番茄转录调控因子基因Pti5植物表达载体的构建及其转化烟草的研究 Construction of a Plant Expression Vector with Tomato Transcription Factor Gene Pti5 and Studies on Transgenic Tobaccos

本实验构建了含有CaMV35S启动子控制下的Pti5-VP16基因的植物双元表达载体pBI121UCH1。通过根癌农杆菌叶盘转化法,将Pti5-VP16基因导入烟草SRI中,经卡那霉素筛选,获得了抗性植株。经PCR和Southern印迹分析,表明抗性植株中整合了Pti5-VP16基因,经抗病性鉴定转基因烟草植株的抗病性明显提高。 Abstract:The plasmid pBI121UCH1 carrying Pti5-VP16 gene under the control of the cauliflower mosaic virus 35s promoter was constructed.Leaf segments of tobacco SRI were infected by Agrobacterium tumefaciens EHA105 with plasmid pBI121UCH1,from which kanamycin resistant plants were obtained.PCR and Southern analysis proved that the Pti5-VP16 gene was integrated into the genomes of the tobacco plants.The disease resistance assay showed that the disease resistance was enhanced in the transgenic tobacco plants.     

马铃薯天冬氨酸蛋白酶抑制剂基因转化烟草研究

将马铃薯天冬氨酸蛋白酶抑制剂基因由重组质粒pAPI189中亚克隆到双元表达载体pGA643的XbaI和KpnI位点之间,构建成高效表达重组质粒pGAPI3。三亲融合法将其转移至农杆菌LB4404。通过叶盘法利用此融合后的含有目的基因的农杆菌转化烟草叶圆片,在含有Km的培养基上筛选抗性芽。将抗性芽接种到生根培养基至长成完整植株后再移栽到土质中以获得转基因植株。通过目的基因的特异引物进行PCR检测以及目的基因的片段为探针进行Southern杂交检测,证实已获得马铃薯天冬氨酸蛋白酶抑制剂基因的转基因烟草植株。 Abstract：An aspartic proteinese inhibitor gene contained in the recombinant plasmid pAPI189 was inserted into the XbaI and KpnI site of binary vector pGA643 for constructing subclone pGAPI.The pGAPI was introduced into Agrobacterium LB4404 by coculturing the mixture of DH5α(pGAPI)、HB101(pRK2013) and Agrobacterium LB4404.We transformed tobacco leaves by coculturing them with Agrobacterium LB4404 which contained aspartic proteinese inhibitor gene and then screening the shoots with T?medium containing kamnamycin (Km,100μg/ml).The shoots resistant to Km were transferred into the rooting medium.When roots were formed the whole plants were transferred into pots.PCR reaction using the primers complementing with potato aspartic proteinese inhibitor gene and Southern hybridization using the the fragments contained aspartic proteinese inhibitor gene as the probe were performed.The results of PCR and Southern hybridization showed that we obtained the transgenic tobacco plants.     

Bt水稻杂交育种中转基因的遗传分析

利用PCR、GUS染色和Western印迹杂交技术检测了Bt水稻杂交后代群体,发现在394株GUS阳性株中,共有392株表达Bt蛋白,协同表达株率达99.49%。由此表明,在杂交后代中报告基因Gus和目的基因crylAb紧密连锁遗传与表达。本试验还发现,在BC1、BC1F2和粳粳交F2群体中转基因呈单基因显性遗传,而在籼粳交F2群体中偏离3:1分离。 Abstract:Improved histochemical staining for GUS activity,PCR and Western blotting were used to detect the population of Bt rice crossed to conventional rice varieties.A total of 392 plants expressing Bt toxin protein were found in 394 GUS positive plants.The result demonstrated that cry1Ab gene closely inherited and expressed with reporter gene gus.Therefore,it is possible to develop GUS-assisted-selection to preliminarily identify the Bt gene and study the inheritance of transgenes in (back)cross breeding.Mendelian segragation of reporter gene Gus was observed in F2,BC1 and BC1F2 progenies.Thus indicated that transgenes inherited as a single dominant gene in the progenies of Bt rice crossed to conventional rice varieties.     

