我国柞蚕(Antheraea pernyi)遗传育种研究进展

柞蚕遗传学研究是新品种选育的前提和基础,品种改良是发展柞蚕业最经济有效的手段,柞蚕遗传学研究及新品种选育工作取得了显著成绩,在柞蚕生产发展上发挥了重要作用。作者概述了柞蚕部分性状的遗传学及育种学的研究概况及分子生物学技术在柞蚕种质资源方面的研究进展,论述了柞蚕遗传学及育种学今后应重点研究的内容。

Fluorescence Tracking of Exogenous DNA in Genetic Transformation of the Chinese Oak Silkmoth Antheraea Pernyi via Sperm-Mediated Gene Transfer

Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( SMGT). Sperms entry into the female reproductive system and eggs were observed using fluorescence microscopy. The ability of A. pernyi sperms to uptake exogenous DNA was confirmed,and transfer of the exogenous DNA was shown by GFP expression in the transgenic eggs. Our result suggested that SMGT could also be used to directly generate transgenic A. pernyi expressing functional genes of interest.

Enhanced CD3^+/CD4^+ T Cell Activities and Modulation of Th1/Th2 Lineage Development in Irradiated Rats Due to Treatment with the Male Zooid of Antheraea Pernyi Extracts

Objective： Cancer patients undergoing large dose radiotherapy exhibit multifaceted defects in their immune capacity that are likely to contribute to an increased susceptibility to infections and disease progression. The immune impairment may also constitute a barrier to effective immunotherapeutic interventions. Here, we evaluate whether supplementation with the male zooid of Antheraea pernyi extracts could enhance immune function in irradiated rats. Methods： Fifty male Wistar rats were randomly divided into a control group, a simple radiation group and a treatment group. The mice in the simple irradiation and treatment groups were given whole-chest irradiation with 6Gy. In the treatment group, the male zooid of Antheraea pernyi extracts was gavaged at the doses of 16.53mg/kg （large dose group）, 2.62mg/kg （medium dose group）, and 0.564mg/kg （small dose group） once a day for 14 days. The thymus and spleen indices were calculated. T cell subsets in peripheral blood were determined by flow cytometry and the expressions of IL-2, IFN-γ, IL-4 and IL-10 in sera were determined by ELISA on the 15th day. Results： The thymus index and spleen index of the high dose treatment group were statistically lower than that of the control group and higher than that of the radiation group （P〈0.01）. CD3＋ and CD4＋ T cells in the peripheral blood were increased in the high dose treatment group and decreased in the radiation group （P〈0.01）. Expression of IL-2 and INF-T in the radiation group was lower than that in control, and significantly increased during therapy. The production of IL-4 and IL-10 could be induced by radiation and was inhibited in the high dose treatment group （P〈0.01）. Conclusion： Our data indicate that the male zooid of Antheraea pernyi extracts may be administrated to improve immune function in irradiated rats and reverse the radial immune inhibition of rats by stimulating the proliferation of Th ceils and inducing the differentiation of Th2 to Th1.

Cloning of Immune System-related Gene ApTOLL1 and Its Expression in Nosema-infected Antheraea pernyi

[ Objective ] This study aimed to identify the immune system-related TOLL-like receptor family gene of Aherea pernyi, to lay the foundation for further investigating the immune mechanism of Antherea perny/. [ Method] Immune system-related TOLL-like receptor family gene of Antherea perny/was cloned for se- quencing and phylogenetic analysis ; in addition, expression variations of TOLL-like receptor family gene in Antherea pernyi infected with Nosema pernyi and Nosema bombycis were detected to analyze the differences in immunological reactivity of Antherea pernyi to Nosema infection. [ Result] Based on cDNA cloning and sequen- cing, an immune system-related gene fragment was isolated from Antherea pernyi, which is the most homologous to the ToUl gene in Toll signaling pathway of Bom- byx mot/according to the sequencing result and phylogenetic analysis, which is named ApTolll gene. Subsequently, Antherea pernyi pupae were injected respective- ly with Nosema bombycis and Nosema pernyi, fluorescent real-time quantitative PCR analysis showed that ApTolll was abundantly expressed in Antherea pernyi pupa 2 h after Nosema bombycis injection, which began to express in Antherea pernyi pupa 11 h after Nosema pernyi injection, indicating that the immune response time induced by various Nosema strains varies in Toll signaling pathway of Antherea perny/. [ Conclusion ] ApTolll gene of Antherea perny/was first cloned in this study, which provides reference for further investigating the immune mechanism of Antherea pernyi.

