EuFPS基因表达载体构建及对杜仲遗传转化的研究

本实验用EcoRⅠ和BamHⅠ双酶切植物表达载体pSH737和含有目的基因的pUC-FPS,定向连接得到重组质粒pSH-FPS,将其导入农杆菌EHA105.采用农杆菌介导法对杜仲进行遗传转化,研究了卡那霉素(kanamycin,Km)浓度、预培养时间、菌液浓度及侵染时间、乙酰丁香酮(acetosyringone,AS)浓度、共培养时间等对杜仲遗传转化效率的影响.结果表明,选择无菌苗苗龄15 d的杜仲下胚轴,卡那霉素浓度50mg/L,农杆菌浓度OD600值0.3-0.6,侵染时间8 min,侵染时菌体重悬液中添加50 μmol/L乙酰丁香酮,共培养时间3 d,抗性芽的获得率最高.对再生植株进行GUS检测发现有45%的植株呈阳性.     

无核白葡萄愈伤组织瞬时转化体系的优化与应用

为了优化根癌农杆菌介导的葡萄愈伤组织瞬时转化体系,该研究以欧洲葡萄品种无核白(Vitis vinifera L.cv.Thompson Seedless)单芽茎段诱导的愈伤组织为材料,探讨重悬液pH、菌液浓度、真空渗透时间等主要因素对葡萄愈伤组织瞬时转化效率的影响。结果表明:(1)以激素组合分别为1.0mg/L BAP、1.0mg/L BAP+0.02mg/L NAA、2.0mg/L BAP+0.02mg/L NAA和4.0mg/L BAP+0.02mg/L NAA的系列培养基更适合无核白葡萄单芽茎段逐步诱导胚性愈伤组织。(2)葡萄愈伤组织瞬时转化体系中,重悬液pH 5.1,菌液浓度OD6001.0,真空渗透20min为转化效率最佳条件。(3)利用优化的瞬时转化体系瞬时转化无核白葡萄的不同组织,发现在不同器官中转化效率存在显著差异。其中以愈伤组织为受体的转化效率显著高于其他器官(65 231.99±3 339.29mU/g),而且愈伤组织的GUS组织化学染色最深,以叶片为受体的转化效率则最低。利用该体系转化质粒载体pCAMBIA0390∷GUS,瞬时表达产物经过GUS蛋白活性检测,结果表明该研究优化的葡萄愈伤组织瞬时转化体系有助于外源基因在葡萄愈伤组织内的表达,为后期通过转基因技术研究目标基因功能奠定了技术基础。     

一种根癌农杆菌介导的耐盐椒样薄荷含芽茎段转化系统

椒样薄荷的转基因技术是提高精油产量和品质的重要手段,为解决耐盐椒样薄荷叶片不定芽诱导过程中易褐化,难分化,不适合作为转基因外植体的问题,利用含芽的椒样薄荷茎段作为外植体建立了一种根癌农杆菌介导的外源基因转化体系。利用正交试验对转化所用的根癌农杆菌菌液浓度、转化时间和共培养时间进行优化,结果显示:3个因素对转化效果的影响作用表现为转化时间(B)菌液浓度(A)共培养时间(C),筛选出的最优转化条件为菌液浓度为OD_(600)=0.8,转化时间为15 min,共培养时间为3 d。对不同转化液组分进行优化筛选,结果获得最适的转化液组分为:1/5MS+0.5 g/L MES(2-(N-吗啡啉)乙磺酸)+1%葡萄糖+2%蔗糖,PH5.4。利用不同植物表达载体进行转化,并通过基因组PCR和GUS染色进行转化植株的阳性检测,结果表明该转化体系适用于多种植物表达载体的转化,容易获得再生植株,为耐盐椒样薄荷的基因转化提供新的方法与体系。     

