中国红豆杉羟化酶基因TcCYP725A22的克隆与功能研究

基于中国红豆杉( Taxus wallichiana var. chinensis (Pilger)Florin)转录组序列,从中扩增到1个新的P450羟化酶基因 TcCYP725A22 ,并对其进行了克隆、表达及功能分析。生物信息学分析结果显示:该基因编码区长1500 bp,共编码499个氨基酸;编码蛋白无信号肽,包含1个跨膜区,具有催化活性中心结构域。进一步将该基因在酿酒酵母WAT11 中进行异源表达功能研究,蛋白质免疫印迹(Western blot)实验结果表明,目的蛋白TcCYP725A22可在WAT11中成功表达;液相色谱-质谱联用(LC-MS)分析结果显示,羟化酶TcCYP725A22对底物紫杉素表现出催化活性。依据质谱结果及反应底物紫杉素的分子结构,判断其催化产物可能为添加了2个羟基的紫杉烷新衍生物。     

中国红豆杉羟化酶基因TcCYP725A22的亚细胞定位及过表达作用分析

抗癌药物紫杉醇生物合成途径中羟化酶的发现是当今研究的热点和难点。文中利用前期分析得到的1个新的中国红豆杉羟化酶基因TcCYP725A22(GenBank登录号:MF448646.1),构建了亚细胞定位载体pCAMIBA1303-TcCYP725A22-EGFP,瞬时侵染洋葱表皮细胞,激光共聚焦显微镜观察结果发现该基因编码蛋白定位在细胞膜。进一步构建了植物表达载体pBI121-TcCYP725A22,经根癌农杆菌AgrobacteriumtumefaciensLBA4404介导转化到中国红豆杉细胞中过表达后,利用荧光定量PCR (qRT-PCR)和液质联用(LC-MS)分析TcCYP725A22过表达对紫杉醇生物合成的影响。结果显示,瞬时转化pBI121-TcCYP725A22的红豆杉细胞中TcCYP725A22基因的转录水平明显高于pBI121空载体对照组。随着TcCYP725A22基因过表达,几个已知的紫杉醇合成关键酶基因的表达量也都有不同程度的上调,且细胞中各紫杉烷的含量普遍提高。这些结果说明羟化酶基因TcCYP725A22极有可能参与紫杉醇的生物合成路径。     

苹果酸度相关基因MdPH1的克隆、表达及亚细胞定位分析

以苹果属（Malus）植物沧江海棠（M.ombrophila Hand.-Mazz）的果实为材料,对其发育过程中苹果酸的含量进行测定,并结合转录组测序的方法筛选控制果实酸度的候选基因。结果显示：MdPH1候选基因的编码区包含2829 bp,编码942个氨基酸;基因组序列全长为4269 bp,包含8个外显子和7个内含子。对10份苹果种质资源中PH1基因序列的分析结果表明,该基因序列中存在22个单核苷酸多态性（SNP）,其中13个位于内含子区,9个位于外显子区;位于最后一个外显子上SNP（G/A）的变异导致了编码氨基酸从缬氨酸变为异亮氨酸。MdPH1蛋白包含8个跨膜结构域,其中蛋白N端包含3个跨膜结构域,C端包含5个跨膜结构域。系统进化分析结果显示,苹果中的PH家族成员与梨（Pyrus communis L.）中的PH家族成员聚集成一簇。组织特异性表达结果发现,MdPH1基因在苹果果实中的表达量最高,其次是叶、花和根,茎中表达量最低。亚细胞定位分析表明MdPH1蛋白定位于液泡膜上。     

紫杉醇生物合成相关酶基因的克隆与表达

以牛儿基牛儿基二磷酸为前体的紫杉醇生物合成大约有20步酶促反应,其反应过程已基本阐明,近一半的相关酶基因已得到克隆与表达。综述了编码参与紫杉醇生物合成的紫杉二烯合酶、紫杉二烯5α羟化酶、紫杉烷10β羟化酶、紫杉烷13α羟化酶、紫杉二烯5α醇O乙酰基转移酶、紫杉烷2α苯甲酰转移酶、去乙酰基巴卡亭Ⅲ10βO乙酰转移酶、3氨基3苯基丙酰转移酶和3N去苯甲酰2脱氧紫杉醇苯甲酰转移酶等9个酶基因的克隆和表达方面的研究情况,并指出随着紫杉醇生物合成的分子生物学研究的不断深入,利用分子生物技术大规模生产紫杉醇将为期不远 。     

