辣椒花器官发育以丹基因和PDS基因VIGS载体的构建及鉴定

以辣椒恢复系121C为材料．采用RT—PCR技术从121C花药中克隆得到控制花器官发育B类基因朋丹的部分序列，片段长379bp，阳性对照八氢番茄红素脱氢酶基因（phytoenedesaturae，PDS），片段长323bp，然后通过酶切分别连接pTRV2质粒，获得重组载体pTRV2-pPAP3和pTRV2-PDS。利用PCR进行初步鉴定后进行序列测定。结果表明，辣椒尉丹基因和PDS基因已插入到pTRV2载体上，VIGS载体构建成功。该研究为下一步分析基因沉默和辣椒花器官的发育提供了试验基础。     

八氢番茄红素合成酶(PSY2)基因果实特异表达载体的构建

八氢番茄红素合成酶是番茄红素合成的关键酶,通过PCR法获取PSY2基因和E8启动子序列,将目的基因和E8启动子序列构建到植物表达载体pBI101.2中,构建了果实特异表达启动子的八氢番茄红素合成酶基因植物表达载体。并采用PCR、限制性内切酶酶切和序列测定分析法,对重组质粒进行鉴定。结果表明,番茄果实特异性表达PSY2蛋白的重组质粒构建成功;通过农杆菌直接转化技术将其成功转入转化农杆菌LBA4404、EHA105,为下一步PSY2蛋白在番茄果实中特异表达奠定了基础。     

番茄果实特异性LYC-B 干扰载体的构建及在不同颜色果实中的表达特异性验证

为了研究番茄LYC-B 干扰对类胡萝卜素合成主要酶和主要代谢产物的影响,构建了果实特异性的番茄红素β- 环
化酶LYC-B 干扰载体,并验证了其在不同颜色番茄果实中的有效性。依据X13437.1 扩增番茄果实特异启动子E8,构建了果
实特异性载体E8-pBI121,其在粉色、红色、绿色和紫色的番茄果实中均能表达。依据X86452.1 扩增番茄LYC-B 从61～861
bp 间长度为801 bp 的片段LYC-B1 和从 480～781 bp 间长度为302 bp 的片段 LYC-B2,构建了以CaMV 35S 为启动子的LYC-B
干扰表达载体pBI121-B1B2,以E8 替换CaMV 35S,构建了果实特异性干扰载体E8-pBI121-B1B2。采用农杆菌注射法分别
侵染番茄叶片和果实,GUS 染色显示,pBI121-B1B2 在叶片、果实和种子中均表达,E8-pBI121-B1B2 只在果实和种子中表达。     

超表达草生欧文氏菌crtB基因促进转基因番茄类胡萝卜素合成的研究

利用重组PCR 技术,将草生欧文细菌八氢番茄红素合成酶基因crtB 与豌豆质体定位序列ts-rbcS 融合,插入质粒载体PMV 中,构建了植物表达载体pBI-ts-rbcS-crtB,并通过根瘤农杆菌EHA105介导转化番茄。PCR 检测、Southern 杂交和RT-PCR 分析表明,外源基因crtB 已整合到番茄基因组中,并在转基因植株中得到表达。转基因番茄果实中的类胡萝卜素总量增加1.3 ~ 2.5 倍,八氢番茄红素、番茄红素、β–胡萝卜素和α–胡萝卜素分别增加了4.3、1.8、2.2 和2.3 倍。转化株系中内源类胡萝卜素合成基因的表达也受到广泛的影响。crtB 基因过量表达有效促进了番茄果实中类胡萝卜素的合成和积累。     

甜瓜蔗糖磷酸合成酶基因全克隆及工程载体的构建

蔗糖磷酸合成酶在甜瓜果实蔗糖合成途径中起关键性的调节作用,克隆该基因并导入甜瓜低糖自交系,可改良甜瓜果实品质创新优良种质。从甜瓜幼苗叶片提取总RNA,利用RT-PCR与Southern blot方法相结合,重组子质粒经限制性内切酶酶切和PCR验证以及测序同源性比较,克隆到基因的5’(2859bp)和3’(852bp)。应用高保真Taq聚合酶PCR拼接蔗糖磷酸合成酶完整cDNA序列3692bp,该片段与GenBank中其它植物基因序列具有97%￣99%的同源性,登录号DQ364058。应用BamHⅠ、KpnⅠ、BglⅡ限制性酶切获得植物双元表达载体pROK2及基因的线性片段,通过T4连接酶反应,构建了以CaMV35S为启动子,以Tnos为终止子的工程载体pROK-SPS,该载体含有Npt-II选择标记基因。     

