家蚕滞育激素基因的克隆

利用放射性同位素３２Ｐ－ｄＣＴＰ标记家蚕滞育激素ｃＤＮＡ作为探针,从家蚕基因库大约４×１０~５个噬菌体斑中筛选得到３０个阳性克隆,其中３个克隆具有相同的限制性内切酶图谱,并测知插入ＤＮＡ约１８ｋｂ,用限制性内切酶ＳａｌＩ、ＥｃｏＲＩ切割,进行次克隆。用ＴａｑＤｙｅｐｒｉｍｅｒ序列测定法,得到部分核苷酸序列,经比较,鉴定了这３个克隆确属滞育激素基因。基因组Ｓｏｕｔｈｅｒｎ分析,推定家蚕滞育激素基因属单基因编码。     

激光诱发家蚕孤雌生殖的研究

采用 CO2激光扩束辐照诱发家蚕孤雌生殖,获得着色卵率约60%,转青卵率约31%,孵化率只占转青率的1.85%,全是雌蚕,其发育机制属非减数分裂型,没有出现畸形蚕,茧质未下降。从激光诱发的孤雌生殖的后代中,初步选出两个新类型的可作育种材料。 Abstrect:In order to induce silkworm(Bombyx mori)parthenogenesis,we irradiated eggs by Co2 laser.The results showed,60% colored eggs,31% average bluish eggs,only 1.85% hatchavility among the bluish eggs,all female from those eggs and so an ameiosis parthenogenesis;no malformed worm,thus similar constitution and quality to their parents;and finally two new strains which can be used to breed new strains and furhther studies.     

Dietary Sterol Preference in the Silkworm,Bombyx mori

Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that β-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: β-sitosterol >> ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouthpart might be different from the sterol recognition mechanism present in sterol metabolism.     

家蚕细胞遗传学及其应用

由于家蚕染色体数目较多、着丝粒弥散, 在较长时期内, 家蚕染色体识别、核型分析、染色体结构和功能的研究都受到很大限制。近年来, 应用比较基因组杂交、基因组原位杂交、基于细菌人工染色体克隆的原位杂交技术建立了家蚕的细胞学图, 综合分子连锁图构建高密度的细胞遗传学图已成为可能。分子细胞遗传学的应用正在推动家蚕染色体结构和功能的研究, 揭示出家蚕W染色体密集地分布着嵌套结构的逆转座子, 染色体端粒由重复序列(TTAGG)n以及端粒特异的非长末端逆转座子TRAS1和SART1组成, TRAS1、SART1具有较高的转录活性, 可能与维持染色体的稳定性有关。     

Comprehensive Profiling of Lysine Acetylome in Baculovirus Infected Silkworm (Bombyx mori) Cells

Bombyx mori is one of the key lepidopteran model species, and is economically important for silk production and proteinaceous drug expression. Baculovirus and insect host are important natural biological models for studying host–pathogen interactions. The impact of Bombyx mori nucleopolyhedrovirus (BmNPV) infection on the proteome and acetylome of Bombyx mori ovarian (BmN) cells are explored to facilitate a better understanding of infection‐driven interactions between BmNPV and host in vitro. The proteome and acetylome are profiled through six‐plex Tandem mass tag (TMT) labeling‐based quantitative proteomics. A total of 4194 host proteins are quantified, of which 33 are upregulated and 47 are downregulated in BmN cells at 36 h post‐infection. Based on the proteome, quantifiable differential Kac proteins are identified and functionally annotated to gene expression regulation, energy metabolism, substance synthesis, and metabolism after BmNPV infection. Altogether, 644 Kac sites in 431 host proteins and 39 Kac sites in 22 viral proteins are identified and quantified in infected BmN cells. Our study demonstrates that BmNPV infection globally impacts the proteome and acetylome of BmN cells. The viral proteins are also acetylated by the host acetyltransferase. Protein acetylation is essential for cellular self‐regulation and response to virus infection. This study provides new insights for understanding the host–virus interaction mechanisms, and the role of acetylation in BmN cellular response to viral infection.     

Genome-Wide Transcriptional Response of Silkworm (Bombyx mori) to Infection by the Microsporidian Nosema bombycis

Microsporidia have attracted much attention because they infect a variety of species ranging from protists to mammals, including immunocompromised patients with AIDS or cancer. Aside from the study on Nosema ceranae, few works have focused on elucidating the mechanism in host response to microsporidia infection. Nosema bombycis is a pathogen of silkworm pébrine that causes great economic losses to the silkworm industry. Detailed understanding of the host (Bombyx mori) response to infection by N. bombycis is helpful for prevention of this disease. A genome-wide survey of the gene expression profile at 2, 4, 6 and 8 days post-infection by N. bombycis was performed and results showed that 64, 244, 1,328, 1,887 genes were induced, respectively. Up to 124 genes, which are involved in basal metabolism pathways, were modulated. Notably, B. mori genes that play a role in juvenile hormone synthesis and metabolism pathways were induced, suggesting that the host may accumulate JH as a response to infection. Interestingly, N. bombycis can inhibit the silkworm serine protease cascade melanization pathway in hemolymph, which may be due to the secretion of serpins in the microsporidia. N. bombycis also induced up-regulation of several cellular immune factors, in which CTL11 has been suggested to be involved in both spore recognition and immune signal transduction. Microarray and real-time PCR analysis indicated the activation of silkworm Toll and JAK/STAT pathways. The notable up-regulation of antimicrobial peptides, including gloverins, lebocins and moricins, strongly indicated that antimicrobial peptide defense mechanisms were triggered to resist the invasive microsporidia. An analysis of N. bombycis-specific response factors suggested their important roles in anti-microsporidia defense. Overall, this study primarily provides insight into the potential molecular mechanisms for the host-parasite interaction between B. mori and N. bombycis and may provide a foundation for further work on host-parasite interaction between insects and microsporidia.     