桦褐孔菌膜二肽酶在巴斯德毕赤酵母中的分泌表达

本研究利用巴斯德毕赤酵母Pichia pastoris蛋白表达体系表达了药用担子菌桦褐孔菌的一个二肽酶基因。该二肽酶基因编码区全长1814bp,包含6个内含子,编码465个氨基酸。生物信息学分析发现,二肽酶基因编码的蛋白中不含信号肽序列,但在第55–77位氨基酸之间存在一个跨膜结构。将含跨膜结构和去跨膜结构蛋白的cDNA序列分别克隆到酵母分泌型表达载体pPICZαA上,电转化至巴斯德毕赤酵母X-33中,用1%(V/V)甲醇诱导重组菌株表达目标蛋白,采用SDS-PAGE和Western-blot检测表达蛋白。结果显示,巴斯德毕赤酵母可表达含跨膜结构的完整基因,但目标蛋白不能分泌到胞外,存在于破碎细胞的沉淀中,且没有催化活性;而去跨膜结构的蛋白则可分泌表达到胞外,并具有催化活性。Ni-NTA纯化去跨膜结构的桦褐孔菌二肽酶浓度可达0.12mg/mL,并发现其在pH 7.3、反应温度50℃、反应时间2h的条件下,以Gly-Gly为底物时,其比活为433U/mg。同时检测到其对Ile-Leu、Trp-Trp和Phe-Phe具有较高的水解活性。     

人分泌磷蛋白2基因的克隆、原核表达与活性检测

提取人肝癌组织RNA,通过RT-PCR扩增人SPP2成熟蛋白编码区基因并克隆至原核表达载体pET-22b（＋）,将测序正确的质粒转化大肠杆菌BL2l（DE3）,IPTG诱导重组蛋白表达,用Ni-NTA柱进行纯化,SDS-PAGE电泳及Western blot检测并证实目的蛋白的表达,通过重组蛋白对木瓜蛋白酶的抑制作用验证其生物学活性。重组质粒测序和酶切结果显示SPP2成熟蛋白基因已成功克隆到pET-22b（＋）。IPTG诱导重组菌后有25kDa大小的目的蛋白表达。优化诱导表达条件,获得可溶性表达的目的蛋白,纯化后纯度达90%,western blot表明其具有His标签抗原活性。重组SPP2成熟蛋白可抑制木瓜蛋白酶的水解作用（酪蛋白为底物）,重量抑制比为1∶3.1,抑制比活性为2511U/mg。     

南方红豆杉紫杉烷13α-羟化酶基因的克隆及序列分析

目的:紫杉烷13α-羟化酶是紫杉醇下游合成途径关键酶之一,负责催化紫杉二烯-5α-醇的C13侧链发生羟基化反应生成紫杉二烯-5α、13α-二醇.该研究从南方红豆杉中克隆出紫杉烷13α-羟化酶基因并对其序列进行生物信息学分析.方法:利用南方红豆杉的总DNA和总RNA为模板,采用PCR和RT-PCR技术克隆出紫杉烷13α-羟化酶基因的DNA序列和cDNA序列,利用swiss-prot、DNAMAN等生物信息学工具对其核酸序列和蛋白序列进行分析.结果:测序结果显示其cDNA序列长度为1 651bp,含有一个1 458bp的开放阅读框,同源性比较分析结果表明,其氨基酸序列与已经报道的蔓地亚红豆杉的紫杉烷13α-羟化酶氨基酸序列的一致性为96%.结论:成功克隆出南方红豆杉紫杉烷13α-羟化酶基因,为利用合成生物学工程技术生产紫杉醇或其前体物质提供了分子基础.     

