马尾松银松素合酶基因转化双子叶植物表达载体的构建及鉴定

以马尾松为材料,在获得马尾松银松素合酶基因的基础上,构建了该基因转化双子叶植物的表达载体,该表达载体含有35S启动子和NOS终止子,能启动基因在双子叶植物中高效地表达.设计并合成分别含有Bgl和BstE酶切位点的上下游引物,通过PCR反应扩增出含有该酶切位点的银松素合酶基因cDNA全序列,酶切后连接到pCAMBIA1301植物表达载体上.经PCR鉴定、酶切分析及DNA测序证实cDNA片段大小、序列以及读码框的正确性.最后通过电击将表达载体转化农杆菌LBA4404,为进一步研究银松素合酶基因的功能奠定基础.     

异戊烯基转移酶基因克隆及植物表达载体构建

根据已知的异戊烯基转移酶基因保守序列设计引物,以根癌农杆菌C58的Ti质粒为模板,采用PCR方法扩增获得了异戊烯基转移酶基因723bp的片段.将该片段回收,连接到pMD18-T载体测序,测序片段经酶切回收后克隆到植物表达载体pBI121中,转化至大肠杆菌感受态细胞DH5α.将阳性质粒转化农杆菌感受态细胞EHA105,经菌液和质粒PCR分析,获得了真核表达载体pBI121-ipt,这为异戊烯基转移酶基因功能的进一步研究提供了基础.     

鱼腥藻铁吸收调节蛋白基因(alr0957)的克隆和表达

依据CyanoBase提供的鱼腥藻PCC7120 furC基因(alr0957)的序列信息设计了一对特异性引物,用Touch-down PCR的方法从基因组DNA中扩增得到大小约450bp的目的片段.通过TA克隆的方法将该片段连接到pMD18-T载体上筛选出重组质粒pMD18-T-fur,然后进行双酶切,纯化furC基因,再连接到原核表达载体pET-28a(+)上,转化表达菌株BL21(DE3).经PCR、双酶切和测序鉴定,对阳性菌株进行IPTG诱导表达,SDS-PAGE检测重组蛋白.结果表明:在25℃条件下经1mmol/L IPTG诱导20h,融合蛋白被成功表达,其分子量约为19 000,为进一步纯化蛋白和对基因的调控功能方面研究奠定了基础.     

烟草反义Mlo基因表达载体的构建

获得烟草反义Mlo基因的植物表达载体pBI 121-Mlo,为进行烟草遗传转化,获得该基因表达的缺陷型植株打下基础.从烟草叶片中提取总RNA,利用RT-PCR技术扩增得到Mlo基因的c DNA,以此为模板设计反义引物,通过PCR扩增出反义Mlo基因,将此反义Mlo基因与T载体连接,测序正确后再将此反义片段与植物表达载体pBI 121连接,构建烟草反义Mlo基因的植物表达载体pBI 121-Mlo.经Kan选择筛选出反义重组菌落,碱裂解法小量提取质粒后,用Xba I和Bam HI双酶切后再进行电泳鉴定.结果表明,目的基因已与植物表达载体pBI 121连接成功.成功构建了烟草反义Mlo基因表达载体pBI 121-Mlo.     

酵母海藻糖合成酶基因植物表达载体的构建

以从原核表达载体中酶切得到的tps1基因为模板，设计适当的引物，利用PCR技术，克隆出核苷酸数大约1.5k的片段，PCR产物经回收后，与PCAMBIAI303载体连接并转化大肠杆菌DH5a，阳性重组子经PCR鉴定，结果表明已获得海藻糖磷酸合成酶基因的植物表达载体。     

矮牵牛ACS2基因正、反义重复表达载体的构建

将矮牵牛ACS2基因的正义重复和反义重复分别与植物表达载体质：pBI-121连接,构建ACS2基因的正义重复和反义重复表达载体,经PCR和酶切鉴定,基因已成功构建到表达载体质粒上,为延长矮牵牛花期的基因工程研究奠定了基础．     

根癌农杆菌介导的绿色荧光蛋白基因在水稻植株中的表达

将改良的绿色荧光蛋白（EGFP）基因插入到植物表达载体中,构建了ubi启动子驱动下的植物表达载体p13UEGFP．通过根癌农杆菌介导转化水稻的胚性愈伤组织,经潮霉素筛选,获得抗性愈伤组织和再生植株．对T2代植株进行PCR分析、激光共聚焦显微镜检测和RT—PCR分析,结果表明,绿色荧光蛋白基因已经在转基因植株中稳定表达．     