Isolation and Expression Pattern Analysis of Two Ferritin Genes in Tobacco

For understanding of the ferritin gene expression pattern and the mechanism of iron homeostasis in tobacco (Nicotiana tabaccum L.) plants, two full-length ferritin cDNAs, NtFerl and NtFer2, were isolated from tobacco seedlings and characterized. These cDNAs are 1 214 and 1 125 bp nucleotides and encode 251 and 259 amino acid residues, respectively. The deduced amino acid sequences showed that two tobacco ferritins share the same characteristics as the plant ferritins from Arabidopsis, soybean, and maize. Southern blotting analysis indicated that both NtFerl and NtFer2 were probably multicopy genes in the tobacco genome. Northern blotting analysis indicated that iron loading of tobacco plantlets increased the ferritin mRNA abundance and that NtFerl expression was higher and more sensitive to iron than NtFer2 expression. Furthermore, NtFerl was expressed in both leaves and roots, whereas NtFer2 was expressed mainly in leaves.     

Co-expression and immunity of Legionella pneumophila mip gene and immunoadjuvant ctxB gene

The mip gene of Legionella pneumophila and the ctxB gene of Vibrio cholerae were amplifiedby PCR respectively.The amplified cDNA was ligated to the pcDNA3.1(+)vector.The recombinant plasmidspcDNA3,1-mip and pcDNA3.1-ctxB were identified by restriction analysis and PCR,and further confirmedby sequencing analysis.NIH3T3 cells were transfected with pcDNA3.1-mip and pcDNA3.1-ctxB accordingto the Lipofection method.Transient and stable products of the co-expression of the mip gene and ctxB genewere detected by immunofluorescence and Western blotting.The results showed that NIH3T3 cells weresuccessfully transfected,and that the transiently and stably co-expressed products can be detected in thetransfected cells.To detect the humoral and cellular immune response in immunized mice induced by the co-immunization of the mip and ctxB genes,female BALB/c mice were immunized intramuscularly with pcDNA3.1-mip and pcDNA3.1-ctxB.The results showed that the specific antibody titer and the cytotoxic T-lymphocyteresponse for pcDNA3,1-mip immunization and co-immunization were increased compared with that ofpcDNA3.1(+) immunization.Furthermore,the specific antibody titer and cytotoxic T-lymphocyte responsefor co-immunization were increased compared with that of pcDNA3.1-mip immunization.Statistical analysisusing one-way analysis of variance(ANOVA)showed that there was a significant difference between thegroups(P<0.01).The results indicated that the ctxB gene enhanced the humoral and cellular immune responseto the mip gene immunization.These findings provide experimental evidence to support the development ofthe L.pneumophila DNA vaccine.     

丙肝病毒融合抗原基因NS3-C定点整合入衣藻 叶绿体基因组的研究

用PCR方法从丙型肝炎病毒(HCV) cDNA文库中克隆了两段DNA片段,即HC基因组非结构NS3区抗原基因(约0.7 kb)和核心抗原C区抗原基因(约0.6 kb)的cDNA片段。在两段cDNA间加入连接肽Ser-Pro-Gly-Ser的密码子序列,构建成融合抗原基因NS3-C。将该融合基因与衣藻叶绿体基因atpA的启动子和rbcL基因的3'末端连接,得到丙肝病毒融合抗原基因NS3-C表达盒,再将该表达盒与选择标记基因aadA表达盒和衣藻叶绿体基因组同源片段连接,构建成衣藻叶绿体转化载体pSS6。基因枪法转化衣藻叶绿体,经壮观霉素筛选获得转化再生的单藻落,对转基因衣藻的PCR和Southern杂交分析表明,融合抗原基因NS3-C已整合到衣藻叶绿体基因组中。 Abstract:　Two DNA fragments encoding the nucleocapsid (C) region protein and the non-structural region 3 (NS3) protein of hepatitis C virus(HCV) were amplified from cDNA library by using PCR method. The 5' terminal of C cDNA fragment was linked up with the 3' terminal of NS3 cDNA fragment by a oligonucleotide linker Ser-Pro-Gly-Ser to form a chimeric gene NS3-C, which was placed under the control of the chloroplast atpA promoter and rbcL 3' region of Chlamydomonas reinhardtii to construct the chimeric gene NS3-C cassette. Then the NS3-C cassette was linked with selectable gene aadA cassette and the chloroplast homologous fragments of Chlamydomonas reinhardtii to generate transformation vector pSS6. Chloroplasts of Chlamydomonas reinhardtii were transformed by particle bombardment. Plastid transformants were selected by their resistance to 100 mg/L of spectinomycin. PCR and Southern hybridization analysis showed that the chimeric gene NS3-C had been integrated into chloroplast genome of Chlamydomonas reinhardtii.     

