Expression of Recombinant Gene P1-2A3C of FMDV Asia I in Different Bombyx mori Varieties

[Objective] The aim was to investigate the expression level of recombinant gene P1-2A3C of FMDV Asia I in different Bombyx mori varieties,so as to screen out the B.mori varieties suitable for the foreign gene expression.[Method] The recombinant B.mori baculovirus rBmNPV（P1-2A3C） are injected into pupae of original species and hybrids of B.mori respectively.Then,the expression of antigen is detected by ELISA method and compared.[Result] The expression level of P1-2A3C significantly differed in different B.mori varieties,in which,the hybrids of Qiufeng×TQ78 and Qiufeng×Sijiaojian could be considered as the effective bioreactors for high-level expression of foreign genes.[Conclusion] This study provided the basis for breeding special B.mori varieties which can highly express target protein of Asia I FMDV.

Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm (Bombyx mori) on Day 1 and Day 4 in the 5th Instar Stage

The posterior silk gland （PSG） of silkworm is an important organ where fibroin is synthesized and secreted exclusively. Because fibroin constitutes 75-80% of the silk filament, the mechanism governing fibroin secretion, quality and yield of cocoon can be elucidated by the study on the PSG. Using two-dimensional gel electrophoresis （2-DE） and image analysis system, the changes in the protein composition in the PSG cell were investigated on the day 1 （D1） and day 4 （D4） in the 5th instar stage from five different strains of silkworm （Bombyx mori）. While differences at protein level between days and strains were far less than those observed at the gene level using EST analysis. The change trends in protein composition from D1 to D4 were diverse among the different strains. The results suggest that the secretion of fibroin is regulated by multiple proteins. The site of regulation and the proteins responsible for the regulation vary with the strain, which leads to differences between strains in the capacity of fibroin secretion in the PSG cell.

Prevalence of Muscardine Disease in Different Silkworm Hybrid, Bombyx Mori L. under Agro-climatic Conditions in Aurangabad （M.S.）, India

A study was carried out in Dr. Babasaheb Ambedkar Marathwada University Campus Aurangabad, Maharashtra State, India for analyzing the prevalence of Muscardine disease during rainy, winter and summer season in bivoltine x bivoltine hybrids viz. CSR2 × CSR4, CSR4 × CSR2 and multi × bivoltine hybrid PM × CSR2. Observation on Muscardine disease was recorded till the onset of spinning. Analysis of results shows that disease prevalence was more in bivoltine x bivoltine hybrids compare to multi × bivoltine hybrids. PM × CSR2 was found to be more resistant towards Muscardine disease compared to other hybrids under agro-climatic conditions of Aurangabad.

桑蚕(Bombyx mori L.)1～2龄人工饲料1回育3～5龄桑叶育及其成本分析

通过春、夏２期桑蚕１～２龄人工饲料１回育３～５龄桑叶育的农村试养，结果表明，试验区的茧丝质成绩春季基本接近全龄桑叶育，夏季优于全龄桑叶育；通过小蚕人工饲料育的成本分析，采用１～２龄人工饲料１回育每盒蚕种可节省７．５３元。

Bombyx mori Pyridoxal Kinase cDNA Cloning and Enzymatic Characterization

Pyridoxal kinase （PLK） （EC 2.7.1.35） catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal-5＇-phosphate （PLP）, an important cofactor for many enzymatic reactions. Bombyx mori, similar to mammals, relies on a nutritional source of vitamin B6 to synthesize PLP. This article describes how a cDNA encoding PLK was cloned from Bombyx mori using the PCR method （GenBank accession number： DQ452397）. The cDNA has an 894 bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33.1 k.Da. The amino acid sequence shares 48.6% identity with that of human PLK, and it also contains signature conserved motifs of the PLK family. However, the protein is 10 or more amino acids shorter than the PLK from mammals and plants, and several amino acid residues conserved in the PLK from mammals and plants are changed in the protein. The cDNA cloned was expressed successfully in Escherichia coli using the T7 promoter/T7 RNA polymerase expression system, and the crude extracts containing the expressed product were found to have strong PLK enzymatic activity with a value of 30 nmol/min/mg, confirming that the cDNA encodes the functional PLK of Bombyx mori. This is the first identification of a gene encoding PLK in insects.

