中华眼镜蛇短链神经毒素cDNA的克隆及在大肠杆菌中的表达

利用RT-PCR 技术从中华眼镜蛇毒腺组织中成功地克隆了短链神经毒素cDNA。测序结果表明,该基因开放阅读框架编码83个氨基酸残基,其中21个为信号肽,成熟肽为62个氨基酸残基。该基因与GenBank 报道的相同物种的神经毒素基因有相当的同源性,不同物种之间的信号肽序列十分保守。将短链神经毒素cDNA再经PCR扩增除去信号肽序列,克隆到pT7ZZ表达质粒中,转化E. coli BL21(DE3)后,经IPTG诱导可高效表达分子量为23kDa②左右的融合蛋白。表达产物占菌体总蛋白的25%左右。 Abstract:A novel short-chain neurotoxin cDNA was cloned from Chinese cobra venom by RT-PCR. The cDNA was cloned into the pGEM-T vector and sequenced. It has a ORF encoding 83 amino acid residues and a 21 residues signal peptide. This neurotoxin gene of Chinese cobra was highly homogeneous to the short-chain neurotoxin gene of similar species reported in GenBank. Among the genes of neurotoxin from different species, the signal peptides were very conserved. The cDNA encoding the mature peptide was amplified by PCR and was cloned into pT7ZZ vector. The recombinant vector was transformed into E. coliBL2(DE3). The E. coli highly expressed the fusion protein whose mollecular weight is 23kDa, after induced by 0.1 mol/L IPTG. The expressed protein was accumulated up to more than 25% of total bacterial protein.     

野油菜黄单胞菌S-152内切葡聚糖酶基因在大肠杆菌中的克隆与表达

以质粒pUC9为载体,大肠杆菌JM83为宿主菌,用鸟枪法克隆到纤维素降解细菌野油菜黄单胞菌S-152的内切葡聚糖酶(CMCase)基因,克隆到的CMCas e基因位于2.7kb的HindⅢ片段上,该重组质粒命名为pUC9H-1。Southern印迹杂交分析结果显示所克隆到的内切葡聚糖酶基因与野油菜黄单胞菌染色体DNA有亲和性。对克隆株的酶学性质分析表明,其CMCase活力为0.310μmol Glu/mg蛋白·分钟,最适作用pH为6.4,最适作用温度为55℃。 Abstract:Using plasmid pUC9 as vector,Escherichia coli JM83 as host strain,an endoglucanase(CMCase)gene has been cloned by Shoot-gun method from cellulose-degrading bacteria Xanthomonas campestris S-152,The recombinant plasmid pCU9H-1 was isolated from the positive transformant producing CMCase.The CMCase gene located in a 2.7kb HindIII DNA fragment.The result of Southern hybridization indicated that the CMCase gene cloned has affinity to chromosome DNA of Xanthomonas campestris S-152.The analysis of enzymatic activity of CMCase positive clone JM83(pUC9H-1)was carried out and the result indicated that the CMCase activity was about 0.310μmol Glu/mg pro·min,the optimum pH was 6.4 and the optimum temperature was 55℃.     

幽门螺杆菌肽脱甲酰基酶基因在大肠杆菌中的可溶性表达与活性检测

幽门螺杆菌肽脱甲酰基酶（PDF）的可溶性表达是基于PDF靶位新药筛选的前提。本研究在克隆肽脱甲酰基酶基因（螂后,构建了其融合表达载体pTrcHisB-def,并在不同宿主菌（大肠杆菌BL21、DH5a和JM109）中进行了诱导表达。结果显示,只有在BL21（DE3）中获可溶性表达,产物经纯化后具有肽脱甲酰基酶活性。以上研究为PDF特性分析及PDF抑制剂筛选奠定了基础。     

牛乳铁蛋白素(1-15)-蜂毒素(5-12)杂合肽基因的合成及在大肠杆菌中的表达

为了构建新型的杂合肽,设计了一种新型杂合抗菌肽牛乳铁蛋白素(1-15)-蜂毒素(5-12),由牛乳铁蛋白素(LfcinB)N端第1~15个氨基酸残基和蜂毒素(Melittin)N端第5~12个氨基酸残基组成。根据大肠杆菌密码子的偏爱性,设计合成了杂合肽LfcinB(1-15)-Melittin(5-12)的基因片段,插入到表达载体pET-32a的NcoI和SalI的酶切位点之间,构建重组表达质粒。重组表达质粒转化到Escherichia coli BL21(DE3)中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,融合蛋白以可溶形式在Escherichia coli BL21(DE3)中获得成功表达,表达量占菌体总蛋白的35%以上,每升培养物可获得35mg融合蛋白。带His标签的融合蛋白经His-Bind纯化试剂盒纯化、肠激酶切割和抑菌实验表明,杂合肽具有明显的抑菌效果。这为利用基因工程方法生产抗菌肽奠定了理论基础。     

