纹枯病菌诱导水稻表达的WRKY基因克隆及分析

[目的]克隆纹枯病菌诱导的水稻上调表达基因.[方法]对应用抑制差减杂交枝术(SSH)获得的纹枯病菌诱导水稻表达的EST序列K16进行电子克隆,根据电子克隆产物设计引物进行RT-PCR、克隆测序,利用生物信息学技术对克隆产物进行功能预测.[结果]克隆出一个长度为1079bp的序列,结构功能分析显示该基因编码236个氨基酸的蛋白,存在多个功能位点,含有2个典型的WRKY结构域和1个C2H2锌指型结构,该基因与水稻WRKY8、WRKY24和WRKY30基因有较高的同源性.[结论]经电子克隆获得的纹枯病菌诱导表达的基因为典型的WRKY家族基因,该基因可能在水稻抗纹枯病中起重要作用.     

水稻PHD锌指蛋白cDNA的克隆及其表达分析

利用cDNA-AFLP分析籼稻明恢86应答稻纵卷叶螟取食基因差异表达,发现1个与植物同源结构域(PHD)锌指蛋白高度同源的TDF,分离获得该TDF对应的粳稻全长cDNA.该全长cDNA与籼稻、玉米、蓖麻、葡萄、拟南芥和大豆等作物PHD锌指蛋白推定氨基酸序列的同源性分别为98.43%、86.01%、54.58%57.02%、57.51%和55.88%.荧光定量PCR研究表明,日本晴(粳稻)叶片中该基因的表达也受稻纵卷叶螟取食的诱导,推测该基因与籼稻和粳稻应答稻纵卷叶螟取食密切相关.     

D-甘露糖异构酶在大肠杆菌中的表达及催化条件的研究

根据已知的放射性土壤杆菌体内的D-甘露糖异构酶(Fmi)氨基酸序列,设计适合在大肠杆菌内表达的Fmi基因序列(1245 bp),采用体外基因拼接合成.构建了表达Fmi的重组大肠杆菌BL21(DE3)/pET30-Fmi,诱导表达的Fmi蛋白经SDS-PAGE验证为可溶性蛋白,分子量44000.通过实验优化了Fmi的催化条件,结果表明Fmi催化的最适pH值为7.5,最适温度为45℃,催化1 h酶活达5.29 U/mL.以25%的果糖溶液为底物时,催化2 h果糖转化率达29.4%.     

前期诱导在解剖教学中的应用探讨

目的:探讨分析前期诱导在解剖学教学中的应用方法及效果.方法:选取84名学生为研究对象,按入学成绩排名的单双数号分为两组,实验组采用前期诱导的方式进行教学,对照组采用传统教学模式,通过同课时的教学后问卷调查对课程的的满意度情况及理论与实验考核成绩情况.结果:实验组课程设计满意率为95.2%,对照组课程满意度为83.3%,两组对课程设计的满意度差异有统计学意义(P<0.05).两组在理论和实验考核的平均成绩及优秀(90分以上)、良好(80～90分)差异有统计学意义(P<0.05).结论:前期诱导在解剖学教学中能够较好的提高学生积极性,减少学生的陌生感和恐惧程度,调整心态,自主掌握发现问题后解决问题的能力,针对内容陌生,与以前接触少的解剖知识教学,学生满意度较高,且对学生的成绩有较好的提升.     

鸡Mx蛋白在293T细胞中的表达及其抗病毒活性

[目的]研究鸡Mx 蛋白在293T 细胞中的表达并检测其抗病毒活性.[方法]将鸡的Mx基因克隆入pEGFP-C1栽体,经筛选鉴定后磷酸钙法转染293T 细胞,并对转染细胞进行荧光分析以及RT-PCR与Western blot鉴定,同时提取细胞蛋白质,微量细胞病变试验检测其抗新城疫病毒的活性.[结果]试验构建的pEGFP-C1-cMx真核表达栽体转染293T 细胞后,在胞浆中可检测到绿色荧光,RT-PCR与Western blot 结果进一步证实,pEGFP-C1-cMx可在293T细胞中表达,微量细胞病变试验显示,表达的融合蛋白可保护鸡胚成纤维细胞免受新城疫病毒感染.[结论]试验构建的pEGFP-C1-cMx 表达载体可在293T细胞中表达具有抗新城疫病毒活性的鸡Mx 蛋白.     

