Relationship between Biological Characteristics of Beauveria bassiana (Bals.) Vuill and Pathogenicity to Bombyx mori L.

[Objective] This study was to investigate the relationship between biological characteristics of Beauveria bassiana （Bals.） Vuill and pathogenicity to Bombyx rnori L, with the aim to provide scientific basis for the control of white muscardine in Bombyx mori L. [Method] The strains isolated and purified from the 6 Beauveria bassiana biocontrol agents from all over the country and the 3 white muscardine silkworms collected from Guangxi provincial silkworm rearing areas were identified by the morphological observation and molecular biology technology. The pathogenicity of B. bassaina to silkworms was determined, and the biological characteristics such as growth diameter, sporulation and the extracellular protease activity of the different B. bassiana strains were compared. [Result] The isolated 9 strains were all B. bassaina （Bals.） Vuillemin, and all strains had high pathogenicity to silkworm, but with different pathogenicities. The growth diameter, sporulation and extracellular protease activity of different B. bassiana strains were also different, and showed correlation with the patheogenicity to silkworms. [Conclusion] B. bassiana spores production amount and exocellular protease activity had significant positive correlation with their pathogenicity to silkworm.

家蚕Bombyx mori L.染色体的G带组型研究

以今井的涂片法制备染色体标本,采用GUG法进行G-带分染,研究了家蚕早双线期染色体的组型。依照染色体相对长度及带型鉴别了家蚕28对染色体,并绘制了染色体组型模式图。利用限性普斑系统W 染色体上的易位片断作标识,观察到不对称配对二价体,推论此异形二价体即性染色体。性染色体在14—16染色体范围内。

Targeting of human aFGF gene into silkworm,Bombyx mori L. through homologous recombination

The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL 1392 used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through homologous recombination.

Molecular Systematic Studies on Chinese Mandarina Silkworm (Bombyx mandarina M.) and Domestic Silkworm (Bombyx mori L.)

Molecular systematic studies on mandarina silkworm (Bombyx mandarina M.) in 11 regions in China and 25 representative strains of domestic silkworm (Bombyx mori L.) were conducted using molecular biology techniques. Results obtained from the analysis of DNA polymorphism and clustering of all the silkworm samples provide new evidence for the view that the domestic silkworm originated from the Chinese mandarina silkworm. On the basis of literature reviewing, a new hypothesis on the origin of the domestic silkworm was put forward. It was thought that the domestic silkworm was most probably domesticated from the Chinese mandarina silkworm of different ecotypes including monovoltinism, bivoltinism and multivoltinism; and that the domestic silkworm had the genetic background of monovoltinism, bivoltinism and multivoltinism at the very beginning of the domestication. The current strains of the domestic silkworm of different voltinism are the evolutionary results of thousands of years of rearing and artificial selections.

辛基酚对家蚕(Bombyx mori L.)生长发育的影响

为探讨辛基酚对鳞翅目模式昆虫家蚕生长发育的影响,使用添加0.01~0.08 g·kg-1辛基酚(4-tert-octylphenol,4-t-OP)的人工饲料饲养家蚕(Bombyx mori L.),研究了4-t-OP对家蚕幼虫和蛹的生长发育速度、生长量、发育整齐度和生命力等影响。结果表明:连续取食0.01 g·kg-1以上浓度4-t-OP的饲料,家蚕幼虫和蛹的生长发育速度(幼虫期和蛹期发育经过时间)、生长量(幼虫期体质量和蛹期茧层量)、生命力(3龄起蚕绝食生命时数、4龄起蚕结茧率、蚕茧的死笼率和羽化率)等都明显下降,抑制眠蚕体质量。4-t-OP对家蚕幼虫生长发育后期、化蛹及羽化等发育变态期的影响更加明显,其在家蚕体内可能有累积效应。添食0.08 g·kg-14-t-OP的家蚕不能完成生活史。研究表明,4-t-OP对家蚕生长发育毒性影响有显著的浓度效应和时间效应。

Analysis of bulked segregants to identify molecular markers linked with cocoon weight and cocoon shell weight in the silkworm Bombyx mori L