碱性β-甘露聚糖酶基因异源表达的研究

亚克隆的碱性β-甘露聚糖酶基因(man)来源于嗜碱芽孢杆菌N16-5,构建了大肠杆菌-枯草芽孢杆菌诱导型表达质粒pDG-man,在大肠杆菌JM109中获得了活性表达,经0.5 mmol/L的IPTG诱导后,可表达5 U/mL碱性β-甘露聚糖酶。重组大肠杆菌DE3-RIL(pDG-man)表达β-甘露聚糖酶水平是大肠杆菌JM109(pDG-man)的2倍。重组枯草芽孢杆菌WB600(pDG-man)可表达19.2 U/mL碱性β-甘露聚糖酶。     

大肠杆菌RNaseⅢ基因的克隆与表达

目的：克隆并在原核表达系统中表达RNaseⅢ基因。方法：提取大肠杆菌JM109株的基因组DNA,以之为模板扩增得到RNaseⅢ基因全序列,将该编码序列克隆到原核表达载体pET-22b（＋）中,转化大肠杆菌BL21（DE3）,IPTG诱导重组RNaseⅢ表达。结果与结论：在大肠杆菌中克隆到了RNaseⅢ的全基因,经测序证明与数据库中报道的序列一致;表达的重组RNaseⅢ主要以包涵体形式存在。     

鸡传染性法氏囊病病毒内蒙古毒株VP2基因的克隆和序列分析

以鸡传染性法氏囊病病毒内蒙古毒株（ＩＢＤＶ-ＮＭ）ｄｓＲＮＡ为模板,用ＲＴ-ＰＣＲ法扩增其主要寄主保护抗原ＶＰ２基因的全长ｃＤＮＡ,克隆于ｐＵＣ１９的ＸｂａＩ／ＫｐｎＩ位点,进行了全序列分析。序列比较发现ＩＢＤＶ-ＮＭ毒株ＶＰ２基因与已报道的其它７个ＩＢＤＶＣＪ８０１ｂｋｆ、Ｃｕ１、ＰＢＧ９８、５２／７０、００２—７３、ＳＴＣ及ＶａｒｉａｎｔＥ毒株之间高度同源,其核苷酸序列的同源率为９１.６％～９６．２％,推测的氨基酸序列的同源率为９６.２％～９８．６％。ＩＢＤＶ-ＮＭ毒株ＶＰ２高变异区的第一个亲水区氨基酸序列与ＣＪ８０１ｂｋｆ、Ｃｕ１、ＰＢＧ９８、５２／７０、ＳＴＣ、００２—７３比较,有一个氨基酸差异,第二个亲水区氨基酸序列与上述６个毒株完全相同。而与ＶａｒｉａｎｔＥ比较,两个亲水区内各有两个氨基酸差异。此外,ＩＢＤＶ-ＮＭ毒株ＶＰ２具有强毒株所特有的７肽保守区：ＳＷＳＡＳＧＳ。这些结果表明,ＩＢＤＶ-ＮＭ毒株为标准血清Ⅰ型ＩＢＤＶ强毒株。     