Breeding of Silkworm Variety "Chuancan No.24" with Strong Vitality for Summer-Autumn Rearing

[ Objective] The aim was to introduce the breeding process of natural colorful cocoon silkworm variety, Chuancan No. 24. [ Method] By cross breeding and pedigree separation, the silkworm variety, Chuancan No. 24, with double sex-limited markings and strong vitality and for sum- mer-Autumn rearing was bred. [ Result] The results of laboratory identification and countryside test in Sichuan Province indicated that the Chuancan No. 24 was strong and could be reared easily. They had uniform development and excellent quality of cocoon and silk. In addition, their sexes could be identified by larval body color and markings. The main indexes are as follows： cocoon yield per 10 003 fourth instar larvae, 19.66 kg; filament length, 1 241.29 m; length of non-broken filament, 941.99 mm; reelability, 75.87% ; raw silk ratio of fresh cocoon, 17.07% ; and neatness, 94.03 points. The main economic features of new variety were better than those of the control. [ Conclusion] The Chuancan No. 24 has been approved by the Sichuan Silkworm Evaluation Commission, and it is suitable to rear this new variety in some regions of the Yangtze River drainage basin, such as Sichuan Province and Chongqing.

Detection of Contamination and Analysis of Vertical Transmission of BmNPV in Eggs and Moths of Bombyx mori

This study reports the molecular detection of Bombyx mori nucleopolyhedrovirus (BmNPV) in silkworm strains of the Universidade Estadual de Maringá Brazilian Germplasm Bank (UBGB). DNA extraction was carried out by using six Bombyx mori female moths of each strain, followed by PCR amplification. A pair of primers was designed based on a specific sequence of the baculovirus genome related to the BmNPV ORF 14. Another pair of primers was used to amplify the silkworm Actin A3 gene segment, which was used as positive control. Twenty gene pools were analyzed, and fifteen revealed a fragment of 443 base pairs (bp), which indicated the presence of the BmNPV. The frequency of contaminated moths was as following: 100% for silkworm strains M18-2, M12-2 and J1;83% for C25, C75 and C24 strains;66% for KR01;50% for M11-A;33% for AS3, B106, M8 and M11 and 16% for C211, E8 and Hindu strains. These are promising results for the identification of contaminated B. mori moths by BmNPV, which may prevent virus proliferation in subsequent generations. We also analyzed DNA samples extracted from B. mori eggs, but the results were not conclusive regarding the detection of the fragments of the expected size (443 bp). The difficulty in detecting BmNPV contamination in B. mori eggs may be due to the low concentration of virus in samples.

家蚕30K蛋白BmLP1的表达模式及其在胚胎中的作用

家蚕30K蛋白是家蚕血液和卵中的高丰度蛋白,参与营养、免疫、抑制细胞凋亡等功能.探究30K蛋白在家蚕体内的表达模式有助于更清楚地了解30K蛋白的功能.利用生物信息学方法发现4种30K蛋白基因Bmlp1,Bmlp2,Bmlp3和Bmlp7都含有信号肽.其中,Bmlp1与其他30K蛋白的序列相似性小于50%.半定量RT-PCR结果表明,Bmlp1从5龄第3 d到化蛹第1 d在脂肪体中表达量都比较高,之后表达量降低.Western blotting结果表明,BmLP1从5龄第5 d到化蛹第4 d在血淋巴中的含量都比较高,之后含量降低.与血淋巴中的变化趋势不同,卵巢中的BmLP1在上蔟第2 d被检测到,之后含量一直增加,化蛾时含量达到最高.这是由于上蔟第2 d卵巢管迅速长大,血液中的BmLP1等营养蛋白逐渐转运至卵巢管的未受精卵并开始积累.免疫组织化学定位结果表明,在胚胎发育前期,BmLP1分布于蚕卵的卵黄颗粒中,在孵化前期,BmLP1被吞入胚胎的肠道内.体外孵育结果表明,蚁蚕粗提物可以降解BmLP1,暗示蚁蚕肠道内可能存在某种蛋白酶可以将大部分BmLP1水解,最终为蚁蚕的孵化提供能量或者氨基酸.系统分析了BmLP1在不同发育时期脂肪体、血液、卵巢和胚胎中的表达特征,揭示了其在胚胎发育过程中的重要作用.

