In vitro ruminal methanogenesis of a hay-rich substrate in response to different combination supplements of nitrocompounds; pyromellitic diimide and 2-bromoethanesulphonate

Sixteen combinations of 5 treatments at 4 levels were designed in a L₁₆(4⁵) orthogonal experimental design to evaluate associative effects of five methanogenesis inhibitors at four dose levels: nitroethane (NE, 0 mM, 5 mM, 10 mM and 15 mM), 2-nitroethanol (NEOH, 0 mM, 5 mM, 10 mM and 15 mM), 2-nitro-1-propanol (NPOH, 0 mM, 5 mM, 10 mM and 15 mM), pyromellitic diimide (PMDI, 0 mM, 0.02 mM, 0.05 mM and 0.07 mM) and 2-bromoethanesulphonate (BES, 0 mM, 0.01 mM, 0.03 mM and 0.05 mM) on in vitro ruminal methane production of the mixed substrate (Chinese wildrye hay:maize meal = 4:1) using a cumulative gas production technique. After 48 h incubation, in vitro dry matter disappearance (IVDMD), total gas production (GP₄₈, ml/g DM) and total volatile fatty acids (VFA) production in various combinations of these inhibitors were decreased by 10.6-56.0, 26.5-44.5 and 20.3-47.6%, respectively (P<0.05). The molar proportion of acetate in the inhibitor combination groups was decreased by 6.6-12.5% while those of propionate and butyrate were increased by 7.0-19.2 and 21.9-56.5% (P<0.01), respectively. Methane proportion (MP) in total gas production was reduced by 79.4-98.5% (P<0.01), and the highest inhibition occurred in the combination of 10 mM NE, 10 mM NPOH, 0.07 mM PMDI and 0.01 mM BES in cultures. The partial correlation coefficients between NE, NEOH, NPOH, PMDI or BES and CH₄ proportion were −0.465 (P<0.01), −0.417 (P<0.01), −0.355 (P<0.05), −0.408 (P<0.01) and −0.345 (P<0.05), respectively, indicating that NE was the most potent inhibitor, followed by NEOH and PMDI, and finally NPOH and BES. In general, VFA production in the inhibitor combinations was substantially shifted to produce much more butyrate and propionate and less acetate. The combination of 15 mM NE, 10 mM NEOH, 5 mM NPOH, 0.07 mM PMDI and 0.01 mM BES in cultures, leading to >95% methane inhibition, may be the optimal application of these inhibitors with less depression of total VFA production. Further feeding trials to validate these combinations is still required on rumen function, methane production, growth performance and milk production.     

Proteomic analysis of BBMV in Helicoverpa armigera midgut with and without Cry1Ac toxin treatment

The crystal proteins of Bacillus thuringiensis (Bt) are widely used for insect control. Helicoverpa armigera is the model insect for Bt studies. In this study, brush border membrane vesicle (BBMV) proteins from fifth instar larvae of Helicoverpa armigera were prepared, and proteomic approaches based on two-dimensional (2D) gel electrophoresis and mass spectrometry were used to elucidate changes in BBMV proteins with and without Cry1Ac toxin treatment. Sixty-one protein bands separated by 1D electrophoresis were cut out from the gel for tryptic digestion and were detected with molecular mass spectrometry (ESI-Q-TOF) and High Capacity Ion Trap Ultra (HCT Ultra). BBMV proteins of interest separated by 2D electrophoresis were excised and digested with trypsin, and the resulting peptides were analyzed by mass spectrometry. Mass fingerprints were compared with the non-redundant NCBI Metazoa (Animals) database. We found a noticeable increase in the level of aminopeptidase N (APN) that is important for detoxification reactions. Additionally, a significant decrease in the level of trypsin-like protease is important during early responses and adaptation of the insect to Bt and exposure to its toxins. Furthermore, the increase in V-ATPase subunits indicate elevated cellular energy profile which is necessary to combat toxin stress. The increased level of actin in larvae provides immediate protection by strengthening the midgut epithelium and enhancing cellular defenses in the tissue. This study presents the differences in the BBMV proteins of Helicoverpa armigera with and without Cry1Ac toxin treatment, and provides a theoretical basis for research on the mechanism of action of Bt toxin.     

大肠杆菌中利用苏云金芽胞杆菌强启动子p1Ac指导Cry1Ac蛋白表达的特性分析

对利用苏云金芽胞杆菌(Bacillus thuringiensis,Bt)强启动子——cry1Ac基因启动子p1Ac指导cry基因在大肠杆菌中的表达进行了研究。结果显示,大肠杆菌中由启动子p1Ac指导表达的Cry1Ac蛋白与苏云金芽胞杆菌来源的Cry1Ac蛋白在碱溶性、胰蛋白酶活化、杀虫活性等方面有较好的一致性,从而解决了目前商业化载体大肠杆菌表达cry基因时形成不易溶解的包涵体问题。同时,还对p1Ac指导的cry1Ac基因在大肠杆菌中表达的发酵条件进行了初步探索。     