广州地区106例汉族人群MICA和MICB微卫星基因座 单体型研究

利用聚合酶链反应和荧光（6－FAM）自动化检测技术对广东地区汉族106例无亲缘关系样本进行MICA基因外显子5和MICB基因内含子1微卫星基因座多态性及其单体型分布调查。根据群体资料估算两者间的单体型频率、连锁不平衡参数、相对连锁不平衡参数。结果显示,广州地区汉族人群MICA和MICB微卫星基因座基因型分布符合Hardy-Weinberg平衡法则,共检出MICA微卫星基因座 5个等位基因, MICB微卫星基因座14个等位基因。其中MICA A5基因频率最高（0.2877）,A4基因频率最低（0.1321）。MICB CA14等位基因频率最高（0.3255）,CA19、CA28等位基因频率最低（0.0047）,未检出CA27。21种MICA－MICB单体型频率大于1%(连锁不平衡参数>0), 其中单体型A5－CA14 (16.73%), A5.1－CA18 (8.75%), A4－CA26(3.76%),A9－CA15(3.66%)和A6－CA21(2.61%)为强连锁常见单体型（χ2>3.84, P<0.05）。广州地区汉族人群MICA和MICB微卫星基因座多态性和单体型分布有其自身特点,MICA和MICB微卫星基因座适合做为遗传标志,用于人类学、遗传疾病基因连锁分析、法医学亲子鉴定和个体识别等研究领域。Abstract: This study is to investigate genetic polymorphisms and haplotypes of microsatellite locus in the exon 5 of the MICA gene and intron 1 of the MICB gene based on 106 samples of Guangzhou Han Population by polymerase chain reaction and fluorescent technique (6-FAM). The corresponding haplotype frequencies, linkage disequilibria values and relative linkage disequilibria values were estimated based on population data. The results show that the genotype distributions of MICA and MICB microsatellite meet Hardy-Weinberg equilibrium in Guangdong Han population. In total, 5 alleles of MICA microsatellite locus and 14 alleles of MICB microsatellite locus were observed. MICA A5 was the most common allele (0.2877), whereas A4 was the least popular one (0.1321). MICB CA14 was the most common allele (0.3255), and CA19 and CA28 were the least popular ones (0.0047). CA27 was not observed. Twenty-one kinds of MICA-MICB haplotypes occurred at frequencies of more than 1% (linkage disequilibria value>0). The common MICA-MICB haplotypes were A5-CA14（16.73％）, A5.1- CA18 (8.75%), A4- CA26(3.76%),A9-CA15(3.66%) and A6-CA21(2.61%)（χ2>3.84, P<0.05）, and they were strong linkage disequilibria. The polymorphisms and haplotypes distributions of MICA and MICB microsatellite locus in Guangzhou Han population have their own genetic characteristics. The microsatellite locus of the exon5 of the MICA gene and intron 1 of the MICB gene could be used as the genetic markers in the studies of anthropology, linkage analysis of genetic disease genes, individual identification and paternity test in forensic medicine.     

广州地区106例汉族人群MICA和MICB微卫星基因座 单体型研究 The Study on the Haplotype of MICA and MICB Microsatellite Locus In Guangzhou Han Population

利用聚合酶链反应和荧光（6－FAM）自动化检测技术对广东地区汉族106例无亲缘关系样本进行MICA基因外显子5和MICB基因内含子1微卫星基因座多态性及其单体型分布调查。根据群体资料估算两者间的单体型频率、连锁不平衡参数、相对连锁不平衡参数。结果显示，广州地区汉族人群MICA和MICB微卫星基因座基因型分布符合Hardy-Weinberg平衡法则，共检出MICA微卫星基因座 5个等位基因， MICB微卫星基因座14个等位基因。其中MICA A5基因频率最高（0.2877），A4基因频率最低（0.1321）。MICB CA14等位基因频率最高（0.3255），CA19、CA28等位基因频率最低（0.0047），未检出CA27。21种MICA－MICB单体型频率大于1%(连锁不平衡参数>0), 其中单体型A5－CA14 (16.73%), A5.1－CA18 (8.75%), A4－CA26(3.76%)，A9－CA15(3.66%)和A6－CA21(2.61%)为强连锁常见单体型（χ2>3.84, P<0.05）。广州地区汉族人群MICA和MICB微卫星基因座多态性和单体型分布有其自身特点，MICA和MICB微卫星基因座适合做为遗传标志，用于人类学、遗传疾病基因连锁分析、法医学亲子鉴定和个体识别等研究领域。Abstract: This study is to investigate genetic polymorphisms and haplotypes of microsatellite locus in the exon 5 of the MICA gene and intron 1 of the MICB gene based on 106 samples of Guangzhou Han Population by polymerase chain reaction and fluorescent technique (6-FAM). The corresponding haplotype frequencies, linkage disequilibria values and relative linkage disequilibria values were estimated based on population data. The results show that the genotype distributions of MICA and MICB microsatellite meet Hardy-Weinberg equilibrium in Guangdong Han population. In total, 5 alleles of MICA microsatellite locus and 14 alleles of MICB microsatellite locus were observed. MICA A5 was the most common allele (0.2877), whereas A4 was the least popular one (0.1321). MICB CA14 was the most common allele (0.3255), and CA19 and CA28 were the least popular ones (0.0047). CA27 was not observed. Twenty-one kinds of MICA-MICB haplotypes occurred at frequencies of more than 1% (linkage disequilibria value>0). The common MICA-MICB haplotypes were A5-CA14（16.73％）, A5.1- CA18 (8.75%), A4- CA26(3.76%)，A9-CA15(3.66%) and A6-CA21(2.61%)（χ2>3.84, P<0.05）, and they were strong linkage disequilibria. The polymorphisms and haplotypes distributions of MICA and MICB microsatellite locus in Guangzhou Han population have their own genetic characteristics. The microsatellite locus of the exon5 of the MICA gene and intron 1 of the MICB gene could be used as the genetic markers in the studies of anthropology, linkage analysis of genetic disease genes, individual identification and paternity test in forensic medicine.     

