利用模板活性染色质转移外源基因的研究

本文首次报道以家蚕(Bombyx mori Linnaeus)为利料,选择五龄幼虫期的黑缟品种的真皮组织及沔阳红品种的丝腺,分离出模板活性染色质,获得了预期的目的基因一Ps及Pk。把它们用显微注射及激光微束技术分别转移到华合(白蚕)、龙角蚕、巴陵黄(黄茧)的受精卵中,观察到黑缟基因(p^s)、红茧基因(Pk)在白蚕(华合)、黄茧蚕(巴陵黄)中的当代表达及在子代中的传递情况。     

家蚕荧光茧色的判性机理

以家蚕荧光茧色判性品种为实验材料,对蚕茧层表面和茧层抽提物的荧光光谱分析表明,蚕茧荧光色不论雌雄都出现黄色和蓝紫色二个荧光发射峰,因两者相对荧光强度不同而引起雌雄茧之间的荧光色差异,通过对家蚕中肠组织的荧光观察和荧光显微观察,发现蚕茧荧光色的雌雄差异或判性是由于雌蚕中肠组织对黄色荧光色素的特异性积累而引起的,5龄4日以后,雌蚕食桑后经过中肠消化与吸收,全部或大部分黄色荧光物质开始积累于中肠后部细胞内直至变态化蛹,所有蓝紫色荧光物质单独或与少量黄色荧光物质一起分泌进入血液,再到达丝腺体而使雌茧呈蓝紫或蓝白荧光色,而雄蚕中肠组织对这二类荧光物质都不积累,全部分泌进入血液再到达丝腺体最后使雄蚕茧呈黄白荧光色,应用硅胶薄层色谱和荧光光谱分析提示,蓝紫荧光色物质至少由5种蓝紫荧光色素成分组成,而黄色荧光物质至少由3种黄色荧光色素成分组成;从雄蚕茧层中提取到的黄色荧光色素与雌蚕后部中肠积累的色素完全相同,紫外光谱分析和AlCl3显色反应表明,3种黄色荧光色素属于黄酮类或黄酮苷类化合物,其中一种黄色荧光色素（Rf=0.82）为主要成分,其荧光发射光谱最大发射峰为533nm,激发光谱峰值为377nm,上述结果充分表明,家蚕荧光判性品种的雌雄荧光茧色差异就是由雌性家蚕中肠尤其是后部中肠对黄色荧光色素的特异性积累而引起的,因此,可以推测,在荧光茧色判性的5龄雌蚕中肠,特别是后部中肠上皮圆筒形细胞中特异表达一种能与黄色荧光色素结合的蛋白。     

家蚕 Fhx/P25 基因的一种新的转录模式分析研究

家蚕 Fhx/P25 蛋白是丝素蛋白的主要成分之一,过去报道只在家蚕后部丝腺特异的转录表达 . 通过对大规模的家蚕 EST 序列分析发现, Fhx/P25 基因不仅在家蚕后部丝腺高效转录,而且在家蚕幼虫五龄第三天的卵巢组织及其他组织也有转录;分析还发现 Fhx/P25 基因在丝腺和卵巢组织中有不同的转录起始位点,在卵巢组织中的转录起始位点比在丝腺中的至少要提前 115 bp 左右 . 用 RT-PCR 和 FQ-PCR 进一步验证,以上分析结果均正确 . 分析还发现 Fhx/P25mRNA 存在选择性拼接 . 以上结果表明 Fhx/P25 基因并不是组织特异转录基因,它的转录表达存在复杂的调控机制,可能还有其他功能 .     

以模板活性染色质转移家蚕基因的研究

一定的组织中仅仅转录一定的基因,组织中那些进行转录的染色质区域称为模板活性染色质.因此,只要选择一定的组织提取模板活性染色质,就可获得预期的基因.我们从红茧蚕(沔阳红带P_k基因)五龄幼虫的丝腺组织中提取模板活性染色质,用显微注射技术转移到黄茧蚕(巴陵黄)的受精卵中,观察到了能结红茧的变异体并已传至F₃代;说明P_k基因被成功地转移并能传递给子代.另外,以普通褐卵(苏春)的活性染色质注射到白卵(沄纹皮斑)受精卵中,也得到了黑卵变异.利用分离模板活性染色质来转移预期基因的技术,可能为真核生物的基因转移提供一个新的途径.     