外源基因在苏云金杆菌染色体上的定点整合及表达

[目的]研究构建稳定表达外源基因、无抗性标记基因的苏云金杆菌(Bacillus thuringiensis简称Bt)工程菌的方法.在构建Bt工程菌时,高拷贝外源质粒的转入导致Bt芽孢数量减少,芽孢形成期延滞,影响Bt菌株的杀虫活力.而且,外源质粒在Bt中的稳定性较差,外源基因容易丢失.将基因整合人染色体是一种构建遗传性状稳定、杀虫活力高的Bt工程菌的有效方法.[方法]本研究采用PCR技术,分两段扩增定位于Bt无晶体突变株XBU001染色体上的trigger factor基因片段作为同源臂,克隆入温度敏感型载体pKSV7,构建了定点整合载体pKTF12.并利用pKTF12质粒将crylAc基因定点整合入XBU001染色体上.[结果]利用载体pKTF12将crylAc定点插入triggerfactor位点,对宿主菌XBU001的正常生长没有影响.重组菌株KCTF12中的crylAc基因能够稳定遗传、表达并形成菱形晶体.与携带高拷贝外源质粒的Bt菌株HTX42相比较,KCTF12具有芽孢数量增多、芽孢形成期提前的优势.[结论]定点整合法是一种构建稳定表达外源基因、无抗性标记基因Bt工程菌的有效方法.     

Effect of water-deficit stress on cotton plants expressing the Bacillus thuringiensis toxin

Bacillus thuringiensis (Bt) crops require a high dosage of Bt toxin to delay development of insect resistance, in particular, when the refuge strategy is applied. This strategy is threatened by plant developmental and environmental factors that might reduce Bt toxin concentration and Bt efficacy in Bt crops. Growth of Bt (Cry1Ac) cotton under prolonged, moderate water deficit as a single stress factor was evaluated. Bt cotton plants were analysed for physiological performance, Bt toxin concentration and Bt efficacy. For performance analysis, leaf and total plant dry weight and leaf area were measured. Bt toxin concentration was determined by an immuno‐assay. Effects of Bt toxin on growth and mortality of African cotton bollworm, Helicoverpa armigera, larvae were measured in different plant organs. Leaves from young plants exposed for 30 days to moderate water deficit had both higher Bt toxin concentrations and were more effective against larvae than leaves, flowers or bolls from mature flowering plants exposed to 60 days of moderate water deficit. Although growth of Bt cotton plants under moderate water‐deficit conditions decreased Bt concentrations in leaves, flowers and bolls, this had no effect on efficacy against first‐instar cotton bollworm larvae. No significant evidence was found that moderate water deficit, as a single stress factor, decreases Bt efficacy in Bt cotton.     

Ac／Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(Ac TPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ae×Ds的杂交组合354个。检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性。结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4％。检测到T-DNA可插入到编码蛋白的基因中。在Ac×Ds的F2代中,Ds因子的转座频率为22.7％。对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制——转座和小完全切离等现象。获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录。探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略。     

陆地棉栽培品种转复合启动子控制下的cry1Ac3基因获得高效抗虫植株(英文)

以根癌土壤杆菌 (Agrobacteriumtumefaciens)介导法分别将植物表达载体pBinMoBc和pBinoBc导入陆地棉(GossypiumhirsutumL .)栽培品种“新陆早 1号”、“晋棉 7号”、“晋棉 12号”和“冀合 32 1”。pBinMoBc携带有高效启动子复合OM启动子控制下的cry1Ac3基因 ,pBinoBc携带有 35S启动子控制下的cry1Ac3基因。经过共培养、卡那霉素筛选抗性愈伤组织及体细胞胚的诱导 ,得到了再生植株。对T2 代的PCR、Southernblotting、ELISA检测及Westernblotting证明cry1Ac3基因已整合入受体棉花基因组并得到表达。抗虫性检测表明转基因后代对棉铃虫 (Heliothisarmigera )具有良好的抗性 ,转pBinMoBcT2 代与转pBinoBcT2 代相比 ,对棉铃虫具有更快的致死速度。本研究建立了一套高效的陆地棉栽培品种转化体系 ;进一步的检测结果表明 ,复合OM启动子可以提高外源基因的表达量从而增强转基因棉的抗虫性。     

蛋白磷酸酶-2Ac在不同倍性鱼6种组织中的分化表达模式

蛋白磷酸酶-2A是最重要的丝氨酸/苏氨酸蛋白磷酸酶之一,对于调控多细胞的生命活动起着非常重要的作用.以异源四倍体鲫鲤及其二倍体父/母本(湘江野鲤/红鲫)和子代三倍体湘云鲫等为实验材料,运用Westernblot技术及荧光免疫组织化学技术等实验手段,得到了Protein PJhosphatase-2A(PP2A)的催化亚基在上述不同倍性鱼体内6种不同组织的表达模式:Protein Phosphatase-2Ac(PP2Ac)在异源四倍体鲫鲤及其二倍体父/母本及子代三倍体湘云鲫不同组织中蛋白水平均有表达,而且出现了明显的种属特异性和组织特异性,如在大脑、肌肉、肝脏三组织中,三倍体湘云鲫中PP2Ae的表达相对最高.而在肾脏组织中,PP2Ac在异源四倍体鲫鲤中的表达水平最高,父本与三倍体湘云鲫中的表达比较相近,且最低;而在性腺组织中则是父本精巢中的表达最高;在心脏组织中,PP2Ae在母本红鲫中的表达相对较高.这种明显的种属之间组织特异性可能说明了子代与父母本之间的变异性.荧光免疫组化实验结果显示,从整体水平来看,4种不同鱼的同一组织中,PP2Ac的相对定位是非常相似的,这可能说明了异源四倍体鲫鲤与其二倍体父/母本及子代三倍体湘云鲫之间的遗传相似性.研究结果为进一步探索PP2Ac在脊椎动物不同组织中的功能提供了实验依据.