用天花粉蛋白基因转化小麦获得转基因植株

取普通小麦品种京411未成熟胚诱导愈伤组织,10天左右,对820个胚性愈伤组织用含有35S启动子的天花粉蛋白（trichosanthin,TCS）基因轰击。2周后,将这些被轰击的愈伤组织转到含潮霉素50mg/L的筛选培养基上,经分化和生根,获得了33棵再生植株,经接饲毒蚜虫抗病性鉴定和PCR、Southern杂交分析,从中筛选出4株含有编码TCS的转基因小麦植株。转化频率为0.49%。 Abstract：Wheat immature embryo calli of 10 days culture from Jing 411,a common wheat variety, Triticum aestivum, were used as the receptor of Trichosanthin gene droven by 35S promotor to be bombardmented.Two weeks after bombardment, 820 embryonic calli were transferred onto selection medium containing 50mg/L hygromycin,and 33 plantlets with healthy roots were regenerated finaly from the calli. By the barley yellow dwarf virus resistance test of inoculating the wheat aphids carrying the virus on the plants, PCR analysis, and Southern blotting analysis, 4 plants were identified to be the transgenic wheats, containing the alien DNA that ecodes TCS gene. The transformed efficiency was around 0.12 percent.     

Regulation of purine biosynthetic genes expression in Salmonella typhimurium Ⅳ O~c mutation site of purG and its function analysis

Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded by purG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu-tamine and ATP for the de novo purine nucleotide biosynthesis. purG gene is negatively regulated by a repressor-oper-ator system. The O purG and OC purG were cloned respectively in vivo. Restriction enzymes analysis of preliminary clones pLBG-1 (O ) and pLBG-2 (OC) were carried out. The hybrid plasmids pLB1933 (O ) and pLB1927 (OC) containing 5 control region of purG were constructed and the DNA sequences were determined respectively. DNA se-quences data showed that Oc mutation of purG occurred at the 3rd position of 16 bp PUR box in the 5' control region ( G→A). Gel retardation experiment indicated that the repressor bound well with O PUR box, but not with Oc PUR box. The result strongly supported the idea that PUR box is the binding region of represser protein and the 3rd posi-tion base G of PUR bo     

Antiporter Gene from Hordum brevisubulatum （Trin.） Link and Its Overexpression in Transgenic Tobaccos

A vacuolar Na^ /H^ antiporter cDNA gene was successfully isolated fromHordeum brevisubulatum (Trin.) Link using the rapid amplification ofcDNA ends (RACE) method. The gene was named HbNHXI and was found to consist of 1 916 bp encoding a predicted polypeptide of 540 amino acids with a conserved amiloride-binding domain. Phylogenetic tree analysis of the Na^ /H^ antiporters showed that the HbNHXI gene shares 55.3%-74.8% similarity with the vacuolar-type Na^ /H^ antiporters. Transgenic tobaccos that contain the HbNHXI gene, integrated by forward insertion into the tobacco genome, were obtained via Agrobacterium tumerfaciens and characterized for the determination of the concentration of Na^ and K^ ions, as well as proline, in the presence of 300 mmol/L NaCl. The T1 transgenic plants showed more tolerance to salt and drought than did wild-type plants. Our data suggest that overexpression of the HbNHXI gene could improve the tolerance of transgenic tobaccos to salt and drought through the function of the vacuolar Na^ /H^ antiporter.     

