家蚕PcG家族分析及BmRYBP的功能研究
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摘要
多细胞有机体一般起源于单个细胞,分化为多种类型的细胞后,虽然每个细胞中都包含相同的遗传信息,但不同组织的细胞行使截然不同的功能。细胞所携带信息中仅有约1%的区域产生蛋白,其余的99%通过DNA的甲基化等作用被抑制,有一群很重要的基因polycomb group (PcG)维持了生命的这种既定的分子机制。PcG蛋白是一群对任何有机体的生长发育都很重要的普遍的转录抑制因子,首先在果蝇中被发现,虽然他们通常在结构域和结构基序上并不相关,却由于行使共同的功能而形成一个家族,在物种间高度保守。果蝇中已有的研究表明,PcG蛋白主要通过形成蛋白复合体的形式行使功能,目前鉴定的蛋白复合体形式主要有三种：Polycomb抑制复合体1 (PRC1)、Polycomb抑制复合体2(PRC2)和Pho抑制复合体(PhoRC)。近二十年来的研究表明,PcG蛋白不仅调控了同源异型基因的正确表达,而且与其他的生命进程密切相关,例如x染色体的失活、细胞的增殖与分化、干细胞的全能性和癌症发生。新的PcG蛋白抑制因子及调控机制的研究已经成为发育生物学以及表观遗传学领域的研究热点,鉴定PcG蛋白成员并进行功能研究对于阐明真核生物基因的表达调控机制具有重要意义。
家蚕是完全变态昆虫,是鳞翅目昆虫的典型代表,蚕的细胞遗传、形态遗传、连锁分析等研究曾和果蝇并驾齐驱,为人类认识生物的遗传和变异规律提供了重要的证据。家蚕也是研究动物发生、分化、生长发育及调节、遗传和变异的模式生物之一,有关蚕的发育生物学研究一直是昆虫学领域研究的热点和重点,阐明家蚕的发育机制对于研究以鳞翅目昆虫为代表的农业害虫防治也具有指导意义。本研究基于家蚕全基因组序列和表达谱数据,在基因组水平上对家蚕PcG家族进行鉴定和分析；并基于GAL4/UAS双元系统构建转基因品系,研究PcG蛋白相关成员BmRYBP (Ring and YY1 Binding Protein)基因的功能,获得的主要结果如下：
1家蚕Polycomb group家族成员的鉴定及表达分析
PcG蛋白参与了几乎所有生物有机体的发育调控。基于家蚕全基因组数据,首先鉴定家蚕的PcG蛋白成员。根据果蝇PcG蛋白成员信息,通过生物信息学方法对9×家蚕基因组数据进行同源检索,鉴定了家蚕中22个PcG同源基因,这些成员在家蚕中均无报道。在PRC1的四个核心亚基中,PC、PH和PSC三个成员在果蝇和家蚕间的相似性都比较低；而PRC2的四个核心亚基中,E(Z)、ESC和SU(z)12三个成员在两物种间都高度保守,推测这可能与PRC2在转录抑制方面的古老功能有关；PhoRC己知的两个核心亚基PHO和SFMBT在两物种间也具有很高的保守性。EST数据分析表明,家蚕的22个PcG蛋白成员中有17个基因具有ESTs证据。
基于全基因组芯片数据,对家蚕PcG成员的表达谱进行分析,有18个基因具有探针序列。对五龄3天幼虫组织芯片进行分析,发现有7个基因在至少一个组织中有表达信号。其中PcG招募蛋白成员Dspl、PRC2成员E(z)和Nurf-55(Caf1)三个基因在所有组织中均高量表达；除dSfmbt基因外,其他六个成员Nurf-55(Caf1)、Esc、E(z)、Sce/dRing, Dspl和Rbf均在生殖腺中出现最高的表达量；dSfmbt基因只在头部、体壁和中肠有较弱表达。家蚕不同发育时期的芯片数据分析表明,有11个基因在至少一个时间点有表达信号,其中三个基因Dspl、E(z)、Nurf-55(Cafl)在所有时间点均持续表达,染色体装配因子Cafl基因的表达值最高；Dspl、E(z)、Nurf-55(Cafl)、Rbf和Rpd3五个基因在蛾期出现明显的雌雄差异表达,雌蛾中的表达值为雄蛾的两倍以上,尤其是Rpd3和Rbf的表达差异最为明显,从上蔟第8天开始出现雌雄差异并一直延续到蛾期,在雌中有明显的表达信号,而在雄中几乎不表达；另外的三个基因Pho、Sce/dRing和Psc在雌蛾中有微弱表达。
2家蚕Ring和RYBP基因的克隆及表达分析
Ring是PRC1的核心成员之一,是组蛋白H2A泛素化的催化亚基。RYBP是在小鼠中鉴定到的与RING相互作用的蛋白,由于RYBP也与YYi转录因子相互作用,因此命名为Ring and YY1 Binding Protein (RYBP), RYBP是推断的PcG蛋白相关成员,该基因行使转录抑制作用的具体功能还不清楚。根据果蝇Ring和RYBP基因的氨基酸序列,检索家蚕基因组数据库,鉴定到了与Ring和RYBP同源的家蚕基因,分别命名为BmRing和BmRYBP。编号为BGIBMGA006985的预测基因是Ring的同源基因,位于第17号染色体nscaf2865上,基因结构分析显示该基因由4个外显子和3个内含子组成,编码377个氨基酸,预测蛋白质分子量为41.8kDa,与其他物种同源基因的多序列比对显示出较高的相似性,N端为典型的环指结构域,c端有尚未被命名的保守的功能结构域。在家蚕基因组数据库中没有预测到RYBP同源序列,通过对家蚕EST数据库进行同源检索,电子克隆得到了一条长882nt的序列,ORF为420bp,编码139个氨基酸,预测分子量15.4kDa,位于第28号染色体的nscaf3097上。将家蚕RYBP基因的氨基酸序列与其它昆虫和人的RYBP/YEAF1,人和鼠的YAF2氨基酸序列进行多序列比对发现,其在N端的锌指结构域高度保守。
利用半定量RT-PCR手段检测了BmRing和BmRYBP基因在家蚕幼虫五龄3天各组织的表达情况,研究结果显示,BmRing基因在精巢、卵巢、头和血液有表达,在其他组织几乎没有表达,与组织芯片分析结果基本一致。BmRYBP基因在生殖腺和头中的表达量较高,在其它组织表达量相对较低。本研究对BmRing和BmRYBP基因进行了克隆,以进行下一步的功能研究。
3家蚕Ring和RYBP的原核表达及多克隆抗体的制备
