少根根霉Δ6-脂肪酸脱氢酶基因在转基因油菜中的表达

 【目的】验证少根根霉△6-脂肪酸脱氢酶基因在转基因油菜中的表达情况，为进一步基因工程化生产γ-亚麻酸打下基础。【方法】将少根根霉△6-脂肪酸脱氢酶基因和植物表达载体pCPN2301连接，构建重组表达质粒pCPNRAD6，采用农杆菌介导的油菜子叶节转化法，将该基因导入甘蓝型油菜H165，获得转基因植株，最后通过气相色谱检测基因的表达情况。【结果】PCR、GUS组织化学染色和Southern杂交分析结果表明，目的基因已经整合到油菜基因组中，Northern杂交分析进一步表明目的基因在RNA水平获得表达，气相色谱分析检测到转基因油菜叶片中γ-亚麻酸和十八碳四烯酸生成，证明目的基因获得功能表达。同样，用改变少根根霉Δ6-脂肪酸脱氢酶基因的转译起始密码子周边序列的基因RAD6-1所构建的转基因酵母中，也得到类似的结果，而且各种目的脂肪酸的含量均有提高。【结论】初步建立了少根根霉△6-脂肪酸脱氢酶基因在转基因油菜中的表达体系。     

大肠杆菌γ-ggt原核表达载体pGEX-4T-1/γ-ggt的构建及其蛋白表达

构建大肠杆菌γ-谷氨酰转肽酶原核表达载体pGEX-4T-1/γ-ggt及表达与鉴定目的蛋白,为进一步利用该酶催化合成茶氨酸等目的产物奠定了基础。以大肠杆菌DH5α总DNA为模板,用PCR法在γ-ggt编码序列上下游引入酶切位点并扩增出γ-谷氨酰转肽酶基因;将γ-ggt编码序列克隆入原核表达载体pGEX-4T-1的相应酶切位点;用PCR鉴定重组质粒pGEX-4T-1/γ-ggt,并对插入基因片断测序;重组质粒pGEX-4T-1/γ-ggt转化大肠杆菌BL21(DE3),经乳糖诱导后用SDS-PAGE分析表达产物,并经Westernblot鉴定。获得大肠杆菌γ-谷氨酰转肽酶基因,并成功构建到原核表达载体pGEX-4T-1上,转化有pGEX-4T-1/γ-ggt工程菌BL21(DE3)经1 g/L乳糖诱导1 h后即开始表达目的蛋白,在诱导6 h后表达蛋白量达到最高,随着时间的延长,目的蛋白没有被降解,此蛋白主要以包涵体形式存在,Western blot鉴定了此目的蛋白。成功构建了大肠杆菌γ-谷氨酰转肽酶原核表达载体pGEX-4T-1/γ-ggt,并进行了目的蛋白的鉴定。     

东北虎γ-干扰素基因的克隆、生物信息学分析及其在毕赤酵母中的表达

本试验旨在通过克隆东北虎γ-干扰素（IFN-γ）基因,研究其分子特征并预测蛋白生物学功能,为后续研究干扰素抗病毒活性做前期准备。通过RT-PCR从ConA诱导过的东北虎血淋巴细胞中扩增东北虎IFN-γ基因并测序,应用生物信息学方法进行序列分析。结果表明：东北虎IFN-γ编码区由504个核苷酸组成,共编码167个氨基酸,蛋白相对分子质量为19.59 ku,等电点为9.03,所编码的蛋白为碱性亲水性蛋白,其中前23个氨基酸可能为信号肽,IFN-γ编码蛋白保守结构域为IFN-γ超家族,且存在跨膜结构,其中1－6位氨基酸为胞内区域,7－28位氨基酸为跨膜区域,29－167位氨基酸为胞外区域;IFN-γ编码蛋白二级结构主要以α-螺旋（58.08%）和无规则卷曲（33.53%）为主,存在5个潜在的B细胞抗原表位,3个潜在的N-糖基化位点;分子进化分析显示,东北虎IFN-γ与GenBank上发表的东北虎、非洲狮、金钱豹、美洲狮、猎豹、家猫、加拿大猞猁、野猪等的核苷酸相似性为80.4%~99.8%,氨基酸相似性为70.5%~100%,东北虎IFN-γ与非洲狮、金钱豹亲缘关系最近,美洲狮、猎豹、家猫、猞猁次之,野猪最远。通过合成改造后的东北虎IFN-γ基因,构建能表达IFN-γ蛋白的重组质粒pPIC9K-IFN-γ,将其导入高效表达系统-毕赤酵母中进行诱导表达,经SDS-PAGE分析,表达蛋白的分子量约17.8 ku,与预期大小相符,表明东北虎IFN-γ成功表达。     