水稻谷氨酸脱羧酶基因(OsGAD3)分子进化与表达分析

利用分子克隆技术从8个水稻品种中克隆到谷氨酸脱羧酶基因OsGAD3,并进行了相关的生物信息学及表达谱分析.结果表明,不同水稻品种OsGAD3的核苷酸序列差异性很小,都在1%以内,在氨基酸序列上都具有保守的结构域,在C末端均具有类似于OsGAD1结合CaM的关键氨基酸位点,但不同于OsGAD2.系统进化树分析表明,不同水稻品种的OsGAD3在进化上可以分为粳稻和籼稻两大类,与水稻常规分类相一致.此外,对不同水稻品种的谷氨酸脱羧酶基因的表达谱分析发现,OsGAD1、OsGAD2、OsGAD3、OsGAD4和OsGAD5在测试的11种水稻叶片中都有表达,但表达特征存在一定的差异性,可能与品种特性相关.     

黄鳝抗菌肽hepcidin基因大肠杆菌表达载体的构建

[目的]构建黄鳝抗菌肽hepcidin基因大肠杆菌表达栽体.[方法]根据Genbank中黄鳝hepcidin基因序列设计引物进行PCR扩增,将测序正确的基因片段连接入栽体pET-28a中.[结果]以黄鳝cDNA为模板,扩增出hepcidin基因片段,长度为291 bp,连接到pET-28a上.经PCR扩增、双酶切验证,证实成功构建原核表达栽体.[结论]成功构建了黄鳝hepcidin基因大肠杆菌表达载体.     

信号转导及转录活化因子3蛋白在乳腺癌组织中的表达及其临床意义

目的:检测人信号转导及转录活化因子3(STAT3)蛋白在乳腺癌组织中的表达,探讨其与乳腺癌患者临床病理参数及预后的关系.方法:应用免疫组织化学SP法检测67例乳腺癌及其配对正常乳腺组织中STAT3蛋白的表达水平,分析具有不同临床病理参数患者间STAT3的表达情况.结果:STAT3蛋白在乳腺癌组织的表达水平明显高于正常乳腺组织(P<0.001),STAT3表达水平与乳腺癌患者年龄、肿瘤大小和组织学类型无关联性(P>0.05),与乳腺癌临床分期、淋巴结转移情况及患者生存时间有关联(P<0.05).临床分期越高的乳腺癌组织中STAT3的表达阳性率越高(P<0.05);有淋巴结转移者STAT3阳性表达率明显高于无淋巴结转移者(P<0.05);STAT3阳性表达的患者生存时间[(51.1±4.4)月]明显短于阴性表达的患者[(86.0±4.9)月](P<0.05).结论:STAT3蛋白的过表达可能在乳腺癌的发生发展过程中发挥了促进作用,并且是乳腺癌患者预后不良的一个重要指标.     

玉米单倍体诱导系及其在育种中的应用研究

利用单倍体诱导系选育玉米自交系这一新的育种方法能够快速提供来自供体亲本的稳定纯系,大大缩短自交系选育年限,加快育种进程,提高选育效率.就玉米单倍体诱导系在育种应用中的3个方面进行了论述与探讨,揭示了提高单倍体诱导系诱导率和建立高效的单倍体加倍方法,以及对单倍体诱导系诱导性状的遗传规律和诱导机制研究等问题是目前单倍体诱导系在玉米育种应用中亟待解决的问题.玉米单倍体诱导系在玉米育种中显示出巨大的应用前景.     

基于灰色关联度的DTOPSIS方法在水稻优选中的应用

针对水稻品种优选的DTOPSIS方法中权重给定的主观性,提出利用灰色关联度来确定权重的方法.所获得的评价矩阵无量纲化,然后计算性状的关联度矩阵,并用列和归一化方法导出权重,进而利用DTOPSIS方法对水稻品种排序、择优.     