家蚕体内细菌感染模型建立及不同菌株的协同致病特征分析

【目的】建立家蚕体内细菌感染模型,揭示不同细菌协同感染家蚕的致病力,明确云南蚕区家蚕细菌病的发病原因及其规律,为指导家蚕生产过程中细菌病的防治提供科学依据。【方法】从家蚕龄期、感染浓度和感染方法3个因素出发构建家蚕体内细菌感染模型,然后选用从云南地区自然发病蚕体中分离获得的5株肠道致病菌[Mammaliicoccus sciuri(葡萄球菌属)、粘质沙雷氏菌(Serratia marcescens)、弗氏柠檬酸杆菌(Citrobacter freundii)、Klebsiella variicola(克雷伯菌属)和Pseudomonas soli(假单胞菌属)],开展5种单菌感染及17种组合协同感染试验。【结果】通过建立家蚕体内细菌感染模型发现,家蚕群体添食细菌比单头添食的存活率低;细菌感染浓度选择时建议二龄期家蚕感染选用5.0 OD/mL、四龄期和五龄期家蚕感染选用10.0 OD/mL。选取的5株细菌对家蚕均具有致病力,整体上表现为随着细菌感染时间的延长,家蚕存活率逐渐降低;小蚕期感染细菌对家蚕的影响更明显,至五龄期有大量家蚕发病死亡,与养蚕过程中的细菌病发生规律相同。无论是小龄期感染还是大龄期感染,细菌协同感染的家蚕存活率总体上低于单菌感染,尤其是粘质沙雷氏菌参与的协同感染,至上蔟期的家蚕存活率均较低。此外,无论是二龄期感染还是四龄期感染,夏季家蚕的存活率总体上均高于春季家蚕,说明云南蚕区夏季家蚕的抗病力优于春季家蚕。【结论】5株分离自云南地区自然发病蚕体的病原菌对家蚕产生明显致病性,其致病性与感染龄期、感染浓度、不同细菌组合及感染季节等均有关,总体上具体表现为小蚕期感染较大蚕期感染对家蚕的影响更明显,细菌协同感染比单菌感染的危害更严重,且夏季家蚕感染后的存活率高于春季家蚕。

桑蚕的强制牵伸抽丝及其纤维性能

为提升蚕丝的力学性能,采用一种圆锥斜面牵伸装置对五龄桑蚕进行强制牵伸抽丝以制备高强牵伸丝,研究不同牵伸速度对桑蚕牵伸丝形貌、力学性能的影响,并将牵伸丝与蚕丝进行对比与分析。结果表明:由强制抽丝获得的牵伸丝表面光滑平坦,丝胶均匀附着;随着牵伸速度的增加,牵伸丝截面逐渐向弓形转变;牵伸丝的综合力学性能显著优于蚕丝,且其断裂强度、弹性模量、断裂比功与断裂伸长率随着牵伸速度的增加均有不同程度的增加;其中,以接近桑蚕自然吐丝速度(1 cm/s)牵伸制备的牵伸丝的断裂强度与蚕丝相当,约为3.00 cN/dtex,但其弹性模量(93.10 cN/dtex)远高于蚕丝(76.90 cN/dtex);当牵伸速度增加至4 cm/s时,可获得力学性能最佳的牵伸丝,其断裂伸长率为22.49%,断裂强度为3.39 cN/dtex,弹性模量为95.76 cN/dtex,断裂比功为0.62 cN/dtex,相比于蚕丝的平均值分别提高了7%、13.76%、24.53%、31.91%。

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。

1-Deoxynojirimycin Content and Alfa-Glucosidase Inhibitory Activity and Heat Stability of 1-Deoxynojirimycin in Silkworm Powder

Silkworm powder containing 1-deoxynojirimycin (DNJ) has α-glucosidase inhibitory activity and is promising as a complementary and alternative medicine (CAM) agent in Japan. Silkworm powder produced in Korea was extracted with 75% ethanol. The extract was derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl), and DNJ-FMOC content was measured by HPLC. Then, alfa-glucosidase inhibition by silkworm and mulberry powder in pig liver crude enzyme was assayed using 4-nitrophenyl-alfa-D-glucopyranoside as a substrate. Silkworm powder DNJ content (0.39 to 0.58%) was higher than that in mulberry powder (0.08 to 0.12%). The alfa-glucosidase inhibitory activity of silkworm powder was more potent than that of mulberry leaves and green tea. Silkworm powder DNJ was stable upon heating to 121?C for up to 15 min.