家蚕一对相对性状的遗传实验

笔者于2005年的春天开始养蚕活动。第1周要来2条蚕,1条黄足蚕,1条是自足蚕。黄足蚕吐黄丝结黄茧,白足蚕吐白丝结白茧。黄足蚕即蚕幼虫的腹足和尾足呈黄色,而白足蚕即腹足和尾足呈白色(比体色稍淡些但不黄)。当幼蚕第2次脱皮后可用手轻轻捉起观察辨认,但这时幼蚕还小,为了避免被捏死,最好等第3次脱皮后观察辨认更清楚。     

基于RNA-Seq分析Ras1~(CA)在家蚕后部丝腺过表达对细胞周期通路基因的影响

【目的】本文旨在挖掘野生型家蚕Bombyx mori和Ras1CA过表达转基因家蚕后部丝腺中的转录本差异,分析和验证细胞周期通路中的差异表达基因,从而探讨Ras1CA过表达转基因家蚕提高蚕丝产量的分子机制。【方法】利用转录组学比较野生型和Ras1CA过表达转基因家蚕后部丝腺中的基因转录本差异,经实时荧光定量PCR(q RT-PCR)验证。【结果】野生型家蚕和Ras1CA过表达转基因家蚕后部丝腺组织中有2 636个差异表达基因,其中细胞周期信号通路中有42个差异表达基因,包括细胞周期依赖性激酶(CDK)、细胞周期素(Cyclin)以及转录因子等。通过q RT-PCR检测cdk1、cyclin D1、cyclin D2、cdc7、cdh1、dp-1,2等6个基因在野生型和Ras1CA过表达转基因家蚕后部丝腺组织中的相对表达量,发现转基因家蚕中的表达量均显著高于野生型(P<0.05),其中cyclin D2的表达差异极显著(P<0.01)。q RT-PCR结果与转录本差异一致,表明Ras1CA过表达后,能够促进细胞周期通路基因的表达。【结论】野生型家蚕和Ras1CA过表达转基因家蚕后部丝腺中有大量差异表达基因,且Ras1CA能够在转录水平上调控细胞周期通路,影响后部丝腺组织的细胞分裂和器官生长,从而促进蚕丝蛋白的合成。     

转植酸酶基因家蚕的制作及表达检测

家蚕Bombyx mori丝腺具有高效合成蛋白质的特性,开发在丝腺特异表达外源蛋白质的生物反应器具有重要的意义。本研究利用piggyBac来源的两种载体pPIGA3GFP和pBac{3×P3-EGFPaf},建立了稳定的家蚕转基因技术体系; 然后,利用一株黑曲霉来源的植酸酶基因,构建了在家蚕后部丝腺特异表达的融合表达载体pBac [3×P3-EGFP+ FibLphyADsRed],注射蚕卵后,在53个G1蛾区中检测到3个有荧光蚕的蛾区。经Southern blot和反向PCR验证,转基因表达盒整合到家蚕染色体上。RT-PCR结果显示,植酸酶基因特异性地在后部丝腺表达,其表达模式与家蚕轻链丝素基因一致。结果表明我们成功获得了在后部丝腺特异表达植酸酶融合蛋白的转基因蚕,这为进一步开发家蚕生物反应器,利用转基因蚕生产各种重组蛋白具有积极的促进作用。     

蚕丝天然丝胶蛋白的紫外吸收能力研究

丝胶蛋白质将广泛应用于食品、化妆品、纤维加工、细胞工程、医用材料等领域,这种预言是基于其在这些领域的利用上具有诸多独特的、有用的功能特性。紫外吸收能力是化妆品利用的重要功能特性之一。本研究对全天然丝胶茧品种"绿S"、"白S"和现行推广普通蚕品种"菁松×皓月"的丝胶蛋白含量及其紫外吸收能力进行了比较分析。结果表明天然的"绿S"蚕丝胶蛋白质具有较强的紫外吸收能力,在化妆品添加剂领域具有极其优越的利用价值。     