为了进一步研究家蚕Ring和RYBP的功能,首先对两个基因进行了原核表达及抗体制备。基于p28载体分别构建BmRing和BmRYBP的原核表达重组质粒BmRing/p28和BmRYBP/p28,将重组质粒转化至Rosetta(DE3)表达菌株中,通过在不同温度和时间条件下的IPTG诱导表达,检测目的蛋白的表达形式。最终选择在37℃,0.2mM浓度的IPTG诱导4h后收集菌体并纯化蛋白。纯化包涵体得到BmRING和BmRYBP的蛋白,免疫成年健康公兔5次后,取耳部静脉血,用间接ELISA法检测抗体效价在1：64000以上,收获血清,纯化得到多克隆抗体。
4利用GAL4/UAS系统实现BmRYBP基因的转基因干涉研究
基于GAL4/UAS基础载体,构建含有BmRYBP基因序列反向重复结构的转基因表达载体pBac[UAS-RYBP~IRSV40,3xp3EGFP],通过显微注射家蚕大造品种的早期胚胎,筛选获得在眼部和神经具有荧光标记的转基因家蚕,建立UAS-RYBP~IR的转基因品系。利用基因组PCR,Southern blot和反向PCR等方法对UAS-RYBP~IR的转基因个体的拷贝数目和插入位点进行分子水平的检测,结果显示在UAS-RYBP~IR转基因品系的大造基因组中插入了外源基因,piggyBac转座子在不同转基因品系的家蚕基因组中分别发生了一次转座事件,插入位点分别位于第9、12和22号染色体上。
红色荧光标记的A4GAL4转基因家蚕与绿色荧光标记的UAS-RYBP~IR转基因家蚕进行杂交,在具有双色荧光的杂交后代中以发夹结构的RNA形成dsRNA实现对BmRYBP的干涉。利用RT-PCR,定量PCR和western blot等检测手段,对A4GAL4与UAS-RYBP~IR的杂交后代进行转录水平和翻译水平的表达检测,在具有双色荧光标记的杂交后代A4GAL4/UAS-RYBP~IR中,BmRYBP的mRNA表达量和蛋白表达量均显著降低,说明在杂交后代中实现了对BmRYBP的特异干涉。转基因RNA干涉抑制BmRYBP表达,同时BmRing的mRNA表达量和蛋白表达量也有显著降低,推测BmRYBP与PcG蛋白核心元件BmRing之间存在调控表达关系,二者可能以协同作用的方式调控家蚕发育基因的表达,具体的调控机制还需要更深入的研究证实。
对杂交后代的表型进行观察,发现A4GAL4/UAS-RYBP~IR杂交后代,出现了不同的表现型：G1代蚕卵转青后出现少数死亡的现象；幼虫生长至五龄,根据眼部发荧光情况,筛选具有双色荧光的G1代转基因个体,待发育到蛾期后,观察到A4GAL4/UAS-RYBP~IR成虫出现小翅表型；对存活到蛾期的A4GAL4/UAS-RYBP~IR转基因阳性个体进行交配制种得到G2代,发现G2代的产卵量明显减少,且蛾圈之间产卵量差异很大,多有未受精卵；蚕卵的孵化率也有差异,部分蚕卵在点青后死亡,孵化后的幼虫出现了陆续死亡的现象；约10%的幼虫在腹足的不同位置中出现畸形,这种畸形现象包括缺失单个或多个腹足,或腹足发育不全等；部分出现畸形的幼虫可以继续存活直至正常化蛾。
Multicellular organisms generally originated from a single cell to differentiate into many types of cells, although each cell contains the same genetic information, cells in different organs execute completely different functions. Only 1%of the genetic information carried by cell can be coded for proteins, the remaining are inhibited by methylation of DNA. Polycomb group(PcG), a group of very important genes, maintain this established molecular mechanism in life. Although the members of PcG proteins have differences in structure and motifs, they form a common family due to perform the common function. PcG proteins regulate transcriptional repression of many important genes related to development, which are highly conserved among species. It has been confirmed in Drosophila that PcG execute function mainly through forming complex proteins. The modality of complex proteins identified at present mainly contain three forms:Ploycomb repression complex 1(PRC1), Ploycomb repression complex2(PRC2) and Pho repression complex(PhoRC). What is especially significant to define the mechanism of PcG proteins is that isolation and identification to the component of complex, as well as verification of function. New regulator of PcG proteins and the regulation mechanism have become to be a hot research field of developmental biology and epigenetics.