甜瓜果实蔗糖磷酸合成酶基因cDNA片段的克隆及表达分析

根据在GenBank中登录的番茄、马铃薯等蔗糖磷酸合成酶基因的保守序列设计引物, 采用RT-PCR方法从甜瓜花后25 d的果实总RNA中扩增出目标cDNA片段, 克隆到pMD18-T载体中。序列分析表明, 该片段与番茄蔗糖磷酸合成酶氨基酸序列同源性为98.9% , GenBank中登记号为DQ355797。通过Northern blot检测其在甜瓜果实不同发育时期的表达变化, 结果表明该基因在甜瓜果实花后25 d开始表达,随着果实的成熟, 表达量升高。     

果实特异启动子调控薄皮甜瓜ACC合成酶cDNA反义表达载体的构建

应用反义RNA技术抑制甜瓜成熟过程中内源乙烯的合成,从而培育耐贮运品种是解决甜瓜延熟保鲜难题的可行新方法。根据GenBank中甜瓜、黄瓜ACC合成酶基因氨基酸保守序列设计引物,从成熟的薄皮甜瓜(齐甜1号)果肉组织中提取总RNA,经RT-PCR扩增得到约0.7kb的ACC合成酶cDNA片段,将其克隆到质粒载体pGEM-TEasy中,测序表明,该基因为777bp,编码258个氨基酸;从番茄(东农706)叶片组织中提取总DNA,经PCR扩增得到约2.2kb的E8基因片段,将其克隆到质粒载体pGEM-TEasy中,测序表明,该基因为2192bp;以pCAM2301为起始植物表达载体,pCAM-GT为中间载体,成功构建了果实特异启动子(E8)调控薄皮甜瓜ACC合成酶cDNA反义表达载体,采用冻融法将其转入根癌农杆菌LBA4404,得到了完整的Ti质粒表达载体系统。     

番茄CRISPR/Cas9介导的多基因编辑技术体系构建与应用

为了构建番茄CRISPR/Cas9介导的多基因编辑体系，用SlU6-2p、SlU6-3p、SlU6-7p、SlU3-5p、SlU3-9p和SlU6-5p启动子分别替换pKSE401和pCBC-DT1T2载体中的拟南芥U6启动子，构建了1个双元载体（p MGET）、2个中间载体（pKC-S2M和pKC-S3M）和3个gRNA模块载体（pCBC-S1、pCBC-S2和pCBC-S3）。为了测试该多基因编辑体系，通过PCR扩增、Goldengate克隆和同尾酶技术，将含有6个番茄果实性状相关基因Green flesh(GF)、Ovate(O)、Locule number(LC)、SlMYB12(Y)、Tangerine(T)、Uniformripening(U)靶点序列的sgRNA聚合构建多基因编辑载体pMGET-OYGTULC和pMGET-TULCOYG，检测6个基因同时被编辑的效率分别为44.00%和11.76%。用携带pMGET-OYGTULC载体的根癌农杆菌转化番茄材料获得2株6个基因均被编辑的植株，序列变异为单个碱基的插入、单个或多个碱基的缺失及大片段的缺失等多种形式。本研究中该多...     

超量表达欧李ChPSY基因促进转基因番茄类胡萝卜素合成的研究

将从欧李果实中分离的ChPSY cDNA经过序列分析、酶切之后,连接到植物表达载体PMV,构建了植物重组载体pBI-ChPSY,并通过根瘤农杆菌EHA105介导转化番茄,获得了12株抗性植株。PCR检测和Southern杂交结果显示,12株抗性植株均为阳性,说明外源基因ChPSY整合到转化植株的基因组中。RT-PCR分析表明,ChPSY基因在转化植株果实中得到表达。HPLC分析表明,转ChPSY基因番茄果实中总类胡萝卜素含量增加了1.62 ~ 3.04倍,八氢番茄红素、番茄红素、β–胡萝卜素和α–胡萝卜素含量分别增加了4.87、2.10、2.59和3.25倍。转化植株中内源类胡萝卜素合成基因的表达也受到广泛的影响。ChPSY基因超量表达有效促进了番茄果实中类胡萝卜素的合成和积累。     

