抗人端粒酶逆转录酶单链抗体基因在烟草中的表达

构建了含抗人端粒酶逆转录酶单链抗体基因ScFv-hTERT的植物表达载体p1304-SH,通过农杆菌介导的叶盘法转化烟草.转基因烟草植株叶片总DNA的PCR,Southern b lot检测结果表明,ScFv-hTERT基因已整合进了转基因烟草植株基因组中;RT-PCR,SDS-PAGE分析证实目的基因已在烟草叶片中成功表达,竞争性ELISA的结果表明,表达的重组抗体与其抗原有良好的结合活性.     

表达千穗谷Ah-AMP基因的转基因烟草抗病性研究

通过PCR从苋属植物千穗谷(Amarathus hypochondriacus)的总DNA中扩增出苋菜抗菌肽(Ah-AMP)的核基因片段,序列分析结果表明该基因长261bp,编码一个由86个氨基酸组成的Ah-AMP前体多肽.在构建Ah-AMP基因的植物表达载体pBinAH916后,通过根癌土壤杆菌介导方法转化了烟草.转化再生植株和T 1 代转基因烟草的PCR和Southern blot分析表明,Ah-AMP基因已整合到烟草的染色体中,并为单拷贝整合.T 1 代转基因烟草的Northern blot分析结果表明,Ah-AMP基因在转基因烟草中至少在mRNA水平上能正常表达.对T 0 代转基因烟草进行烟草青枯病的抗病性试验,筛选出两株抗性较强的植株.对T 1 代抗青枯病的统计分析发现,其抗病性比起始品种SR1分别提高了2.24和1.62个级别;其病情指数比起始品种降低49.6%和37.3%.对黑胫病也表现一定的抗性,主要表现在推迟发病时间,减缓发病速度上.这些结果表明Ah-AMP基因在植物抗病基因工程研究中可能是一个有潜在应用价值的基因.     

转蔗糖:蔗糖-1-果糖基转移酶基因提高烟草的耐旱性

蔗糖: 蔗糖-1-果糖基转移酶（sucrose: sucrose 1-fructosyltransferase, 1-SST）以蔗糖为底物催化生成蔗果三糖等低聚合度的果聚糖.将从莴苣中克隆的1-SST基因重组到pCAMBIA1300-als中,构建了在CaMV 35S启动子调控下的植物表达载体,利用农杆菌介导的叶盘转化法将1-SST基因导入烟草中,PCR和Southern杂交检测表明获得了转基因植株,RT-PCR结果表明该基因在烟草中正常表达. 对T₀代转基因烟草进行的耐旱性分析结果表明,干旱胁迫6d的转基因植株丙二醛含量和电解质渗漏率显著低于未转基因对照,叶片相对含水量下降速度也明显比对照慢. 对转基因植株叶片糖分分析表明,转基因烟草植株积累果聚糖,并在干旱胁迫后含量明显增加,而未转基因对照植株不积累果聚糖. 在14%PEG溶液中未转基因烟草种子的萌发率仅为转基因烟草种子的一半;在附加200mmol/L甘露醇的培养基中未转基因烟草种子根的生长明显受到抑制,而转基因烟草根的生长发育正常. 以上研究结果表明,转1-SST基因烟草植株耐旱性的提高可能与该基因的表达有关.     

苦瓜McAG2启动子的克隆与表达研究

文中从苦瓜基因组中克隆得到长为1417 bp的McAG2基因5′上游片段并进行了DNA序列分析.通过PCR得到了其缺失片段,将其插入pBI121载体替换CaMV35S启动子,得到了McAG2基因5′侧翼缺失表达载体.并利用农杆菌介导转化烟草,建立了相应的转基因烟草植株,以研究其在不同器官组织中的表达特性. β-glucuronidase(GUS)染色结果显示该启动子在转基因烟草叶片和根组织中没有表达活性.     

H5N1型禽流感病毒HA基因在烟草中的表达

禽流感病毒H5N1是可以直接感染人类的甲型流感病毒,发展植物源口服疫苗是疫苗研究的方向之一.本研究通过农杆菌介导的方法将禽流感病毒H5N1的HA基因转化烟草.共获得38株潮霉素抗性植株,经PCR和Southern-blotting检测,目的基因已整合到转基因植株的基因组中.Western-dotting检测结果表明,目的基因在转基因烟草中得到表达,具有免疫原性,获得了能够表达HA基因的植物口服疫苗候选植株.     