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.

不同家蚕品种五龄幼虫期与蛹期中肠细菌组成与功能差异分析

【目的】分析生命力及茧层率不同的家蚕Bombyx mori品种间中肠细菌组成的差异及其对生命力与蚕茧量等性状的影响。【方法】基于长期的家蚕资源饲养调查成绩,选取了高生命力家蚕品种932G和高丝量品种2041J作为实验材料,收集5龄幼虫和蛹的中肠,采用高通量测序平台对中肠细菌16S rDNA进行测序分析,比较不同品种及不同发育阶段家蚕中肠细菌组成及其功能的差异。【结果】分别获得932G和2041J 5龄幼虫期中肠细菌399和453个可操作分类单元(operational taxonomic unit,OTU),蛹期OTU数目分别为138和162个。5龄幼虫期中肠优势菌门是变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)及放线菌门(Actinobacteriota),优势属是甲基杆菌属Methylobacterium,丰度最高,其次为葡萄球菌属Staphylococcus。有多个菌属的相对丰度在932G和2041J间差异显著,其中德沃斯菌属Devosia、罗尔斯通菌属Ralstonia、硝化螺旋菌属Nitrospira、短杆菌属Brachybacterium、罗氏菌属Rothia、葡萄球菌属及劳氏菌属Lawsonella等在932G 5龄幼虫中肠中的丰度显著高于在2041J 5龄幼虫中肠中的,而假单胞菌属Pseudomonas、甲基杆菌属、不动杆菌属Acinetobscter、管道杆菌属Cloacibacterium、明串珠菌属Leuconostoc、丙酸杆菌属Propionibacteriaceae、嗜冷杆菌属Psychrobacter、鞘氨醇菌属Sphingobium和拟杆菌属Bacteroides等在2041J 5龄幼虫中肠中的相对丰度显著高于932G 5龄幼虫中肠中的。932G和2041J 5龄幼虫中肠细菌分别有77%和78%的功能基因富集在KEGG代谢通路上,其次是富集在环境信息处理和遗传信息处理通路的功能基因;932G的5龄幼虫中肠中硝酸盐还原、氮气呼吸、硝酸盐呼吸、亚硝酸盐呼吸及固氮等功能菌群占比高于2041J 5龄幼虫中肠中的,2041J 5龄幼虫中肠中化学异养、尿素分解、甲醇氧化等功能菌群占比高于932G 5龄幼虫中肠中的。5龄幼虫中肠细菌组成与蛹的显著不同,蛹中肠变形菌门欧文氏菌属Erwinia为优势属,且蛹期两个品种间欧文氏菌属相对丰度无显著差异;932G蛹中肠细菌氨基酸转运与代谢、胞外分泌与转运、碳水化合物转运与代谢和无机盐转运与代谢等功能基因相对丰度显著高于5龄幼虫中的。【结论】家蚕5龄幼虫期中肠细菌组成和预测功能在家蚕高生命力品种932G与高丝量品种2041J间差异显著,蛹期中肠细菌组成与功能与5龄期显著不同,且蛹期中肠细菌的相对丰度在两个品种间无显著差异。这些研究结果可以为进一步探讨肠道微生物在家蚕抗逆、耐药、抗病及其蛋白质合成与转化等方面的作用及其家蚕品种选育提供参考。

抗血液型脓病家蚕新品种‘明·湖×春·江’多地农村试验初报

‘明·湖×春·江’是浙江省农业科学院蚕桑与茶叶研究所育成的一对抗血液型脓病家蚕新品种,2022年分别通过浙江省主要农作物品种审定委员会、国家畜禽遗传资源委员会审定。2023年春期,四川、江西、陕西、山东等地进行了‘明·湖×春·江’的引进试验,结果显示,新品种适应性强,在各地均表现孵化眠起齐一,食桑旺盛,强健好养,蚕体和茧形大,产茧量明显高于各地对照品种,健蛹率高,解舒优,茧丝长和解舒丝长长,洁净与万米吊糙优,适于各试验省区推广饲养。