家蚕黄血抑制基因的SSR定位

家蚕黄茧性状主要由3个基因控制, 分别是黄血基因(Yellow blood, Y), 黄血抑制基因(Yellow inhibitor, I)和黄茧基因(Out-layer yellow cocoon, C)。I基因阻止类胡萝卜素从中肠上皮细胞到血淋巴的转运, 是天然黄茧形成过程中的重要控制基因。利用家蚕雌性不发生交换的特点, 采用黄血黄茧品系KY和白血白茧品系巴格达特(Ba)组配正反交群体(Ba×KY)×KY和KY×(Ba×KY), 分别记作BC1F和BC1M, 根据已经构建的家蚕SSR分子标记连锁图谱对I基因进行了定位及连锁分析。筛选出3个与I基因连锁的SSR标记。BC1F群中的所有白血个体均表现出与(Ba×KY) F1相同的杂合型带型; 而所有黄血个体带型与亲本KY一致, 为纯合型。利用另一个群体BC1M构建了关于I基因的遗传连锁图, 连锁图的遗传距离为38.4 cM, 与I基因最近的引物为S0904, 图距为7.4 cM。     

利用同源重组改变家蚕丝心蛋白重链基因

在家蚕丝心蛋白重链基因5‘和3‘端序列之间插入以绿色荧光蛋白（green fluorescent protein,GFP）基因（gfp）与人工合成丝心蛋白样基因的融合基因,利用电穿孔方法导入蚕卵中,卵孵化、发育和结茧后,用紫外灯检查,在约5400个茧中有73个“亮茧”,茧蛋白在ELISA应中可以与GFP的多克隆抗体反应。“亮茧”对应的蚕蛾进行交配、制种。对其后代进行了基因鉴定,Southern杂交的结果表明,gfp基因和人工合成丝心蛋白样基因都存在于家蚕基因组DNA中且发生了预期的同源重组事件。上述结果说明“亮茧”这一表型能用于筛选转基因蚕,融合基因已通过同源重组进入家蚕基因组。     

A carotenoid-binding protein (CBP) plays a crucial role in cocoon pigmentation of silkworm (Bombyx mori) larvae

We examined the role of carotenoid-binding protein (CBP) in yellow cocoon pigmentation. First, using yellow or white cocoon races, we investigated the linkage between the yellow pigmentation and CBP expression. CBP was expressed only in the silk gland of the yellow cocoon races, which utilize carotenoids for cocoon pigmentation. Furthermore, CBP expression in the silk glands of day 1-7 fifth instar larvae matched the period of carotenoid uptake into the silk gland. Finally, we gave double-stranded CBP RNA to Bombyx mori (B. mori) larvae to induce RNA interference. The significantly reduced expression of CBP in the silk gland of fifth instar larva was confirmed on day 4 and a decrease in yellow pigmentation was observed in the cocoon. We showed that CBP plays a key role in the yellow cocoon pigmentation caused by carotenoids.     

BmStart1, a novel carotenoid-binding protein isoform from Bombyx mori, is orthologous to MLN64, a mammalian cholesterol transporter

Carotenoid-binding protein (CBP) from the silkworm Bombyx mori is an essential molecule for carotenoid dependent cocoon pigmentation. We identified a novel isoform of CBP, Start1 of B. mori (BmStart1). BmStart1 contains a membrane-spanning MENTAL domain in its N-terminus and a lipid-binding START domain in its C-terminus. This domain architecture is identical to the mammalian MLN64 and Start1 of Drosophila melanogaster (DmStart1), both of which have been implicated to function in cholesterol transport and regulation of steroidogenesis. BmStart1 is expressed in both white and yellow cocoon strains of B. mori, while CBP is only detected in the yellow cocoon strain. BmStart1 mRNA abundance in the prothoracic gland, the main ecdysteroidogenic tissue, positively correlates with changes in the hemolymph ecdysteroid level. Genomic sequence analysis revealed that BmStart1 and CBP are generated from the same gene locus by alternative splicing. Splice site comparison and homology search indicate that BmStart1 is orthologous to both MLN64 and DmStart1. This study implies that alternative splicing of the BmStart1/CBP gene generates unique protein isoforms whose endogenous ligands, sterol or carotenoid, are structurally different.     