Immunization of mice with concentrated liquor from male zooid of Antheraea pernyi

AIM: To study the effects of concentrated liquor from male zooid of Antheraea pernyi on immunological mice.METHODS: For each experiment, 40 mice were randomly divided into normal saline group (control group) and three tested groups that were administered different dosages of concentrated liquor from male zooid of A. pernyi and food for 15 d. The typical FSR and HC50 value, monocyte-phagocytic exponent Kand emendated monocyte-phagocytic exponent α were determined and calculated respectively.RESULTS: After 24 and 48 h, the FSR values of the three tested groups improved significantly in comparison to the control group by variance analysis. The HC50 values showed a significant difference between the high dosage group and the control group, as well as between the high dosage group and other two tested groups. The monocyte-phagocytic exponent Kand emendated exponent α showed rising tendencies, but no significant differences were found by variance analysis.CONCLUSION: The concentrated liquor from male zooid of A. pernyi can significantly enhance cellular and humoral immune function in mice, but has no distinct influence on the monocyte-phagocytic system in mice.

Enhancement of CD4^+ T cell activities and modulation of Th1/Th2 lineage development in radiated tumor-bearing rats treated with male zooid of Antheraea pernyi extracts

AIM: To investigate whether supplementation of male zooid of Antheraea pernyi extracts (MZAPE) could enhance immune function of radiated tumor-bearing rats. METHODS: Eighty male Wistar rats were randomly divided into a control group, a simple radiation group, a MZAPE group, and a radiation plus MZAPE group. With the tumor model established by implanting Walker-256 ascites tumor cells, tumor weight and tumor control rate were calculated. The rats in the simple radiation and radiation plus MZAPE groups were underwent to radiation at 10 Gy within 2 d. In the MZAPE and radiation plus MZAPE groups, the MZAPE was gavaged at a dose of 16.53 mg/kg once a day for 7 d. T cell subsets in peripheral blood were determined by flow cytometry and the expression of IL-2, IFN-γ, IL-4 and IL-10 in sera were determined by ELISA on the 8th d. RESULTS: The tumor weight of simple radiation group, MZAPE group and radiation plus MZAPE group was lower than that of control group (P < 0.01) and tumor control rates were 63.08% ± 6.43%, 69.86% ± 7.12% and 35.30% ± 7.67%, respectively. CD4+ T and CD8+ T cells in the peripheral blood of the simple radiation group were fewer than in control group. In the MZAPE and radiation plus MZAPE groups, the number of CD4+ T cells was higher while CD8+ T cells was lower than in the control and simple radiation groups. Expression of IL-2 and INF-γ in the radiation group was lower than in control group, and significantly enhanced during MZAPE therapy (P < 0.05). Expression of IL-4 and IL-10 in the radiation group had no significant changes compared with the control group, and decreased significantly after MZAPE treatment (P < 0.01). CONCLUSION: MZAPE administration may help improve the immune function of the radiated tumor-bearing rats and reverse the radiation-induced immune inhibition by promoting the proliferation of T helper cells and inducing the transdifferentiation from Th2 to Th1.

Variation and Characterization Analysis of Partial Fragment of Fibroin Gene From Silkworm, Antheraea pernyi

A 1.4Kb DNA fragment containing 3’ flanking sequence of fibroin gene of silkworm, Antheraea pernyi, was obtained from the silk gland’s mRNA of 5 th larva. Analysis of this sequence with another A.pernyi fibroin protein (accession No. D83241) revealed that it consists of a completely open reading frame (ORF), which includes 14 polyalanine-containing units (motifs) and 100bp 3’-UTR. The sequence of the predicted amino acid reveals the highest level of overall identity (90%) with D83241. It was found that it loses a repeat region at the upstream of TAA codon and some mutations. A putative polyadenylation signal AATAAA tail was found in position 1300, which follows the termination codon.