甘蓝型油菜和Lesquerella fendleri fad2基因片段克隆及hpRNA植物表达载体的构建

首先利用PCR从甘蓝型油菜(Brassica napus L.)和Lesquerella fendleri基因组DNA中分别扩增出Δ12-脂肪酸去饱和酶fad2基因片段,再以扩增出的片段为模板设计引物从一端扩增出相应的小片段,然后将同一基因的大小片段反向连接,插入到种子特异表达载体2300-nap多克隆位点的napin启动子和nos终止子之间,构建成可以在种子中转录表达发夹RNA(Hairpin RNA,hpRNA)结构的植物表达载体.     

Regulatory effect of cotton leaf curl virus AC2 on virion sense promoter

The AC2 gene of cotton leaf curl virus (CLCuV) was obtained by polymerase chain reaction (PCR) . The total DNA of the CLCuV infected tomato leaves was used as template, and the amplified DNA fragment was inserted into a cloning vector. Transient expression vectors were constructed by inserting the AC2 gene into downstream region of CaMV 35S promoter. These constructs were delivered into tobacco and cotton leaf cells for transient expression by particle bombardment. The results indicated that the virion sense promoter was activated by AC2 and its activity increased remarkably. However, the activity of transactivated virion sense promoter was still lower than that of the complementary sense promoter. The expression pattern of transactivated virion sense promoter was similar to that of the complementary sense promoter, namely with high activity in both mesophyll and vascular tissues. The possibility of application of AC2 in plant genetic manipulation was also explored.     

白藜芦醇合酶基因的克隆及对毕赤酵母的转化

目的建立白藜芦醇合酶基因的毕赤酵母表达系统。方法通过PCR、限制性内切酶消化、连接、电激等方法,构建表达载体,转化毕赤酵母GS115。结果从葡萄基因组DNA中扩增得到了 1 200 bp目的基因,并克隆至pBS-T栽体;成功构建了表达载体pPIC3．5K／RS;目的基因成功整合进毕赤酵母GS115基因组中。测序及PCR鉴定证明,白藜芦醇合酶基因的毕赤酵母表达系统建立成功。结论所得方法无需高质量的RNA,从而降低了试验条件,达到了快速简便的目的。     

pQE30-bgln原核表达质粒的构建和鉴定

构建重组原核表达质粒pQE30-bgln使之可以在大肠杆菌中表达以获得β-葡糖苷酶,用于提高从植物中提取白藜芦醇的得率.以克隆质粒pUCP67为模板,用降落PCR的方法扩增β-葡糖苷酶基因片段(bgln).利用原核表达载体pQE30构建pQE30-bgln重组质粒,用酶切电泳验证重组结果的正确性,测序检测质粒重组后序列情况.PCR结果显示扩增片段2.2 kb,与预期相同.重组质粒酶切后显示其大小约5.6kb,大小及酶切图谱与预期相同.经测序发现插入片段读码框正确.可用于原核表达的pQE30-bgln质粒构建成功.     

Function of resveratrol derived from transgenic plant expressing resveratrol synthase gene

Two genes from grapevine coding for resveratrol synthase, named RS1 and RS2, were cloned by RT-PCR. AnEscherichia coli expression vector was constructed by insertion of RS1 into pBV221. A specific protein with the same molecular weight (42 ku) as the resveratrol synthase was expressed and used to prepare the rabbit antiserum. A plant expression vector was constructed by inserting the RS1 gene into pBin438 downstream of the doubled CaMV 35S promoter and TMV-Ω fragment. PCR-positive transgenic tobacco plants were obtained after transformation withAgrobacterium tumefaciens LBA4404 harboring the plant expression vector. Southern blot analysis demonstrated that the foreign gene was integrated into the tobacco genome. The results of RT-PCR and Western blot indicated that the RS1 gene was transcribed and expressed. Formation of resveratrol in transgenic tobacco was further determined by thin-layer chromatography of silica gel and HPLC. Increased accumulation of human breast adenocarcinoma cells in G₀ and G₁ phases of cell cycle was observed in cells treated with resveratrol purified from transgenic tobacco as compared to the untreated cells.     

