棉花半胱氨酸蛋白酶抑制剂基因的克隆、表达与活性研究

通过从已构建好的突变体湘棉-18的cDNA文库中筛选分离出一个半胱氨酸蛋白酶抑制剂基因的全长cDNA序列,经测序和序列分析,发现该片段包含一个完整的开放阅读框,长378个碱基,编码125个氨基酸,氨基酸序列中包含了一个半胱氨酸蛋白酶抑制剂家族的保守区段Gln-Val-Val-Ala-Gly;信号肽预测发现该序列包含一个N端信号肽,保守区分析该基因编码的氨基酸包含一个半胱氨酸蛋白酶抑制剂相似区域.半胱氨酸蛋白酶抑制剂在大肠杆菌中表达重组蛋白,在条件为30℃,IPTG 5mmol/L,时间24～36h时,CPI实现最优表达,Western Blotting分析表明表达的蛋白正确.通过木瓜蛋白酶活性抑制实验,结果表明该重组CPI具有一定的酶活抑制作用.     

BmNPV半胱氨酸蛋白酶基因的核苷酸序列研究

对家蚕核型多角体病毒苏州株（BmNPVsu）半胺氨酸蛋白酶基因（CP）的序列分析表明，该基因读码框为972个核苷酸，编码323个氨。同源性分析表明，BmNPVsu CP的氨基酸序列与不同来源的木瓜蛋白酶超家庭的CP具有较高的同源性，特别与Trpanosomabrucei的CP具有较高的一致性，达32％。在组织蛋白酶B、H、L、S以及木瓜蛋白酶的36个保守氨基酸残基中有31种出现在BmNPUsu的CP中，BmNPVsuCP同其他杆状病毒CP一样，可看作木瓜蛋白酶超家庭成员。     

半胱氨酸蛋白酶基因RNA干扰表达载体的构建及油菜的遗传转化

将半胱氨酸蛋白酶基因(Bncp5)cDNA序列片段正反向插入表达载体pFGC5941中,构建了RNA干扰表达载体pFGC-Bncp5-RNAi.采用根癌农杆菌介导法,转化甘蓝型油菜下胚轴.在5mg/L BastaMS培养基中筛选,PCR鉴定结果显示,获得Bncp5转基因植株28株.半定量PCR分析结果显示,半胱氨酸蛋白酶基因在转基因油菜中表达受到一定程度的抑制,表明Bncp5的干扰载体在油菜中得到表达.     

Programmed cell death during terminal bud senescence in a sympodial branching tree, Eucommia ulmoides

Eucommia ulmoides Oliv. is a typical sympodial branching tree. The apical bud of the branch ages and dies every year, replaced by the nearby axillary bud in the second year. Structural assays and a series of biochemical analyses were performed to analyze the senescence mechanism in the apical bud. It was revealed that most cells of the apical bud underwent the programmed cell death (PCD) during the senescence: the chromosomes were congregated and the nuclear contents were condensed, as shown by 4′,6-diamidino-2-phenylindole (DAPI) fluorescence. DNA fragmentation was detected during senescence using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end in situ labeling (TUNEL) method, coincident with the appearance of a DNA ladder. Moreover, a 20 kD DNase related to fragmentation was found. PCD was initiated first in the young leaves, leaf primordia and peripheral zone cells, then in the central mother cells and initial layer cells in the apical meristem. The terminal buds remain in vegetative growth during senescence, in contrast to buds of many annual plants.     

棉花GhGAI1基因克隆及其功能的初步分析

DELLA蛋白是赤霉素信号通路中重要的蛋白质作用因子,负调节植物体中赤霉素的水平,同时受到生长素、脱落酸和乙烯的共同调节。根据已知的生物信息数据,本研究克隆了棉花GhGAl1基因,序列分析表明它编码DELLA蛋白。构建GFP与GhGAl1融合蛋白重组质粒,利用根癌农杆菌介导的花浸泡转基因方法转化拟南芥columbia生态型植株,进行过量表达实验。结果表明,GFP—GhGAl1融合蛋白定位于细胞核并且在施加赤霉素后迅速降解,表明该基因编码蛋白参与赤霉素信号通路。     

Identification of a senescence-related protease in coriander leaves

Senescence-related protease may play an important role in leaf senescence. By improved SDS-Gela-tin-PAGE assay, a 63 ku senescence-related protease (63 SRP) in coriander leaves was identified. Activity of 63 SRP was increased in parallel to the advance of coriander leaf senescence, and inhibited by treating the leaf with gibberellic acid, and enhanced by ethylene treatment. The 63 SRP was suggested to be a serine protease based on the fact that its activity was inhibited by the protease inhibitor PMSF. The optimal temperature for the activity of the 70 ku protease was 50℃. The maximal activity was observed at pH 6-9, some activity could be observed on the gel slices incubated at pH 5 or 11. The 63 SRP was partly purified by the way of ammonium sulfate precipitation and then gel slicing after gel elec-trophoresis.     