Silkworm is a complete metamorphosis insect and the typical representive of Lepidoptera insects. The research of linkage analysis and so on in silkworm once kept pace with those in Drosophila and provided significant evidences for human to cognitive the regularity of heredity and variation in organisms. Silkworm is also a model in research of development,differentiation, growth and regulation, heredity, variation in animals. Stepping into this century, function of generous known and unknown genes are urgent to be resolved followed the fine silkworm genome map was finished. GAL4/UAS system is a useful tool to investigate gene function, which is generally under application in Drosophila but not in silkworm. In order to accomplish the application of this system in silkworm, we utilize the system to study the function of RYBP (Ring and YYl binding protein) which is a component of PcG proteins. On the one hand, we establish the transgenic interference system based on GAL4/UAS system in silkworm. On the other hand, we study the function of members in silkworm PcG proteins. The main results are as follows:
1 bioinformatical analysis and expression profile of silkworm Polycomb group
23 drosophila PcG homologs were indentified in silkworm genome by BLAST, all the members haven't been reported yet. Those genes were highly conserved between fruitfly and silkworm, especially the polycomb repressor complex 2 components, which were supposed to be related with its ancient function of transcriptional repression.18 of them were supported by EST data.
18 out of 23 genes were detected based on whole genome microarray data.7 of them expressed in at least one tissue of day3 fifth instar larvae. PcG recruiter Dspl, PRC2 component E(z) and Nurf-55(Cafl) revealed high expression in all organs; Nurf-55 (Cafl), Esc, E(z), Sce/dRing, Dsp1, Rbf highly expressed in gonad, while rarely expressed in other organs; dSfmbt revealed low expression in head, body wall and midgut.11 out of 18 detectable genes were found to express in at least one stage during metamorphosis. Dspl, E(z) and Nuf-55(Cafl) persistently expressed in all stages, especially the chromosome assembly factor Cafl,which represented the highest expression level; Dspl, E(z), Nurf-55(Cafl), Rbf and Rpd3 exhibited a conspicuously different expression pattern between male and female moth, which was 2-fold in female than male, above all, sex-dependent differential expression pattern of Rpd3 and Rbf began at 8th day mounting, and continued till moth stage, which was notably expressed in female, rarely in male; the other three Pho, Sce/dRing and Psc were constrainedly detected in female.