Cre-lox重组系统介导转基因烟草中外源基因删除的研究

对Cre在转基因个体中介导的重组效率进行了研究。构建了含有Cre 基因(p35S-Cre)和 GUS 基因侧翼含同向loxP 位点的(loxP-p35S-GUS-loxP)两种植物表达载体。以共转化的技术将两种基因元件同时转化烟草得到转基因植株,根据对共转化植株GUS 基因的活性分析、分子检测、PCR检测及对重组后扩增DNA片段进行序列分析表明：Cre-loxP 重组系统在转基因烟草中能精确高效地介导转基因的删除,但也存在部分植株不能完全删除的现象。     

一个水稻类受体蛋白激酶OsRPK2的过量表达分析

为了解类受体蛋白激酶Os RPK2在水稻发育中的作用,采用反向遗传学方法构建了该蛋白激酶基因的过表达载体,将其导入野生型水稻中获得转基因植株后,观察植株的表型变化以及该基因在水稻植株不同部位的表达情况.结果发现,Os RPK2的转基因植株产生白化现象,q RT-PCR分析表明白化植株中Os RPK2的表达量明显增加,说明水稻转基因植株的白化表型是由Os RPK2基因的过表达造成的.Os RPK2的组织特异性表达结果表明,Os RPK2在水稻的不同组织器官中均有表达,但在水稻幼嫩的分生组织和幼龄叶片中表达量较高,反映了Os RPK2在水稻植株发育及建成中具有重要作用.     

油菜ICE1基因表达载体构建及转化烟草

从油菜中克隆得到了ICE1基因的开放阅读框序列.构建了以CaMV35S为启动子的植物表达载体,经农杆菌介导法转化烟草,获得转基因植株.通过PCR检测,目的基因成功转入烟草中.将转油菜ICE1基因的烟草和非转基因烟草置于2℃处理7 d,非转基因烟草出现萎蔫,转基因烟草没有明显变化,非转基因烟草丙二醛浓度明显高于转基因烟草.将转基因烟草与非转基因烟草分别置于0、-2、-4℃各1 h后,转基因烟草APX、SOD活性明显高于非转基因烟草.     

基于过表达技术的烟草NtGGPPS1基因功能的研究

将NtGGPPS1-T质粒和spCAMBIA1300载体分别双酶切(SalⅠ和KpnⅠ)后连接,转化,挑取单克隆,筛选得到植物超量表达载体spCAMBIA1300-NtGGPPS1,并通过农杆菌介导侵染栽培烟草K326,在获得阳性转基因烟草后,进行定量RT-PCR以定量分析烟草牻牛儿基牻牛儿基焦磷酸合成酶基因NtGGPPS1在转基因植株中的转录积累.同时,通过LC-MS和GC-MS检测转基因植株中质体色素和萜类化合物含量的变化.结果显示,与对照组相比,NtGGPPS1基因在编号为Nt1-OE1、2、3、4、7植株中表达上调,阳性转基因烟草中新黄质、紫黄质、叶黄素、叶绿素a、叶绿素b和β-胡萝卜素6种质体色素,以及烟叶中代表性二萜类物质α-西柏三烯二醇(α-CBD)和β-西柏三烯二醇(β-CBD)的含量明显增加.以上结果表明,NtGGPPS1基因参与了烟草中质体色素和二萜类化合物的生物合成.     

葡萄糖氧化酶基因的克隆及其在转基因烟草中的表达

通过PCR扩增,克隆了黑曲霉编码葡萄糖氧化酶的GO基因.构建了CaMV35S启动子驱动的GO基因植物表达载体,经农杆菌介导的遗传转化,获得了转基因烟草植株.经Southern,Northern和Western杂交分析,证明了GO基因在转基因烟草中的整合、转录和翻译.转基因烟草中H2O2的含量最高可达610μmol/L,比对照提高近5倍.嫁接试验证实H2O2可由砧木远距离运输至接穗,但不能由接穗转运至砧木,说明H2O2是通过导管由下向上运输.非转基因接穗嫁接至转基因砧木上3周,接穗上不同节位叶片中H2O2的含量不存在浓度梯度,说明这是一种主动运输而不是被动扩散.     

