抗速灭威单链抗体基因在巴斯德毕赤酵母中的表达及性质研究

选用巴斯德毕赤酵母（Pichia pastoris）系统表达特异性抗速灭威单链抗体（scFv）基因,以为速灭威特异性抗体的大量制备奠定基础。设计引物扩增阳性克隆scFv基因,亚克隆至表达载体pPICZαC,获得重组酵母表达质粒pPICZαC-scFv,线性化pPICZαC-scFv并高效电转P.pastoris（X-33）,对转化子进行抗性梯度筛选得到一株高效表达的X-33-Pp-SMW-12-6菌株。对获得的菌株先后进行表达条件的优化、优化条件下的诱导表达及单链抗体性质研究。结果表明：P.pastoris-scFv的质量接近其亲本E.coli-scFv的质量,X-33-Pp-SMW12-6在优化条件下的表达产量达28mg/L,比未优化前的产量提高了约8mg/L,经纯化后抗体纯度可达85%以上。因此,利用P.pastoris表达系统制备抗速灭威单链抗体比细菌表达系统更有效、更经济。     

弹性蛋白酶基因(PAE)的克隆及在毕赤酵母中的表达

以1株产弹性蛋白酶的铜绿假单胞菌(Pseudomonas aeruginosa)基因组DNA为模板,经PCR扩增得到的铜绿假单胞菌弹性蛋白酶(P.acruginosa elastase,PAE)基因,与GenBank中的序列对比发现同源性为99%.成功地构建了重组表达载体pPIC3.5K/PAE,莺组质粒Sac Ⅰ线性化后转化毕赤酵母(Pichia pastoris)菌株KM71中,通过PCR和表型鉴定表明,PAE基因已经整合到毕赤酵母染色体上.经大量筛选获得48株含高拷贝的重组毕赤酵母转化子.在甲醇诱导下,经过毕赤酵母高密度发酵进行PAE的表达,经SDS-PAGE分析.结果表明,在培养基上清中含有一明显特异性蛋白条带,大小为34kD.活性检测结果,酶活为1 060 U/mL,是出发菌株的26倍.     

木糖还原酶基因克隆及重组表达载体的构建

木糖还原酶是木糖醇生物合成的关键酶,催化木糖合成木糖醇。以实验室筛选并经分子生物学鉴定为季也蒙毕赤酵母的木糖还原酶基因为基础,以酵母表达载体pPICZαA成功构建了含有木糖还原酶的重组表达载体pPICZαA-xyl,为进一步研究真核表达木糖还原酶和木糖醇的生产奠定基础。     

Disulphide structure of high-molecular-weight (HMW-) gliadins as affected by terminators

This study aimed at elucidating SS-bonds of HMW-gliadins (HGL) from wheat with the focus on terminators of glutenin polymerisation. HGL from wheat flour extracts non-treated or treated with the S-alkylation reagent N-ethylmaleinimide (NEMI) were compared. HGL from wheat flour Akteur were isolated, hydrolysed with thermolysin and the resulting peptides pre-separated by gel permeation chromatography and analysed by liquid chromatography/mass-spectrometry using alternating electron transfer dissociation/collision-induced dissociation. Altogether, 22 and 28 SS-peptides from samples without and with NEMI treatment, respectively, were identified. Twenty-six peptides included standard SS-bonds of α- and γ-gliadins, high-molecular-weight and low-molecular-weight glutenin subunits. Eleven SS-bonds were identified for the first time. Fifteen peptides unique to HGL contained cysteine residues from gliadins with an odd number of cysteines (ω5-, α- and γ-gliadins). Thus, gliadins with an odd number of cysteines, glutathione and cysteine had acted as terminators of glutenin polymerisation. Decisive differences between samples without and with NEMI treatment were not obvious showing that the termination of polymerisation was already completed in the flour. The two HGL samples, however, were different in the majority of ten peptides that included disulphide-linked low-molecular-weight (LMW) thiols such as glutathione and cysteine with the former being enriched in the non-treated HGL-sample.     