2 Cloning, sequence analysis of Ring and RYBP
Ring is a core member of the family of PRC1, which function as a subunit to catalyze the ubiquitination of histone protein H2A. RYBP is a protein reported both in Drosophila melanogaster and Mus musculus, which can interact with RING and predicted to be a member of PcG protein family, but its function remains undiscoved. Two homologous genes of Ring and RYBP of silkworm were identified from the silkworm genome database based on the amino acid sequence of Ring and RYBP reported from drosophila. Among these results, a sequence which is homologous to Ring and located on the nscaf2865 of No.17 chromosome with a number of BGIBMGA006985, was identified from silkworm prediction genome database, prediction of gene structure indicates that this gene consists of 4 exons and 3 introns, encodes a 41.8kDa protein with 377 amino acids, and is highly similar to the homologous gene from other species, the N-teminal of this protein contains a typical Ring-domain, and C-teminal also contains a conserved function domain which has not been named yet. Whereas, the sequence which is homologous to RYBP has not been identified from the silkworm prediction genome database, but we electronic-clone a gene sequence of 882nt from silkworm EST database in which contains a ORF of 420bp, encodes a 15.4kDa protein with 139 amino acids, this gene locates on the nscaf3097 of No.28 chromosome and its N-teminal zinc finger domain is high conserved to both RYBP/YEAF1 of human and YAF2 of human and mouse.
The results of RT-PCR by which to investigate the expressions of Ring and RYBP of silkworm on 3~rd day fifth instar indicate that the Ring remarkably expressed in testis, ovary, head and hemocytes, and had a lower expression level in fat body, no signal was detected in other tissues, this was consistent with the mircroarray data. Meanwhile, RYBP expressed almost in all tissues with a high expression levels gonad and head and a lower expression levels in other tissues.
3 Prokaryotic expression and preparing polyclonal antibody of the Ring and RYBP
In purpose of further functional analysis, we finished the prokaryotic expression of BmRing and BmRYBP. We first transformed the recombination plasmid Ring/p28and RYBP/p28 into the strain Rosetta(DE3), and then detected the expression of the two proteins under the different induced temperature and IPTG concentration. At last, we purified the proteins in the inclusion body to go on the backward experiments by IPTG inducement four hours under the temperature 37℃.
We used the two purified proteins to immunize the adult male rabbits for five times, and then detected the antibody titer of serum by ELISA. When the antibody titer is up to 1:64000,we obtain the serum and purified the two polyclonal antibodies.
4 Interference of BmRYBP by GAL4/UAS system in the transgenic silkworm
GAL4/UAS system is a powerful way tool of founctional gene research. It can control the expression of the target genes in different tissues and different times and make gene overexpressed and transgentical interfered. With the broad application of RNAi technique in the reverse genetics, this system has more and more been used in the functional research. We have constructed the transgenic expression construct pBac[UASRYBP~IRSV40-3xp3EGFP], and by the strandard method of transgenic inject we have gotten the UAS-RYBP~(IR)transgenic silkworm and established the separate silkworm transgenic lines. To induce the RNA interference and detect the founctional deletion phenotype of RYBP gene, we crossed the A4GAL4 lines marked by red fluorescence with UAS-RYBP~(IR)lines marked by green fluorescence and then successfully interfered the target gene in the double-fluorescence crossbreed by double-strand RNA forming by RNA hairpin structure.
We used PCR, Southern blot and Inverse-PCR to detect the copy number and insertion sites of the transgenic individuals of UAS-RYBP~(IR) at the molecular level. Then, using RT-PCR, quantitative PCR and western blot to detect transcription and expression level of the hybrid progeny of UAS-RYBPIR and A4GAL4 idividuals, the results showed that the mRNA and protein expression of BmRYBP gene decreased in the hybrid progeny,which means specific interference of RYBP gene happened. Also, the mRNA and protein expression of BmRing decreased. It's speculated that BmRing may be regulated by BmRYBP. Besides, there are different phenotypes in hybrid offspring of different strains.