野葛异黄酮合酶基因的分离与功能验证

异黄酮是野葛（Pueraria lobata）中的主要活性成分,而异黄酮合酶（IFS）是催化异黄酮生物合成的第一步关键酶,尽管野葛的IFS基因已被分离,但其功能还未得到任何验证。本研究以中国安徽省郎溪县的野葛为材料,利用RT-PCR技术成功克隆到野葛IFS基因,命名为PlIFS,PlIFS开放阅读框大小为1566 bp,编码521个氨基酸,将该基因克隆到GAL1启动子控制下的酵母表达载体pESC-TRP上,得到重组质粒pESC-TRP-PlIFS,通过LiAc/ssDNA/PEG方法将其转化进酿酒酵母（Saccharomyces cerevisiae）WAT11中进行异源表达,并在酵母体内对其活性进行验证,结果显示PlIFS能催化甘草素生成大豆苷元,表现出异黄酮合酶活性特征。荧光定量PCR分析显示,PlIFS基因主要在野葛的根中表达,这与活性物质异黄酮主要在野葛根中的积累模式一致。     

苦荞糖基转移酶基因的克隆及活性鉴定

糖基化修饰在调控各种小分子的溶解度、稳定性及生物活性中具有重要的作用。该研究基于苦荞转录组数据,克隆获得2条糖基转移酶基因（FtUFGT4和FtUFGT5）,并对其在大肠杆菌中的表达产物进行酶催活性鉴定。结果表明：（1）获得的苦荞糖基转移酶基因cDNA分别为1 434和1 470 bp,其编码蛋白同属于拟南芥糖基转移酶E类群,可能参与黄酮类化合物的糖基化。（2）多重序列比对表明,FtUFGT4和FtUFGT5蛋白C端都具有PSPG框,其催化活性位点分别是H17和H16;FtUFGT4和FtUFGT5都是典型的植物糖基转移酶GT B结构,二者的蛋白模型能与矢车菊素和UDP进行分子对接。（3）FtUFGT4和FtUFGT5在大肠杆菌中获得了可溶性表达,薄层层析实验表明二者均具有催化矢车菊素糖基化为矢车菊素 3 O 葡萄糖苷的活性。     

桔小实蝇线粒体基因组全序列及其分析

桔小实蝇Bactrocera dorsalis线粒体基因组全序列对研究实蝇分子系统进化具有重要意义。本研究通过DNA测序和克隆技术,对桔小实蝇mtDNA全序列进行了测定和分析。结果表明：桔小实蝇线粒体基因组全长15 915 bp（GenBank序列号: DQ845759）。基因组碱基组成为39.3%A,16.2%C,10.2%G,34.3%T,由13个蛋白编码基因、22个tRNA基因、2个rRNA基因以及一个非编码的控制区域（A+T-rich区）组成。7个蛋白编码基因和13个tRNA基因从J链编码,其余6个蛋白编码基因和9个tRNA基因从N链编码。位于J链上的蛋白编码基因具有近似的A、T含量,而位于N链上的蛋白编码基因的A的含量明显高于T的含量。以mtDNA COⅠ基因为例,比较了桔小实蝇与其他14种实蝇的亲缘关系,结果显示其与同亚属（果实蝇亚属Bactrocera）内的其他近缘种相互间的同源性很高。     

Metallothionein gene expression and secretion in white adipose tissue

White adipose tissue (WAT) has been examined to determine whether the gene encoding metallothionein (MT), a low-molecular-weight stress response protein, is expressed in the tissue and whether MT may be a secretory product of adipocytes. The MT-1 gene was expressed in epididymal WAT, with MT-1 mRNA levels being similar in lean and obese (ob/ob) mice. MT-1 mRNA was found in each of the main adipose tissue sites (epididymal, perirenal, omental, subcutaneous), and there was no major difference between depots. Separation of adipocytes from the stromal-vascular fraction of WAT indicated that the MT gene (MT-1 and MT-2) was expressed in adipocytes themselves. Treatment of mice with zinc had no effect on MT-1 mRNA levels in WAT, despite strong induction of MT-1 expression in the liver. MT-1 gene expression in WAT was also unaltered by fasting or norepinephrine. However, administration of a beta(3)-adrenoceptor agonist, BRL-35153A, led to a significant increase in MT-1 mRNA. On differentiation of fibroblastic preadipocytes to adipocytes in primary culture, MT was detected in the medium, suggesting that the protein may be secreted from WAT. It is concluded that WAT may be a significant site of MT production; within adipocytes, MT could play an antioxidant role in protecting fatty acids from damage.     

Fucoxanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues

Mitochondrial uncoupling protein 1 (UCP1) is usually expressed only in brown adipose tissue (BAT) and a key molecule for metabolic thermogenesis to avoid an excess of fat accumulation. However, there is little BAT in adult humans. Therefore, UCP1 expression in tissues other than BAT is expected to reduce abdominal fat. Here, we show reduction of abdominal white adipose tissue (WAT) weights in rats and mice by feeding lipids from edible seaweed, Undaria pinnatifida. Clear signals of UCP1 protein and mRNA were detected in WAT of mice fed the Undaria lipids, although there is little expression of UCP1 in WAT of mice fed control diet. The Undaria lipids mainly consisted of glycolipids and seaweed carotenoid, fucoxanthin. In the fucoxanthin-fed mice, WAT weight significantly decreased and UCP1 was clearly expressed in the WAT, while there was no difference in WAT weight and little expression of UCP1 in the glycolipids-fed mice. This result indicates that fucoxanthin upregulates the expression of UCP1 in WAT, which may contribute to reducing WAT weight.     

Morphological characteristics of liver and white adipose tissue during development of melanocortin obesity in C57Bl/6J Agouti yellow mutant mice

The changes of liver and white adipose tissue (WAT) morphology during development of melanocortin obesity in female Agouti yellow (genotype A Y/a) C57Bl/6J mice have been investigated. Mouse strain of a/a genotype was used as a control. Results have been compared with the hormonal and metabolic changes during development of obesity and type 2 diabete in Agouti yellow mice. The tissues obtained from mice of 8-, 11-, 15- and 22-week old have been analyzed. The morphology of liver and WAT of A(Y) and control animals did not differ during 15 weeks of age. 40% of A(Y) mice revealed liver steatosis (fatty liver) at the age of 22 weeks. In addition, elevation in the inflammatory and proliferating processes in the liver and severe inflammation in WAT has been observed in these animals. Since as early as 15 weeks old A(Y) mice demonstrated the appearance of insulin resistance characteristics we conclude that hormonal and metabolic abnormalities could play a role of the primary factor of pathological reorganization of liver and WAT morphology.     

NGF gene expression and secretion in white adipose tissue: regulation in 3T3-L1 adipocytes by hormones and inflammatory cytokines

The sympathetic nervous system plays a central role in lipolysis and the production of leptin in white adipose tissue (WAT). In this study, we have examined whether nerve growth factor (NGF), a target-derived neurotropin that is a key signal in the development and survival of sympathetic neurons, is expressed and secreted by white adipocytes. NGF mRNA was detected by RT-PCR in the major WAT depots of mice (epididymal, perirenal, omental, mesenteric, subcutaneous) and in human fat (subcutaneous, omental). In mouse WAT, NGF expression was observed in mature adipocytes and in stromal vascular cells. NGF expression was also evident in 3T3-L1 cells before and after differentiation into adipocytes. NGF protein, measured by ELISA, was secreted from 3T3-L1 cells, release being higher before differentiation. Addition of the sympathetic agonists norepinephrine, isoprenaline, or BRL-37344 (beta(3)-agonist) led to falls in NGF gene expression and secretion by 3T3-L1 adipocytes, as did IL-6 and the PPARgamma agonist rosiglitazone. A substantial decrease in NGF expression and secretion occurred with dexamethasone. In contrast, LPS increased NGF mRNA levels and NGF secretion. A major increase in NGF mRNA level (9-fold) and NGF secretion (相似文献   

Obesity modulates the expression of haptoglobin in the white adipose tissue via TNFalpha.