家蚕雌性附腺及其Ng突变体蛋白质组双向电泳图谱分析

分别对家蚕(Bombyx mori．L)正常及Ng突变体雌蛾件附腺分泌部组织的蛋白质进行提取,并采用双向凝胶电泳和计算机辅助分析方法,对提取的蛋白质混合物进行分离和比较分析。用银染的方法,平均每张电泳图谱可以分离约700个蛋白质点,其中大部分的蛋白质点分布在pH4～8范围内,在分子量上主要集中在30～70kD区域：比较分析发现,有4种蛋白只在正常性附腺组织中特异表达,而有2种蛋白只在Ng突变体的组织中特异表达。另外约有29种蛋白在正常性附腺分泌部组织中的表达水平明显高于Ng突变,而约有15种蛋白在Ng突变体的分泌部组织中表达水平较高。这些差异蛋白质可能与Ng突变的形成和导致这种突变体的性附腺不能正常分泌粘性蛋白的性状有关。     

家蚕BmST2基因的克隆与转录活性检测

通过生物信息学分析和生物学试验获得了家蚕糖转运蛋白基因BmST2(GenBank登录号:GQ871755),基因位于家蚕27号染色体,开放阅读框(ORF)长1398 bp,编码465个氨基酸,预测蛋白序列有典型的Sugar_tr结构域和11个疏水的跨膜结构域,与家蚕BmST1蛋白相似性和一致性分别达79%和64%,与登录号为EAT47626、EDS35465、EAA11457和EFA05337的同源蛋白相似性在50%以上。RT-PCR检测基因在5龄第3天家蚕幼虫的9种组织中转录活性,结果显示,BmST2基因除在脂肪体没有表达外,其他组织均有表达。最后成功构建了基因的酵母穿梭表达质粒pG-BKT7-BmST2。     

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。     

新霉素抗性基因在家蚕中的插入和表达

构建含新霉素抗性基因（ｎｅｏｍｙｃｉｎｒｅｓｉｓｔａｎｃｅｇｅｎｅ,ｎｅｏＲ）的重组质粒ｐＦＮ,经ＨｉｎｄＩＩ酶切后,用基因枪将ＤＮＡ片段导入家蚕早期受精卵中（Ｇ０代）。孵化的Ｇ１、Ｇ２代蚁蚕均经含新霉素的人工饲料添食２４ｈ后,筛选出新霉素抗性的个体（能正常生长发育的）改为桑叶饲养。于Ｇ２代的５龄第二天从后部丝腺抽提总ＤＮＡ,再以ｎｅｏＲ的ｃＤＮＡ为探针进行Ｓｏｕｔｈｅｒｎ杂交检测。结果表明ｎｅｏＲ基因已转入家蚕ＤＮＡ中,获得了含ｎｅｏＲ的转基因蚕。     

差异蛋白质组学在家蚕中的研究进展

差异蛋白质组是蛋白质组学的一个重要分支,通过对蛋白质组表达谱的比较,揭示细胞生理或病理状态的进程与本质,发现具有关键作用的蛋白。近年来,家蚕差异蛋白质组学发展迅速且涉及面广,已然成为家蚕蛋白质组学研究的热点。对差异蛋白质组学的主要研究方法,及在家蚕中的研究进展做一简要评述。     

Silkworm pupae (Bombyx mori) and locusts as alternative protein sources for high-energy biscuits

This study explored the possibility of replacing skimmed milk – a core ingredient of High-energy biscuit (HEB), with edible-insects as inexpensive and sustainable alternative sources. HEB was formulated to meet the USAID 2016 (STD) nutritional standards and powdered locust and mulberry silkworm pupae (LP and SWP) at 15% inclusion, were used as alternative protein sources. Preliminary nutrients composition of the insect powders were examined. Physicochemical, sensory and microbial qualities of insect-enriched biscuits (LPB and SWPB) were evaluated and compared with that of skimmed milk (SMB) and STD. Spread ratios of the biscuits were between 7.8 and 11.6 – a technically acceptable range for biscuits. SWP possessed impressive amounts of protein (60.7 g/100 g), fat (23.5 g/100 g) and energy contents (498.93 kcal/100 g), while LP had slightly more fibre (1.4 g/100 g), ash (2.1 g/100 g) and carbohydrate (31.5 g/100 g). LPB and SWPB had relatively similar proximate compositions as SMB with slight variations, and all the HEBs were above USAID minimum standard. SWPB was marginally below requirements for Ca and Mg minerals, but both (SWPB and LPB) had more than twice the recommended pro-vitamin A (918.44 and 957.65 µg/100 g) and vitamin C contents (102.17 and 98.51 mg/100 g). All the samples received reasonably good sensorial rankings; but SMB’s aroma was preferred. Similarly, microbial quality assessment of the products reflected remarkable hygienic sample preparation. These results demonstrated that HEB of significant nutritional, sensorial and microbial qualities could be developed using edible-insects as alternative protein sources.