豌豆顶芽衰老过程中的显微、超微结构和nDNA的变化

显微、超微结构研究表明 ,短日照条件下豌豆顶芽的衰老过程是从营养生长锥向花芽的转化 ,而用DNA原位末端标记 (TUNEL)、Caspase 8WesternBlot和 14 0bpDNA片断积累的试验结果证明 ,转化为花芽的整个生长锥细胞发生了编程性死亡 (PCD) ,而且其最顶端部分细胞首先发生PCD ,而顶端周围的分生组织细胞逐渐分化出花芽的各部分 ,但顶芽最后并没有发育成为完整的花 ,所有细胞就都发生PCD ,从而顶芽衰老     

Cloning and expressional characterization of soybean GmLls1 gene

In higher plants, green leaf is a very important part involved in the photosynthesis process. Good develop-ment of the leaves is the guarantee of high yields and quality. Leaf senescence that occurs in the last stage of leaf development is a genetically p…     

Structure and function of bak gene and its apoptosis-inducing mechanism

The Bcl-2 family plays an important role in the regulation of apoptosis. Some members in the family prevent apoptosis while others promote it. The newly discovered bcl-2 homolog, bak, promotes the apoptosis induced by a variety of stimuli. It can interact with the anti-apoptotic members through its BH3 domain and antagonize their activity. Investigation results suggest that the expression of bak gene becomes altered in some diseases, such as cancer. The preferential stimulation of Bak-induced apoptosis, therefore, may be involved in the mechanisms of some an-titumor drugs.     

Fine mapping of the red plant gene R_1 in upland cotton (Gossypium hirsutum)

Sub 16 is a substitution line with G. hirsutum cv. TM-1 genetic background except that the 16th chro-mosome (Chr. 16) is replaced by the corresponding homozygous chromosome of G. barbadense cv. 3-79, and T586 is a G. hirsutum multiple gene marker line with 8 dominant mutation genes. The R1 gene for anthocyanin pigmentation was tagged in Chr. 16 in T586. The objective of this research was to screen SSR markers tightly linked with R1 by using the F2 segregating population containing 1259 plants derived from t...     

Cloning and sequence analysis of a gene encoding polygalacturonase-inhibiting protein from cotton

Polygalacturonase-inhibiting proteins (PGIP) play important roles in plant defense of pathogen, especially fungi. A pair of degenerated primers is designed based on the conserved sequence of 20 other known pgip genes and used to amplify Gossypium barbadense cultivation 7124 cDNA library by touch-down PCR. A 561 bp internal fragment of the pgip gene is obtained and used to design the primers for rapid amplification of cDNA ends. A composite pgip gene sequence is constructed from the products of 5′ and 3′ RACE, which are 666 bp and 906 bp respectively. Analysis of nucleic acid sequence shows 69.2% and 68.7% similarity to Citrus and Poncirus pgip genes, respectively. Its open reading frame of the gene encodes a polypeptide of 330 amino acids, in which 10 leucine-rich repeats arrange tandemly. A new set of primers is designed to the 5′ and 3′ ends of the gene, which allows amplification of the full-length gene from the cotton cDNA library. Genomic DNA analysis reveals that this gene has no intron.     

Cloning and molecular characterization of a gene encoding MAP kinase from maize and its expression in E. coli

A new MAPK gene, ZmSIMK1 （Zea mays L. salt-induced mitogen-activated protein kinase 1）, is isolatod from a maize eDNA library. The full-length ZmSIMK1 gene contains 1636 bp and an open reading frame of 1122 nucleotides capable of eneoding 373 amino acid polypeptides with a predicted molecular mass of 42.3 kda and pI of 6.01. The putative ZmSIMK1 protein contains all 11 conserved subdomains that are characteristics of serine/threonine protein kinases and the TEY motif, which is the putative phosphorylation site. Northern blot analysis shows that ZmSIMK1 is ubiquitously expressed in roots, stems, and leaves of maize seedlings and its mRNA accumulation is observed in maize seedlings treated with 30 mmol/L PEG-6000 and 137 mmol/L NaCl, but the expression of ZmSIMK1 is not significantly affected by 4℃ treatment. The expression vector pET-ZmSIMK1 is constructed by inserting the coding region of ZmSIMK1 eDNA into pET-42a（＋）, and transformed into E. coli strain BL21（DE3）. A 77kda fusion protein is induced by the further culture at 37℃ after addition of 1 mmol/L IPTG.     