施γ-聚谷氨酸对桃树生长发育和15N吸收利用及损失的影响

以1年生桃树盆栽实生苗为试材,底肥为尿素3 g(其中15N标记0.4 g)、磷酸二氢钾3 g,设CK为对照,T1、T2、T3分别添加γ-PGA 10,80,150 mg,探究不同浓度γ-聚谷氨酸(γ-PGA)对桃树植株生长与氮素吸收利用的影响。结果表明:施用中量和高量γ-PGA能显著提高土壤脲酶与过氧化氢酶活性以及土壤碱解氮含量;施用中、高量γ-PGA能促进桃实生苗根系生长,尤其是细根的生长。与对照相比,施用中量γ-PGA桃实生苗根系总长度、分支数、根尖数、交叉数和根系总表面积分别增加51.95%,40.53%,30.72%,35.21%,45.23%;施用中、高量γ-PGA能显著提高植株净光合速率和叶绿素SPAD值,以及干物质积累量。施用中、高量γ-PGA能显著提高桃实生苗根系活力、硝酸还原酶、谷草转氨酶和谷丙转氨酶活性。施用中、高量γ-PGA提高了桃实生苗氮素吸收利用率和氮素残留率,降低了氮素损失率,与对照相比,氮素吸收利用率分别提高27.80%和27.07%,氮素残留率分别提高14.00%和19.04%,氮素损失率分别降低16.43%和19.49%,且差异显著。可见施用γ-PGA可改善桃根区土壤理化性状,提高植株氮素吸收利用率和土壤氮素残留率,降低氮素损失率,促进了桃实生苗的生长。     

Effects of dietary γ-aminobutyric acid supplementation on antioxidant status,blood hormones and meat quality in growing-finishing pigs undergoing transport stress

γ-Aminobutyric acid (GABA) is a natural nonprotein amino acid distributed in animals, plants and microbes. GABA is an inhibiting neurotransmitter which takes great effect in mammalian central nervous system. We carried out the research to study the influence of GABA on blood hormone concentrations, antioxidant status and meat quality in fattening pigs after transportation. The 72 pigs with a starting weight of approximately 32.67 ± 0.62 kg were randomly allocated to 2 groups based on dietary treatments, containing 6 replicates with 6 pigs in each. The pigs were fed dietary supplementation of GABA (0 or 30 mg/kg of diets) for 74 days. Twelve pigs were randomly selected from each group and assigned to the either 1 hr of transport (T group) or no transport (N group), resulting in two-factor factorial design. Compared to the control, GABA supplementation increased average daily gain (ADG) (p < .01) and decreased feed–gain ratio (F/G) (p < .05). The pH_45 min was lower and drip loss was greater in the longissimus muscles (LM) of post-slaughter of transported pigs (p < .05). The pH_45 min of 0/T group (group with 0 mg/kg GABA and transport) was significantly lower than the pH_45 min of the 30/T group (diet × transport; p < .05). GABA supplementation significantly increased serum glutathione peroxidase (GSH-Px) concentration (p < .05) before transportation. Following transport, pigs fed GABA had decreased concentrations of serum malonaldehyde (MDA), adrenal cortical hormone and cortisol (p < .05). The results indicate that feeding GABA significantly increased the growth performance of growing-finishing pigs. The transportation model negatively impacted meat quality, antioxidant indexes and hormone parameters, but dietary supplementation of GABA could suppress the rise of drip loss of LM, ACTH and COR and suppress the drop of pH_45 min of LM after transportation stress in growing-finishing pigs. Feeding GABA alleviated transportation stress in pigs.     