Two silkworm strains viz, B20 A (high cocoon shell ratio) and C.Nichi (low cocoon shell ratio) were sib mated for 10 generations to determine the homozygosis. Both bulked segregant analysis(BSA) and near isogenic lines (NIL) studies were done to identify the RFLP markers closely linked to cocoon shell parameters. Three hundred and fifty two random clones were identified as the low copy number sequence and used for identification of Restriction Fragment Length Polymorphic (RFLP) marker linked to cocoon weight and cocoon shell character. In the bulk segregant analysis, DNA from the parents (B20 A, C.Nichi), F 1 and F 2 progeny of high shell ratio (HSR) and low shell ratio (LSR) were screened for hybridization with the random clones. Polymorphic banding pattern achieved through southern hybridization with different probes indicated the probable correlation of polymorphism with high and low cocoon shell character which are possible landmarks in identifying the putative marker(s) for the cocoon shell character. Out of the 100 probes tried with parents, F 1, F 2 and their bulks, 10 probes were found to be closely linked to cocoon shell characters.

Effect of vertebrate hormones and prostaglandins on growth, silk quality and metabolic activities of Bombyx mori L

The economic importance of silkworm has moved biologists to explore various intricate mechanisms of the action of vertebrate hormones. The dietary administration of several vertebrate hormones and prostaglandins enhanced both developmental and metabolic processes of silkworm, Bombyx mori L. The main objective of sericulture research is to apply the results to achieve superior quality silk and greater output, to apply lab findings to achieve desirable ecenomic results.

Biotransformation Effect of Bombyx Mori L. May Play An Important Role in Treating Diabetic Nephropathy

Compared with herbal drugs, medicine processed from animals(animal medicine) was thought to have more bioactive substances and higher activities. Biotransformation effect often plays an important role in their effect. However, researches about effect of animal medicine on diabetic nephropathy and applying animal medicine as natural bio-transformer were seldom reported. The purpose of this paper was to reveal the use of Bombyx Mori L. on diabetic nephropathy from ancient to modern times. The classical literature indicated that Saosi Decoction(缫丝汤), which contains Bombyx Mori L. or silkworm cocoon, was applied to treat disorders congruent with modern disease diabetic nephropathy from the Ming to Qing Dynasty in ancient China. Modern studies showed that Bombyx Mori L. contains four main active constituents. Among these, 1-deoxynojirimycin(1-DNJ) and quercetin showed promising potential to be new agents in diabetic nephropathy treatment. The concentrations of 1-DNJ and the activities of quercetin in Bombyx Mori L. are higher than in mulberry leaves, because of the biotransformation in the Bombyx Mori L. body. However, these specific components need further human and mechanistic studies to determine their therapeutic potential for this challenging condition.

Analysis of ESTs and gene expression patterns of the posterior silkgland in the fifth instar larvae of silkworm, Bombyx mori L.

The fibroin gene expression pattern and regulation of the posterior silkgland were studied by means of expressed sequence tags (ESTs) using the first and fifth day larvae of the fifth instar of silkworm, Bombyx mori L (strain: C 108). The results showed that there were 911 repetitive ESTs and 1950 single sequences (Singlets) among total 2861 consentient sequences, which were spliced. 1335 sequences were identified and the other 1526 were unknown. 5560 sequences (55.89%) in the posterior silkgland cell of the silkworm were new ESTs without homology with EST data published by Mita et al. The number of repetitive ESTs and single sequences from the first day larvae of the fifth instar was double more than that of the fifth day of the same instar in the silkworms. The unigenes which were more than 50 in repetitive EST size (contig size) came to only about 0.5% in total consentient sequences. There were significant differences between gene expression frequencies, and expressed genes were related to fibroin synthesis and its secretion and fibroin composition. Comparing the fifth day with the first day of the fifth instar, the genes-expressed quantity of fibroin heavy-chain gene was 18 fold higher, fibroin light-chain gene 9 fold and fibroin P52 gene 8 fold. 508 genes functioned for cellular component and 315 for enzyme after function tracing. These results implied that the gene expression of the first day was mainly for preparation for fibroin synthesis except for the growth of silkgland cells, and the gene expression of the fifth day of the fifth instar was mainly for synthesizing and excreting fibroin. Because the ratio of heavy chain, light chain and p25 of fibroin was not 6:6:1 as theoretically expected, or its special H-chain structure, the H-chain gene was not easy to detect through EST technique. Most of genes among total 2861 consentient sequences functioned for fibroin synthesis and secretion. This suggested the fibroin synthesis and secretion procedure of the posterior silkgland was more complex than the knowledge we have.