Studies on Insect Resistance of Bt Transplastomic Plants and the Phenotype of Their Progenies

Insecticidal protein gene CrylA (c) from Bacillus thuringiensis (Bt toxin gene) was placed under the control of psbA5'- and 3'- regulatory regions of rice (Oryza sativa L. ) chloroplast to construct Bt expression cassette, which was ligated with selectable marker aadA cassette and homology regions of tobacco ( Nicotiana tabacum L. ) chloroplast genome to generate transformation vector pTRS8. Leaves of tobacco plant cv. NC89 were transformed with particle bombardment method, plastid transformants were selected by their resistance to 500 mg/L of spectinomycin. Some transplastomic plants were toxic to the third-instar larvae of Helicoverpa zea, and the growth of the survived insects was remarkably inhibited. Genetic and molecular analyses of T1 and T2 progenies of plants with highly efficient insect resistance showed that Bt toxin gene had been inherited in progenies, and spectinomycin resistance was inherited maternally.     

Bt水稻中crylAb基因的遗传分析

用PCR、GUS染色和Western点杂交技术检测了Bt水稻杂交后代群体,在394株GUS阳性株中,有392株表达Bt蛋白,协同表达株率达99.49%。由此表明,在杂交后代中报告基因gus和目的基因crylAb紧密连锁遗传与表达。GUS组织染色和Southern杂交检测表明Bt水稻中的crylAb基因呈单位点显性基因遗传,且在有性世代中能稳定传递,还发现,在BC1、BC1F2和粳粳交F2群体中crylAb基因呈单位点显性基因遗传,而在籼粳交F2群体中偏离3：1分离。     

两种凝集素基因在转基因烟草中表达的研究

构建了含尾穗苋凝集素基因(ACA)的cDNA序列和改造后的雪花莲凝集素基因(GNA)的植物表达载体pBACG。在此表达载体中,ACA和GNA基因的表达分别由35S启动子和CoYMV启动子控制。通过农杆菌介导,将ACA和GNA基因转化到烟草中,经卡那霉素筛选获得60株转化再生植株。对PCR检测呈阳性的50株植株进行接蚜虫实验,结果表明,其平均抑虫率达83．9％。Southern blotting分析表明,ACA和GNA基因都已整合到烟草基因组中。Western blotting结果显示这两个基因在不同植株中都可表达其相应的蛋白质,但表达水平不同。部分Western blotting分析呈阳性植株的抗蚜性与T0代相近,达85．3％,说明这两个基因的抗蚜功能可以稳定遗传。     

Dynamic expression of green fluorescent protein and Bacillus thuringiensis Cry1Ac endotaxin in interspecific hybrids and successive backcross generations (BC1 and BC2) between transgenic Brassica napus crop and wild Brassica juncea

The persistence and stability of a transgene encoding a Bacillus thuringiensis (Bt) Cry1Ac insecticidal protein was investigated in hybrids between crop Brassica napus and a recurrent wild Brassica juncea population. Interspecific hybrids (F₁) and backcross progenies (BC₁, BC₂) containing green fluorescent protein (GFP) and Bt genes were successfully produced in the greenhouse. Stable Bt toxin levels were found in hybrid and advanced backcross progenies formed in wild B. juncea. Bt Cry1Ac concentration was significantly lower in BC₂ plants than in transgenic B. napus, F₁, BC₁, while no significant differences were detected among the latter three plant genotypes. A GFP marker gene was used as a scorable marker and indicator of Bt transgene expression. GFP fluorescence intensity was significantly correlated with Bt Cry1Ac concentration at the flowering stage and the pod formation stage in both transgenic oilseed rape hybrids and backcrossed progenies (BC₁, BC₂). It was demonstrated that GFP was a suitable marker for Bt protein in the backcross of B. juncea, which could facilitate the detection of gene flow and is useful in biosafety management.     