Some of Gl embryos died during embryogenesis. A4GAL4/UAS-RYBPIR moths, which were screened at 5th instar according to the dual-fluorescence marker, formed relatively smaller wings compared with the wild-type, and also, they showed a low and unstable oviposition capacity, lots of the eggs were unfertilized; some of the embryos died before hatching or in the 1 st instar.10%of the dead larvae exhibited polymorphic abnormalities in their proleg, including absence of one or more prolegs and lacking of the suctorial disk. Some of the diastrophic larvae remained alive until eclosion. It's suspected that those diastrophic phenotypes of G2 silkworms may be related with the maternal expression of most PcG proteins, decreased PcGs in the G1 moth caused developmental disorder of its posterities.
家蚕是完全变态昆虫,是鳞翅目昆虫的典型代表,蚕的细胞遗传、形态遗传、连锁分析等研究曾和果蝇并驾齐驱,为人类认识生物的遗传和变异规律提供了重要的证据。家蚕也是研究动物发生、分化、生长发育及调节、遗传和变异的模式生物之一,有关蚕的发育生物学研究一直是昆虫学领域研究的热点和重点,阐明家蚕的发育机制对于研究以鳞翅目昆虫为代表的农业害虫防治也具有指导意义。本研究基于家蚕全基因组序列和表达谱数据,在基因组水平上对家蚕PcG家族进行鉴定和分析；并基于GAL4/UAS双元系统构建转基因品系,研究PcG蛋白相关成员BmRYBP (Ring and YY1 Binding Protein)基因的功能,获得的主要结果如下：
1家蚕Polycomb group家族成员的鉴定及表达分析
PcG蛋白参与了几乎所有生物有机体的发育调控。基于家蚕全基因组数据,首先鉴定家蚕的PcG蛋白成员。根据果蝇PcG蛋白成员信息,通过生物信息学方法对9×家蚕基因组数据进行同源检索,鉴定了家蚕中22个PcG同源基因,这些成员在家蚕中均无报道。在PRC1的四个核心亚基中,PC、PH和PSC三个成员在果蝇和家蚕间的相似性都比较低；而PRC2的四个核心亚基中,E(Z)、ESC和SU(z)12三个成员在两物种间都高度保守,推测这可能与PRC2在转录抑制方面的古老功能有关；PhoRC己知的两个核心亚基PHO和SFMBT在两物种间也具有很高的保守性。EST数据分析表明,家蚕的22个PcG蛋白成员中有17个基因具有ESTs证据。
基于全基因组芯片数据,对家蚕PcG成员的表达谱进行分析,有18个基因具有探针序列。对五龄3天幼虫组织芯片进行分析,发现有7个基因在至少一个组织中有表达信号。其中PcG招募蛋白成员Dspl、PRC2成员E(z)和Nurf-55(Caf1)三个基因在所有组织中均高量表达；除dSfmbt基因外,其他六个成员Nurf-55(Caf1)、Esc、E(z)、Sce/dRing, Dspl和Rbf均在生殖腺中出现最高的表达量；dSfmbt基因只在头部、体壁和中肠有较弱表达。家蚕不同发育时期的芯片数据分析表明,有11个基因在至少一个时间点有表达信号,其中三个基因Dspl、E(z)、Nurf-55(Cafl)在所有时间点均持续表达,染色体装配因子Cafl基因的表达值最高；Dspl、E(z)、Nurf-55(Cafl)、Rbf和Rpd3五个基因在蛾期出现明显的雌雄差异表达,雌蛾中的表达值为雄蛾的两倍以上,尤其是Rpd3和Rbf的表达差异最为明显,从上蔟第8天开始出现雌雄差异并一直延续到蛾期,在雌中有明显的表达信号,而在雄中几乎不表达；另外的三个基因Pho、Sce/dRing和Psc在雌蛾中有微弱表达。
2家蚕Ring和RYBP基因的克隆及表达分析
Ring是PRC1的核心成员之一,是组蛋白H2A泛素化的催化亚基。RYBP是在小鼠中鉴定到的与RING相互作用的蛋白,由于RYBP也与YYi转录因子相互作用,因此命名为Ring and YY1 Binding Protein (RYBP), RYBP是推断的PcG蛋白相关成员,该基因行使转录抑制作用的具体功能还不清楚。根据果蝇Ring和RYBP基因的氨基酸序列,检索家蚕基因组数据库,鉴定到了与Ring和RYBP同源的家蚕基因,分别命名为BmRing和BmRYBP。编号为BGIBMGA006985的预测基因是Ring的同源基因,位于第17号染色体nscaf2865上,基因结构分析显示该基因由4个外显子和3个内含子组成,编码377个氨基酸,预测蛋白质分子量为41.8kDa,与其他物种同源基因的多序列比对显示出较高的相似性,N端为典型的环指结构域,c端有尚未被命名的保守的功能结构域。在家蚕基因组数据库中没有预测到RYBP同源序列,通过对家蚕EST数据库进行同源检索,电子克隆得到了一条长882nt的序列,ORF为420bp,编码139个氨基酸,预测分子量15.4kDa,位于第28号染色体的nscaf3097上。将家蚕RYBP基因的氨基酸序列与其它昆虫和人的RYBP/YEAF1,人和鼠的YAF2氨基酸序列进行多序列比对发现,其在N端的锌指结构域高度保守。
利用半定量RT-PCR手段检测了BmRing和BmRYBP基因在家蚕幼虫五龄3天各组织的表达情况,研究结果显示,BmRing基因在精巢、卵巢、头和血液有表达,在其他组织几乎没有表达,与组织芯片分析结果基本一致。BmRYBP基因在生殖腺和头中的表达量较高,在其它组织表达量相对较低。本研究对BmRing和BmRYBP基因进行了克隆,以进行下一步的功能研究。