农杆菌介导的墨兰遗传转化

转基因育种是快速定向改良兰花育种目标性状的有效方法,但迄今未见有关墨兰转基因育种的研究报道。试验以‘企剑白墨’墨兰Cymbidium sinensis cv.‘Qijianbaimo’的根状茎为受体材料,研究了影响农杆菌介导墨兰遗传转化效率的因素,以建立有实用价值的墨兰遗传转化技术体系。结果表明,受体的预培养时间、乙酰丁香酮的添加方式及浓度、农杆菌工程菌液浓度(OD600)、侵染时间和共培养时间均对‘企剑白墨’根状茎的GUS瞬时表达率有显著影响。以预培养39 d的根状茎尖为材料,在添加200μmol/L乙酰丁香酮,OD600为0.9的工程菌液中侵染35 min后,转入添加200μmol/L乙酰丁香酮的共培养基中培养7 d时,‘企剑白墨’根状茎的GUS瞬时表达率最高,为11.67%。采用上述条件对‘企剑白墨’墨兰进行遗传转化,经PCR鉴定和GUS染色检测,从400株再生植株中获得了3株转基因植株,转化率为0.75%。研究表明通过农杆菌介导法对墨兰进行遗传改良是可行的。     

蝴蝶兰花瓣瞬时转化体系建立

蝴蝶兰花器官中基因功能的研究受遗传转化效率低和遗传转化周期长的制约,而花瓣瞬时表达体系是一种快速分析基因功能的有效手段。该研究以蝴蝶兰‘大辣椒’花瓣和萼片为实验材料,通过农杆菌介导的瞬时转化方法,分析了侵染的菌液浓度、侵染时间、乙酰丁香酮浓度和共培养时间等4个因素对β-葡糖醛酸酶(GUS)报告基因表达效率的影响,以探寻其瞬时表达的最佳条件;并将查尔酮合成酶(chalcone synthase,CHS)基因RNAi干扰载体瞬时转化蝴蝶兰花瓣,共培养3d后观察转化材料中花色表型以及色素的变化,并利用半定量RT-PCR来检测CHS基因转录水平的表达。结果表明:(1)农杆菌菌液OD600为0.6、侵染时间60s,在重悬液中添加150μmol/L乙酰丁香酮,共培养3d,GUS瞬时表达率最高(85.01%)。(2)转基因蝴蝶兰花瓣颜色明显变淡,色素含量降低。(3)半定量PCR检测表明,CHS基因的转录活性相比于对照组显著降低。该实验成功的在蝴蝶兰花器官中建立了一种快速基因功能验证方法,为后期蝴蝶兰基因功能研究和育种工作提供技术支持。     

超声波辅助农杆菌介导CP基因转化番木瓜(Carioca papaya L.)的有效方法

本研究探索了通过农杆菌介导,超声波辅助处理,转化番木瓜胚性愈伤组织,获得转基因植株的有效方法.分别将含有日本PLDMV外壳蛋白基因(PTi-Epj-TL-PLDMV)和含有台湾PRSV菌株、美国夏威夷PRSV菌株、泰国PRSV菌株及日本PLDMV菌株的多元外壳蛋白基因编码序列(PTi-NP-YKT)插入双元载体质粒pGA482G,借助于农杆菌系LBA4404将双元载体上的外壳蛋白基因和新霉素磷酸转移酶基因(nptⅡ)转移到番木瓜品种Sunset的胚性愈伤组织中,从而获得抗卡那霉素的转化再生植株.试验着重在转化方法上进行探索.结果表明,农杆菌过夜培养后,用高渗透压培养液(1/2 MS+6%蔗糖+1%葡萄糖,pH 5.7)调整至光密度OD600nm=0.15-0.20,然后用该菌液感染材料30min,其间辅以超声波处理,可以大大提高转化效率.用15m1无菌离心管装载胚性愈伤材料进行15s的超声波处理,在80块被转化的胚性愈伤中获得21个CP基因G转化系(26.3%),而在对照处理64块胚性愈伤中仅获得1个转化系(1.6%);在经过15s的超声波处理48块被转化的胚性愈伤中获得8个CP基因B转化系(16.7%),而在对照处理25块胚性愈伤中未出现转化系.上述操作方法用在两种CP基因转化上均表现出相似的效果.试验还表明120mg/L是卡那霉素抗性筛选的最佳浓度.抗性筛选9个月后,在421块胚性愈伤组织中产生了42个抗卡那霉素的转化系.所获得的转基因植株分别用PCR和Southern印迹杂交进行了鉴定.     