含VHb基因和GFMcryIA基因的双价基因植物高效表达载体的构建

将人工合成的、密码子优化后的透明颤菌血红蛋白基因与人工合成的GFMcryIA基因构建成双价基因植物高效表达载体PGBI4ASVHBBt,vgbM基因表达盒中含2个增强子的35S启动子、Ω前导序列、Kozak序列、多联终止密码子及Nos终止子;Bt基因表达盒中,除含有以上提高转录和翻译的调控元件外,还包含有正确切割、加工序列、Poly(A)信号序列。利用根癌农杆菌介导转化烟草,获得了转基因植株;PCR及Southern blot检测,证实了双价基因在烟草基因组中的整合;Western blot检测证实了vgbM基因在转基因烟草中的表达;杀虫实验表明GFMcryIA基因也表达出活性毒蛋白。     

苏云金芽孢杆菌(Bt)晶体毒蛋白基因在烟草叶绿体中的表达

将全长３．５ｋｂ的Ｂｔ基因３’端缺失,得到长为２．１ｋｂ、１．８ｋｂ的基因。分别将这３个长度（１．８ｋｂ、２．１ｋｂ、３．５ｋｂ）的基因置于水稻叶绿体ｐｓｂＡ基因的启动子和终止子调控之下,并与选择标记基因ａａｄＡ（编码氨基糖苷－３’－腺苷酸转移酶,具壮观霉素抗性）表达盒相连;以烟草叶绿体基因ｔｒｎＨ－ｐｓｂＡ－ｔｒｎＫ为同源片段,构建成叶绿体转化载体ｐＢＴ３、ｐＢＴ８和ｐＢＴ２２。用基因枪把Ｂｔ基     

Stable Bacillus thuringiensis (Bt) toxin content in interspecific F1 and backcross populations of wild Brassica rapa after Bt gene transfer

Stable expression of a transgene may lead to increased fitness for wild plants after acquiring the transgene via crop-weed hybridization. Here, we investigate the stability of Bt toxin content in wild Brassica rapa acquiring the Bt gene from Bt Brassica napus. The Bt toxin content in nine Bt-expressing B. napus lines was 0.80-1.70 micro g/g leaf tissue throughout the growing season. These nine lines were crossed with three accessions of wild B. rapa and the Bt gene was successfully transferred to interspecific hybrids (F1) and successive backcross generations (BC1 to BC4). The Bt toxin level in F1 and BC progenies containing the Bt gene remained at 0.90-3.10 micro g/g leaf tissue. This study indicates that the Bt gene can persist and be stably expressed in wild B. rapa.     

转基因抗虫棉Bt基因插入区碱基组成分析

利用TAIL－PCR的方法克隆不同来源的转基因抗虫棉中外源基因插入区的侧翼序列并对其进行序列和结构分析,结果表明,同一个较基因的单构自交得到的不同株系中外源基因插入区的两侧DNA序列完全相同,不同的转基因抗虫棉虫的外源基因插入位置各不相同,不同来源的转基因品种外源基因插入的上游侧翼片段含有一段残留质粒片段,外源基因插入的下游侧翼片段为富含AT碱基结构,其中泗棉3号转基因抗虫品系中下游侧翼片段的AT碱基高达92％,Southern杂交结果显示这些侧翼序列为高AT含量的多拷贝序列,序列中没有发现拓扑异构酶的结合位点。     

苏云金杆菌毒素调控基因plcR的特性与表达

根据蜡状芽胞杆菌plcR基因和papR基因序列设计特异引物,对6个Bt菌株（WB1、WB7、WB9、HD98、8010、8311）及5个Bc菌株（6A1、6A2、6A3、6A4、6S1）进行了PCR检测.结果显示,3个Bt菌株及4个Bc菌株含有plcR-papR基因.克隆了Bt8010、Bc6A2和6A3的plcR、papR基因,核苷酸序列分析表明,三个菌株的plcR、papR基因与NCBI数据库中的Bt、Bc及Ba相应序列都有很高的相似性.Bt8010的plcR基因编码框由846个核苷酸组成,可编码282个氨基酸;papR基因的编码框由144个核苷酸组成,可编码48个氨基酸.推导的氨基酸序列分析表明,Bt8010 的PapR有21个氨基酸的信号肽序列,PlcR没有信号肽序列.与Bc6A2、6A3和Bc 569相比,Bt8010 的PlcR和PapR在氨基酸序列上与Bc 相应序列存在相对较大的差异.将plcR-papR基因连接到表达载体pHT304中,并转化至大肠杆菌JM109中成功进行了表达,为研究Bt plcR基因的功能奠定了基础.