Isolation,characterization, and cDNA sequence of a carotenoid binding protein from the silk gland of Bombyx mori larvae

A carotenoid binding protein (CBP) has been isolated from the silk glands of Bombyx mori larvae. The protein has an apparent molecular mass of 33 kDa and binds carotenoids in a 1:1 molar ratio. Lutein accounts for 90% of the bound carotenoids, whereas alpha-carotene and beta-carotene are minor components. Immunological analysis demonstrated the presence of CBP only in the yellow-colored tissues of the silk gland, midgut, testis, and ovary. Several phenotypes of B. mori mutants linked to carotenoid transport have been utilized to characterize CBP. The Y (yellow hemolymph) gene controls uptake of carotenoids from the midgut lumen into the midgut epithelium, and larvae with the +(Y) gene lack this property. Immunoblotting analysis confirmed the presence of CBP in mutants with the dominant Y gene only. Immunohistochemistry verified the localization of CBP in the villi of the midgut epithelium, indicating that CBP might be involved in absorption of carotenoids. A cDNA clone for CBP encoding a protein of 297 amino acids has been isolated from the B. mori silk gland cDNA library. The deduced amino acid sequence revealed that CBP is a novel member of the steroidogenic acute regulatory (StAR) protein family with its unique structural feature of a StAR-related lipid transfer domain, known to aid in lipid transfer and recognition. Lutein-binding capacity of the recombinant CBP (rCBP) determined by incubating rCBP with lutein followed by immunoprecipitation using anti-CBP IgG conjugated to protein A-Sepharose, demonstrated the formation of a lutein-rCBP complex. Sequence analyses coupled with binding specificity suggest that CBP is a new member of the StAR protein family that binds carotenoids rather than cholesterol.     

Diversity in copy number and structure of a silkworm morphogenetic gene as a result of domestication

The carotenoid-binding protein (CBP) of the domesticated silkworm, Bombyx mori, a major determinant of cocoon color, is likely to have been substantially influenced by domestication of this species. We analyzed the structure of the CBP gene in multiple strains of B. mori, in multiple individuals of the wild silkworm, B. mandarina (the putative wild ancestor of B. mori), and in a number of other lepidopterans. We found the CBP gene copy number in genomic DNA to vary widely among B. mori strains, ranging from 1 to 20. The copies of CBP are of several types, based on the presence of a retrotransposon or partial deletion of the coding sequence. In contrast to B. mori, B. mandarina was found to possess a single copy of CBP without the retrotransposon insertion, regardless of habitat. Several other lepidopterans were found to contain sequences homologous to CBP, revealing that this gene is evolutionarily conserved in the lepidopteran lineage. Thus, domestication can generate significant diversity of gene copy number and structure over a relatively short evolutionary time.     

Analysis of a silkworm F1 hybrid with yellow cocoon generated by crossing two white-cocoon strains: Further evidences for the roles of Cameo2 and CBP in formation of yellow cocoon

In this report, we examined the gene expression related to carotenoid transport for a silkworm F₁ hybrid with yellow cocoon generated by crossing two white-cocoon strains, Qiubai and 12-260. Our results showed that, in Qiubai, Cameo2, a transmembrane protein gene belonging to the CD36 family genes, was expressed normally in the silk gland, but no intact carotenoid-binding protein (CBP) mRNA (only the truncated CBP mRNA) was detected in the midgut. In 12-260, we detected the intact CBP mRNA expression in the midgut, but no Cameo2 expression in the silk gland. Regarding the F₁ hybrid from crossing Qiubai and 12-260, both Cameo2 and intact CBP mRNA expressed normally in the silk gland and midgut. HPLC detection confirmed that in the F₁ hybrid the carotenoids could be absorbed from dietary mulberry leaves through the midgut and transferred to silk gland via the hemolymph, which eventually colored cocoons into yellow. We also identified four CBP mRNA isoforms expressed in the midgut of the F₁ hybrid, subsequently named as variants 5–8. Our results provide further evidences for the roles of Cameo2 and CBP in the formation of yellow cocoon of silkworm.     

A CD36-related Transmembrane Protein Is Coordinated with an Intracellular Lipid-binding Protein in Selective Carotenoid Transport for Cocoon Coloration

The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.     

Silk fibroin film from golden‐yellow Bombyx mori is a biocomposite that contains lutein and promotes axonal growth of primary neurons

The use of doped silk fibroin (SF) films and substrates from Bombyx mori cocoons for green nanotechnology and biomedical applications has been recently highlighted. Cocoons from coloured strains of B. mori, such as Golden‐Yellow, contain high levels of pigments that could have a huge potential for the fabrication of SF based biomaterials targeted to photonics, optoelectronics and neuroregenerative medicine. However, the features of extracted and regenerated SF from cocoons of B. mori Golden‐Yellow strain have never been reported. Here we provide a chemophysical characterization of regenerated silk fibroin (RSF) fibers, solution, and films obtained from cocoons of a Golden‐Yellow strain of B. mori, by SEM, ¹H‐NMR, HPLC, FT‐IR, Raman and UV‐Vis spectroscopy. We found that the extracted solution and films from B. mori Golden‐Yellow fibroin displayed typical Raman spectroscopic and optical features of carotenoids. HPLC‐analyses revealed that lutein was the carotenoid contained in the fiber and RSF biopolymer from yellow cocoons. Notably, primary neurons cultured on yellow SF displayed a threefold higher neurite length than those grown of white SF films. The results we report pave the way to expand the potential use of yellow SF in the field of neuroregenerative medicine and provide green chemistry approaches in biomedicine. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 287–299, 2016.     

