SILKWORM 30K PROTEIN INHIBITS ECDYSONE‐INDUCED APOPTOSIS BY BLOCKING THE BINDING OF ULTRASPIRACLE TO ECDYSONE RECEPTOR‐B1 IN CULTURED Bm5 CELLS

This study investigates the mechanism through which increased 30K protein inhibits ecdysone‐induced apoptosis in the Bm5 silkworm ovarian cell line. Treatment of Bm5 cells with 20‐hydroxyecdysone (20E) after transfection with the pIZT/V5‐His control vector triggered apoptosis, but 20E treatment did not trigger apoptosis in Bm5 cells transfected with the pIZT/30K/V5‐His vector. To confirm its inhibitory effect on apoptosis, 30K protein was first purified from Escherichia coli transformed with a 30K expression vector and used to generate specific antibodies in mice. Anti‐30K antiserum was used to confirm synthesis of the 30K protein in pIZT/30K/V5‐His‐transfected Bm5 cells and to detect 30K protein binding to the ecdysone receptor‐B1 (EcR‐B1). Anti‐30K antiserum was used to immunoprecipitate protein complexes containing 30K from Bm5 cells transfected with pIZT/30K/V5‐His vector and treated with 20E. We observed that 30K proteins bound primarily to the EcR‐B1 and not to ultraspiracle (USP). Reciprocal immunoprecipitation of EcR‐B1‐containing complexes from Bm5 cells transfected with control pIZT/V5‐His vector and treated with 20E showed that EcR‐B1 bound to USP in the absence of 30K but did not bind to USP in pIZT/30K/V5‐His‐transfected Bm5 cells. These results demonstrate that 30K proteins block USP binding to EcR‐B1 through formation of a 30K/EcR‐B1 complex, resulting in inhibition of 20E‐induced Bm5 cell apoptosis.     

RNAi KNOCKDOWN OF BmRab3 LED TO LARVA AND PUPA LETHALITY IN SILKWORM Bombyx mori L.

Rab3 GTPases are known to play key a role in vesicular trafficking, and express highest in brain and endocrine tissues. In mammals, Rab3 GTPases are paralogs unlike in insect. In this study, we cloned Rab3 from the silk gland tissue of silkworm Bombyx mori, and identified it as BmRab3. Our in silico analysis indicated that BmRab3 is an isoform with a theoretical isoelectric point and molecular weight of 5.52 and 24.3 kDa, respectively. Further, BmRab3 showed the C‐terminal hypervariability for GGT2 site but having two other putative guanine nucleotide exchange factor/GDP dissociation inhibitor interaction sites. Multiple alignment sequence indicated high similarities of BmRab3 with Rab3 isoforms of other species. The phylogeny tree showed BmRab3 clustered between the species of Tribolium castaneum and Aedes aegypti. Meanwhile, the expression analysis of BmRab3 showed the highest expression in middle silk glands (MSGs) than all other tissues in the third day of fifth‐instar larva. Simultaneously, we showed the differential expression of BmRab3 in the early instar larva development, followed by higher expression in male than female pupae. In vivo dsRNA interference of BmRab3 reduced the expression of BmRab3 by 75% compared to the control in the MSGs in the first day. But as the worm grew to the third day, the difference of BmRab3 between knockdown and control was only about 10%. The knockdown later witnessed underdevelopment of the larvae and pharate pupae lethality in the overall development of silkworm B. mori L.     

CHARACTERIZATION OF ADAPTOR PROTEIN COMPLEX‐1 IN THE SILKWORM,Bombyx mori

To investigate the function of adaptor protein complex‐1 (AP‐1) in the silkworm, we characterized AP‐1 in the silkworm by RNAi technique and co‐localization methods. As a result, AP‐1 was found to exist as cytosolic form and membrane‐bound form distinguished by phosphate status, showing molecular mass difference. There was relatively more cytosolic form of AP‐1 than its membrane‐bound counterpart in the silkworm. However, AP‐1 distributed predominantly as cytosolic form in BmN cells. Interruption of AP‐1 expression via DsRNA was more efficient in BmN cells than in the insect larval, which led to a tendency to dissociation between subcellular organelles like the Golgi apparatus and the mitochondria. Environmental condition changes like relatively higher temperature and treatment with dimethyl sulfoxide can lead to expression variance of AP‐1 both in mRNA and protein level. In BmN cells, both the heavy chain γ and light chain σ could clearly co‐localize with AP‐1 β, mostly forming pits in cytoplasm. Two isoforms of AP‐1 σ corresponded to distinct subcellular distribution pattern, possibly due to C‐terminal amino acids difference.     