Analysis and expression of the polyhedrin gene of Antheraea pernyi nucleopolyhedrovirus (AnpeNPV)

The polyhedrin (polh) gene is often used to analyse evolution of baculovirus. In this report, the polh of Antheraea pernyi nucleopolyhedro-virus (AnpeNPV) was cloned and sequenced. The Open reading frame (ORF) of the AnpeNPV consists of 738 nucleotides encoding 245 amino acids with molecular masses of 29 kDa. The deduced amino acids were significant homol-ogy with other baculoviruses, such as Attacus ricini NPV (ArNPV) and Autographa californica NPV (AcNPV). A strongly hydrophilic region was predicted at positions from 30 to 50 of the An-peNPV Polh protein by bioinformatics analysis. Expression of the polh gene of AnpeNPV in E. coli was examined by SDS–PAGE, Western blot and Mass-spectrum analysis. The result showed that the bacterium expression system was suitable for the virus gene expression. It indi-cated that the products of the polh gene ex-pressed in this system can be easier to use for raising antibodies.

3D Printed Gelatin Scaffold with Improved Shape Fidelity and Cytocompatibility by Using Antheraea pernyi Silk Fibroin Nanofibers

Gelatin(G)is a commonly used natural biomaterial owing to its good biocompatibility and easy availability.However,using pure gelatin as a bioink can barely achieve an ideal shape fidelity in 3D printing.In this study,Antheraea pernyi silk fibroin nanofibers(ASFNFs)with arginine-glycine-aspartic acid(RGD)peptide and partial natural silk structure are extracted and combined with pure gelatin bioink to simultaneously improve the shape fidelity and cytocompatibility of corresponding 3D printed scaffold.Results show that the optimum printing temperature is 30℃ for these bioinks.The printed filaments using 16G/4ASFNFs bioink(16wt%gelatin and 4wt%ASFNFs)demonstrate better morphology and larger pore size than those printed by pure gelatin bioink(20G,20wt%gelatin),thus successfully improve the shape fidelity and porosity of the 3D printed scaffold.The 16G/4ASFNFs scaffold also demonstrate higher swelling ratio and faster degradation rate than the pure gelatin scaffold.Moreover,the cell viability and proliferation ability of Schwann cells cultured on the 16G/4ASFNFs scaffold are significantly superior than those cultured on the pure 20G scaffold.The ASFNFs enhanced 16G/4ASFNFs scaffold reported here are expected to be a candidate with excellent potential for biomedical applications.

Gelation behavior of Antheraea pernyi silk fibroin

The sol-gel transition behavior of Antherae pernyi silk fibroin(Ap-SF) has not been systematically investigated.In this work,the influence of environmental temperature,pH,the concentration of Ap-SF,K+ and Ca2+ on the gelation time,and the structural changes of Ap-SF in sol-gel transformation were studied.The results indicated that the gelation time of the Ap-SF aqueous solution decreased with the increase of the Ap-SF concentration and environmental temperature.The sol-gel transformation of Ap-SF was much more rapid than that of Bombyx mori silk fibroin under the same conditions.The Ap-SF was sensitive to changes in the concentration of Ca2+ and K+.Upon gelation,the random coil structure of the Ap-SF was significantly transformed into the β-sheet structure.

SYNTHESIS OF GENE FOR ANTIBACTERIAL PEPTIDE D FROM CHINESE OAK SILKWORM (Antheraea pernyi)

The antibacterial peptide D (ABP-D) was induced from the haemolymph of the Chinese oak silkworm (Antheraea pemyi). It has a marked inhibition effect on some human, insect and plant pathogenic bacteria. In order to synthesize a large amount of ABP-D in microorganism by recombinant DNA technology, here we report the synthesis of the ABP-D gene.

AProteinase Responsible for Degrading Yolk Proteins in Tussah（Antheraea pernyi）

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柞蚕羧酸酯酶活性与经济性状的相关性分析及杂种优势表现

选择3个柞蚕亲本以及6个杂交组合的五龄柞蚕幼虫,测定不同组织中羧酸酯酶的活性,利用生物数学方法分析羧酸酯酶活性与经济性状杂种优势的关系,探讨羧酸酯酶活性用于柞蚕杂种优势预测的可能性。结果表明,不同柞蚕幼虫组织羧酸酯酶活性的杂种优势表现不同。不同组织中羧酸酯酶活性与经济性状的相关性不同,脂肪体中羧酸酯酶活性与茧层率均呈极显著负相关,而且与其杂种优势率也呈极显著负相关;血淋巴中羧酸酯酶活性与生命力性状(死笼率、虫蛹生命率)有显著的相关关系,且杂种优势率也显著相关。因此,柞蚕幼虫脂肪体羧酸酯酶活性可作为预测茧质性状(茧层率)杂种优势、血淋巴中羧酸酯酶活性可作为预测柞蚕生命力性状(死笼率、虫蛹生命率)杂种优势的一种生化指标。