Characterization of the carotenoid-binding protein of the Y-gene dominant mutants of Bombyx mori

Carotenoids play important and diverse roles in insects. Recently, we purified and partially characterized a carotenoid-binding protein (CBP) from the wild type of Bombyx mori. In this report, we utilized immunoblotting, ELISA and immunocytochemistry to further characterize and localize the expression of CBP in the larval midgut and silk gland obtained from the wild type and four naturally occurring mutants linked to carotenoids transport. CBP was expressed throughout the 5th stadium, with highest expressions on days 4-5 in the silk gland and days 3-5 in the midgut. Immunoblotting analyses demonstrated the presence of CBP along the middle part of the midgut. Microscopic immunocytochemistry demonstrated that the 33 kDa CBP was uniformly expressed along the brush border of columnar cells in the epithelium of the midgut typifying its function in aiding absorption of dietary carotenoids. Similarly, CBP was highly expressed along the distal membrane of the middle part of the silk gland demonstrating its function in uptake of carotenoids from lipophorin. When the middle silk glands and midguts of the four mutants were incubated with rabbit anti-CBP antibody, only proteins of the Y-gene dominant mutants cross reacted with the antibody further accentuating the hypothesis that the CBP is a Y-gene dependent protein.     

Conservation of a pair of serpin 2 genes and their expression in Amphiesmenoptera

Silk secreted by the larvae of Hydropsyche angustipennis (Trichoptera) contains serpins HaSerp2A and HaSerp2B that are homologous to serpin 2 known from several lepidopterans and some other insects. The gene HaSerp2A is 2684 bp downstream from the HaSerp2B gene. The genes possess identical exon/intron segmentation (9 exons) and their sequences are nearly identical: only 8 out of 1203 nt differ in the coding region, 4 out of 567 nt in the introns and 2 out of 52 nt in 3' UTR. Both genes are highly expressed in the silk glands whereas expression in larval carcass devoid of the silk glands is hard to detect. Translation products of the genes consist of 401 amino acids, are 98.8% identical, and are secreted as 45 kDa proteins into silk. Homologous genes in similar tandem arrangement occur on chromosome 15 of Bombyx mori (Lepidoptera). The upstream gene BmSerp2B is modified in several exons and does not seem to produce functional mRNA. The gene BmSerp2A contains two copies of exon 9, of which only the second one is used. One kind of mRNA does and the other does not include exon 1, which encodes a signal peptide. The mRNA yielding secreted BmSerp2A is expressed in the posterior, and that encoding the cytoplasmic BmSerp2A in the middle silk gland region; both kinds are strongly expressed in the anterior region. The data indicate that (1) A duplication of serpin 2 gene occurred either before Trichoptera and Lepidoptera diverged as separate orders or independently in early phylogeny of either order; (2) In the caddisfly H. angustipennis, both genes are expressed specifically in the silk glands and generate proteins deposited in the silk; (3) Only one gene seems to be functional in B. mori and is expressed in a cytoplasmic and in a secreted forms in diverse organs, including the silk glands.     

In-situ observation of tryptophan in larval cocoon silk of the hornet Vespa simillima xanthoptera cameron by ultraviolet resonance raman spectroscopy

We performed in-situ ultraviolet resonance Raman (UVRR) spectroscopy of the larval cocoon silk of the hornet, Vespa simillima xanthoptera Cameron, and compared the result with that of the silkworm, Bombix mori. The UVRR spectrum of the hornet cocoon differed markedly from that of the B. mori cocoon: peaks attributable to tyrosine (Tyr) were observed strongly, and tryptophan (Trp) peaks weakly, in the spectrum of the B. mori cocoon, whereas peaks attributable to Trp exclusively appeared in the spectrum of the hornet cocoon.