家蚕抗血液型脓病斑纹全限性品种锦绣3号的育成

家蚕品种洞·庭×碧·波曾是长江流域及其他区域的主推品种之一,但存在茧丝质等性状退化和自身抗频发性血液型脓病能力偏弱的现象。以洞、庭、碧、波4个原种及其改良品系作为受体,以家蚕血液型脓病抗性材料HKC作为供体,采用杂交和连续回交方法将抗性基因导入受体品种,同时通过插入杂交方式导入优良血缘以改良丝质。采用系统分离、添毒筛选、活蛹缫丝等技术,育成了对家蚕血液型脓病具有高度抵抗性的家蚕新品系,并组配成优质、高产新组合7521改K·秋丰BK×7522NK·854BNK(华·康×湘·泰)。国家品种试验结果表明,新品种万蚕产茧量18.24 kg、虫蛹率97.69%、茧丝长1186.2 m、解舒率80.41%、洁净95.64分、鲜毛茧出丝率17.37%,各项指标均优于对照品种秋丰×白玉。BmNPV对新品种的LD50≥2.92×10^(9)mL^(-1),新品种的抗病力显著增强。该品种于2022年12月通过国家畜禽遗传资源委员会审定,适合在长江和黄河流域夏秋季饲养,审定名为锦绣3号。

家蚕1-2龄不同人工饲料育对生长发育和饲料效率的影响

本试验以优食一号蚕品种为材料,以全龄桑叶育为对照,探讨了1—2龄颗粒饲料育及1—2龄蒸煮饲料育、3—5龄桑叶育,对全龄生长发育、茧质及5龄期消化吸收和饲料效率的影响。结果表明,该品种对颗粒饲料和粉体蒸煮饲料的摄食性优良,但颗粒饲料育家蚕的发育经过比桑叶育和蒸煮饲料育延长,两种饲料育1—2龄的蚕体重均低于桑叶育,但3龄改为桑叶育之后,饲料育家蚕的生长发育加快,体重增长倍数明显高于全龄桑叶育,熟蚕体重基本达到对照区水平。小蚕饲料育的小区产茧量和全茧量与全龄桑叶育无明显差异,但茧层量略低于全龄桑叶育,茧层率则以1—2龄蒸煮饲料育最高。1—2龄饲料育试验区的5龄食下量、消化量、消化率均低于全龄桑叶育对照区,但5龄体重转化效率、蚕茧生产效率、蚕茧转化效率、茧层生产效率及茧层转化效率等各项饲料效率指标,均是小蚕饲料育试验区高于对照区,其中以1—2龄颗粒饲料育最高。本试验结果说明,小蚕饲料育提高了家蚕的代偿生长能力和饲料效率,主要是由于5龄期食下量减少,而体重转化效率显著提高所致。

不同品种桑蚕牵伸丝的结构与性能

利用自主研制的圆锥斜面强制牵伸抽丝装置对不同品种五龄桑蚕进行强制抽丝,通过拉伸测试、傅里叶红外光谱及X射线衍射对不同品种牵伸丝与相应原茧丝的结构与性能进行表征与分析。结果表明:两种牵伸丝的力学性能均优于其对应茧丝,黄色雄蚕牵伸丝的力学性能更佳,其初始模量、断裂强度、断裂比功与伸长率的平均值分别为117.35、4.55、0.75 cN dtex与26.54%。牵伸作用会导致丝纤维晶粒尺寸减小、结晶度提高,从而赋予丝纤维更为优异的力学性能。

两种不同剂型人工饲料饲养家蚕的比较试验

为了比较不同剂型人工饲料对家蚕的饲养效果,本试验以‘优食一号’为试验品种,采用1~2龄饲料育、3~5龄桑叶育的饲养模式,研究粉体饲料和膨化颗粒饲料对家蚕生长发育的影响。结果表明:两种剂型的人工饲料饲育家蚕的疏毛率基本一致,低于桑叶育;2种剂型的人工饲料饲育家蚕的体重和龄期经过无显著差异,均显著低于桑叶育;颗粒饲料育家蚕的生长发育略低于粉体饲料育和桑叶育家蚕;2种剂型的人工饲料的茧丝长、茧丝纤度和解数率比桑叶组低,茧丝质量无显著性差异。表明2种剂型的人工饲料均可用于小蚕期使用,大蚕转桑后补偿效应显著。