家蚕血液过氧化氢酶活力及其与蚕体抗逆性的关系

家蚕召Bombyx mori(L.)血液过氧化氢酶(CAT)活力随发育时期呈现有规律的动态变化。在幼虫期,CAT活力以龄初、龄末较高,而龄中较低;化蛹后,CAT活力迅速上升,至中期达到峰值,随后迅速下降直至羽化。不同性别间CAT活力均以雄性较高;不同蚕品种间CAT活力有很大差异;饲料对CAT活力有一定影响,人工饲料育蚕CAT活力略大于桑叶育的;高温冲击和氟化物添食都能引起CAT活力的显著变化,但变化的幅度因品种、性别、时期和添食剂量而有所不同。研究认为,家蚕血液CAT活力与其发育变态、体内代谢均具有密切关系,逆境条件下CAT活力变化幅度的大小可以作为家蚕抗逆性强弱的一个重要生理指标。     

基于超高效液相色谱-质谱技术对野蚕茧和家蚕茧化学成分进行比较

鉴定并比较野蚕茧与家蚕茧的化学成分对于理解家蚕的驯化具有重要的意义。利用高温高压结合甲醇-水提取的方法获得蚕茧中的化学成分,利用UHPLC-MS技术对野蚕、家蚕大造品种和皓月品种3种蚕茧丝中的小分子成分进行鉴定和比较分析。通过阳离子模式和阴离子模式的UHPLC-MS获得了野蚕、大造和皓月蚕茧丝的代谢指纹图谱,对鉴定到的高丰度化合物进行注释,发现其中包括了氨基酸、黄酮、生物碱、萜类、有机酸和木脂素等成分。PLS-DA的得分图表明,野蚕、家蚕大造品种和皓月品种的3种蚕茧的代谢组存在显著差异。发现脯氨酸、亮氨酸/异亮氨酸和苯丙氨酸在大造茧中的含量显著高于在野蚕和皓月茧中的含量,黄酮类植物次生代谢物在大造茧中的含量显著提高,包括槲皮素、异槲皮素、槲皮素-3-O-槐糖苷、槲皮素-3-O-L-鼠李糖苷、槲皮素-3-O-芸香糖苷和山奈酚;而神经碱、白桥楼碱、毛果芸香次碱、美洲豚草内酯、线叶泽兰素和中缅木莲素等生物碱、萜类和木脂素类的植物次生代谢物在野蚕茧中的含量显著高于在家蚕茧中的含量。在紫外光的激发下观察黄酮的绿色荧光发现家蚕大造茧中的黄酮含量最高,家蚕皓月茧中的黄酮含量最低,而野蚕茧中的黄酮含量居中。生物碱和有机酸是良好的抗虫抗菌剂,它们在野蚕茧中的含量较高,能够提高野蚕茧的防护能力。黄酮类物质在家蚕大造茧中的含量较高,是导致家蚕大造茧呈黄绿色的主要原因。     

雌雄蚕蛹近红外反射光谱的差异及其模式识别

采用6250型近红外光谱分析仪,从波长6印到1 235nm分别对日系蚕蛹217颗、中系蚕蛹174颗、杂交种蚕蛹330颗做了扫描测试,结果表明：雌雄蚕蛹的近红外光谱有极显著的差异。从日系217颗蚕蛹中随机选取雌雄各50颗作为学习样本,采用逐步判别方法从其二阶导数光谱中抽取5个特征波长作为观测向量,利用二次型判别函数对其余621颗蚕蛹的性别进行识别,其准确率达98.7%。该方法明显优于以蚕蛹重量或大小识别蚕蛹雌雄的方法。     