Increase in adipose mass results in obesity and modulation of several factors in white adipose tissue (WAT). Two important examples are tumor necrosis factor alpha (TNFalpha) and leptin, both of which are upregulated in adipose tissue in obesity. In order to isolate genes differentially expressed in the WAT of genetically obese db/db mice compared to their lean littermates, we performed RNA fingerprinting and identified haptoglobin (Hp), which is significantly upregulated in the obese animals. Hp is a glycoprotein induced by a number of cytokines, LPS (Lipopolysaccharide), and more generally by inflammation. A significant upregulation of WAT Hp expression was also evident in several experimental obese models including the yellow agouti (/) A(y), ob/ob and goldthioglucose-treated mice (10-, 8-, and 7-fold, respectively). To identify the potential signals for an increase in Hp expression in obesity, we examined leptin and TNFalpha in vivo. Wild type animals treated with recombinant leptin did not show any alteration in WAT Hp expression compared to controls that were food restricted to the level of intake of the treated animals. On the other hand, Hp expression was induced in mice transgenically expressing TNFalpha in adipose tissue. Finally, a significant downregulation of WAT Hp mRNA was observed in ob/ob mice deficient in TNFalpha function, when compared to the ob/ob controls. These results demonstrate that haptoglobin expression in WAT is increased in obesity in rodents and TNFalpha is an important signal for this regulation.     

Up-regulation of genes related to the ubiquitin-proteasome system in the brown adipose tissue of 24-h-fasted rats

The functional balance between brown adipose tissue (BAT) and white adipose tissue (WAT) is important for metabolic homeostasis. We compared the effects of fasting on the gene expression profiles in BAT, WAT and liver by using a DNA microarray analysis. Tissues were obtained from rats that had been fed or fasted for 24 h. Taking the false discovery rate into account, we extracted the top 1,000 genes that had been differentially expressed between the fed and fasted rats. In all three tissues, a Gene Ontology analysis revealed that the lipid and protein biosynthesis-related genes had been markedly down-regulated. The whole-body fuel shift from glucose to triacylglycerol and the induction of autophagy were also observed. There was marked up-regulation of genes in the 'protein ubiquitination' category particularly in BAT of the fasted rats, suggesting that the ubiquitin-proteasome system was involved in saving energy as an adaptation to food shortage.     

GLUT4 protein is differently modulated during development of obesity in monosodium glutamate-treated mice

The aim of the present study was to investigate the GLUT4 protein expression during the development of obesity in monosodium glutamate- (MSG) treated mice. Control (C) and neonatally MSG-treated 2-month-old (2-mo), 4-month-old (4-mo) and 7-month-old (7-mo) mice were analyzed. Anthropometric data, basal glycemia and insulinemia were measured; and the GLUT4 protein was assessed by Western blotting in white adipose tissue (WAT), skeletal muscle gastrocnemius (SM) and heart (H). Compared to age-matched C mice, the 2-mo and 4-mo MSG mice were already obese, but metabolically they showed increased or preserved whole-body insulin sensitivity, respectively. At these ages they showed unchanged total GLUT4 content in SM and H. However, in plasma membrane fraction from WAT, the MSG showed increased GLUT4 content at both 2- (by 60%) and 4-month (by 45%) of age. When the GLUT4 protein was expressed by unit of adipocyte surface area the protein amount was increased by 36 and 220% in 2-mo and 4-mo MSG mice, respectively. At 7 months of age, obesity was fully established in MSG mice, showing a strongly insulin resistant condition. Additionally, in the 7-mo MSG-mice the GLUT4 protein was reduced in SM (by 40%), H (by 28%), PM and M fractions of WAT (by approximately 70%), and PM expressed by unit of adipocyte surface area (by 92%). The data demonstrate that early, during the accelerated development of obesity in MSG-treated mice, the GLUT4 content was increased in WAT, and that may play a key role in the development of obesity. Later on, when obesity is fully established, the GLUT4 protein was reduced in SM, heart and WAT, and that may be involved in the insulin resistance present in this condition.