春柞蚕蒿柳高效养殖技术调查研究

通过对蒿柳扦插模式、树型养成的探索,开展春柞蚕“半龄式”和“全龄式”蒿柳养殖技术研究,调查并统计柞蚕幼虫保苗率、全龄经过时间和蚕茧产量。结果表明:0.5~2.0 cm直径的蒿柳进行直接扦插,成活率在98.0%以上;而用生根剂浸泡后扦插,成活率为100%。采用株距30 cm,墩距4 m,每墩3株,“W”型扦插,通过剪梢后的树型养成,能够显著增加枝条数量,提高柞蚕饲养效率。采用“半龄式”和“全龄式”蒿柳养殖能够显著提高柞蚕养殖过程中的幼虫保苗率,达60%以上,减少了劳动强度并增加蚕茧产量。

柞蚕微孢子虫感染后柞蚕卵转录组及免疫相关基因功能分析

【目的】柞蚕微孢子虫Nosema pernyi引发柞蚕Antheraea pernyi微粒子病,威胁柞蚕种质资源的保育及生产安全,并且能够经卵垂直传播,因此需要寻找柞蚕体内应对柞蚕微孢子虫侵染后的免疫基因,为后续寻找和培育抗微粒子病新品种提供帮助。【方法】构建柞蚕微孢子虫侵染后柞蚕卵转录组,进行GO功能注释和KEGG分析确定免疫相关基因;PCR克隆柞蚕谷胱甘肽S-转移酶(glutathione S-transferase,GST)基因ApGSTo1并进行生物信息学分析;运用RT-qPCR检测ApGSTo1在柞蚕微孢子虫侵染柞蚕产出后0-10 d卵中的表达量。【结果】在感染与未感染柞蚕微孢子虫柞蚕卵转录组检测到的表达基因数为17453,其中显著差异表达基因数目为89个;GO功能注释中细胞解剖实体和催化活性相关基因数量最多,KEGG分析中参与运输和分解代谢、信号转导及消化系统这3个通路的基因数量最多。基于柞蚕微孢子虫侵染后柞蚕卵转录组数据分析,将ApGSTo1作为目的基因,PCR克隆及系统进化分析结果证明ApGSTo1属于omega家族GST;感染柞蚕微孢子虫柞蚕卵中ApGSTo1表达量比正常对照组的高,且在产卵后2 d时的表达量极显著高于正常对照组的。【结论】获得了柞蚕微孢子虫侵染后柞蚕卵转录组数据,找到了1个免疫相关基因ApGSTo1,从分子生物学和转录水平初步证明了ApGSTo1参与柞蚕卵内的抗柞蚕微孢子虫侵染,还发现了抗柞蚕微孢子虫侵染反应的关键时间为产卵后2 d时,这些结果为后续研究ApGSTo1在抗逆性和氧化还原等功能方面提供研究基础。

柞蚕综合利用研究进展

我国柞蚕资源丰富,是世界柞蚕产业大国。目前柞蚕综合利用研究涉及生物农药、医疗保健、新型材料、功能食品等方面,本文总结了柞蚕卵、幼虫、茧、成虫4个不同时期的综合利用研发内容及科技成果转化现状,旨在明确柞蚕产业综合利用研究方向,促进柞蚕综合利用产业高质量发展。

4个柞蚕品种对人工饲料的适应性及其SOD、CAT酶活性差异分析

为了探究不同柞蚕(Antheraea pernyi)品种对人工饲料的适应性,为选育适宜人工饲料饲育的品种奠定基础。以9906、抗大、辽蚕527和大9共4个柞蚕(Antheraea pernyi)品种/品系为材料进行人工饲料适应性调查,并测定取食人工饲料后柞蚕幼虫的SOD、CAT活性。饲育方式为1~2龄人工饲料饲育,3~5龄鲜叶饲育(以下简称小蚕饲料育)。结果表明,不同柞蚕品种对柞蚕人工饲料的适应性存在差异,其中抗大和大9对人工饲料的适应性优于其他品种,抗大饲育成绩最佳,各品种间小蚕人工饲料育成绩均比鲜叶饲育成绩低。不同饲育形式柞蚕幼虫超氧化物歧化酶(SOD)与过氧化氢酶(CAT)活性分析表明,抗大、辽蚕527与大9在小蚕人工饲料育时体内2种抗氧化酶活性较高,相关性分析表明,2龄酶活性与2龄起青率、5龄酶活与结茧率皆呈显著正相关,说明这3个品种人工饲料育适应性较强。另外试验初步筛选出适宜人工饲料育的3个柞蚕品种,可以为下一步柞蚕人工饲料适应性品种选育提供育种材料。