甜瓜果实酸性转化酶基因cDNA片段的克隆

 根据在GenBank中登录的番茄、胡萝卜和柑桔等酸性转化酶基因的保守序列设计引物，采用RT-PCR方法，从甜瓜果实总RNA中扩增出目标cDNA片段，克隆到pMD18-T载体中。序列分析表明，它与其它植物的酸性转化酶基因的同源性很高，与番茄氨基酸序列同源性为99％，说明已经成功克隆到甜瓜果实酸性转化酶基因cDNA片段，在GenBank中登记号AF490425。     

Virus-induced PpCHLH gene silencing in peach leaves (Prunus persica)

Summary

Compared with other model plants or crops, studies on the molecular biology of fruit trees have lagged behind due to technical difficulties in gene transformation and manipulation. Therefore, developing an efficient system for gene manipulation is of particular significance in fruit trees. Here, we report on a method for virus-induced gene silencing (VIGS) by syringe-infiltrating a tobacco rattle virus (TRV) vector containing a specific target gene sequence into peach (Prunus persica) leaves to analyse gene function. The target gene (PpCHLH) was a 4,445 bp sequence encoding the H subunit of magnesium chelatase and was first cloned as a cDNA. This gene (PpCHLH) is reported to be related to chlorophyll biosynthesis, and any loss of function leads to a decrease in chlorophyll content, with concomitant yellow or white colour changes in the leaves. To silence the PpCHLH gene, a 1:1 mixture of Agrobacterium tumefaciens strain GV3101 cultures containing pTRV1 or a pTRV2 vector construct with a 650 bp cDNA fragment of the PpCHLH gene was infiltrated into leaves of 4 – 5 week-old peach seedlings. After 15 d, the inoculated areas of the green leaves faded and finally turned yellow or white. Loss of PpCHLH gene function was confirmed by semi-quantitative RT-PCR, real-time qRT-PCR, and siRNA northern blot analysis. The virus-induced gene silencing (VIGS) technique developed here could be used for further molecular studies on fruit trees.     

Detection,cloning and expression of bone morphogenetic protein-1 from human osteosarcoma cell lines

AIM:To manufacture recombinant protein of the highly conserved domain in human bone morphogenetic protein-1(BMP-1) using gene engineering methods as antigen for making wide spectrum antibody to BMP-1.METHODS:We analyzed the gene sequences and protein structures of BMP-1 and its related proteins, and chose a highly conserved fragment as target gene. Total RNA was prepared from human osteosarcoma cell line Saos-2, then the target gene was amplified with RT-PCR. The PCR product was cloned into prokaryotic expression vector pMAL c2 to get recombinant vector BMP-1(322-588aa)-pMAL c2. After transforming the recombinant plasmid into DH5-alpha and screening, several prositive clones were got for sequencing. Finally the transformed cells was induced with IPTG to get fusion protein.RESULTS:The BMP-1 gene fragment was successfully cloned into vector pMAL c2, and was able to express efficiently with IPTG inducement. The amount of expressed fusion protein is about 66%-72% in total volume of bacterial proteins.CONCLUSIONS:The recombinant protein contains several key domains(2 CUB domains and 1 EGF domain), which are shared by BMP-1 and its related proteins. Specific wide spectrum antibody to human BMP-1 and its related proteins may be generated with this recombinant protein antigen.     