Cloning and characterization of a gene encoding phage-related tail protein (PrTP) of endosymbiont Wolbachia

Wolbachia is an obligatory, maternally inherited intracellular bacterium, known to infect a wide range of arthropods. It has been implicated in causing cytoplasmic incompatibility (CI), parthenogenesis, the feminization of genetic males and male-killing in different hosts. However, the molecular mechanisms by which this fastidious bacterium causes these reproductive abnormalities have not yet been determined. In this study, we report on the cloning and characterization of the gene encoding phage-related tail protein (PrTP) from Wolbachia in Drosophila melanogaster CantonS (wMelCS) and from Wolbachia in Drosophila melanogaster yw67c23 (wMel) by representational difference analysis (RDA) and ligation-mediated PCR (LM-PCR). The functionality of a bipartite nuclear localization signal sequence (NLS) of the gene was also successfully tested in Drosophila S2 cells. PrTP expression in various strains of Wolbachia was investigated. Our results suggest that PrTP may not induce CI directly. However, the existence of prtp provided direct evidence of phage-mediated horizontal gene transfer (HGT) that might play an important role in a variety of reproductive abnormalities of Wolbachia.     

Cloning and characterization of a gene encoding phagerelated tail protein (PrTP) of endosymbiont Wolbachia

Wolbachia is an obligatory, maternally inherited intracellular bacterium, known to infect a wide range of arthropods. It has been implicated in causing cytoplasmic incompatibility (CI), parthenogenesis, the feminization of genetic males and male-killing in different hosts. However, the molecular mechanisms by which this fastidious bacterium causes these reproductive abnormalities have not yet been determined. In this study, we report on the cloning and characterization of the gene encoding phage-related tail protein (PrTP) from Wolbachia in Drosophila melanogaster CantonS (wMelCS) and from Wolbachia in Drosophila melanogaster yw67c23 (wMel) by representational difference analysis (RDA) and ligation-mediated PCR (LM-PCR). The functionality of a bipartite nuclear localization signal sequence (NLS) of the gene was also successfully tested in Drosophila S2 cells. PrTP expression in various strains of Wolbachia was investigated. Our results suggest that PrTP may not induce CI directly. However, the existence of prtp provided direct evidence of phage-mediated horizontal gene transfer (HGT) that might play an important role in a variety of reproductive abnormalities of Wolbachia.     

Cloning of murine BRI3 gene and study on its function for inducing cell death

To understand the molecular mechanism of TNFα effects, the cDNA of murine BRI3 gene was cloned from the total RNA of murine brain endothelial cells (bEnd.3)treated with hTNFα by using the suppression subtractive hybridization (SSH) and the RT-PCR method. The fusion expression vector harbouring BRI3 gene and enhanced green fluorescence protein (EGFP) thus obtained were designated as pEGFP/I3. Then pEGFP/I3 was transiently transfected into L929 cells and the fusion protein EGFP/I3 was localized in cytoplasm. It is found that the expression of EGFP/I3 could induce cell death in L929 cells detected by TUNEL method and flow cytometry. And the overexpression of Bci-2 in L929 cells can block cell death induced by EGFP/I3, indicating that murine BRI3 gene might related to the TNFα mediated cytotoxicity.     

Cloning of a novel gene encoding human thioredoxin-like protein

mRNA differential display (DDRT-PCR) has been used to analyze different human fetal brain tissues of different developmental stages (13- and 33-week). According to the sequence of one EST obtained in this assay, a pair of primers have been designed to screen the arrayed human fetal brain cDNA library. A1 .2-kb cDNA clone has been found. This cDNA consists of an 867 bp open reading frame, a 132 bp 5' untranslated sequence and a 209 bp 3' untranslated sequence with a typical polyadenylation signal. The coding region predicts a protein of 289 amino acids. Its N-terminal of 105 residues is highly homologous to human thioredoxin, while no homology has been found in the databases with its C-terminal of 184 residues. Its N-terminal region also contains the conserved active site sequence CGPC (Cys-Gly-Pro-Cys) of thioredoxin. It was named human Thioredoxin-like gene (hTRXL).     

Cloning and characterization of a new actin gene from Oryza sativa L.

Using Rho family member osRACD as bait, a new member of actin gene family -Act was isolated from Oryza sativa by yeast two-hybrid system. The full-length cDNA was cloned with 5' RACE technology, which contains an open reading frame of 1134 bp with a predicted protein of 377 amino acids. Sequence alignment revealed 96% to 81.8% identities with some known actin proteins in plants. The method of bioinformatics was used to analyze the protein modification sites, structure and evolution of the gene. Southern blot analysis showed that Act is a single-copy gene in the genome. The result of RT-PCR showed it is ubiquitously expressed in root, shoot, callus and panicle in a temporal fashion. The relationship between Rho family and actin family in evolution and function was also studied.     

器官发育及细胞程序化死亡的基因调节研究

用线虫实验模型系统研究调节器官发育及细胞程序化死亡的基因，且发现这种基因在人体中也存在，这一成果获得了2002年诺贝尔生理学医学奖。“程序性细胞死亡”机理的发现为艾滋病、肿瘤和癌症等疾病的治疗提供了寻找新方法的可能，将在人类战胜疾病过程中发挥重要作用。