3家蚕Ring和RYBP的原核表达及多克隆抗体的制备
为了进一步研究家蚕Ring和RYBP的功能,首先对两个基因进行了原核表达及抗体制备。基于p28载体分别构建BmRing和BmRYBP的原核表达重组质粒BmRing/p28和BmRYBP/p28,将重组质粒转化至Rosetta(DE3)表达菌株中,通过在不同温度和时间条件下的IPTG诱导表达,检测目的蛋白的表达形式。最终选择在37℃,0.2mM浓度的IPTG诱导4h后收集菌体并纯化蛋白。纯化包涵体得到BmRING和BmRYBP的蛋白,免疫成年健康公兔5次后,取耳部静脉血,用间接ELISA法检测抗体效价在1：64000以上,收获血清,纯化得到多克隆抗体。
4利用GAL4/UAS系统实现BmRYBP基因的转基因干涉研究
基于GAL4/UAS基础载体,构建含有BmRYBP基因序列反向重复结构的转基因表达载体pBac[UAS-RYBP~IRSV40,3xp3EGFP],通过显微注射家蚕大造品种的早期胚胎,筛选获得在眼部和神经具有荧光标记的转基因家蚕,建立UAS-RYBP~IR的转基因品系。利用基因组PCR,Southern blot和反向PCR等方法对UAS-RYBP~IR的转基因个体的拷贝数目和插入位点进行分子水平的检测,结果显示在UAS-RYBP~IR转基因品系的大造基因组中插入了外源基因,piggyBac转座子在不同转基因品系的家蚕基因组中分别发生了一次转座事件,插入位点分别位于第9、12和22号染色体上。
红色荧光标记的A4GAL4转基因家蚕与绿色荧光标记的UAS-RYBP~IR转基因家蚕进行杂交,在具有双色荧光的杂交后代中以发夹结构的RNA形成dsRNA实现对BmRYBP的干涉。利用RT-PCR,定量PCR和western blot等检测手段,对A4GAL4与UAS-RYBP~IR的杂交后代进行转录水平和翻译水平的表达检测,在具有双色荧光标记的杂交后代A4GAL4/UAS-RYBP~IR中,BmRYBP的mRNA表达量和蛋白表达量均显著降低,说明在杂交后代中实现了对BmRYBP的特异干涉。转基因RNA干涉抑制BmRYBP表达,同时BmRing的mRNA表达量和蛋白表达量也有显著降低,推测BmRYBP与PcG蛋白核心元件BmRing之间存在调控表达关系,二者可能以协同作用的方式调控家蚕发育基因的表达,具体的调控机制还需要更深入的研究证实。
对杂交后代的表型进行观察,发现A4GAL4/UAS-RYBP~IR杂交后代,出现了不同的表现型：G1代蚕卵转青后出现少数死亡的现象；幼虫生长至五龄,根据眼部发荧光情况,筛选具有双色荧光的G1代转基因个体,待发育到蛾期后,观察到A4GAL4/UAS-RYBP~IR成虫出现小翅表型；对存活到蛾期的A4GAL4/UAS-RYBP~IR转基因阳性个体进行交配制种得到G2代,发现G2代的产卵量明显减少,且蛾圈之间产卵量差异很大,多有未受精卵；蚕卵的孵化率也有差异,部分蚕卵在点青后死亡,孵化后的幼虫出现了陆续死亡的现象；约10%的幼虫在腹足的不同位置中出现畸形,这种畸形现象包括缺失单个或多个腹足,或腹足发育不全等；部分出现畸形的幼虫可以继续存活直至正常化蛾。
Multicellular organisms generally originated from a single cell to differentiate into many types of cells, although each cell contains the same genetic information, cells in different organs execute completely different functions. Only 1%of the genetic information carried by cell can be coded for proteins, the remaining are inhibited by methylation of DNA. Polycomb group(PcG), a group of very important genes, maintain this established molecular mechanism in life. Although the members of PcG proteins have differences in structure and motifs, they form a common family due to perform the common function. PcG proteins regulate transcriptional repression of many important genes related to development, which are highly conserved among species. It has been confirmed in Drosophila that PcG execute function mainly through forming complex proteins. The modality of complex proteins identified at present mainly contain three forms:Ploycomb repression complex 1(PRC1), Ploycomb repression complex2(PRC2) and Pho repression complex(PhoRC). What is especially significant to define the mechanism of PcG proteins is that isolation and identification to the component of complex, as well as verification of function. New regulator of PcG proteins and the regulation mechanism have become to be a hot research field of developmental biology and epigenetics.