Construction and identification of eukaryotic dicistronic vector expressing TCA-12-2/TCB-7.1

AIM: To construct the recombinant dicistronic eukaryotic expression vector pDC315-TCA-12-2-TCB-7.1, which containing T cell antigen receptor (TCR) genes TCA-12-2 and TCB-7.1, and to transfer this recombinant vector into 293 cells to investigate the expression of TCA-12-2 and TCB-7.1. METHODS: The TCA-12-2 was obtained by RT-PCR from the T cells and the TCB-7.1 was amplified by PCR from plamid pcDNA3.1-TCB-7.1 that we constructed before. TCA-12-2 and TCB-7.1 was cloned into vector pIRES2-AcGFP1 firstly, then subcloned into vector pDC315. The recombinant plasmid pDC315-TCA-12-2-TCB-7.1 was verified by restriction enzyme digestion and sequencing, the positive recombinant plasmid was transferred into 293 cells using Lipofectamine 2000. The expressions of gene TCA-12-2 and TCB-7.1 were identified by RT-PCR and flow cytometry. RESULTS: Both TCA-12-2 and TCB-7.1 genes were constructed into eukaryotic expression vector pDC315 and the expressions of genes in 293 cells were detected successfully with RT-PCR and flow cytometry. CONCLUSION: The dicistronic expression vector pDC315-TCA-12-2-TCB-7.1 is successfully constructed and expressed.     

Construction and in vitro analysis of recombinant adenovirus vector carrying red fluorescent protein reporter gene

AIM: To provide important tools for gene therapy and gene vaccine research by constructing an adenovirus vector containing red fluorescent protein ( RFP ) reporter gene with the approach of in vitro recombinant ligation. METHODS: The RFP gene fragment of pTurboRFP-N was digested and ligated into pShuttle transfer vector to construct recombinant vector pShuttle-TurboRFP-N. I- Ceu I/PI- Sce I were used to double digest recombinant vector pShuttle-TurboRFP-N and backbone of vector pH5'040.pkGFP-II. The target fragment was collected and ligated, and recombinant adenovirus vector AdH5'.040.CMV.RFP-N was obtained. After linearization, the vector was transfected into AD293 cells by liposome for virus packaging. The efficiency of virus packaging and RFP expression level in AD293 cells were examined using fluorescent microscope. In addition, the biological activity and titer of the virus were tested. Human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were infected with recombinant adenovirus vector AdH5'.040.CMV.RFP-N and control adenovirus vector AdH5.CMV.EGFP respectively. The infection efficiencies of the 2 vectors to different cell lines were compared by evaluating the expression levels of RFP and enhanced green fluorescent protein (EGFP). RESULTS: The recombinant adenovirus vector AdH5'.040.CMV.RFP-N was correctly constructed and confirmed by enzyme digestion. The virus was packaged by the vector in AD293 cells and had the ability to infect the target cells. The target gene in eukaryotic cells was also expressed. The number of recombinant adenoviruses and the titer of the virus after amplification and purification were 3.6×10¹⁵ vp/L and 1×10¹³ pfu/L,respectively. The infection efficiencies of recombinant adenovirus vector Ad5'.040.CMV.RFP-N to human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were higher than those in control adenovirus vector AdH5.CMV.EGFP (P<0.05). CONCLUSION: We have constructed recombinant virus vector carrying RFP reporter gene and provide an important tool for gene therapy and gene vaccine research. The reporter gene can be highly expressed in AD293 cells and has high infection efficiency to cancer cells. RFP is a good substitution and supplement to green fluorescent protein.     

Construction of recombinant adenovirus expressing BDNF and its expression in expanded rat mesenchymal stem cells in vitro

AIM:To construct recombinant adenovirus vector containing brain derived neurotrophic factor, (BDNF) gene using bacterial homogenous recombination, and investigate the expression in expanded rat mesenchymal stem cells (rMSC) in vitro.METHODS:BDNF gene and proBDNF gene were subcloned into adenovirus shuttle plasmid pAdTrack-CMV containing enhanced green fluorescent protein gene (EGFP) expression cassette, forming shuttle vector of pAdTrack-BDNF, and pAdTrack-proBDNF, and co-transformed into BJ5183 bacterial cells with adenovirus backbone vector pAdEasy-1 using chemical transformation. After the recombinant adenovirus vector was obtained, the identified recombinant adenovirus plasmid DNA was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. rMSC were infected by recombinant adenovirus and EGFP expression was detected using fluorescent microscope. Infection efficiency was assessed by flow cytometrics. Western blotting identified expression of Ad -proBDNF and Ad-BDNF in rMSC. rMSC infected with Ad -proBDNF and Ad-BDNF were induced to differentiate into neuron-like cells. rMSC infected with Ad -proBDNF and Ad-BDNF were injected into nude mice and assessd in vivo.RESULTS:We successfully constructed the recombinant adenovirus Ad -proBDNF and Ad-BDNF that expressed in expanded rMSC in vitro.CONCLUSION:Recombinant adenovirus high-effectively mediates Ad -proBDNF and Ad-BDNF expression in expanded rMSC in vitro and in vivo.     