农杆菌介导的大豆子叶节非组织培养遗传转化体系优化

本研究以GUS基因在子叶节区的瞬时表达为依据,通过探讨影响农杆菌转化效率的因素,优化了大豆子叶节非组织培养遗传转化体系;利用该体系对冀豆16号进行Bar基因的遗传转化,并使用针刺法对转基因植株进行草铵膦筛选.结果表明,侵染液中附加3％蔗糖、OD600=0.6、以脱脂棉作为菌液附着介质同时不添加表面活性剂Silwet L-77、侵染1次的GUS阳性率最高达到62.13％.草铵膦抗性植株经PCR检测获得T0阳性植株10个,转化率为2.5％.经PCR和RT-PCR鉴定共获得3株T1阳性植株,初步证明目的基因已整合到大豆基因组中.     

TRV介导的溪荪VIGS转化体系的构建与鉴定

病毒诱导基因沉默（Virus-induced gene silencing,VIGS）技术为缺乏稳定遗传转化体系的植物进行基因功能分析提供了可能，为解决缺少稳定遗传转化体系的植物进行基因功能验证的需求，以单子叶植物溪荪（Iris sanguinea）为材料，克隆IsPDS基因的特异性片段并构建其VIGS重组载体pTRV2-IsPDS，通过注射法侵染溪荪叶片，结果显示：pTRV1和pTRV2-IsPDS的菌液OD600调至1.8～2.0后重悬，重悬液OD600调至0.8～1.0，通过注射器叶脉注射方式侵染溪荪叶片效果最佳。在室外温度为15～20℃的下午6～8时进行，用1 mL注射器针头扎伤溪荪叶片外表皮，沿着平行脉缓慢注射重悬液1 mL至溪荪叶片维管束中，14 d左右会出现明显的白化表型。在出现表型变化的植株和空载组中均检测到TRV1和TRV2的病毒载体，白化植株的IsPDS表达量显著低于对照组。侵染液配制中通过提高携带病毒载体的农杆菌的浓度提高侵染效率，且接种后无需遮光。     

根癌农杆菌介导大花蕙兰遗传转化的研究

以大花蕙兰原球茎(PLBs)为外植体,采用EHA105和LBA4404 2种根癌农杆菌菌株与pCAMBIA1301质粒构建工程菌介导,以建立大花蕙兰遗传转化体系,并比较不同受体处理方式、菌液浓度和侵染方式等对大花蕙兰转化的影响.结果表明:(1)以切成3 mm左右的PLBs小块作为受体材料,用OD600值为0.6的LBA4404根癌农杆菌菌株,并用MS+1.0 mg/L BA+200μmol/L AS(乙酰丁香酮)的液体培养基将菌液等体积稀释侵染,转化率可达62.5%.(2)大花蕙兰对潮霉素(Hyg)十分敏感,5 mg/L Hyg对转化后的PLBs有较好的筛选效果,筛选后最高成活率为13.0%.(3)PCR检测初步证明,通过根癌农杆菌介导的方法获得了2株转基因大花蕙兰植株.     

花粉管通道法介导的铁皮石斛转基因技术

该研究以含有GFP和GUS基因的质粒和农杆菌为载体,采用花粉管通道法对铁皮石斛进行转基因技术研究。结果表明:(1)铁皮石斛种子萌发和原球茎生长对卡那霉素的最低致死浓度分别为90和150 mg·L~(-1)。进一步研究证实,在筛选转化种子和原球茎时,可分别向培养基中添加100和150 mg·L~(-1)的卡那霉素进行选择培养。(2)以携带GFP和GUS基因的质粒(pSuper1300和pBI121)和农杆菌为载体,用无菌去离子水重悬质粒pSuper1300和pBI121至浓度为100 ng·μL~(-1),用2%蔗糖+1/2MS+0.1%silwet-77+0.1%AS或5%蔗糖+0.1%silwet-77+0.1 mmol·L~(-1)AS重悬携带质粒pSuper1300和pBI121的农杆菌至菌液浓度为OD_(600)=0.7~0.8;在授粉后0.5~2.5 h内使用柱头滴加法导入携带外源基因的质粒或农杆菌溶液,收集成熟的转化种子,经选择培养及PCR检测发现,几乎所有处理的转化材料均能检测出外源GFP和GUS基因片段。另外,与农杆菌相比,以质粒为载体进行转化,可获得更高的结实率。该研究结果为铁皮石斛的基因工程育种提供了参考。     