Silkworm is a complete metamorphosis insect and the typical representive of Lepidoptera insects. The research of linkage analysis and so on in silkworm once kept pace with those in Drosophila and provided significant evidences for human to cognitive the regularity of heredity and variation in organisms. Silkworm is also a model in research of development,differentiation, growth and regulation, heredity, variation in animals. Stepping into this century, function of generous known and unknown genes are urgent to be resolved followed the fine silkworm genome map was finished. GAL4/UAS system is a useful tool to investigate gene function, which is generally under application in Drosophila but not in silkworm. In order to accomplish the application of this system in silkworm, we utilize the system to study the function of RYBP (Ring and YYl binding protein) which is a component of PcG proteins. On the one hand, we establish the transgenic interference system based on GAL4/UAS system in silkworm. On the other hand, we study the function of members in silkworm PcG proteins. The main results are as follows:
1 bioinformatical analysis and expression profile of silkworm Polycomb group
23 drosophila PcG homologs were indentified in silkworm genome by BLAST, all the members haven't been reported yet. Those genes were highly conserved between fruitfly and silkworm, especially the polycomb repressor complex 2 components, which were supposed to be related with its ancient function of transcriptional repression.18 of them were supported by EST data.
18 out of 23 genes were detected based on whole genome microarray data.7 of them expressed in at least one tissue of day3 fifth instar larvae. PcG recruiter Dspl, PRC2 component E(z) and Nurf-55(Cafl) revealed high expression in all organs; Nurf-55 (Cafl), Esc, E(z), Sce/dRing, Dsp1, Rbf highly expressed in gonad, while rarely expressed in other organs; dSfmbt revealed low expression in head, body wall and midgut.11 out of 18 detectable genes were found to express in at least one stage during metamorphosis. Dspl, E(z) and Nuf-55(Cafl) persistently expressed in all stages, especially the chromosome assembly factor Cafl,which represented the highest expression level; Dspl, E(z), Nurf-55(Cafl), Rbf and Rpd3 exhibited a conspicuously different expression pattern between male and female moth, which was 2-fold in female than male, above all, sex-dependent differential expression pattern of Rpd3 and Rbf began at 8th day mounting, and continued till moth stage, which was notably expressed in female, rarely in male; the other three Pho, Sce/dRing and Psc were constrainedly detected in female.