柞蚕ApMLEC的重组表达和功能分析

对柞蚕Malectin(ApMLEC)进行克隆、蛋白表达纯化和初步功能分析。基于已知的柞蚕cDNA数据库,通过PCR克隆ApMLEC基因,生物信息学分析序列信息;将基因连接至pET-28a原核表达载体,通过大肠杆菌BL21(DE3)表达系统和亲和层析法纯化重组蛋白,探究该蛋白结合多糖、凝集细菌和抑菌能力;通过将基因连接至转移载体pApBacDual-egfp探究其抗病毒作用。结果表明:基因由798个碱基构成,编码266个氨基酸,纯化得到分子质量约为33 ku、带有组氨酸标签的重组蛋白;ApMLEC能够与麦芽糖、脂多糖、肽聚糖结合;凝集细菌;抑制大肠杆菌和金黄色葡萄球菌的生长,且最小抑菌质量浓度均为250μg/mL;并抑制ApNPV病毒的复制。ApMLEC初步功能分析结果表明其可能在柞蚕天然免疫中发挥重要作用,为今后开展柞蚕免疫系统的研究奠定基础。

柞蚕5龄幼虫主要营养成分含量及相关酶活性测定

为研究抗大、9906、辽蚕582和辽蚕988柞蚕品种5龄幼虫不同生长期体内主要营养成分及相关酶活性变化,测定并比较了4个柞蚕品种5龄幼虫生长第2天、第7天、第14天和营茧第3天体内总蛋白、糖原、海藻糖及溶菌酶、脂肪酶、抗氧化酶(超氧化物歧化酶、过氧化氢酶、过氧化物酶、谷胱甘肽过氧化物酶)等含量及活性。结果表明,柞蚕5龄幼虫体内均含有总蛋白、糖原、海藻糖及溶菌酶、脂肪酶、抗氧化酶等生物活性物质,同一品种各生长期体内总蛋白、糖原、海藻糖及溶菌酶、脂肪酶等生物活性物质差异显著,并且4个柞蚕品种5龄幼虫体内抗氧化酶活性水平较高。研究结果为柞蚕资源进一步开发利用提供了理论依据。

不同饲料育条件下柞蚕幼虫中肠细菌的多样性分析

为了探讨不同饲料饲育条件下柞蚕(Antheraea pernyi)幼虫肠道细菌群落的变化情况,采用新鲜柞叶育(DJY)和人工饲料育(DJS)2种模式饲育柞蚕1~2龄幼虫,收集柞蚕3龄幼虫中肠样品,通过16S rRNA高通量测序技术分析其肠道菌群的组成和多样性差异。结果显示,在门水平上,人工饲料育与鲜柞叶育柞蚕幼虫肠道优势菌组成相似,均为变形杆菌门(Proteobacteria)、厚壁菌门(Firmicutes)和放线菌门(Actinobacteria),但占比不同,DJS组柞蚕中肠内三者占比分别为83.55%、5.90%和4.77%,DJY组柞蚕中肠内则为90.96%、1.48%和2.04%;在属水平上,二者共同的优势菌以不动杆菌属(Acinetobacter)为主,DJS组柞蚕中肠内其他优势菌属按占比由高到低依次为葡萄球菌属(Staphylococcus)、假单胞菌属(Pseudomonas)、红细菌属(Mangrovibacter)、贝氏谷氨酸杆菌属(Glutamicibacter)和肠杆菌属(Enterobacter),DJY组柞蚕中肠中则为泛菌属(Pantoea)、肠杆菌属(Enterobacter)、弧菌属(Vibrio)、贝氏谷氨酸杆菌属(Glutamicibacter)和葡萄球菌属(Staphylococcus)。此外,菌群数目和丰富度分析表明,DJS组柞蚕中肠细菌物种组成的丰富度和物种多样性比DJY组更高。菌群差异性分析结果显示,DJS组细菌丰富度存在显著差异的有假单胞菌属某菌(Pseudomonas-sp-20-2)和约氏不动杆菌(Acinetobacter johnsonii),DJY组细菌丰富度存在显著差异的有醋酸钙不动杆菌(Acinetobacter calcoaceticus)和古氏不动杆菌(Acinetobacter guillouiae)。