LAT52启动子在甘蓝花药中的表达模式

[目的]验证花药特异性启动子LAT52能否在甘蓝花药中发挥功能。[方法]从番茄中扩增得到LAT52核心序列608 bp,进行序列分析,连接LAT52启动子到双元表达载体p BI121,转化农杆菌GV3101,用农杆菌浸染法侵染甘蓝幼苗下胚轴,获得2株阳性植株,对转基因甘蓝植株的花蕾、花药及花粉进行GUS染色。[结果]LAT52核心序列608 bp具多个与花粉特异性表达相关的元件,浸染的LAT52启动子仅在花药及花粉中表达出蓝色,其他部位未染上蓝色。[结论]LAT52启动子仅在甘蓝花药及花粉中特异性表达。     

夜香树DXS2基因的克隆与表达分析

[目的]克隆夜香树(Cestrum nocturnumL.)萜类香气物质代谢途径关键限速酶DXS2(1-deoxy-D-xylulose-5-phosphate synthase 2)基因全长cDNA,并对该基因进行生物信息学分析,检测该基因在开花不同时间及叶片中表达量的动态变化。[方法]采用RTPCR与RACE相结合的技术,获得夜香树DXS2的全长核苷酸序列,并进行生物信息学分析,qRT-PCR分析其在盛花期不同时间花和叶中的表达量。[结果]获得CnDXS2基因全长序列2 370 bp,其中ORF 2 145 bp,编码714个氨基酸,含DXP-synthase-N、TPP-PYRDXS-TK-like和Transketolase-C等保守结构域,与绒毛状烟草(Nicotiana tomentosiformis L.)DXS2蛋白同源性达93%,推测属于Ⅱ类DXS蛋白;qRT-PCR分析表明,CnDXS2在叶中表达量总体高于花,花中22∶00表达量最高,14∶00表达量最低,具有节律性。[结论]萜类MEP代谢途径基因CnDXS2的克隆与表达分析为从分子方面揭示CnDXS2基因在萜类香气代谢途径中的功能提供了依据,有助于夜香树花香萜类代谢途径基因功能和花香基因工程的研究。     

茶树短穗扦插2夹1膜覆盖保温技术研究

[目的]研究茶树短穗扦插2夹1膜覆盖保温技术。[方法]在原有双层覆盖的基础上,加盖1层塑料薄膜,形成2层塑料薄膜中间1层遮阳网——2夹1膜的覆盖保温方式。[结果]越冬期间,2夹1膜覆盖日最低温度平均值为2.32℃,高于无膜覆盖2.29℃,高于双膜覆盖1.79℃;区试期间,极端最低温度无膜覆盖为-11.51℃,双膜覆盖为-9.47℃,2夹1膜覆盖为-6.47℃;2夹1膜覆盖日平均温度为10.30℃,高于无膜覆盖4.93℃,高于双膜覆盖1.78℃;2夹1膜日最高温度平均值为28.12℃,高于双膜覆盖4.30℃,高于无膜覆盖14.30℃;采用2夹1膜覆盖技术,育苗成活率比双膜覆盖高4.48百分点,增值12 750元/hm2。[结论]2夹1膜具有很好的保温、增温能力;可消除越冬时的冻土抬苗现象,提高育苗的安全系数。     

植原体免疫主导膜蛋白Imp基因在大肠杆菌中表达条件优化(英文)

[目的]为了提高植原体膜蛋白Imp基因在E.coliBL21(DE3)中的表达量,优化Imp基因的原核表达条件。[方法]通过设计正交试验,考察不同的培养条件对工程菌E.coliBL21(DE3)-pET-28a(+)-Imp的影响。在获得最佳培养条件的基础上考察不同诱导条件对Imp蛋白表达量的影响。利用SDS-PAGE和GeneTools凝胶分析软件分析融合蛋白Imp的表达量。[结果]表达条件优化结果表明,最佳培养条件为:温度37℃,pH7.0,装液量20%,振荡速度200r/min。最佳诱导条件为:温度37℃,起始OD600≈1.5,IPTG终浓度0.1mmol/L,诱导培养时间6h。[结论]在最佳条件下Imp表达量达到70.98mg/L,确定了Imp融合蛋白在大肠杆菌的优化表达条件。     

菜豆荚斑驳病毒外壳蛋白基因的克隆与原核表达

[目的]克隆菜豆荚斑驳病毒(BPMV)外壳蛋白(CP)基因,并对其进行原核表达。[方法]利用RT-PCR方法克隆BPMV CP基因,将其连接至pMD19-T Simple载体后对阳性克隆进行测序,检测其与已知病毒外壳蛋白基因序列的同源性;将CP基因定向插入双酶切的pET30a,构建其原核表达载体并转化大肠杆菌BL-21表达重组蛋白。[结果]试验克隆得到的CP基因大小为1 122 bp,与NCBI中目标基因的相似度达99%以上;试验成功构建了原核表达载体pET30a-BPMV CP,发现重组蛋白在37℃、1.0 mmol/L IPTG诱导4 h条件下表达量最高。[结论]该研究为BPMV抗血清的制备与液相芯片检测方法的建立奠定了基础。     