饲料防腐剂对家蚕生长发育的影响及防腐效果

筛选适宜江西省小蚕人工饲料育的饲料防腐剂,为家蚕(Bombyx mori L.)人工饲料育技术推广应用提供理论支撑。选择纳他霉素、富马酸二甲酯、山梨酸钾、丙酸钙、多菌灵5种防腐剂添加到家蚕配合饲料中,以常规饲料为对照,开展不同饲料防腐剂对小蚕生长发育的影响及防腐效果研究。结果表明,人工饲料不同防腐剂对家蚕生长发育有一定影响,24 h疏毛率以添加山梨酸钾的处理最高,达94.54%,其他处理之间差异不显著;3龄眠蚕体重以添加丙酸钙和山梨酸钾的处理较高;蚁蚕结茧率以添加山梨酸钾的处理最高;死笼率以添加山梨酸钾的处理最低;各处理1龄眠蚕体重、2龄眠蚕体重和茧质之间差异不显著;人工饲料不同防腐剂防霉效果不同,家蚕饲养过程中以添加富马酸二甲酯、山梨酸钾、丙酸钙和对照的饲料霉变程度较轻,自然条件下以添加0.25%山梨酸钾饲料霉变程度最轻且保质期最长。人工饲料中添加0.25%山梨酸钾其防腐效果好、饲料保质期长,且有利于家蚕的生长发育。

家蚕与桑树相互作用的研究现状及进展

栽桑养蚕是世界农耕文明的一大壮举。专食桑叶的家蚕由其祖先野桑蚕经人工驯化而来,是第一个完成全基因组测序的鳞翅目昆虫。本文综述家蚕与桑树协同进化方面的研究证据,包括家蚕蛋白酶与桑树蛋白酶抑制子的互作、家蚕对桑树次生代谢物质的适应以及桑树尿素酶对家蚕氮代谢的意义等,为今后深入解析家蚕与桑树的互作提供新见解,并丰富植食性昆虫与宿主植物互作的研究信息。

家蚕丝素蛋白轻链基因(fib-L)启动子序列的克隆及其活性分析

家蚕Bombyx mori丝素蛋白轻链(fibroin light-chain,fib-L)基因fib-L具有在后部丝腺组织专一性、高效性表达的特点。为了利用其启动子构建能够表达外源基因的丝腺生物工厂,本实验对fib-L启动子活性进行了研究。通过PCR法克隆了fib-L启动子元件,序列分析显示fib-L启动子由位于-33^-25处的TATA盒元件和位于-128^-121处的特征性序列GTCAATTT共同组成。用fib-L启动子控制报告基因DsRed进行家蚕BmN细胞和蚕体内的瞬时表达研究,结果表明fib-L启动子可以驱动DsRed报告基因在BmN细胞和家蚕后部丝腺组织中瞬时表达。

家蚕茧丝相关性状的性连锁QTLs定位与分析

家蚕的茧丝性状存在明显的性别差异。以茧丝性状成绩差异较大的家蚕品种兰10和菁松为亲本组配回交1代分离群体(BC1M),在前期研究的基础上,利用Mapmaker软件构建含有17个SSR或SNP分子标记的家蚕性染色体的遗传连锁图,并对茧丝量、全茧量、茧层量、蛹体质量、茧丝长等性状进行数量性状位点(QTL)扫描,获得与上述5项茧丝相关的QTL位点,其LOD值分别为11.175、6.196、11.036、5.156和8.717。研究结果为家蚕茧丝相关QTL位点的精细定位与克隆奠定了基础。