孔雀活化素基因(activin)ßA 亚基成熟肽序列的分子克隆及其对白孔雀起源与分类的佐证分析

参考已经克隆的活化素(activin)基因βA亚基成熟肽序列,设计一对兼并引物,从绿孔雀(pavo muticus)、蓝孔雀(pavo cristatus)和白孔雀基因组中克隆到活化素基因βA亚基成熟肽序列。测序结果表明,活化素基因βA亚基成熟肽序列长345bp,编码115个氨基酸。序列分析表明,蓝孔雀与绿孔雀核苷酸同源性为98.0%,而蓝孔雀与白孔雀核苷酸同源性为98.8%。NCBI收索结果显示,活化素基因βA亚基成熟肽序列在不同物种间都非常保守。氨基酸功能位点分析表明,活化素βA亚基成熟肽可能在细胞信号传递过程中发挥了很重要的作用。另外,利用活化素基因βA亚基成熟肽序列构建了三种孔雀的限制性酶切图谱及系统发生树。结果显示,白孔雀与蓝孔雀的亲源关系比与绿孔雀的亲源关系近。我们认为,白孔雀来源于蓝孔雀,很可能是蓝孔雀一个杂交后代或亚种,而不是人们通常所认为的仅仅是蓝孔雀的一个颜色突变体。Abstract: The sequences of activin geneβA subunit mature peptide have been amplified from white peafowl, blue peafowl (pavo cristatus) and green peafowl (pavo muticus) genomic DNA by polymerase chain reaction (PCR) with a pair of degenerate primers. The target fragments were cloned into the vector pMD18-T and sequenced. The length of activin gene βA subunit mature peptide is 345bp, which encoded a peptide of 115 amino acid residues. Sequence analysis of activin gene βA subunit mature peptide demonstrated that the identity of nucleotide is 98.0% between blue peaflowl and green peafowl, and the identity of that is 98.8% between blue peaflowl and white peafow. Sequences comparison in NCBI revealed that the sequences of activin geneβA subunit mature peptides of different species are highly conserved during evolution process. In addition, the restriction enzyme map of activins is high similar between white peafowl and blue peafowl. Phylogenetic tree was constructed with Mega 2 and Clustalxldx software. The result showed that white peafowl has a closer relationship to blue peafowl than to green peafowl. Considered the nucleotide differences of peafowls’ activin geneβA subunit mature peptides, a highly conserved region, we supported that white peafowl was derived from blue peafowl, and it is more possible the hybrid but just the product of color mutation, or maybe as a subspecies of Pavo genus.     

Characterisation of SMN hybrid genes in Spanish SMA patients: de novo, homozygous and compound heterozygous cases

Autosomal recessive spinal muscular atrophy (SMA) is classified, by age of onset and maximal motor milestones achieved, into type I (severe form), type II (intermediate form) and type III (mild/moderate form). SMA is caused by mutations in the survival motor neuron telomeric gene (SMN1) and a centromeric functional copy of this gene (SMN2) exists, both genes being located at 5q13. Homozygous deletion of exons 7 and 8 of SMN1 has been detected in approx 85% of Spanish SMA patients regardless of their phenotype. Nineteen cases with the sole deletion of exon 7 but not exon 8 (2 cases of type I, 13 cases of type II, four cases of type III) were further analysed for the presence of SMN2-SMN1 hybrid genes. We detected four different hybrid structures. Most of the patients were carriers of a hybrid structure: centromeric intron 6- centromeric exon 7- telomeric exon 8 (CCT), with or without neuronal apoptosis-inhibitor protein (NAIP). In two patients, a different hybrid structure, viz. telomeric intron 6- centromeric exon 7- telomeric exon 8 (TCT), was detected with or without NAIP. A phenotype-genotype correlation comparing the different structures of the hybrid alleles was delineated. Type I cases in our series are attributable to intrachromosomal deletion with a smaller number of SMN2 copies. Most cases with hybrid genes are type II occurring by a combination of a classical deletion in one chromosome and a hybrid gene in the other. Type III cases are closely associated with homozygozity or compound heterozygozity for hybrid genes resulting from two conversion events and have more copies of hybrid genes and SMN2 than type I or II cases.     