毛白杨4CL基因启动子的克隆及初步功能分析

4CL(4-coumarate:CoA ligase,4-香豆酸:辅酶A连接酶)在植物木质素合成途径中催化羟基香豆酸生成羟基肉桂酰CoA,主要在木质部中表达,对植物木质素生物合成具重要调控作用.为研究4CL基因启动子在转基因植物中的表达特性,探索其在植物基因工程研究中的潜在应用价值,利用PCR方法从毛白杨基因组DNA中扩增得到了4CL启动子片段.序列分析表明与美洲山杨(P.tremuloids)的4CL启动子同源性为95%.采用生物信息学方法对该序列进行分析.与GUS基因融合构建双元表达载体,转化烟草的瞬时表达检测可见明显GUS活性.     

含内蛋白子融合表达载体的构建与人神经营养因子-3的初步表达

为了构建一个含蛋白质剪接元件的表达载体 ,将 3.38kb的 p Ex Sec 的多克隆位点 (Eco R /Bam H )处插入一个含多个酶切位点的 49bp寡聚核苷酸。然后将 p MYB12 9中 intein- CBD基因片段移插入上述经改造的p Ex Sec I中 ,构建成含内蛋白子的表达载体 p Ex IC。根据人神经营养因子 - 3(h NT- 3)的已知 DNA序列 ,设计含 NdeI/Xho I酶切位点的引物 ,用 PCR法从人全血总 DNA中扩增 h NT- 3,再插入 p Ex IC载体中 ,构建成 p Ex IC- h NT- 3重组质粒。经转化后 ,工程菌 E.coli BL 2 1(DE3) /p Ex IC- h NT- 3在 L B培养基中获得融合表达 ,根据 intein的自剪切原理 ,在还原剂 DTT作用下 ,初步获得了约 14k D的 h NT- 3目的蛋白。     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize (Zea mays L.)

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutin1 gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors (pUUOAH). The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of Cry1Ah protein in the construct containing the ubi1 intron (pUUOAH) was 20% higher than that of the intronless construct (pUOAH). Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubi1 intron was higher than that of the intronless construct. These results indicated that the maize ubi1 intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently     

大黄欧文菌中蔗糖异构酶基因的克隆表达及应用

以大黄欧文菌(Erwinia rhapontici)NX-5基因组DNA为模板,PCR扩增得到编码蔗糖异构酶(SIase)的基因palⅠ,构建克隆载体pUC18-palⅠ。经测序正确后,将palⅠ亚克隆至表达载体pET-22b(+)上,并在E.coliBL21(DE3)中成功表达相对分子质量约为66 000的可溶性蛋白。通过Ni-NTA柱对表达产物进行纯化,纯酶的比活为40 U/mg。转化条件研究表明:重组菌能够高效转化质量分数为50%的蔗糖溶液,转化液中异麦芽酮糖得率为85%。     

人白介素-3乳腺表达载体的构建

人白介素-3是一种造血系统和免疫系统调节剂,在治疗造血系统疾病、肿瘤、先天或获得性免疫功能缺陷等方面发挥着重要作用.应用牛乳腺生物反应器生产人白介素-3的研究具有重要的临床应用和经济价值.研究选用具有红色荧光蛋白报告基因(R ed2)和新霉素抗性基因(neor)表达框架的pD sR ed2-1质粒为骨架构建人白介素-3乳腺表达载体.通过PCR方法分别扩增牛β-酪蛋白基因5′端上游调控序列、人IL-3基因以及CM V启动子序列,将它们按先后顺序分别定向克隆于质粒pD sR ed2-1的多克隆位点内,使牛β-酪蛋白基因调控序列位于人IL-3基因的上游,指导人IL-3基因在乳腺组织中特异性表达,而CM V启动子位于红荧光蛋白基因的上游,指导红荧光蛋白基因在所有的组织中非特异性表达.限制性酶切片段分析及部分DNA序列鉴定结果表明,所构建载体结构正确.     

棉铃虫组织蛋白酶B在杆状病毒表达系统中的表达及鉴定

以棉铃虫（Helicoverpa armigera）组织蛋白酶B作外源基因重组构建克隆载体，自重组菌株HCB-DH10Bac、Histag-HCB-DH10Bac中提取穿梭质粒，脂质体法转染草地贪夜蛾卵巢细胞系Sf21细胞，转染液再感染细胞，收集感染3～4d的上清液，提取芽生病毒的DNA，PCR法鉴定外源HCB基因，感染上清进行SDS-PAGE、Western-blotting、蛋白酶活性检测．结果：感染上清中的芽生病毒的DNA作模板扩增出预期的1700bp片段，SDS-PAGE、westem-blotcing检测均在28ku处有明显表达产物，且表达产物有蛋白水解活性．