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.     

生长素结合蛋白研究进展

植物细胞感受生长素的信号是由生长素结合蛋白和生长素分子相结合。从而引起生长素信号传递的一系列级联反应而完成的．生长素与其结合蛋白的结合是生长素引发生理反应的第一步，也是必需的过程．     

Expression of lysine-rich protein gene and analysis of lysine content in transgenic wheat

Expression vector pBPC102, which carries winged bean lysine-rich protein (wblrp) gene and dihydropicolinate synthase (DHDPS) gene, was transferred into hexaploid winter wheat cv. Jinghua No.l, Jing411, You899 and Yangnongl5 explants of immature inflorescence and immature embryos by particle bombardment. More than 100 transgenic plants were obtained under the selection of s-(2-aminoethyl)-L-cysteine (AEC). Confirmed transgenic plants of To and TI generation by PCR and PCR-Southern blotting analyses showed successful integration of wblrp gene into wheat genome. Analysis of transgenic plant lines of T2 by Northern dot-blotting showed good expression of wblrp gene in offspring seed. The content of free lysine in leaves, contents of bound lysine and total proteins in seeds of T2 transgenie wheat lines were determined and analyzed. Among 34 tested transgenic lines, levels of free lysine content in leaves of 9 transgenic lines are 2~3times higher than un-trans-formed wild-type cultivars. Among 17 analyzed transgenic lines, bound lysine content of 4 transgenic lines is more than 10% higher than that of wild-type cultivars. Our research suggests that introducing wblrp gene into wheat is an effective way to improve its nutrition quality.     

菠菜43 kD叶绿素a结合蛋白编码基因克隆及原核表达条件优化

将菠菜43 kD叶绿素结合蛋白编码基因亚克隆至原核表达载体pET-28a上,构建重组表达质粒pET-28a-psbC转化感受态E.coli BL21(DE3),经IPTG诱导实现了外源基因的可溶性表达.探讨了含组氨酸标签融合蛋白的最佳表达条件(包括表达单克隆、时间、温度对表达的影响).利用Ni2+-Sepharose 4 Fast Flow亲合层析纯化出His-apo CP43蛋白.体外重组实验证实 His-apo CP43能特异结合叶绿素a,经过温和电泳分离纯化所得体外重组色素蛋白复合物具有与提取自类囊体膜的天然CP43相似(但不完全相同)的荧光特性.     

人Rab蛋白cDNA的克隆和表达

从人胎脑cDNA文库中克隆到一种新的Rab cDNA,全长920bp,以编码213个氨基酸残基,该蛋白预测的分子质量为24567u,等电点7．34,经同源比较,该cDNA与GenBank数据库中登录号为X14964的Rab蛋白有83％的相似性和76％的相同性,将该cDNA克隆到经改造的PBV220表达质粒,转化DH5a菌株诱导表达出该蛋白,取24种不同组织的总cDNA各100ng,用该基因序列设计引物作PCR,结果在胎肝组织中检测到有明显条带,表明该Rab基因相对在胎肝有高表达。     

Generation of transgenic wheat resistant to wheat yellow mosaic virus and identification of gene silence induced by virus infection

The plasmid containing the promoter Act1, the coat protein (cp) gene of wheat yellow mosaic virus (WYMV) and the selectable bar gene, was delivered via particle bombardment, directly into immature embryos of a wheat cultivars. PCR and PCR-RFLP were employed to screen the existence of the cp gene in T0 and T1 generations. Seeds from the positive T1 plants were sowed in fields heavily contaminated with WYMV to detect their resistance. In field trial of virus infection, one of the transgenic wheat lines, P8-T2, exhibited highly disease-resistance. Western blot and RT-PCR analysis showed that the expression level of cp gene in the resistant transgenic line was reduced greatly compared to those susceptible to WYMV infection. This provided evidence to presume that the resistance obtained by the transgenic wheat line was stimulated by the mechanism of the virus induced gene silencing.     

Isolation and cloning of a new gene encoding maize initiation factor

It was demonstrated that parental gene expression of maize in hybrid genetic background was different from that in their inbred lines. RT-PCR revealed a cDNA overexpressed in hybrid maize. The cDNA was reamplified and used to screen a corresponding cDNA library. A full length of a cDNA insert ZH01 was isolated and shown to have an open reading frame encoding 414a. a. Sequence comparison shows that ZH01 has extremely high similarity with the previously reported plant initiator factor elF-4A and distinct difference from another maize elF-4A gene MIF4A. Ovcrexpression of ZH01 in hybrid may involve vigorous growth of hybrid maize seedlings.     