γ-氨基丁酸代谢酶的研究进展

谷氨酸脱羧酶催化产生的γ-氨基丁酸具有调节血压、促进精神安定和改善脑机能等重要功效。富集高γ-氨基丁酸成为目前研究的热点。该研究对有关γ-氨基丁酸代谢的关键酶的研究概况进行了综述。通过激活或抑制酶活性,促进γ-氨基丁酸的富集,为开发新型保健产品提供参考。     

黄曲霉毒素B1对雏鸡外周血T淋巴细胞亚群及 血清IL–2和IFN–γ的影响

为探明黄曲霉毒素B1(AFB1)对雏鸡细胞免疫功能的影响,将100只1日龄艾维茵健康公雏鸡随机分为4组,分别喂以基础日粮(玉米–豆粕型基础日粮,对照)和AFB1日粮(基础日粮中AFB1的添加量分别为0.15、0.3、0.6 mg/kg,分别记为AFB1Ⅰ、Ⅱ、Ⅲ组),试验期21 d。以流式细胞术检测雏鸡外周血T淋巴细胞亚群变化;用酶联免疫吸附试验(ELISA)检测雏鸡血清IL–2和IFN–γ含量变化。结果显示,与对照组相比,AFB1Ⅱ和AFB1Ⅲ组7、14和21日龄雏鸡外周血CD3+,CD3+CD4+和CD3+CD8+T淋巴细胞百分比下降明显(P0.05或P0.01),血清中IL–2和IFN–γ含量降低(P0.05或P0.01),AFB1Ⅰ组雏鸡14日龄的血清IL–2和7日龄的IFN–γ含量降低(P0.05),表明摄食含黄曲霉毒素B1的日粮可导致雏鸡细胞免疫功能下降。     

Berberine hydrochloride attenuates cyclooxygenase-2 expression in rat small intestinal mucosa during acute endotoxemia

The effect of berberine hydrochloride (BBR) on inducible cyclooxygenase-2 (COX-2) in small intestinal mucosa and related mechanisms was investigated in a rat model of acute endotoxemia. The results showed that lipopolysaccharide (LPS) increased COX-2 expression, whereas SB202190 and BBR curtailed it. LPS increased phosphorylation of mucosal p38 MAPK and ATF2 as well as production of ATF2, whereas BBR attenuated these effects. LPS upregulated mucosal peroxisome proliferator-activated receptor gamma (PPARγ), but BBR reduced this receptor. GW9662 aggravated LPS-induced and reversed BBR-attenuated COX-2 expression. The findings showed that BBR ameliorated COX-2 overexpression partially via modulation of p38 and PPARγ pathways during acute endotoxemia.     

空间环境和地面γ辐照对水稻诱变的差异

研究了不同类型的水稻品种空间搭载和地面⁶⁰Co γ辐照两种条件下的诱变敏感性和诱变效应。结果表明，诱变的敏感性均以粳稻强于籼稻，推广种强于农家种，籼稻中以中籼和晚籼>早籼，而不同粳稻气候生态型间的差异较小。对γ辐照不敏感的品种大多数对空间诱变也不敏感，而对γ辐照敏感的品种中仅有1/4对空间诱变敏感。空间搭载对54.2%的品种的处理当代秧苗生长具有加速作用，有的同时提高结实率。M₂代研究表明，两种诱变处理均能诱发株高和抽穗期突变，但其突变频率差异较大。空间搭载处理的M₂代矮秆、高秆和早熟的突变频率与M₁代总生理损伤存在显著正相关。表明空间环境与地面γ辐照的诱变机制可能不完全相同。本研究结果对开展水稻空间诱变育种具有一定的指导意义。