FT及其同源基因在植物发育调控中的多功能效应

FT是植物开花调控途径的重要整合因子和调控开花的关键基因之一,也是植物可传导开花物质“成花素”的编码基因.FT蛋白是成花素的必需组分,FT mRNA是成花素的潜在组分;FT及其同源基因不仅参与株龄影响及逆境诱导的开花,还参与营养生长、器官形态建成、源库关系和器官脱落调控.该文对近年来国内外有关FT及其同源基因在植物发育调控中的多种功能效应及作用方式进行综述.     

成花素FT的调控运输及生理功能的研究进展

开花作为植物从营养生长到生殖生长的转折点,受外界环境变化影响,对植物发育和繁殖有着至关重要的作用。成花素(flowering locus T FT)蛋白作为拟南芥开花信号核心分子,在开花调控中扮演重要角色。拟南芥叶片在感应外界温度光照等因素变化后,通过生理钟(constants,CO)等蛋白及部分Mico-RNA正负协调调控筛管伴胞中的FT基因表达,FT蛋白通过筛管从叶片运输到茎顶端分生组织后,与一个碱性亮氨酸拉链(basic leucine zipper,b ZIP)蛋白FD结合调控茎顶端分生组织(shoot apical meristem,SAM)中的花组织形成相关基因表达,继而诱导开花。对近年来FT基因在叶片中的调控、FT蛋白的运输及其在顶端分生组织中的开花诱导机理进行综述,为进一步完善FT表达调控及功能研究提供参考。     

甘蓝型油菜BnCOP1基因的原核表达和纯化

COP1蛋白是植物光信号调节网络中介导光信号转导的一个关键因子,对植物生长发育起重要调控作用.通过RT-PCR方法从甘蓝型油菜中克隆BnCOP1基因编码区全长序列,并将其连接到冷诱导原核表达载体pCold TF上,构建原核重组表达载体pCold TF/BnCOP1,转化大肠杆菌BL(21)star.通过IPTG法低温诱导表达融合蛋白.结果表明,重组质粒在大肠杆菌中获得高效表达,融合蛋白分子质量约为128 kD,融合蛋白在上清液和包涵体中均有表达,可溶性部分经亲和层析纯化和Western blotting检测证实获得了高纯度的重组蛋白,这些结果为进一步研究油菜BnCOP1的结构和功能奠定了基础.     

毛白杨NBS型基因PtDRG01原核表达研究

为了分析毛白杨NBS型抗病基因PtDRG01(EF157840)编码蛋白的特性,研究构建了该基因的原核表达载体pGEX-Pt01,并导入大肠杆菌XA90,经IPTG诱导表达后,SDS-PAGE电泳分析证明,该特异蛋白的分子量约为79kD。对原核表达体系以及融合蛋白的表达特性进行优化与分析表明:最佳诱导表达条件为1mmol/L IPTG在37℃下诱导4h,所表达的融合蛋白为胞内分泌的可溶性蛋白。利用谷胱苷肽-琼脂糖亲和层析柱纯化获得了电泳纯级的目标蛋白,为PtDRG01基因编码蛋白的功能鉴定研究奠定了基础。     

痢疾志贺菌侵袭素IpaC的原核表达及纯化复性

目的为进一步研究痢疾志贺菌侵袭素IpaC蛋白的侵袭活性,必须先获得IpaC重组融合蛋白。方法将IpaC基因亚克隆至表达载体pET-24α(+)中,转化至E.coli BL21(DE3)表达宿主菌中,采用IPTG优化诱导表达,然后纯化复性重组蛋白。结果重组蛋白最佳表达条件为0.50mmol/L IPTG、30℃诱导6h,获得分子量约为33kDa的IpaC重组蛋白。Western blotting证实了重组蛋白的特异性。IpaC重组蛋白主要以包涵体形式在宿主菌中表达,通过纯化后得到单一的目的蛋白。结论成功构建IpaC原核表达体系,并获得了蛋白的高效表达及优化。初步建立了IpaC重组蛋白的纯化方案。为进一步研究IpaC的侵袭性作用奠定了基础。     