家蚕V型ATP酶B亚基的克隆及表达特征

V型ATP酶(Vacuolar-type ATPase)是一种定位于细胞膜和细胞器膜上的氢离子转运酶。它利用ATP水解的能量将氢离子转运到液泡、囊泡或者胞外,从而维持细胞内正常的酸碱环境。V型ATP酶B亚基(V-ATPase B)作为ATP的催化位点,也有着非常重要的作用。为了探讨家蚕V-ATPase B(Bm V-ATPase B)的功能,首先从家蚕五龄幼虫的中肠c DNA中克隆了Bm V-ATPase B基因并构建原核表达载体进行原核表达,获得了重组蛋白,经质谱鉴定正确后,通过镍柱亲和层析的方法纯化了该蛋白并制备了多克隆抗体;最后分析了该蛋白在家蚕丝腺中的表达特征并利用免疫荧光对其在丝腺中的表达位置进行了定位。结果显示Bm V-ATPase B基因序列全长1 473 bp,预测蛋白分子量55 k Da,预测等电点5.3。通过Western blotting对家蚕5龄第3天和上蔟第1天幼虫丝腺的不同区段进行Bm V-ATPase B蛋白的表达特征分析,发现在两个时期该蛋白均在前部丝腺高量表达,而在中部丝腺和后部丝腺表达量相对较低。进一步对两个时期丝腺的不同区段进行免疫荧光定位,发现该蛋白在两个时期的前部丝腺、中部丝腺和后部丝腺均定位于细胞层。利用激光共聚焦显微镜对该蛋白进行进一步的定位,发现该蛋白主要在丝腺的细胞膜表达。研究结果明确了该蛋白在丝腺中的表达模式,为深入研究该蛋白在蚕丝纤维形成中的作用奠定了基础。     

POSITIVE SELECTION DRIVES FASTER‐Z EVOLUTION IN SILKMOTHS

Genes linked to X or Z chromosomes, which are hemizygous in the heterogametic sex, are predicted to evolve at different rates than those on autosomes. This “faster‐X effect” can arise either as a consequence of hemizygosity, which leads to more efficient selection for recessive beneficial mutations in the heterogametic sex, or as a consequence of reduced effective population size of the hemizygous chromosome, which leads to increased fixation of weakly deleterious mutations due to genetic drift. Empirical results to date suggest that, while the overall pattern across taxa is complicated, systems with male heterogamy show a faster‐X effect attributable to more efficient selection, whereas the faster‐Z effect in female‐heterogametic taxa is attributable to increased drift. To test the generality of the faster‐Z pattern seen in birds and snakes, we sequenced the genome of the lepidopteran silkmoth Bombyx huttoni. We show that silkmoths experience faster‐Z evolution, but unlike in birds and snakes, the faster‐Z effect appears to be attributable to more efficient positive selection. These results suggest that female heterogamy alone is unlikely to explain the reduced efficacy of selection on vertebrate Z chromosomes. It is likely that many factors, including differences in overall effective population size, influence Z chromosome evolution.     

INHIBITION OF CASPASE‐LIKE ACTIVITIES PREVENTS THE APPEARANCE OF REACTIVE OXYGEN SPECIES AND DARK‐INDUCED APOPTOSIS IN THE UNICELLULAR CHLOROPHYTE DUNALIELLA TERTIOLECTA1

When the chlorophyte alga Dunaliella tertiolecta Butcher is placed in darkness, a form of programmed cell death with many similarities to apoptosis is induced, including the induction of caspase‐like proteases. Many uncertainties about the regulation and mediators that participate in the process remain. To examine the relationship between caspase‐like activities and different apoptotic events (i.e., phosphatidylserine [PS] translocation), increases in membrane permeability and numbers of dead cells revealed by SYTOX‐green staining, and the generation of reactive oxygen species (ROS), we used the broad‐range caspase inhibitor Boc‐D‐FMK to block the activity of the whole class of caspase‐like proteins simultaneously. In the presence of the inhibitor, ROS were not produced, and cells did not die. Loss of membrane asymmetry, indicated by external labeling of PS by annexin V, was apparent at midstages of light deprivation, although it did not conform to the typical pattern for PS exposure observed in metazoans or vascular plants, which occurs at early stages of the apoptotic event. Thus, we have evidence for a link between ROS and cell death involving caspase‐like enzymes in an alga. The fact that caspase‐like inhibitors prevent not only cell death, but also ROS and loss of cell membrane integrity and asymmetry, suggests that caspase‐like proteases might have regulatory roles early in cell death, in addition to dismantling functions.     