水稻杂种一代与亲本幼苗基因表达差异的分析

杂种优势是一种普遍存在的生物学现象,其形成的原因十分复杂。本世纪初,Bruce和Shull相继提出的杂种优势形成的显性互补假设和超亲优势假设至今仍作为一种理论模型而缺乏实验证实。水稻杂种优势的利用自70年代三系配套技术建立得到了广泛的应用,但水稻杂种优势形成的遗传学基础目前还知之甚少。在水稻杂种优势形成机理研究中,分别从生理生化代谢、同工酶分析、DNA限制性片段多态性和DNA含量差异进行了分析,但杂种优势形成的分子机理仍未得到阐明。杂种优势的形成是与异质化相关的过程,它涉及到两个遗传背景不同的体系的相互作用。因此,在相互作用过程中,亲本基因的表达与调控就决定了杂种一代的基因表达类型和特性。因此,我们从分析基因表达与调控入手,运用mRNA差异展示技术分析了玉米杂种一代与亲本基因表达的差异,揭示了不少有意义的现象。本研究以水稻籼型杂交组合(汕优63:珍汕97A×明恢63)为材料,探讨水稻杂种一代与亲本基因表达的差异,揭示了杂种优势形成过程中的一些重要现象。     

Herpes simplex virus type 1/adeno-associated virus rep(+) hybrid amplicon vector improves the stability of transgene expression in human cells by site-specific integration

Herpes simplex virus type 1 (HSV-1) amplicon vectors are promising gene delivery tools, but their utility in gene therapy has been impeded to some extent by their inability to achieve stable transgene expression. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). A rep(-) HSV/AAV hybrid amplicon vector was made by inserting a transgene cassette flanked with AAV ITRs into an HSV-1 amplicon backbone, and a rep(+) HSV/AAV hybrid amplicon was made by inserting rep68/78 outside the rep(-) vector 3' AAV ITR sequence. Both vectors also had a pair of loxP sites flanking the ITRs. The resulting hybrid amplicon vectors were successfully packaged and compared to a standard amplicon vector for stable transduction frequency (STF) in human 293 and Gli36 cell lines and primary myoblasts. The rep(+), but not the rep(-), hybrid vector improved STF in all three types of cells; 84% of Gli36 and 40% of 293 stable clones transduced by the rep(+) hybrid vector integrated the transgene into the AAVS1 site. Due to the difficulty in expanding primary myoblasts, we did not assess site-specific integration in these cells. A strategy to attempt further improvement of STF by "deconcatenating" the hybrid amplicon DNA via Cre-loxP recombination was tested, but it did not increase STF. These data demonstrate that introducing the integrating elements of AAV into HSV-1 amplicon vectors can significantly improve their ability to achieve stable gene transduction by conferring the AAV-like capability of site-specific genomic integration in dividing cells.     

Strategy and utilization of a herbicide resistance gene in two-line hybrid rice

Hybrid rice plays an important role in China's aim to improve rice production as it accounts for some 50% of rice planting area but produces about 60% of the total rice grain. However, the existing three-line system used in hybrid rice production has its limitations. The two-line system, which makes use of photoperiod-sensitive genic male-sterile (PGMS) and thermo-sensitive genic male-sterile (TGMS) lines to generate the male-sterile parental line, was developed to overcome some of these limitations. The sterility of the male-sterile line of two-line hybrid rice, however, fluctuates when the temperature-sensitive phase of fertility encounters abnormal low temperatures during hybrid seed production, which induces selfing and decreases the purity of hybrid. We describe here the strategy of utilizing a herbicide resistance gene in two-line hybrid rice to eliminate this fluctuation in the sterility of the P/TGMS lines during hybrid seed production and reports the development of the herbicide resistance restorer line Bar68-1 and its herbicide-resistant early season hybrid rice Xiang125s/Bar68-1. When the restorer line and its derived hybrid are herbicide resistant, the selfed seeds can be removed easily from the hybrid by herbicide spraying. A herbicide resistance gene bar was transferred into a restorer line by particle bombardment. The resulting transgenic restorer line Bar68-1 and its hybrid Xiang125 s/Bar68-1 inherited stable herbicide resistance. The purity of Xiang125s/Bar68-1 was increased by spraying the seed bed with herbicide, which resulted in a significant increase in yield, grain quality, and disease resistance in comparison to the controls in a regional trial.     