黄瓜过敏性诱导反应蛋白基因(CsHIR1)原核表达及其多克隆抗体制备

该试验用黄瓜霜霉菌侵染黄瓜幼苗,并通过PCR方法克隆其过敏性诱导反应蛋白(HIR)的基因CsHIR1,构建原核表达载体pET28a-CsHIR1,实现在大肠杆菌(E.coli)BL21(DE3)中的高效表达;对诱导表达的时间和IPTG的浓度进行了优化;利用钴离子螯合层析纯化了重组蛋白并制备高效价多克隆抗血清。结果表明:该黄瓜过敏性反应诱导蛋白以包涵体的形式表达,最佳诱导时间和IPTG浓度分别为4h和0.5mmol·L-1;经纯化,得到高纯度的分子量为34kD重组蛋白CsHIR1。Western blotting显示CsHIR1的抗体具有较好的特异性。原核表达体系的建立和多克隆抗体的制备为进一步研究CsHIR1基因在黄瓜中的功能奠定了基础。     

小麦细胞分裂素氧化酶基因TaCKX1原核表达载体的构建及表达

目的:构建小麦细胞分裂素氧化酶基因TaCKXI原核表达载体并进行表达,以期得到大量的His标签融合蛋白.方法:根据GenBank中的TaCKXI序列以及pET-24a载体中的多克隆位点设计引物,以含有TaCKXl编码基因的批pMD-QRCKXI重组质粒为模板,经PCR扩增得到TaCKXI基因的DNA片段.将所得的片段与pET-24a载体连接,转化DH5α大肠杆菌,筛选阳性克隆,其测序结果与原序列一致,表明原核表达载体pET-TaCKXI已构建成功.提取per-TaCKXI质粒转化到BL21(DE3)pLysS表达菌株中,经IPTG诱导后收集菌体进行SDS-PAGE电泳鉴定,并优化其表达条件.结果:在大肠杆菌中获得TaCKXI基因融合表达,主要以包涵体形式存在;融合蛋白的分子量为58.91kD;IPTG终浓度为0.5、1.0、1.5.2.0mmol/L时,诱导融合蛋白产量相差不大.选用0.5mmol/L诱导15h获得大量的融合蛋白.经用原核表达蛋白纯化试剂盒纯化,得到了单一的融合蛋白.结论:小麦TaCKXI基因在大肠杆菌中获得了高效表达,为今后TaCKXI蛋白多克隆抗体的制备奠定了基础.     

肺炎克雷伯菌调控蛋白RcsAB的构建表达及活性鉴定

为了探讨转录调控子Rcs AB对靶基因的转录调控作用,构建肺炎克雷伯菌RcsA和RcsB的重组质粒,之后诱导蛋白表达,提取纯化后测定其活性。PCR得到rcsA、rcsB片段,分别将两DNA片段克隆至表达载体pMAL-C5X、pET28a,构建重组质粒。再将重组质粒导入E. coli BL21(DE3)菌株中,经IPTG诱导后收集菌体,超声破碎。破碎后的上清过柱、透析纯化,得到高纯度的蛋白,通过EMSA进行蛋白活性鉴定。RcsA,RcsB蛋白成功表达纯化,并能够与靶基因结合,初步证明蛋白具有生物学活性。成功制备有生物学活性的RcsA、RcsB蛋白,为进一步研究RcsAB蛋白复合物特异的生物学功能提供物质基础。     

拟南芥酪蛋白激酶1A的原核表达及纯化

拟南芥酪蛋白激酶1A(Arabidopsis casein kinase 1A,At CK1A)属于丝氨酸/苏氨酸蛋白激酶家族,其可能参与了蓝光信号转导并调控植物开花时间。通过对At CK1A的全长序列进行生物信息学分析得到了其激酶区;采用RT-PCR的方法从拟南芥c DNA中扩增At CK1A激酶区,并构建至p ET28a原核表达载体;将表达载体导入BL21(DE3)菌株后,经IPTG诱导表达和Ni亲和层析、阳离子交换层析以及凝胶过滤层析纯化,得到高纯度的重组蛋白;最后利用His标签抗体对重组蛋白进行了验证。     

SDHB蛋白的表达、纯化及多克隆抗体制备

SDHB(succinate dehydrogenage complex,subunit B)基因可能介导呼吸链生物功能和调控细胞生长.采用PCR技术扩增出SDHB基因,并将其连接到pGEX-4T-1原核表达载体中,经酶切及测序鉴定后,转化BL21细菌,并用IPTG诱导表达融合蛋白,谷胱甘肽琼脂糖珠亲和纯化,将纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western blot检测抗体.获得了SDHB原核表达重组融合蛋白及高效价的特异性兔抗SDHB多克隆抗体,为SDHB进一步的功能研究奠定了基础.     

