基于Super-GBS简化基因组测序技术的柞蚕SNP位点挖掘

基于Super-GBS基因分型(super-genotyping-by-sequencing)技术开展柞蚕单核苷酸多态性标记(single nucleotide polymorphism,SNP)位点挖掘研究,旨在开发性状关联SNP分子标记,为柞蚕种质资源评价鉴定、遗传图谱构建、QTL定位提供数据支撑。以白体色小白蚕为母本、黄体色H04为父本,获得BC1M群体(110个)、F_(1)(3个)、P_(1)(1个)、P_(2)(1个)总计115个柞蚕材料,利用PstⅠ-HF/MspⅠ双酶切基因组构建Super-GBS文库并测序,使用BWA软件将过滤后的序列数据比对到柞蚕参考基因组,采用GATK软件开发SNP标记,使用SnpEff软件注释SNP位点及R语言分析遗传结构。测序共产生序列数据量为106.39 Gb,过滤后的平均读长为0.89 Gb,Q30测序质量值平均为90.11%,比对率平均为98.48%。共得到有效SNP位点141100个,主要位于基因间隔区、内含子区、基因下游、上游,其中C/T、A/G变异类型最多,转换与颠换的比例为1.296187∶1。SNP位点变异主要为同义突变和错义突变,产生修饰(MODIFIER)影响。主成分与聚类分析将115个样品分为4组(P_(1)、P_(2)、F_(1)、BC_(1)M),反映了样品的遗传背景及亲缘关系,群体遗传分化指数F_(ST)在0.0003777~0.8272间,群体遗传距离DR分布在0.0004~1.7556,F_(ST)与DR均表现出明显的遗传背景上的聚类。结果表明,Super-GBS技术能够获得大量柞蚕SNP位点信息,可用于SNP标记开发,且开发的SNP标记能够对115个样品的亲缘关系、体色演变进行解释,为今后柞蚕种质资源评价与鉴定、分子标记辅助育种工作奠定基础。

4个柞蚕品种蚁蚕耐饥饿能力的比较

饥饿是影响柞蚕生长发育、防病害能力和繁殖习性的重要因子。为探索柞蚕的耐饥饿能力,以柞蚕品种辽蚕527、9906、抗大和吉青为试验材料,对蚁蚕采取低温(6℃)饥饿的方法进行耐饥饿能力测试。结果表明,辽蚕527蚁蚕耐饥饿能力最强,其次是吉青,耐饥饿能力最差的品种是9906和抗大;辽蚕527蚁蚕耐饥饿平均时长为10.26 d,总时长达18 d,分别比抗大长1.93 d、6 d;辽蚕527蚁蚕平均饥饿死亡率为9.51%,比抗大低3.59百分点,差异达显著水平;辽蚕527蚁蚕饥饿死亡率最高峰出现的时间最晚(第10天),分别比吉青、抗大和9906晚1 d、2 d和3 d,并且最高峰的死亡率也最低;辽蚕527蚁蚕的LT_(50)和LT_(95)分别为9.22 d和15.43 d,均极显著长于其他3个品种;同时,相关分析表明柞蚕蚁蚕的耐饥饿能力与卵重无关。综上表明,辽蚕527蚁蚕具有较强的耐饥饿能力,可通过低温饥饿手段筛选耐饥饿能力强的蚁蚕个体,为抗逆品种的选育奠定基础。

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。

柞蚕微孢子虫(Nosema pernyi)对柞蚕卵巢初代培养细胞的侵染与增殖

将精制的柞蚕微孢子虫孢子用0.2mol.L-1KOH处理后,接种感染生长状态良好的柞蚕蛹卵巢初代培养细胞,调查其感染增殖规律。接种1h后,孢子的发芽率为53.8%,培养细胞的初期感染率为30%。接种24h后,可观察到裂殖体的数量急速增加,72h达最高峰。接种96h后可观察到短极丝孢子发芽后形成的空孢壳和二次感染体。接种5d后形成成熟孢子,每个细胞平均产100个孢子。在其生活史中,柞蚕微孢子虫呈双核或四偶核,并表现出孢子二型性,具有典型的Nosema属特征。

柞蚕蛹虫草不同培养时间中的活性物质代谢组分析

为解析柞蚕蛹虫草不同培养时间中活性代谢物的种类及积累规律,运用广泛靶向代谢组学技术对培养0、10、20、30、40 d的柞蚕蛹虫草中的代谢物进行鉴定。结果表明,柞蚕蛹虫草不同培养时间中的主要活性物质种类大体相同,在各试验培养期中分别鉴定出419、419、420、417和417个代谢物,主要包括氨基酸及其衍生物、核苷酸及其衍生物、酚酸、有机酸、糖及醇类、脂类、生物碱等多种化合物。其中,首次检测到3-吲哚丙酸、吡哆素。柞蚕蛹虫草培养过程中差异代谢物的相对含量差异显著,所有的差异代谢物聚为9个簇,大致分为5种类型,其中,有61个代谢物在试验期内呈现持续上调趋势,包括虫草素、2-脱氧腺苷等。对比主要活性物质中6种核苷类成分及鲜味氨基酸含量,培养40 d的柞蚕蛹虫草品质更佳。