Expression of nuclear hormone receptors in Escherichia coli

If streptococci and staphylococci remain the main bacteria responsible for infective endocarditis (80%), the emergence of Streptococcus bovis associated with intestinal lesions, the confirmation of the role of Coxiella burnetii, and the discovery of the responsibility of Bartonella sp in case of negative blood culture endocarditis have been the principal microbiological modifications during the last few years. Blood cultures performed under the best technical conditions and the examination and culture of the valve after surgery remain the better means for diagnosing infective endocarditis. In case of negative blood culture (11% of the infective endocarditis), the serologic tests against C. burnetii and Bartonella should decrease the rate of diagnostic uncertainty. The development of bacteriological techniques, particularly molecular methods, should improve the microbiology of infective endocarditis lead to the discovery new aetiological agents.     

Expression of regulated cardiac troponin I in Escherichia coli

The study of the functional effects of troponin isoform changes would be greatly aided by the development of a strategy permitting protein engineering and mutational analysis. To assess the role of troponin isoforms in regulating myofibrillar ATPase activity, we have expressed rat cardiac troponin I (cTnI) in E. coli and purified the protein to near homogeneity. We utilized the inducible expression vector pGEX-KG to create a glutathione-S-transferase fusion protein which can be cleaved with thrombin. Approximately 6 mg of cTnI can be purified from 1 l of culture. Ca2+Mg2+ ATPase activity was measured using the bacterially synthesized cTnI and the remaining components of the regulated actomyosin complex (troponin T, troponin C, tropomyosin, actin, and myosin) purified to homogeneity from mammalian hearts. In the presence of free Ca2+ ranging from 10(-2) to 10(-8) M, bacterially synthesized cTnI exhibits specific activity similar to that observed for control cTnI isolated from rat hearts. The bacterially synthesized protein is capable of stoichiometric phosphorylation and demonstrates appropriately regulated specific activity. These results establish the feasibility of using bacterial expression to study functional consequences of changes in expression of troponin isoforms.     

Expression of a neutral horseradish peroxidase in Escherichia coli

Many measures of depression severity appear confounded by including depressive sub-typing features. We report the design of a brief (11 item) self-report scale of depression severity (the AUSSI), assessing both mood state and social impairment domains, and designed to be independent of sub-typing features. Mood severity and functional impairment scores demonstrated some independence in a sample of 360 patients. Patients with a 'melancholic' depressive type (categorised by four differing systems) differed from residual 'non-melancholic' depressed patients by having higher impairment scores, but the assigned groups did not differ, in the main, by mood severity scores. Advantages of the measure are summarised.     

Protein expression in response to folate stress in Escherichia coli

Interruption of folate metabolism by trimethoprim results in the elevated expression of folate stress proteins in Escherichia coli. E. coli grown in culture medium supplemented with the folate-dependent metabolites glycine, methionine, and the purine nucleoside inosine shows reduced expression of folate stress proteins. The folate stress proteins include the universal stress protein, the ferric uptake regulatory repressor, and possibly, lipoamide dehydrogenase, the L protein component of the glycine cleavage enzyme complex.     

Expression of the Streptomyces griseus alpha-amylase gene in Escherichia coli

The amy gene of Streptomyces griseus was not expressed in Escherichia coli cells due to the lack of recognition of the amy promoter by the E. coli RNA polymerase, as confirmed by using promoter-probe vectors. The expression of the amy gene in E. coli was detected only when the promoter-less gene was placed under the control of the lacZ promoter and was dependent on the level of IPTG added to the medium. The extracellular alpha-amylase detected in the culture broth seems to be released by cellular lysis. When the amy gene lacking both leader peptide and promoter was transcribed from the lacZ promoter, no alpha-amylase activity was detected but larger E. coli cells and inclusion bodies were observed.     

Expression of active human tissue-type plasminogen activator in Escherichia coli

The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 microgram of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins in E. coli without the need for in vitro refolding.     