具有丙酮酸脱氢酶功能的家蚕Bm-l(1)基因的克隆及序列结构与表达研究

丙酮酸脱氢酶(PDH)是丙酮酸脱氢酶复合体(PDC)中的前件酶,参与生成柠檬酸循环(TCA)的起始物乙酰辅酶A,决定生物体内营养成分的分配。利用电子克隆、RT-PCR和cDNA末端快速扩增(RACE)等方法克隆了与果蝇lethal(1)G0334基因类似的具有丙酮酸脱氢酶功能的家蚕Bm-l(1)基因。Bm-l(1)基因的cDNA全长为1 630 bp,由1 200 bp的完整ORF序列、186 bp的5'-UTR和207 bp的3'-UTR组成,含8个外显子和7个内含子,编码蛋白为399个氨基酸残基,分子质量43.93kD,pI 8.07。Bm-l(1)基因编码蛋白的69-365氨基酸残基为E1-dh结构域,该结构域为硫胺素焦磷酸依赖性脱氢酶所特有。蛋白质二级结构预测结果表明α螺旋占28.8%,β折叠占12.0%。采用Clustal W进行多序列比对发现,Bm-l(1)基因编码蛋白与赤拟谷盗等昆虫的PDH具有63%以上的序列相似性,且保守区域高度一致。Bm-l(1)基因mRNA在家蚕整个卵期、幼虫期、蛹期和刚羽化的成虫中,以及5龄3 d幼虫的头部、丝腺、生殖腺、脂肪体、中肠和血液6种组织中都有较高的转录水平,且存在较小的组织差异性。

家蚕AFLP分子进化研究

为进一步探讨家蚕的起源分化和其系统发育,为家蚕遗传育种提供科学依据,利用AFLP技术和统计学分析对具有代表性的9个不同的地区家蚕品种进行了分子系统学研究。家蚕的DNA多态性分析及其聚类分析结果进一步证实家蚕可能起源于不同地方(多起源中心)、由多种生态类型(包括一化、二化、多化)混杂的野桑蚕驯化而来,其驯化之初就已拥有一化、二化、多化的遗传背景,而且一化和二化品种间的遗传距离小,分化程度小,表明它们应该有共同的起源中心。

利用Balb/c小鼠构建家蚕蛹食物过敏模型的研究

利用Balb/c小鼠构建家蚕蛹过敏模型并研究其致敏作用。48只雌性Balb/c小鼠分别用灌胃和皮下注射家蚕蛹蛋白的方法进行致敏。小鼠致敏后通过尾静脉注射伊文斯蓝考察其对血管通透性的影响;光镜和扫描电镜下观察致敏小鼠空肠组织的病理切片;测定血清中IgE、组胺、IFN-γ、IL-4和IL-6等的含量;免疫印记反应测定小鼠血清特异性抗体与家蚕蛹变应原的结合度。结果表明:小鼠发生过敏反应时,血清中产生与家蚕蛹蛋白特异性结合的IgE抗体,血清中组胺、IL-4和IL-6等的含量显著上升,而IFN-γ含量显著下降,同时各致敏试验组小鼠血管通透性增加,空肠组织出现糜烂和结构受损症状。该小鼠致敏模型的成功构建对于后续研究家蚕蛹变应原及其致敏的抗原表位等均具有重要基础作用。