基于amy基因的中国野桑蚕遗传多样性及其与家蚕的系统发育关系

为探索中国野桑蚕Bombyx mandarina的遗传多样性及其与家蚕B. mori的系统发育关系, 采用PCR产物直接测序法（少数样本克隆测序）获得34个家蚕和野桑蚕样本淀粉酶基因amy序列片段（715 bp）。分析发现56个多态性位点, 鉴定出28种单倍型（haplotype）; 核苷酸多样性π=0.01390±0.00103, 单倍型多样度Hd=0.988±0.011。核苷酸不配对分析（mismatch analysis）和Fu’s Fs 检测表明中国野桑蚕曾发生过种群扩张。分子方差分析（AMOVA）表明, 遗野桑蚕传差异主要在种群内, 种群间和地理组群间差异不显著。聚类树上34个样本聚为3枝/3蔟, 野蚕和家蚕都不按地理区域或系统（类型）聚类, A枝由来自不同地区的野蚕和不同类型的家蚕混合构成, 并且进一步分成3个亚枝, 每一亚枝也同时包含家蚕和野蚕, B枝由3个家蚕和1个野蚕混合构成, C枝全部由来自不同地区的野蚕构成。网络分析没有发现“祖先单倍型”和优势单倍型。结果提示, 淀粉酶基因是一个多态性丰富的分子标记, 中国野桑蚕遗传多样性十分丰富, 据此推测家蚕起源于多种生态类型混杂的野桑蚕。     

木黄酮对人催乳素瘤细胞增殖及其凋亡影响的实验研究

目的：观察木黄酮（genistein,GST)对培养的人垂体催乳素瘤细胞增殖和凋亡的影响。方法：将CST、β-雌二醇（E2）作用于体外培养的人催乳素瘤细胞,测定MTT值及^3H-TdR掺入量,流式细胞仪测定细胞周期,并用TUNEL法观察细胞凋亡情况。结果：不同浓度的GST可抑制催乳素瘤细胞的增殖,并存在着剂量效应,10^-5mol.L^-1GST可使G1期的细胞比例从对照组的55.3%上升为90.3%;不同浓度的E2以剂量依赖方式刺激催乳素瘤细胞的增殖,并使G2期的细胞比例从对照组的15.6%上升为41.8%,GST和E2的共同作用仍可抑制催乳素瘤细胞的增殖,但抑制程度降低,不同浓度的GST均明显促进催乳素瘤细胞的凋亡,E2对GST的促凋亡作用无明显影响。结论：GST在体外能明显抑制培养人催乳素瘤细胞的增殖,并促进其凋亡,E2可部分拮抗GST的抑制增殖作用,但对其促凋亡作用无明显影响。     

Effects of overexpression and inhibited expression of thymosin,an actin‐interacting protein from Bombyx mori,on BmNPV proliferation and replication

Previous study showed that exogenously applied recombinant thymosin from Bombyx mori (BmTHY) reduces B. mori nucleopolyhedrovirus (BmNPV) proliferation in silkworm. Which stands to reason that BmTHY in B. mori is crucial for the defense against BmNPV. However, little is known about the effect of endogenously overexpressed or repressed BmTHY on B. mori resistance to virus infection. To study this issue, we constructed an overexpression and inhibited expression systems of BmTHY in BmN cells. The viral titer and the analysis from the quantitative real‐time polymerase chain reaction (PCR) revealed that overexpression of BmTHY decreased the copies of BmNPV gene gp41, which goes over to inhibit the proliferation of BmNPV in BmN cells, while the inhibited expression of BmTHY significantly enhanced viral proliferation in infected BmN cells. These results indicated that endogenous BmTHY can inhibit BmNPV proliferation and replication in infected BmN cells. Furthermore, Co‐IP showed that BmTHY could bind to actin in BmN cells. Also, the overexpression or inhibited expression of BmTHY shifted the ratio of F/G‐actin in infected BmN cells. Lastly, the BmTHY, an actin‐interacting protein, might be one of the key host factors against BmNPV, which inhibits viral proliferation and replication in BmN cells.     