Expression of bovine mitochondrial tRNASer GCU derivatives in Escherichia coli

By replacing a stretch of five A-U base pairs in the acceptor stem with G-C pairs, mitochondrial tRNA-SerGCU lacking a D arm could be expressed in Escherichia coli cells in considerable amounts. The expressed tRNA with no modified nucleoside was serylated in vitro with the mitochondrial enzyme. The tRNASerGCU derivatives carrying identity elements for alanine tRNA and the related anticodons were expressed. However, this expression event did not affect cell growth, probably because the expression started from the late log phase, which suggests that these mitochondrial tRNA derivatives are not involved in E.coli gene expression systems. Although there are some restrictions in the secondary structure of tRNAs that can be expressed by this method, it could prove useful for preparing large amounts of heterologous tRNAs in vivo.     

Expression of mutant horse cytochrome c genes in Escherichia coli

Here we describe genetically engineered constructs for the expression in Escherichia coli of genes for horse cytochrome c mutants. These constructs allow the expression of the cytochrome c genes together with hemeligase, an enzyme which covalently links heme to cytochrome. Careful selection of producer strains and the adjustment of the conditions of expression provided for expression levels of 10-15 mg of protein per liter of culture. This is by an order of magnitude greater than the expression previously achieved in yeast. A series of horse cytochrome c mutants were obtained in this way.     

Expression and purification of HIV-I p15NC protein in Escherichia coli

M2 is a minor component of the influenza A virus envelope. The cytoplasmic tail of the M2 protein is posttranslationally modified in the infected cell by palmitylation and phosphorylation. The primary site for phosphorylation of the M2 cytoplasmic tail is serine 64, which is highly conserved yet not required for the activity of the M2 ion channel. Using an exogenous incorporation assay, we have shown that incorporation of M2 into virus particles is type-specific and does not require phosphorylation of the cytoplasmic tail. In addition, phosphorylation of the cytoplasmic tail is not required for the directional transport of M2 in polarized MDCK cells. Using a reverse genetics and reassortment procedure, we generated a virus (Ra) specifically mutated in segment 7 such that the M2 cytoplasmic tail could no longer be phosphorylated. The virus was found to grow as well as wild-type virus in tissue culture and in eggs, was stable on passage in these systems, and possessed no second-site mutations in the engineered RNA segment. In vivo Ra replicated in Balb/c mice at least as well as the parent strain A/WSN/33. These studies indicate that phosphorylation of the M2 cytoplasmic tail is not required for in vitro or in vivo replication of influenza A virus.     

Expression of ribonucleolytic toxin restrictocin in Escherichia coli: purification and characterization

OBJECTIVES: To define the incidence and severity of ovarian hyperstimulation syndrome (OHSS) occurring in oocyte donors. METHODS: Women (n = 149) aged 31.3 +/- 4.8 years (mean +/- S.D., range 21-41 years) participated as designated oocyte donors and underwent 400 consecutive cycles of controlled ovarian stimulation using human menopausal gonadotropin following pituitary downregulation with gonadotropin-releasing agonist. Patients were monitored by serial transvaginal ultrasound examinations and serum estradiol (E2) determinations. Oocytes (15.6 +/- 7.5 per aspiration; range 2-57) were harvested by ultrasound-directed transvaginal follicle aspiration 36 h following the intramuscular injection of human chorionic gonadotropin (hCG). Follow-up examination occurred 1 and 2 weeks post-aspiration. RESULTS: On the day of hCG injection E2 levels ranged from 512 to 13,502 pg/ml (mean 2902.7 +/- 1486.9 pg/ml). Over the next few weeks the degree of hyperstimulation in donors was staged: mild 65% (grade I, n = 98; grade II, n = 162); moderate 33.5% (grade III, n = 120; grade IV, n = 14); severe 1.5% (grade V, n = 6; grade VI, n = 0). Associated preaspiration E2 levels were: grade I, 1120 +/- 424 pg/ml; grade II, 2084 +/- 613 pg/ml; grade III, 3785 +/- 1713 pg/ml; grade IV, 5370 +/- 1264 pg/ml; grade V, 4286 +/- 1100 pg/ml. Worsening OHSS was associated with increasing levels of E2. There were no serious complications and hospitalization was not required. All symptoms resolved within 30 days of aspiration, disappearing by the time of the first menstrual flow in women of grade-III or lower stage. CONCLUSION: Although oocyte donors commonly experienced exaggerated levels of serum E2 they rarely (< 2%) developed severe OHSS. This may be attributable to their lack of embryo transfer which avoids exacerbating the illness.