家蚕组织蛋白酶L(BmCathepsin L)基因鉴定、表达及其功能分析

【目的】鉴定克隆家蚕组织蛋白酶L基因BmCathepsin L(BmCat L),分析其序列和表达特征。分析家蚕组织蛋白酶L在大肠杆菌诱导下m RNA表达量的变化趋势,为进一步研究家蚕组织蛋白酶L的功能打下基础。【方法】基于家蚕基因组数据库(Silk DB)中序列BGIBMGA006893设计引物,利用RACE技术克隆家蚕组织蛋白酶L基因的cDNA全长序列,通过在线工具对基因结构和蛋白质分子量及等电点进行预测;在NCBI数据库下载其他物种组织蛋白酶L同源序列,利用Clustalx(1.83)和MEGA 6.0软件进行同源比对并构建进化树。运用RT-PCR和qRT-PCR对其时空表达特征进行分析。选取该基因的特异性片段,经PCR扩增,测序验证后将其连接至pET-28载体,转化至Rosseta感受态表达菌株,经IPTG在适宜温度条件下诱导,获得组织蛋白酶L重组蛋白。然后再利用镍离子亲和层析法对该蛋白进行纯化,获得纯度较高的重组蛋白。最后利用大肠杆菌对家蚕血液系统进行攻毒,检测家蚕组织蛋白酶L基因的表达水平。【结果】通过查询家蚕基因组数据库和生物信息学分析得出,家蚕组织蛋白酶L基因定位于家蚕第10号染色体上,基因编号为BGIBMGA006893,nscaf2860。基因开放阅读框全长687 bp,编码228个氨基酸,含有保守的Pept_C1结构域。通过在线网站预测,得出该蛋白酶的分子量为26.132 k D,等电点为4.57。进化分析表明该基因与同为鳞翅目昆虫的草地贪夜蛾和黑脉金斑蝶亲缘关系最为接近。RT-PCR显示该基因集中表达于血细胞,在血液不同龄期也较高表达。用His抗体检测经纯化后的蛋白,最终成功孵育出纯化后的蛋白条带;大肠杆菌个体攻毒免疫试验检测显示,其mRNA表达水平随攻毒时间呈现抛物线式的显著性变化规律,从而揭示出BmCat L与家蚕免疫系统的关联性。【结论】克隆获得家蚕组织蛋白酶L基因的cDNA序列,发现其特异性高表达于血细胞。通过原核表达和蛋白纯化成功获得了该基因的重组蛋白。大肠杆菌的刺激能够显著上调其表达,推测组织蛋白酶L可能参与家蚕免疫反应。

Analysis of SSR Fingerprints in Introduced Silkworm Germplasm Resources

Thirty-five SSR markers were used to construct 96 silkworm races fingerprint. All the SSR markers were polymorphic and unambiguously separated silkworm strains from each other. A total of 467 alleles were detected with a mean value of 13.34 alleles/locus （range 3-28）. The mean polymorphism index content （PIC） was 0.71 （range 0.299-0.919）. UPGMA cluster analysis of Nei＇s genetic distance grouped silkworm strains on the basis of their origin. The results indicated that SSR markers are efficient tools for fingerprinting cultivars and conducting genetic diversity studies in the silkworm.

Bmo-miR-375-3p对家蚕丝素蛋白轻链基因BmFib-L表达的调控作用

【目的】探究家蚕Bombyx mori miRNAs对丝素蛋白轻链基因(fibroin light chain gene,Bm FibL)和P25蛋白基因Bm P25表达的调控作用。【方法】分别以Bm Fib-L和Bm P25 mRNA 3'UTR为靶序列,应用在线预测软件RNAhybrid从前期家蚕4和5龄幼虫后部丝腺(posterior silk gland)sRNA高通量测序获得的29个差异表达bmo-miRNAs中,筛选对Bm Fib-L和Bm P25具有调控作用的候选bmo-miRNAs;采用半定量RT-PCR方法在mRNA水平分析候选bmo-miRNAs及其靶基因Bm Fib-L和Bm P25在4和5龄幼虫期以及5龄第3天幼虫不同组织的表达水平;利用双荧光报告基因检测系统在家蚕细胞Bm N中验证候选bmo-miRNAs对Bm Fib-L和Bm P25表达的调控作用。【结果】筛选到一个在Bm Fib-L和Bm P25 mRNA 3'UTR上都有靶位点的bmo-miR-375-3p(曾称bmo-miR-375*)。在后部丝腺中bmo-miR-375-3p和其预测靶基因Bm Fib-L和Bm P25都有较高的表达水平,提示bmo-miR-375-3p具备调控Bm Fib-L和Bm P25表达的时空条件。miR-375-3p重组表达载体与融合了Bm Fib-L 3'UTR的萤火虫荧光素酶(luciferase)报告基因(luc)表达载体共转染Bm N细胞,报告基因luc的表达水平显著下降;而在miR-375-3p重组表达载体与融合了Bm P25 3'UTR的luc表达载体共转染Bm N细胞中报告基因luc的表达水平下降不显著。【结论】miR-375-3p显著下调Bm Fib-L基因的表达,而对Bm P25基因的表达无明显调控作用。

利用CLSM检测GFP基因在家蚕的瞬时表达

将绿色荧光蛋白（GFP）基因导入家蚕蛹（G0）的睾丸，利用激光共聚焦扫描显微镜（CLSM）系统检测，得到GFP基因在家蚕刚孵出未进食的幼虫(蚁蚕)（G1）瞬时表达的荧光图像。