β葡聚糖酶杂合基因bglHAM的克隆及在P.pastoris中的表达

采用PCR的方法,以Bacillus macerans总DNA为模板,构建出β-1,3-1,4葡聚糖酶杂合基因bglHAM,与巴斯德毕赤酵母表达载体pPIC9K重组,得到重组质粒pPIC9K-HAM,电脉冲转化法转化酵母菌GS115,KM71,在MM,MD平板上筛选表型,YPD-G418平板上筛选多拷贝重组子,重组菌株经甲醇诱导后,刚果红平板染色法检测到β-葡聚糖酶活性,SDS-PAGE证明表达产物的分子量约为24kD,摇瓶诱导培养筛选到的重组菌株,胞外每mL发酵液最高酶活达240U。     

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).     

羊布鲁菌外膜蛋白OMP2b的克隆，原核表达及纯化

研究羊布鲁菌外膜蛋白 OMP2b 基因的克隆,原核表达,以及纯化。根据羊布鲁菌 M5株外膜蛋白 OMP2b 蛋白基因序列设计引物,扩增出大小约为1130bp 的目的基因片断,克隆入融合表达载体 pGEX-4T-1,构建重组质粒 pGEX-4T-1-omp2b。在大肠埃希菌中将该蛋白表达并用亲和层析法纯化。用 Western-blot 分析方法鉴定纯化蛋白。结果成功构建了 pGEX-4T-1-omp2b 原核表达载体并在大肠埃希菌中表达了 omp2b 基因,纯化后所获得的蛋白与目的蛋白大小一致且与兔抗布鲁菌血清发生特异性反应,说明其为布鲁菌 OMP2b 蛋白。本研究成功构建了 pGEX-4T-1-omp2b 原核表达载体,并且在大肠埃希菌中进行表达,纯化的 OMP2b 蛋白具有良好的免疫原性。     

AtGT-3b基因克隆及原核表达与纯化

GT-3b转录因子是一个受NaCl和病原体诱导表达的GT-1-like转录因子,它能与GT-1 cis-element( GAAAAA)相互作用,促进下游基因的表达,在植物耐盐中起着重要的调节作用.通过分离了拟南芥(Arabidopsis thaliana)AtGT-3b基因,克隆到原核表达载体pCold TF中,并在大肠杆菌(Escherichia coli)BL21中进行融合表达;通过纯化得到AtGT-3b融合蛋白,以期用于研究其与GT-1顺式作用元件在体外的相互作用.     

人血管生成素基因的克隆及表达

利用RI-PCR方法从培养的人黑色素瘤细胞系A375中扩增得到了人血管生成素cDNA片段,测序正确后克隆入表达载体pET-28a( )中并转化于E.coli BL21宿主菌中.经IPTG诱导,表达了N端融合6个组氨酸标签(6His-tag)的血管生成素融合蛋白.利用6His-tag与过渡态金属离子Ni2 高亲和力结合的性质,经镍柱纯化,获得了高纯度的血管生成素融合蛋白,为进一步研究其生物活性及应用奠定了基础.     

The relationship of differential expression of genes in GA biosynthesis and response pathways with heterosis of plant height in a wheat diallel cross

Heterosis in internode elongation and plant height is commonly observed in hybrid plants, but the molecular basis for the increased internode elongation in hybrids is unknown. In this study, midparent heterosis in plant height was determined in a wheat diallel cross involving 16 hybrids and 8 parents, and real-time PCR was used to analyze alterations in gene expression between hybrids and parents. Significant heterosis of plant height and the first internode in length were observed for all 16 hybrids, but the magnitude of heterosis was variable for different cross combinations. Analysis revealed that the heterosis of the first internode was significantly correlated to that of plant height （r = 0.56, P 〈 0.05）, suggesting that the increased elongation of the first internode is the major contributor to the heterosis in plant height. Real-time PCR analysis exhibited that significant difference in heterosis of gene ex- pression was observed among all cross combinations. Moreover, heterosis of the first internode in length was correlated significantly and positively with expression heterosis of KS, GA3ox2-1, GA20ox2, GA20ox1D, GA-MYB and GID1-1, but significantly and negatively with expression heterosis of GAI and GA2ox-1, which is consistent with our recently proposed model of GAs and heterosis in wheat plant height, suggesting the alteration of GA biosynthesis and response pathways might be responsible for the observed heterosis in plant height.     

Cloning and expression of the Chinese wheat mosaic virus RNA2 coat protein readthrough and 19 ku cysteinerich domains and localization of these proteins

The 5′-terminal (RTn) and 3′-terminal (RTc) halves of the coat protein readthrough domain and the 19 ku cysteine-rich protein of Chinese wheat mosaic virus (CWMV) were amplified by RT-PCR, cloned and expressed in E. coli. Antisera and monoclonal antibodies against these proteins were prepared by immunising these purified proteins to mice. Detection of RTn, RTc and 19 ku proteins in CWMV infected wheat sap and leaf tissue indicated that the RTn and RTc proteins were distributed on the surface of virus particles whereas the 19 ku protein was in the cytoplasm of the infected wheat cells.