2 Cloning, sequence analysis of Ring and RYBP
Ring is a core member of the family of PRC1, which function as a subunit to catalyze the ubiquitination of histone protein H2A. RYBP is a protein reported both in Drosophila melanogaster and Mus musculus, which can interact with RING and predicted to be a member of PcG protein family, but its function remains undiscoved. Two homologous genes of Ring and RYBP of silkworm were identified from the silkworm genome database based on the amino acid sequence of Ring and RYBP reported from drosophila. Among these results, a sequence which is homologous to Ring and located on the nscaf2865 of No.17 chromosome with a number of BGIBMGA006985, was identified from silkworm prediction genome database, prediction of gene structure indicates that this gene consists of 4 exons and 3 introns, encodes a 41.8kDa protein with 377 amino acids, and is highly similar to the homologous gene from other species, the N-teminal of this protein contains a typical Ring-domain, and C-teminal also contains a conserved function domain which has not been named yet. Whereas, the sequence which is homologous to RYBP has not been identified from the silkworm prediction genome database, but we electronic-clone a gene sequence of 882nt from silkworm EST database in which contains a ORF of 420bp, encodes a 15.4kDa protein with 139 amino acids, this gene locates on the nscaf3097 of No.28 chromosome and its N-teminal zinc finger domain is high conserved to both RYBP/YEAF1 of human and YAF2 of human and mouse.
The results of RT-PCR by which to investigate the expressions of Ring and RYBP of silkworm on 3~rd day fifth instar indicate that the Ring remarkably expressed in testis, ovary, head and hemocytes, and had a lower expression level in fat body, no signal was detected in other tissues, this was consistent with the mircroarray data. Meanwhile, RYBP expressed almost in all tissues with a high expression levels gonad and head and a lower expression levels in other tissues.
3 Prokaryotic expression and preparing polyclonal antibody of the Ring and RYBP
In purpose of further functional analysis, we finished the prokaryotic expression of BmRing and BmRYBP. We first transformed the recombination plasmid Ring/p28and RYBP/p28 into the strain Rosetta(DE3), and then detected the expression of the two proteins under the different induced temperature and IPTG concentration. At last, we purified the proteins in the inclusion body to go on the backward experiments by IPTG inducement four hours under the temperature 37℃.
We used the two purified proteins to immunize the adult male rabbits for five times, and then detected the antibody titer of serum by ELISA. When the antibody titer is up to 1:64000,we obtain the serum and purified the two polyclonal antibodies.
4 Interference of BmRYBP by GAL4/UAS system in the transgenic silkworm
GAL4/UAS system is a powerful way tool of founctional gene research. It can control the expression of the target genes in different tissues and different times and make gene overexpressed and transgentical interfered. With the broad application of RNAi technique in the reverse genetics, this system has more and more been used in the functional research. We have constructed the transgenic expression construct pBac[UASRYBP~IRSV40-3xp3EGFP], and by the strandard method of transgenic inject we have gotten the UAS-RYBP~(IR)transgenic silkworm and established the separate silkworm transgenic lines. To induce the RNA interference and detect the founctional deletion phenotype of RYBP gene, we crossed the A4GAL4 lines marked by red fluorescence with UAS-RYBP~(IR)lines marked by green fluorescence and then successfully interfered the target gene in the double-fluorescence crossbreed by double-strand RNA forming by RNA hairpin structure.
We used PCR, Southern blot and Inverse-PCR to detect the copy number and insertion sites of the transgenic individuals of UAS-RYBP~(IR) at the molecular level. Then, using RT-PCR, quantitative PCR and western blot to detect transcription and expression level of the hybrid progeny of UAS-RYBPIR and A4GAL4 idividuals, the results showed that the mRNA and protein expression of BmRYBP gene decreased in the hybrid progeny,which means specific interference of RYBP gene happened. Also, the mRNA and protein expression of BmRing decreased. It's speculated that BmRing may be regulated by BmRYBP. Besides, there are different phenotypes in hybrid offspring of different strains.
Some of Gl embryos died during embryogenesis. A4GAL4/UAS-RYBPIR moths, which were screened at 5th instar according to the dual-fluorescence marker, formed relatively smaller wings compared with the wild-type, and also, they showed a low and unstable oviposition capacity, lots of the eggs were unfertilized; some of the embryos died before hatching or in the 1 st instar.10%of the dead larvae exhibited polymorphic abnormalities in their proleg, including absence of one or more prolegs and lacking of the suctorial disk. Some of the diastrophic larvae remained alive until eclosion. It's suspected that those diastrophic phenotypes of G2 silkworms may be related with the maternal expression of most PcG proteins, decreased PcGs in the G1 moth caused developmental disorder of its posterities.
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