中国特有小麦中杂种黄化基因Chl和提型胞质育性恢复基因的分布研究

本研究以Ｔ型不育系ＱＡ１１０４（具有杂种黄化基因Ｃｈ２）和Ｋｈａｐｌｉ（具有杂种黄化基因Ｃｈ１）为测验种,对中国特有小麦等六倍体小麦类型和一些四倍体小麦类型中的Ｔ型胞质育性恢复基因和杂种黄化Ｃｈ１基因的分布进行了研究。结果表明：中国白麦子类型、西藏半野生小麦、云南铁壳麦、圆锥小麦（矮兰麦）一节节麦人工合成双二倍体以及中国圆锥小麦等类型中未发现Ｔ型育性恢复基因和杂种黄化基因Ｃｈ１;在斯卑尔脱小麦杜哈米林类型和野生二粒小麦中发现有Ｔ型育性恢复基因的存在,但是不存在杂种黄化基因Ｃｈ１。在新疆稻麦和中国波兰小麦中未发现有Ｔ型胞质育性恢复基因的存在。但在新疆稻麦中普遍含有杂种黄化基因Ｃｈ１,在波兰小麦中一些居群有Ｃｈ１基因、一些居群无。这暗示：新疆稻麦可能来源于含有Ｃｈ１基因的波兰小麦类型,而且可能是起源于波兰小麦与节节麦的天然杂交并经过双二倍体化途径而形成的。     

一种新融合抗菌肽Hex-Mag基因的克隆、表达及其抗菌活性的研究

通过在Magainin1-12(GIGKFLHSAKKF)的N端添加碱性氨基酸片段Hexapeptide(RRWQWR)以增强其对细胞膜的吸附能力来提高Magainin1-12的抗菌活性。利用Hexapeptide和Magainin1-12的基因序列,结合酵母偏爱密码子设计出新的融合基因Hex-Mag,通过重叠区扩增基因拼接法(Gene splicing by overlap extension,gene SOEing)利用PCR扩增出基因片段,再将融合基因进行酶切并纯化后导入穿梭质粒pPIC9中,构建受乙醇氧化酶1基因(AOX1)的启动子与转录终止区控制的酵母表达质粒,转化GS115毕赤酵母宿主菌,经表型筛选,阳性克隆用甲醇诱导表达。表达出的融合肽Hex-Mag分子量约2.3kDa,其耐热性强,在100℃条件下,其活性可维持3h以上。琼脂糖孔穴扩散法检测显示Hex-Mag对多种革兰氏阴性菌和阳性菌具有抑制活性,与Magainin1-12相比,其活性有明显增强,N端正电荷增加的预期效应得到初步体现。     

TA29-barnase基因转化甘蓝产生雄性不育植株

用PCR技术从烟草革新1号品种的总DNA中扩增了TA29基因的启动子和从解淀粉芽孢杆菌的总DNA中扩增了核糖核酸水解酶基因(barnse),将其构建成融合基因,并克隆于pCAMBIA2301载体上。通过根癌农杆茵介导转化甘蓝下胚轴,经Km选择压下连续选择、扩繁和进行生根培养,获得了甘蓝转基因植株。经GUS、PCR和Southernblot检测,证明TA29-bar-nase融合基因已经整合至转基因植株的染色体中。经花器官观察,转基因植株中有雄蕊退化的雄性不育和半不育植株出现。用正常花粉对不育株进行人工授粉,不育株能正常结实,这表明转基因不育植株的雌性器官发育正常,其不育性与TA29-barnse融合基因在转基因植株中的表达有关。     

Highly efficient gene expression of a fibrinolytic enzyme (subtilisin DFE) in Bacillus subtilis mediated by the promoter of α-amylase gene from Bacillus amyloliquefaciens

Subtilisin DFE is a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4. The promoter and signal peptide-coding sequence of alpha-amylase gene from B. amyloliquefaciens was cloned and fused to the sequence coding for pro-peptide and mature peptide of subtilisin DFE. This hybrid gene was inserted into the Escherichia coli/Bacillus subtilis shuttle plasmid vector, pSUGV4. Recombinant subtilisin DFE gene was successfully expressed in B. subtilis WB600 with a fibrinolytic activity of 200 urokinase units ml(-1).