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。

Purification and Characterization of a Cathepsin B-Like Proteinase from Eggs of Antheraea Pernyi

A cathepsin B-like proteinase was purified from eggs of Antheraea pernyi by 5 steps of purification. The molecular mass of the proteinase are estimated to be 47 kDa by using SDS-polyacrylamide gel electro-phoresis. The activity of the purified proteinase were strongly inhibited by E-64 and Leupeptin. The optimum pH is 3.5 as determined by using the bovine hemo-globin as substrate. The activity and quantity of the proteinase during embryo development were studied. The results suggested that the proteolytic activities during embryo development at least partially came from this proteinase.

应用生物素标记和蛋白质组学方法分离鉴定柞蚕微孢子虫(Nosema pernyi)的表面蛋白

建立灵敏、特异、稳定的柞蚕微孢子虫(Nosema pernyi)表面蛋白分离鉴定技术,对研究其侵染机制和早期检测技术非常重要。采用生物素Sulfo-NHS-SS-biotin能特异性标记柞蚕微孢子虫的表面蛋白;经生物素标记后的微孢子虫用0.01 mol/L NaOH和2%SDS的混合液裂解结合超声波破碎的方法能提取到更多的微孢子虫总蛋白质;用30%乙腈和70%甲酸配合沸水浴处理可有效洗脱微孢子虫表面蛋白。对得到的柞蚕微孢子虫表面蛋白检测样品进行LC-MS/MS质谱鉴定,共获得了8个假定的表面蛋白,其中分子质量为26.6 k D(Np SWP12)、25.7 k D(Np SWP26)、32.0 kD(Np SWP30)的3个假定蛋白经间接免疫荧光分析确证为定位在孢子表面的蛋白质。序列分析显示,NpSWP12、NpSWP26、NpSWP30与家蚕微孢子虫(Nosema bombycis)孢壁蛋白NbSWP12、NbSWP26、NbSWP30的氨基酸序列相似度分别为100%、93%、79%,其中Np SWP26无N-糖基化位点和O-糖基化位点,但C端具有介导孢子粘附宿主细胞的肝素结合基序。分离鉴定的3个表面蛋白可以作为柞蚕微孢子虫侵染机制研究和早期检测的靶标蛋白。

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. CD^4＋ T and CD^8＋ 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 CD^4＋ T cells was higher while CD^8＋ T cells was lower than in the control and simple radiation groups. Expression of IL-2 and INF-y 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.

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 pernyion 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. pernyiand food for 15 d. The typical FSR and HC50 value, monocybe-phagocytic exponent Kand emendated monocyte-phagocytic exponent were determined and calculated respectively.RESULTS： Alter 24 and 48 h, the FSR values of the three tested groups improved significantly in comparison to the conlyol group by variance analysis. The HCso 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 bested 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. pemyican significantly enhance cellular and humoral immune function in mice, but has no distinct influence on the monocyte-phagocytic system in mice.

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.

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.

TEUPERNIN D: A CHLORINE-CONTAINING NEO-CLERODANE DITERPENE FROM TEUCRIUM PERNYI

A new chlorine-containing neo-clerodane diterpene, teupernin D, was isolated from the aerial parts of Teucrium pernyi. The structure for teupernin D was established as (12S) -15, 16-epoxy-8β-hydroxy-17-chloro-19-nor10α-neo-cleroda-4, 13(16), 14-triene-18,6β;20,12-diolide on the basis of spectral evidence. The absolute configuration was established by CD spectrum and confirmed by X-ray crystallographic analysis.

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.

TEUPERNIN A,B AND C,THREE ENT-CLERODANE DITERPENOIDS FROM TEUCRIUM PERNYI FRANCH

Three furanoid ent-clerodane diterpenoids,teupernin A,B and C have been isolated from the acetone extract of Teucrium pernyi Franch.Their structures were established by spectroscopic means(UV,IR,MS,~1H NMR,^(13)C NMR,H-H COSY,C-H COSY and NOE)and by comparison with related compounds.

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。

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

A proteinase responsible for degradation of yolk proteins has been discovered in oocyte of Antheraea pernyi which was found to found to hydrolyze vitelluin effectively in acidic pH. Using bovine hemoglobin as substrate,the optimum pH was determine3d at 3.5.Unaffected by EDTA and DipF, the activity of the proteinase was strongly inhibited by E64 and other thiol blocking reagents. It is thus very likely to be a cysteine proteinase. Activity of the proteinas. was found to be increasing in developing oocyte.