus.
(4) The interaction factor of BmUsh:In order to find the interaction factor of BmUsh, the full-length expression vector of BmUsh was constructed and transfected into BmE-SWU3. The BmUsh-DsRed were highly expressed and clustered in nucleus. Accoring to the known NLS site, we mutated the456and458arginines of full-length BmUsh and found that the mutant BmUsh could not enter into nucleus. Direct evidence showed the locus indeed played the role of the nuclear localization signal in the full-length protein of BmUsh. Another hematopoiesis regulation factor BmLz was clone into full-length expression vector and cotransfected with BmUsh and mutant BmUsh. The results showed that BmLz was co-located with BmUsh in same site of nucleus, and the BmLz could gather the mutant BmUsh into nucleus or enter into cell cytoplasma to co-locate with mutant BmUsh. BmUsh and BmLz were simultaneously overexpressed by Bac-to-Bac/BmNPV expression system. The immunoprecipitation assay by recombinant bacmid illustrates that BmUsh and BmLz as a protein-protein interaction performs functions in nucleus.
(5) The hematopoiesis regulation function of BmUsh:BmUsh was overexpressed in primary culture of circulating hemocytes with the full-length expression vector of BmUsh. BmUsh protein was distributed in nucleus of hemocytes. Comparision between the control plasmid and BmUsh plasmid, the BmUsh could promote the plasmatocyte differentiating into the oenocytoid. Larvae on L5D5were injected with synthetic double-stranded RNA, and the expression of BmUsh was successful knock down. Meanwhile, the expressions of PPO1, PPO2, PPAE, and BAEE in hemocytes were also down. The melanization response of RNAi larvae was not processed. The overexpression and knowndown assay show that BmUsh regulate the differentiation of oenocytoid.
(6) BmUsh involve the immune response:Injection L5D3larvae with heat-inactivated E.coli could induce the phagocytosis response of the hemocytes. The results showed that at6h, the expression level of BmUsh increased20-fold; at12h-24h, the expression level of BmUsh already were close to normal level. All these indicate that BmUsh is involved in the melanization response by regulating the differentiation of oenocytoid.
引文
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