乳糖诱导木聚糖酶基因在大肠杆菌中的可溶性表达

以构建好的大肠杆菌工程菌BL21(DE3)/xylanase为研究对象,研究了以IPTG和乳糖作为诱导剂时重组蛋白的表达规律。在摇瓶发酵条件下研究了诱导剂浓度、诱导时机、诱导培养时间和诱导培养温度对目标蛋白表达的影响。实验结果表明,乳糖作为诱导剂时,重组菌产酶活力33.9 U/mg略高于IPTG作为诱导剂时重组菌产酶活力28.10 U/mg,这为乳糖作为诱导剂应用于重组大肠杆菌生产木聚糖酶提供了参考依据。     

Optimization of an extracellular zinc-metalloprotease (SVP2) expression in Escherichia coli BL21 (DE3) using response surface methodology

In this work, SVP2 from Salinivibrio proteolyticus strain AF-2004, a zinc metalloprotease with suitable biotechnological applications, was cloned for expression at high levels in Escherichia coli with the intention of changing culture conditions to generate a stable extracellular enzyme extract. The complete ORF of SVP2 gene was heterologously expressed in E. coli BL21 (DE3) by using pQE-80L expression vector system. In initial step, the effect of seven factors include: incubation temperature, peptone and yeast extract concentration, cell density (OD600) before induction, inducer (IPTG) concentration, induction time, and Ca(2+) ion concentrations on extracellular recombinant SVP2 expression and stability were investigated. The primary results revealed that the IPTG concentration, Ca(2+) ion concentration and induction time are the most important effectors on protease secretion by recombinant E. coli BL21. Central composite design experiment in the following showed that the maximum protease activity (522 U/ml) was achieved in 0.0089 mM IPTG for 24h at 30 °C, an OD600 of 2, 0.5% of peptone and yeast extract, and a Ca(2+) ion concentration of 1.3 mM. The results exhibited that the minimum level of IPTG concentration along with high cell density and medium level of Ca(2+) with prolonged induction time provided the best culture condition for maximum extracellular production of heterologous protease SVP2 in E. coli expression system.     

HER2胞外段的克隆与原核表达

应用RT-PCR技术从人乳腺癌细胞系SK-BR-3中克隆出人表皮生长因子受体2(human epidermal growth factorreceptor 2,HER2)基因的胞外段,并插入到表达载体pET-30a中,得到重组表达载体pET30-HER2(Ex)。将该载体转化至大肠杆菌BL21(DE3)细胞中,加入IPTG进行诱导表达,成功获得HER2胞外段蛋白。分别提取培养液上清、大肠杆菌周质腔、细胞质可溶性及不可溶性组分蛋白进行SDS-PAGE电泳分析,确定目的蛋白定位于大肠杆菌细胞质包涵体中。通过改变诱导温度、诱导物浓度、诱导起始菌体密度和诱导时间,寻找最佳表达条件,使目的蛋白的表达量达到最高。结果表明,在37℃下,OD600达到1.0时,经终浓度为0.1 mmol/L的IPTG诱导4 h,目的蛋白的表达量最高。将重组表达菌进行超声破碎,分离出包涵体组分,经Ni2+亲和层析纯化后获得了纯度>90%的HER2胞外段蛋白,从而为抗HER2抗体的制备及肿瘤疫苗的研究奠定了基础。     

拟南芥中柯浩体蛋白Atcoilin的克隆、表达及纯化

旨在制备柯浩体的标志蛋白——Atcoilin蛋白,利用pET-28a与目的基因构建重组表达质粒,经DNA测序证实插入序列与设计完全一致后,将重组质粒转化大肠杆菌BL21(DE3),用IPTG进行诱导表达,产物用SDS-PAGE及Western blotting分析鉴定。通过分别改变IPTG的浓度、培养时间、培养温度等来优化Atcoilin蛋白的表达条件。表达出的重组蛋白经过镍柱、分子筛进行纯化。结果显示,原核表达载体pET28a-At1g13030成功构建,可在大肠杆菌BL21(DE3)中诱导表达,得到相应的重组蛋白经Western blotting鉴定正确。在IPTG浓度为0.7 mmol/L,18℃培养20 h的条件下,目的蛋白表达量最高。经过SDS-PAGE分析鉴定,过镍柱、分子筛后得到的重组蛋白纯度较高。     

Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage

The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage.     