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.     

Disease-tolerance of transgenic tobacco plants expressing Ah-AMP gene of Amaranthus hypochondriacus

An antimicrobial peptide gene from Amaranthus hypochondriacus, Ah-AMP, was amplified by PCR and cloned. Sequence analysis results revealed that this gene is 261 bp in length encoding a precursor polypeptide of 87 amino acid residues. Ah-AMP gene was inserted in the binary vector pBin438 to construct a plant expression vector pBinAH916. Leave explants of Nicotiana tabacum var. SR1 were transformed with Agrobacterium tumefaciens LBA4404 harboring the above expression vector. Results from PCR, Southern and Northern blot analyses confirmed that the Ah-AMP gene had been integrated into the tobacco genome and was transcribed at mRNA level. Two bacterial-resistant transgenic plants were selected by inoculating the plants with Pseudomonas solanacearum and statistic analysis of two T1 lines showed that the resistance increased by 2.24 and 1.62 grade and the disease index decreased by 49.6% and 37.3% respectively when compared with the non-transformed control plants SR1. The results from challenging the plants with inoculums of Phytophthora parasitica showed that the symptom development was delayed and disease index was significantly reduced. These results suggest that Ah-AMP gene may be a potentially valuable gene for genetic engineering of plant for disease-resistance.     

泛素和PR1a信号肽融合基因植物表达载体的构建

泛素（Ubiquitin）融合蛋白策略在近年来已被应用于在植物中提高外源蛋白的产量。信号肽（Signal Peptide）使外源蛋白定向运输到细胞内的特定部位。本研究是在植物高效表达载体pBin438的基础上，采用三引物PCR法（TP-PCR）技术，将拟南芥（Ambidopsis）泛素基因与烟草病程相关蛋白PR1a基因的信号肽序列体外重组；并将重组的融合基因定向克隆到植物表达载体pBLG中，获得了含有泛素和PR1a信号肽序列的植物表达载体pBLG-UP。这为进一步验证外源基因在植物体内的高效表达奠定了基础。     

多基因双T-DNA植物表达载体的构建及拟南芥转化

多基因植物表达载体用于植物遗传转化是培育具有多种优良品质作物的有效策略. 双T-DNA系统是实现筛选完成后选择标记基因删除的一种简便可行的方式. 为培育高度抗逆或去除标记基因的农作物,构建了多基因双T-DNA植物表达载体2T-bbgdD,其中含有一个抗除草剂基因bar, 3个抗逆相关基因(DREB1A, Na+依赖性Pi转运体基因（d5）, betA)和一个报告基因gfp. 利用农杆菌介导法将该载体转入拟南芥,获得了多基因共转化及去除标记基因的转基因拟南芥. 可将此植物表达载体进一步用于作物的遗传转化.     

Obtaining High Pest-resistant Tobacco Plants Carrying B.t. insecticidal Gene

To increase the expression level of CryIA(c) gene in transgenic plants, a plant expression vector pBinMoBc carrying the CryIA(c) gene under control of chimeric OM promoter and Ω factor was constructed. As a control, pBinoBc carrying the CryIA(c) gene with the CaMV 35S promoter was also constructed. The vectors were transferred into tobacco plants respectively via Agrobacterium-mediated transformation. ELISA assay showed that the expression level of the CryIA(c) gene in pBinMoBc transgenic tobacco plants was 2.44-times that in pBinoBc transgenic tobacco plants, and it could be up to 0.255% of total soluble proteins. Bioassay showed that pBinMoBc transgenic tobacco plants had more notable insecticidal effect than pBinoBc transgenic tobacco plants. The above results showed that the chimeric OM promoter was a stronger promoter than CaMV 35S promoter that was widely used in plant genetic engineering, and this is very useful in pest-resistant plant genetic engineering.     