Construction of recombinant adenoviruses expressing the helicobacter pylori CagA antigen

AIM: To construct a replication-defective recombinant adenovirus, which expresses the CagA gene. METHODS AND RESULTS: The CagA gene was amplified by PCR. This heterogeneous gene was cloned into shuttle vector pAdTrack-CMV. The recombinant adenovirus DNA was obtained by the homologous recombination between the shuttle vector and adenovirus DNA in E.coli 5183. After linearization， the recombinant adenovirus DNA was transfected into 293 cells and the recombinant adenovirus was obtained. Through this technique, the replication- defective recombinant adenovirus AdEasyCagA was constructed. CONCLUSIONS: The replication-defective recombinant adenoviruses AdEasyCagA was constructed successfully. The work will make a good foundation for studying the effects of the replication-defective recombinant adenovirus on Th1/Th2 balance in asthma and be useful for finding a new pathway to prevent and cure asthma.     

大白菜渗透压应激活化蛋白激酶基因SRK2F的克隆与表达

以不耐盐、不耐旱的大白菜自交系SY-14-06为试材,提取根部总RNA,反转录为c DNA。根据大白菜SRK2F基因设计引物,PCR扩增SRK2F基因CDS序列1044bp。SRK2F编码347个氨基酸,预测分子量为39.3k D,理论等电点为4.88。利用p EASY-E1原核表达载体构建原核表达质粒p EASY-E1-SRK2F,转化表达菌株Transetta(DE3),通过SDS-PAGE检测该蛋白的表达。经Smart-embl预测其具有丝/苏氨酸激酶特有结构域,位于第4~260位氨基酸处。经Clustal X2比对,其与拟南芥同源性最高。最后利用镍离子金属螯合亲和层析介质对该蛋白进行纯化,得到了纯化的融合蛋白。     

Establishment of nasopharyngeal carcinoma (NPC) cell lines stable expressing NPC-derived LMP1

AIM: To establish nasopharyngeal carcinoma(NPC) cell lines stable expressing NPC-derived latent membrane protein 1(LMP1) gene.METHODS:General expression vector and epithelium-specific expression vector of NPC-derived LMP1 gene were constructed by using recombinant techniques, then transfected these vectors into a poor differentiated NPC cell line named CNE-2 ,integration and expression of N-LMP1 in CNE-2 cells were detected by PCR,RT-PCR and Western blot. RESULTS:(1) General expression vector and epithelium-specific expression vector of NPC-derived LMP1 gene were constructed successfully.(2) It showed that N-LMP1 gene expressed in CNE-2 cells correctly.CONCLUSION: The first NPC cell lines which stable express NPC-LMP1 were established. The cell lines obtained will provide important basis for exploring the role of NPC-LMP1 in nasopharynx carcinogenesis.     

Cloning and expression of NK4 gene in Raji cells

AIM: To clone NK4 gene and to construct recombinant eukaryotic expression vector for observing its expression in transfected Raji cells. METHODS: Total RNA was extracted from human hepatic tissue. NK4 gene cDNA was amplified by RT-PCR, and then cloned into vector pVITRO2-mcs to construct the recombinant eukaryotic expression vector pVITRO2-mcs-NK4. Raji cells were transfected by recombinant vector pVITRO2-mcs-NK4 and screened by homomycin B. The stable strain of NK4 gene expression was screened by real-time fluorescent quantitative PCR, ELISA, immunocytohistochemistry and semisolid culture. RESULTS: The specific DNA fragment was detected by RT-PCR in Raji cells transfected with NK4 gene. The transfected Raji cells expressed NK4 mRNA and protein stably, which inhibited Raji cell proliferation, metastasis and invasion. CONCLUSION: NK4 gene is cloned and recombined to construct recombinant eukaryotic expression vector pVITRO2-mcs-NK4 successfully. NK4 gene in Raji cells expresses stably.