单链抗体2F3表达条件的优化及其提纯和性质研究

将构建好的单链抗体 2F3表达载体pTMF2F3ScFv转化到大肠杆菌BL21(DE3)。先挑选出表达量高的单克隆 ,然后让其在 37℃进行表达 ,并将表达时的培养条件进行优化。实验结果表明 :最佳诱导条件为开始诱导时的菌体密度OD_590nm =1.0～ 1.8,所加异丙基β-D 硫代半乳糖苷 (IPTG)的浓度为 0.3～0.5mmol/L ,诱导时间 7h ,优化后目的蛋白表达量占菌体总蛋白的20% ,并用发酵罐成功地进行了扩大培养 ,筛选了洗涤包涵体的最佳条件。采用两步法对包涵体复性进行了研究 ,用Westernblotting及ELISA法检测了所表达的单链抗体及其生物活性 ,并成功制备了含硒单链抗体酶.     

Polycationic amino acid tags enhance soluble expression of Candida antarctica lipase B in recombinant Escherichia coli

Lipase (EC 3.1.1.3) is a popular enzyme used as an ingredient in detergents and biocatalyst in many biochemical reactions. Lipase is usually expressed in Escherichia coli as an inactive inclusion body and at a low level. In this study, Candida antarctica lipase B (CalB) was fused with various polycationic amino acid tags and expressed in E. coli in order to increase a soluble expression level. By induction with 1.0 mM IPTG, the authentic and fused CalBs were expressed at 27-56% of total protein. The 10-arginine and 10-lysine tags fused at the C-terminal of CalB significantly increased the solubility of CalB by five- to ninefold, relative to the case of the authentic CalB expressed in a recombinant E. coli Origami 2? (DE3) strain. Among a series of the C-terminal poly-arginine tags, the recombinant CalB combined with the 10-arginine tag (CalB-R10) possessed the highest lipase specific activity of 9.5 ± 0.03 U/mg protein, corresponding to a fourfold enhancement compared with the authentic CalB.     

用E．coli表达Canstatin—N及其表达条件优化

以重组质粒DET—CN为模板设计引物CASN1N和CASN2,PCR方法扩增约267bp的人血管能抑素N端1～89氨基酸基因片段,用EcoRI和Sal I双酶切将其克隆进pET-22b（＋）载体获得重组表达质粒pET-22b（＋）一CN,转化E．coliBL21（DE3）,用IPTG诱导表达Canstatin-N,产物以包涵体形式存在。本文在摇瓶发酵条件下研究了诱导剂浓度、诱导培养时间对目标蛋白表达的影响,结果表明IPTG的最佳诱导浓度为0．1mmol／L;37℃下诱导培养2h时产物表达量最高。纯化获得的融合hiS6的重组Canstatin—N具有免疫和抑制鸡胚绒毛尿囊膜（CAM）新生血管生成活性。     

Enhanced expression of bovine growth hormone gene by different culture conditions in Escherichia coli

To optimize the production of bovine growth hormone (bGH) in E. coli, the cells harboring pUBJ10 plasmid, which contains the modified 59-coding region of bGH cDNA under the control of trc promoter, was induced to express under various culture conditions such as medium (LB or M9CA), temperature, induction stage, expression time, IPTG concentration, and hosts. Induction stage was effective at early logarithmic phase. The expression levels of bGH were not largely affected by IPTG concentrations, slightly greater in LB medium than in M9CA medium, and efficient in 4 to 6 h of expression time. The highest level of bGH production was obtained in E. coli BL21 strain.     

乳糖代替IPTG诱导己糖激酶在大肠杆菌中的表达

构建了己糖激酶GLK高效表达的工程菌株BL21(DE3)/pET-glk,考察了乳糖代替IPTC诱导己糖激酶GLK在大肠杆菌BL21(DE3)中表达的可行性.实验结果表明,工程菌对数生长中期(OD<,600>约为1.0)添加终浓度为10 g/L的乳糖于25℃的条件下诱导6 h能获得最大量的目的蛋白和菌体量,目的蛋白表达...