Responses of glutamine synthetase activity and gene expression to nitrogen levels in winter wheat cultivars with different grain protein content

Glutamine synthetase (GS) plays a central role in plant nitrogen (N) metabolism, which improves crops grain protein content. A pot experiment in field condition was carried out to evaluate GS expression and activity, and grain protein content in high (Wanmai16) and low grain protein (Loumai24) wheat cultivars under two N levels (0.05 and 0.15 g N kg⁻¹ soil). High nitrogen (HN) resulted in significant increases in GS1 and GS2 expression at 10 days after anthesis (DAA), and higher GS activity during the entire grain filling stage. HN also significantly increased yield, grain protein content and protein fraction (except for glutenin of Luomai24) in two wheat cultivars, which indicated that it increased grain yield and protein content by improving nitrogen metabolism. Wanmai16 showed higher grain protein content, gliadin and glutenin content, and had higher expression level of GS2 both in flag leaves and grains at early grain filling stage. However, Luomai24 had greater yield and higher expression level of GS1. The difference expression of GS2 and GS1 genes indicates they had various contributions to the accumulation of protein and starch in wheat grains, respectively. The results suggest that GS2 would be serving as a potential breeding target for improving wheat quality.     

糖化酶工业株的鉴定及糖化酶基因的克隆

对4种糖化酶工业株的rDNA片段进行克隆,在对比其rDNA-ITS1序列与GenBank数据库的基础上,结合形态学观察对菌株进行了鉴定。结果显示,4种菌株分别为黑曲霉、无花果曲霉、米曲霉和灰色小克银汉霉。通过Me-gAlign得到的进化树表明,前3种菌株亲缘关系较近,三者与灰色小克银汉霉的亲缘关系较远。进一步克隆了黑曲霉、无花果曲霉和米曲霉的糖化酶基因,并在毕赤酵母中实现了分泌表达。     

黑曲霉NRRL3135菌株植酸酶基因在毕赤酵母GS-115系统中的表达

将黑曲S（Aspergillus riger）NRRL3135中的植酸酶基因转入毕赤酵母GS-115中,并整合到染色体DNA上,然后进行诱导表达。通过SDS-PAGE对表达产物进行鉴定,发现重组毕赤酵母GS-115分泌了1个约100kD的蛋白,分泌的蛋白较大。用脱糖基化酶endoglycosidase H降解后,形成了约50kD的蛋白,与植酸酶基因产物的理论值相符。通过对表达产物的酶学性质研究,发现该表达产物在pH2．5和pH5．5时具有较高植酸酶活性,同时该表达产物的最适温度约为55℃。这说明植酸酶得到正确的分泌表达。     

细菌表达 dsRNA 介导桔小实蝇 flightin基因的 RNA 干扰

【目的】探索饲喂细菌表达目标基因dsRNA介导桔小实蝇flightin基因RNAi的可行性。【方法】将桔小实蝇flightin基因的相应干扰片段构建至L4440干扰载体,并转化大肠杆菌HT115(DE3)菌株,利用IPTG诱导表达flightin基因对应的dsRNA,命名为flightin-dsRNA。【结果】通过饲喂表达flightin-dsRNA的大肠杆菌10倍浓缩菌液,桔小实蝇flightin基因的表达普遍出现了不同程度的上调,其中,雌虫饲喂后5、10、20 d,雄虫饲喂后5 d与对照差异显著,雄虫饲喂后15 d诱发约43%的下调;该方法对桔小实蝇飞行能力及胸部肌肉发育未产生明显影响。【结论】通过饲喂表达flightin-dsRNA的大肠杆菌对桔小实蝇flightin基因进行RNA干扰不可行或干扰效果不明显。     

油菜生长后期有机氮再利用关键酶活性的变化动态

采用砂培试验，在正常供氮条件下测定油菜花后不同时期叶片蛋白水解酶、谷氨酰胺合成酶（gluta-mine synthetase，GS）和籽粒谷氨酸合酶（glutamate：z-oxoglutarate aminotransferase，即glutamate synthase，GOGAT）活性以及韧皮部汁液游离氨基酸和L-谷氨酰胺含量。结果表明，随着花后植物体的衰老，叶片蛋白水解酶活性虽有所波动但总体上呈下降趋势；叶片GS活性在角果发育初期和中后期维持较高水平，此后均有下降，有效地保证了籽粒发育初期酶蛋白形成的需要及籽粒发育后期氮素再分配的顺利进行；随着籽粒的发育进程，籽粒GOGAT活性逐渐升高，到角果发育后期达最高值，此后又下降。比较两个油菜品种以上3种酶的活性表明，蛋白水解酶活性品种间差异很小；GS活性角果发育前期742高于汇油50，角果发育后期则742低于汇油50；GOGAT活性，汇油50的酶活性强但维持时间短，742的酶活性弱而维持时间长。在花后有机氮再分配过程中，韧皮部汁液中游离氨基酸含量忽升忽降变化不定，而L-谷氨酰胺含量两品种均呈快速下降的趋势。游离氨基酸含量两个品种间的差异变化较大，且L-谷氨酰胺含量742高于汇油50。