卡那霉素在转基因芥菜中的应用

为了找出芥菜 (Brassica juncea Coss.) 遗传转化中最佳的卡那霉素(Kan)筛选浓度, 将芥菜的子叶接种于含有不同浓度Kan的分化培养基中, 当Kan浓度达到 30 mg/L时, 外植体的分化完全受到抑制。将芥菜种子播种于含有不同浓度Kan的培养基中, 当Kan浓度达到200 mg/L时, 长出的幼苗完全白化; 利用叶片涂抹方法, 将不同浓度的Kan涂抹于田间生长的植株叶片上, 当Kan浓度达到200 mg/L时, 被处理的叶片完全变白。为了对转基因芥菜后代中外源基因的分离情况进行遗传学分析, 分别用200 mg/L的Kan处理以npt-Ⅱ基因为选择标记基因的转基因芥菜的种子和转基因芥菜后代植株的叶片, 利用χ2测验分析试验结果, 4个含有单拷贝外源基因的转基因株系后代, 对Kan的抗感分离都符合3︰1的分离规律; 而2个含有双拷贝外源基因的转基因株系, 其中一个对Kan的抗感分离符合3︰1而不符合15︰1, 另一个对Kan的抗感分离既符合3︰1也符合 15︰1, 双拷贝外源基因在转基因芥菜中的整合方式有待进一步的研究。最后, 用PCR分析证实了该方法的准确性, 因此, 利用Kan对转基因芥菜后代进行筛选是可行的。     

Transgenic Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) plants expressing an <Emphasis Type="Italic">Arabidopsis</Emphasis> phytochelatin synthase (<Emphasis Type="Italic">AtPCS1</Emphasis>) exhibit enhanced As and Cd tolerance

Phytochelatins (PCs) are post-translationally synthesized thiol reactive peptides that play important roles in detoxification of heavy metal and metalloids in plants and other living organisms. The overall goal of this study is to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A FLAG-tagged AtPCS1 gDNA, under its native promoter, is expressed in Indian mustard, and transgenic pcs lines have been compared with wild-type plants for tolerance to and accumulation of cadmium (Cd) and arsenic (As). Compared to wild type plants, transgenic plants exhibit significantly higher tolerance to Cd and As. Shoots of Cd-treated pcs plants have significantly higher concentrations of PCs and thiols than those of wild-type plants. Shoots of wild-type plants accumulated significantly more Cd than those of transgenic plants, while accumulation of As in transgenic plants was similar to that in wild type plants. Although phytochelatin synthase improves the ability of Indian mustard to tolerate higher levels of the heavy metal Cd and the metalloid As, it does not increase the accumulation potential of these metals in the above ground tissues of Indian mustard plants.     

豇豆胰蛋白酶抑制剂基因转化芥菜及抗虫鉴定

用农杆菌介导将豇豆胰蛋白酶抑制剂 (CpTI)基因导入芥菜 ,获得了Kan抗性植株 .经PCR扩增、PCR Southern印迹和Northern印迹分析 ,转化再生植株大部分呈阳性 ,而非转化的再生植株均为阴性 ,证明CpTI基因已存在于芥菜基因组中 .在室内进行了喂虫试验 ,结果表明转基因芥菜抗虫性明显高于对照 ,转基因植株之间存在抗虫性差异     

Competitiveness of transgenic oilseed rape

The competitiveness of two transgenic oilseed rape (Brassica napus ssp.napus) lines and their fertile transgenic hybrid was tested in field trials in Belgium and Denmark. The lines contained genes for male sterility, restoration of fertility and herbicide resistance. The competitiveness of the three transgenic lines was related to three non-transformed commercially-grown oilseed rape varieties: Drakkar, Topas and Line. As a reference of a more aggressive crucifer, white mustard (Sinapis alba) was also included in the experiment. The experimental design was a complete block design with two locations, monocultures and mixtures with barley (Hordeum vulgare), three plant densities, four harvest times and four blocks. The yield density relationship of the transgenic oilseed rape lines was not different from that of the non-transgenic varieties in either location. The first harvest times showed a vigorous biomass production of white mustard, which in turn produced a significant difference in the competitive ability between oilseed rape and white mustard. Later, this difference decreased, and in Belgium there was no difference at the last harvest time. Variations within populations may blur actual differences between lines and varieties, and it is argued that unless the experimental design covers a range of competitiveness for which it is possible to detect significant differences, test results reporting a lack of difference between transgenic and non-transgenic plants are of little value.     