CHITIN SYNTHASE B: A MIDGUT‐SPECIFIC GENE INDUCED BY INSECT HORMONES AND INVOLVED IN FOOD INTAKE IN Bombyx mori LARVAE

Chitin synthase (CHS) is the key regulatory enzyme in chitin synthesis and excretion in insects, and a specific target of insecticides. We cloned a CHS B gene of Bombyx mori (BmChsB) and showed it to be midgut specific, highly expressed during the feeding process in the larva. Knockdown of BmChsB expression in the third‐instar larvae increased the number of nonmolting and abnormally molting larvae. Exposure to nikkomycin Z, a CHS inhibitor, reduced the amount of chitin in the peritrophic membrane of molted larvae, whereas abnormally elevated BmChsB mRNA levels were readily detected from the end of molting and in the newly molted larvae. Exogenous 20‐hydroxyecdysone (20E) and methoprene, a juvenile hormone analogue, significantly upregulated the expression of BmChsB when the levels of endogenous molting hormone (MH) were low and the levels of endogenous juvenile hormone (JH) were high immediately after molting. When levels of endogenous MH were high and those of endogenous JH were low during the molting stage, exogenous 20E did not upregulate BmChsB expression and exogenous methoprene upregulated it negligibly. When the endogenous hormone levels were low during the mulberry‐leaf intake process, BmChsB expression was upregulated by exogenous methoprene. We conclude that the expression of BmChsB is regulated by insect hormones, and directly affects the chitin‐synthesis‐dependent form of the peritrophic membrane and protects the food intake and molting process of silkworm larvae.     

MECHANISMS OF THE INDUCTION OF APOPTOSIS IN HUMAN HEPATOMA CELLS BY TUMOUR NECROSIS FACTOR-α

Tumour necrosis factor alpha (TNF-α) at 20 ng/ml induced apoptosis in human hepatoma cellsin vitro . The effect of TNF-α-induced apoptosis was exacerbated by the hypoxanthine-xanthine oxidase (HX/XO) system and cycloheximide (CHX), but alleviated by superoxide dismutase (SOD), suggesting that TNF-α-induced apoptosis may be due to oxidative stress, and independent of protein synthesis. TNF-α elevated free Ca²⁺concentration, triggered lipid peroxidation and decreased the expression of bcl-2 protein. The findings suggest that TNF-α-induced apoptosis may be involved in stimulating Ca²⁺-dependent endonuclease activity and increasing membrane lipid peroxidation. Bcl-2 may play a pivotal role in serving as a Ca²⁺regulator or antioxidant, preventing lipid peroxidation in the process.     

APOPTOSIS,REDIFFERENTIATION AND ARRESTING PROLIFERATION SIMULTANEOUSLY TRIGGERED BY OXIDATIVE STRESS IN HUMAN HEPATOMA CELLS

The effects of oxidative stress (ascorbic acid—ferrous system) on the proliferation, differentiation and apoptosis of the human hepatoma cell SMMC-7721 were studied. Oxidative stress significantly inhibited cell proliferation and induced morphological differentiation. Whatever the indices related with cell malignancy, such as α-fetoprotein and c-glutamyltranspeptidase or the index related with cell differentiation, such as tyrosine-α-ketoglutarate transaminase, all inclined evidently to normalization. The tumour's clonogenic potential decreased significantly. Moreover, together with differentiation, the phenomenon of apoptosis was found by the appearance of apoptotic bodies, detached cells, and apoptotic morphological feature. Although, their DNA was not degraded into oligonucleosomal fragmentation, the DNA was cut into larger fragments (about 21.2kbp) of a size associated with chromatin loops. These findings indicated that oxidative stress can induce both differentiation and apoptosis simultaneously in tumour cells. All the results showed that oxidative stress may initiate the tumour cells reverse transformation. The possible mechanism of the differentiation and apoptosis induced by oxidative stress may be related to the lipid peroxidation of cell membrane.     

Three vital RNA functions and interactions in the process of silk gland apoptosis in silkworm Bombyx mori

The Silkworm Bombyx mori is an important insect in terms of economics and a model organism with a complete metamorphosis. The economic importance of silkworms is dependent on the functions of the silkgland, a specialized organ that synthesizes silk proteins. The silk gland undergoes massive degeneration during the larval to pupal stage, which involves in cell apoptosis. In this paper, high throughput sequencing was used to detect the expression of messenger RNA (mRNA), long noncoding RNA (lncRNA), and microRNA (miRNA) from silk glands of Day 3 in the fifth instar larvae (L5D3) and the spinning 36h (sp36h). We analyzed the Gene Ontology (GO) functions of target genes of the differentially expressed lncRNAs and miRNAs. We investigated the regulations of mRNA, lncRNA, and miRNA on silk gland apoptosis in L5D3 and sp36h. In total, 10,947 lncRNAs were detected in the silk gland and the index number TCONS‐00021360 lncRNA may be involved in the process of apoptosis. In addition, 344 miRNAs targeted 285 mRNAs were related to the death process under GO entry. The results indicated that miRNAs play an important role in the molecular regulation of the silk gland apoptosis compared with that of lncRNAs. Finally, we screened 746 lncRNAs and 20 miRNAs that might interact with BmDredd, and drew an interaction network among them.