低温对2种紫草科植物△6 脂肪酸脱氢酶基因表达的影响

以紫草科植物新疆软紫草(Arnebia euchroma)和假狼紫草(Nonea capsica(willd.)G.Don)为材料,翻译延伸因子EF1a为内参基因,利用半定量逆转录聚合酶链式反应(semi-RT-PCR)技术,对2种紫草植物中γ-亚麻酸合成关键酶基因△6-脂肪酸脱氢酶基因的表达进行研究。结果表明,△6-脂肪酸脱氢酶基因在紫草植物的叶、茎、根中均表达,在低温诱导不同时间后紫草植物叶片中△6-脂肪酸脱氢酶基因的表达有所增强。气相色谱分析显示,其产物γ-亚麻酸在低温下积累,推测紫草科植物叶片中△6-脂肪酸脱氢酶基因表达量增加可能与适应低温胁迫的逆境有关。     

含LacZ重组逆转录病毒的制备及其在NIH3T3细胞的表达

构建了含有3．7kbLacZ的重组逆转录病毒载体,转染包装细胞系PA317后,经G418筛选,得到了G418的抗性的产毒细胞系,用收获的含有重组逆转录病毒粒子的浓缩病毒上清,感染NIH3T3细胞。经X-gal染色检测,发现表达了LacZ的NIH3T3细胞呈蓝色。     

Transport of nutrients and electrolytes across the intestinal wall in pigs

In recent years, intestinal transport processes have been studied in detail regarding both, functional and structural aspects. For monosaccharides different systems have been demonstrated for apical uptake: this includes the high-affinity SGLT1 as a distinct d-glucose system and GLUT5 for fructose. Specifically in pigs a low affinity, high-capacity system for d-glucose and d-mannose with no preference for Na⁺ over K⁺ and a very low affinity system are suggested as further uptake systems. As in other species, basolateral extrusion is mediated by GLUT2. The distributions of monosaccharide transport along the gastrointestinal axis as well as the potential role of paracellular monosaccharide absorption have not yet been clarified.

Amino acids can principally be absorbed by the paracellular and transcellular pathway whereas transcellular transport can either be mediated by facilitated diffusion or secondary active Na⁺-coupled transport. This includes different transport systems for neutral, anionic and cationic acids. In addition, the presence of the di-/tripeptides transport system PEPT1 which depends on an inwardly directed H⁺-gradient has also been confirmed for the pig small intestine, its quantitative proportion is still under debate.

Short chain fatty acids (SCFA) are the major end products of microbial carbohydrate fermentation which occurs along the gastrointestinal tract with the highest production rates in the large intestines. At least two uptake mechanisms have to be assumed, i.e., non-ionic diffusion and anionic exchange via SCFA⁻/HCO₃⁻-exchange. Controversial views still exist to what extent SCFA are metabolized within the epithelial tissue.

Segmental differences between small and large intestines have been demonstrated for Na⁺ absorption. Whereas in the small intestines the major part of Na⁺ absorption is mediated by coupled nutrient transport systems, aldosterone sensitive Na⁺ channels and Na⁺/H⁺-exchange are the dominant mechanisms in the hindgut. For Cl⁻ paracellular transport and anionic Cl⁻/HCO₃⁻-exchange are the major absorptive mechanisms. Cl⁻ secretion is mediated by apical channels which may be activated by toxins of different origin. Different types of Cl⁻ channels have been identified, such as Cystic Fibrosis Transmembrane Regulator (CFTR), Ca-activated Cl⁻ channels (CLCA) and Outwardly Rectifying Cl⁻ Channels (ORCC). Whereas CFTR has clearly been shown for jejunal and colonic epithelial and goblet cells controversy still exists on the relevance of CLCA and ORCC in pigs.

For Ca²⁺ there is evidence that both recently published channels TRPV5 and TRPV6 are also expressed in pig intestinal tissues, however, this has not yet been shown on protein level. From several functional approaches it was demonstrated that phosphate uptake can be mediated by both, a Na⁺-dependent transcellular component and paracellularly. On a molecular basis it is uncertain whether the transport protein of transcellular mechanism belongs to the NaPi-IIb cotransporter family.     

三倍体梨新品种‘龙园洋红’

 龙园洋红’是三倍体梨新品种, 属短枝型, 亲本为56-5-20和乔玛, 抗寒力强, 平均单果质量185.5 g, 最大可达650.6 g, 果实为不规则长椭圆形, 果型指数1.13。果皮底色黄, 有红晕。果肉白色,细软多汁, 可溶性固形物16.05% , 可溶性糖11.68%。可滴定酸0.81% , 果实9月15日左右成熟, 风味酸甜适度、有香味。     

果实特异启动子调控薄皮甜瓜ACC合成酶cDNA反义表达载体的构建

应用反义RNA技术抑制甜瓜成熟过程中内源乙烯的合成,从而培育耐贮运品种是解决甜瓜延熟保鲜难题的可行新方法。根据GenBank中甜瓜、黄瓜ACC合成酶基因氨基酸保守序列设计引物,从成熟的薄皮甜瓜(齐甜1号)果肉组织中提取总RNA,经RT-PCR扩增得到约0.7kb的ACC合成酶cDNA片段,将其克隆到质粒载体pGEM-TEasy中,测序表明,该基因为777bp,编码258个氨基酸;从番茄(东农706)叶片组织中提取总DNA,经PCR扩增得到约2.2kb的E8基因片段,将其克隆到质粒载体pGEM-TEasy中,测序表明,该基因为2192bp;以pCAM2301为起始植物表达载体,pCAM-GT为中间载体,成功构建了果实特异启动子(E8)调控薄皮甜瓜ACC合成酶cDNA反义表达载体,采用冻融法将其转入根癌农杆菌LBA4404,得到了完整的Ti质粒表达载体系统。