绿色荧光蛋白基因转化大岩桐的研究

利用农杆菌介导法 ,用含有绿色荧光蛋白基因的二元双价表达载体pBINm -gfp5 -ER转化大岩桐 ,并得到卡那霉素 (Kanamycin ,Kan)抗性再生植株 .对其进行初步PCR检测 ,结果表明 ,K2 0 0 (含Kan 2 0 0mg/L)培养基上的绿苗中有 3株PCR结果呈阳性 .对PCR阳性的植株进行了点杂交分析 ,均表现出较强的杂交信号 ,这说明外源基因已整合转入到大岩桐基因组中 .在荧光显微镜下观察转基因大岩桐 ,发现部分花、叶细胞均发出一定强度的绿色荧光     

Functional analysis of DNA 4 coding region from a Chinese Zhangzhou isolate of banana bunchy top virus

The DNA 4 coding region of banana bunchy top virus from a Chinese Zhangzhou isolate (BBTV-ZZ) is cloned by PCR. The sequencing analysis shows that it is 351 nucleotides long and it putatively encodes a protein of 116 amino acids. On the basis of a plant binary vector pBin438, the plant expression vector pBBTV-4B harboring the BBTV-ZZ DNA 4 coding region has been constructed and then transferred to tobacco (Nicotiana tobacum cv. Xanthi nc) by a Agrobacterium-mediated procedure. Under insect-free condition, movement-defective mutant of CMV-Fny strain (CMV-Fny-△MP) is mechanically inoculated on the lower leaves of transgenic plants. Systemic symptoms with different degrees of severity are developed in the upper uninoculated leaves of transgenic plants at 12 days postinoculation (dpi), while no symptoms can be seen in the uninoculated leaves of untransformed plants at any time. Accumulation of CMV-Fny is detected on the upper uninoculated leaves of transgenic plants, but is not on that of untransformed plants by indirect double antibody sandwich enzyme-link immunosorbent assay (DAS-ELISA). The results reveal that transgenic plants have acquired the property of cell-to-cell movement and systemic spread of CMV-Fny-△MP. This suggests that the protein encoded by BBTV-ZZ DNA 4 might have function of viral movement protein.     

一种带有表型标记基因的诱导型抗旱植物表达载体的构建

为了克服组成型表达转录因子基因影响转基因植物性状的缺点,并构建一种具有级联放大作用并带有表型标记的诱导型植物双价表达载体。研究采用PCR方法从拟南芥克隆获得冷诱导转录因子CBF3基因,蜡质合成相关WIN1基因,干旱诱导RD29A基因启动子和冷诱导的LEA14基因启动子,并用CBF3转录因子所调控的下游RD29A基因启动子和LEA14基因启动子分别驱动CBF3基因和W1N1基因表达,构建了双价植物表达载体RD29AP-CBF3／LEA14P—WIN1／pcAMBIA2201。我们预测在转基因植物中,该表达系统可在干旱等逆境信号存在条件下,通过级联放大的方式诱导表达,在增加植物抗逆性的同时,增加叶片表层蜡质的积累,从而易于表型识别。本研究为利用花粉管通道法转化棉花,提高抗逆转基因棉花田间筛选的效率奠定了基础。     

Construction of a new plant expression vector containing two insect resistant genes and its expression in transgenic tobacco plants

A new plant expression vector (pBS29K-BA) containing two insect resistant genes, a synthetic chimeric gene BtS29K encoding the activated insecticidal protein Cry1Ac and a gene API-BA encoding the arrowhead (Sagittaria sagittifolia L.) proteinase inhibitor (API) A and B, is constructed. Transgenic tobacco plants expressing these two genes are obtained through Agrobacterium-mediated transformation of tobacco leaf discs. The average expression levels of Cry1Ac and API-BA proteins in transgenic plants are of 3.2 μg and 4.9 μg per gram fresh leaf respectively. The results of insecticidal assay of transgenic plants indicate that the pBS29K-BA transformed plants are more resistant to insect damage than the plants expressing the Cry1Ac gene or API-BA gene alone.     

抗汉滩病毒单抗3G1 scFv植物表达载体的构建

利用PCR方法，从含有3G1 scFv基因的重组质粒中扩增出抗体基因，并使基因两端携带合适的限制性酶切位点．将其克隆人植物表达载体pBI121，构建获得3G1 scFv-pBI121重组质粒．酶切鉴定及测序结果均证明重组质粒构建成功，将重组植物表达载体转入农杆菌LBA4404，为进一步构建转基因植物的研究奠定了基础．