Exploring the selenium phytoremediation potential of transgenic Indian mustard overexpressing ATP sulfurylase or cystathionine-gamma-synthase

Indian mustard (Brassica juncea) plants overexpressing ATP sulfurylase (APS transgenics) were previously shown to have higher shoot selenium (Se) levels and enhanced Se tolerance compared to wild type when supplied with selenate in a hydroponic system. Other transgenic Indian mustard overexpressing cystathionine-gamma-synthase (CGS) showed a higher Se volatilization rate, lower shoot Se levels, and higher Se tolerance than wild type, also in hydroponic studies. In the present study, these APS and CGS transgenics were evaluated for their capacity to accumulate Se from soil that is naturally rich in Se. Wild-type Indian mustard and the Se hyperaccumulator Stanleya pinnata were included for comparison. After 10 weeks on Se soil, the APS transgenics contained 2.5-fold higher shoot Se levels than wild type Indian mustard, similar to those of S. pinnata. The CGS transgenics contained 40% lower shoot Se levels than wild type. Shoot biomass was comparable for all Indian mustard types and higher than that of S. pinnata. These results obtained with these transgenics on soil are in agreement with those obtained earlier using hydroponics. The significance of these findings is that they are the first report on the performance of transgenic plants on Se in soil and show the potential of genetic engineering for phytoremediation.     

Nonspecific binding of monoclonal anti-FLAG M2 antibody in Indian mustard (Brassica juncea)

Chimeric constructs with the hydrophilic octapeptide FLAG epitope (DYKDDDDK) have been widely used as multipurpose tags for identification, detection, and purification of FLAG fusion proteins. Constructs consisting of C-terminal FLAG-tagged genomic and cDNA clones of anArabidopsis phytochelatin synthase gene,AtPCS1, were used in developing transgenic lines of Indian mustard. Presence and expression ofAtPCS1 in transgenic lines were confirmed by using PCR and Northern blot analyses. However, immunoblot analysis revealed strong nonspecific binding of a monoclonal anti-FLAG M2 antibody to an endogenous protein in both shoot and leaf tissues of wild-type Indian mustard (85-kDa) that masked presence of the phytochelatin synthase (PCS) protein of interest (55-kDa). Further analysis revealed absence of a nonspecific protein in root tissues of transgenic plants, thus allowing detection of the FLAG-tagged PCS protein.     

The isochorismate pathway is negatively regulated by salicylic acid signaling in O<Subscript>3</Subscript>-exposed <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phytochelatins (PCs) are heavy metal binding peptides that play an important role in sequestration and detoxification of heavy metals in plants. In this study, our goal was to develop transgenic plants with increased tolerance for and accumulation of heavy metals from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A 35S promoter fused to a FLAG–tagged AtPCS1 cDNA was expressed in Indian mustard, and transgenic lines, designated pc lines, were evaluated for tolerance to and accumulation of Cd and Zn. Transgenic plants with moderate AtPCS1 expression levels showed significantly higher tolerance to Cd and Zn stress, but accumulated significantly less Cd and Zn than wild type plants in both shoot and root tissues. However, transgenic plants with highest expression of the transgene did not exhibit enhanced Cd and Zn tolerance. Shoots of Cd-treated pc plants had significantly higher levels of phytochelatins and thiols than wild-type plants. Significantly lower concentrations of gluthatione in Cd-treated shoot and root tissues of transgenic plants were observed. Moderate expression levels of phytochelatin synthase improved the ability of Indian mustard to tolerate certain levels of heavy metals, but at the same time did not increase the accumulation potential for Cd and Zn.     

Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens

Defensins are small positively charged, antimicrobial peptides (~5 kDa in size) and some of them exhibit potent antifungal activity. We have cloned the complete cDNA containing an ORF of 243 bp of a defensin of mustard. The deduced amino acid sequence of the peptide showed more than 90% identity to the amino acid sequence of the well-characterized defensins, RsAFP-1 and RsAFP-2 of Raphanus sativus. We have generated and characterized transgenic tobacco and peanut plants constitutively expressing the mustard defensin. Transgenic tobacco plants were resistant to the fungal pathogens, Fusarium moniliforme and Phytophthora parasitica pv. nicotianae. Transgenic peanut plants showed enhanced resistance against the pathogens, Pheaoisariopsis personata and Cercospora arachidicola, which jointly cause serious late leaf spot disease. These observations indicate that the mustard defensin gene can be deployed for deriving fungal disease resistance in transgenic crops.