小麦抗白粉病相关基因的转化

利用玉米花青素苷合成调节基因C1-Lc作为报告基因, 通过瞬间表达后愈伤组织表面红色斑点的统计分析, 优化了小麦幼胚愈伤组织的基因枪转化参数。小麦Beclin1类似基因TaTBL和硫代硫酸硫转移酶基因TaTST是2个在白粉菌诱导条件下具有增强表达特性的抗病相关基因。本实验进一步利用基因枪将ubi强启动子控制下的2个基因导入到小麦品种扬麦158的幼胚愈伤组织细胞中, 使用除草剂经两轮选择培养基上的筛选和再生获得抗性植株, 进一步通过抗性植株的PCR分析获得转TaTBL基因植株5株, 转TaTST基因植株6株。转基因植株离体叶片的人工接种实验表明, 外源基因的导入不同程度上增强了植株的白粉病抗性, 表现为延缓了白粉菌的发育。利用玉米花青素苷合成调节基因C1-Lc作为报告基因,通过瞬间表达后愈伤组织表面红色斑点的统计分析,优化了小麦幼胚愈伤组织的基因枪转化参数。小麦Beclin1类似基因TaTBL和硫代硫酸硫转移酶基因TaTST是两个在白粉菌诱导条件下具有增强表达特性的抗病相关基因。本实验进一步利用基因枪将ubi强启动子控制下的两个基因导入到小麦品种扬麦158的幼胚愈伤组织细胞中,使用除草剂经两轮选择培养基上的筛选和再生获得抗性植株,进一步通过抗性植株的PCR分析获得转TaTBL基因植株5株,转TaTST基因植株6株。转基因植株离体叶片的人工接种实验表明,外源基因的导入不同程度上增强了植株的白粉病抗性,表现为延缓了白粉菌的发育。     

农杆菌介导玉米胚性愈伤的遗传转化研究

利用3种不同类型的农杆菌菌株C58、LBA4404和EHA105携带外源GUS基因分别侵染玉米自交系齐319和18(红)胚性愈伤.结果显示,不同的菌株和自交系间的搭配,其遗传转化效率差异很大,GUS瞬时表达率呈极显著差异(F=24.92**),抗性愈伤率也呈极显著差异(F=19.43**).其中,EHAl05-齐319组合遗传转化效率最高,其GUS瞬时表达率平均为55.5%,最高可迭71.1%;其抗性愈伤率平均为14.4%,最高可达20%;对22株转基因To代抗性植株进行PCR检测,其中PCR呈阳性植株有11株,阳性率为50%.进一步对此22株To代抗性植株进行叶片组织化学染色分析,结果显示,PCR呈阳性的植株中均有GUS基因表达.从而证明,外源GUS基因在转基因玉米To代植株中得到稳定表达,而且验证了PCR检测结果和GUS表达分析结果的一致性.     

超声波介导铁蛋白基因转化苹果的研究

以嘎拉苹果的叶片为转化受体,应用超声波法将菜豆铁蛋白基因导入受体中.实验表明,以0.5 W/cm2的声强、25℃、20 min超声波处理的转化效果最佳,抗性愈伤组织的比例高达24%.抗性愈伤组织经诱导获得了16个苹果抗性植株,有11株表现出GUS阳性,阳性比率为2.75%.经PCR检测,11株GUS阳性植株中有6株能扩增出目的条带;而Southern blotting实验表明,菜豆铁蛋白基因仅在2个苹果转基因株系的基因组中实现了完整插入,并且杂交的信号较强;进一步RT-PCR检测实验显示,外源菜豆铁蛋白基因在上述2株转基因植株中并未转录,发生了基因沉默.     

用无启动子的GUS报告基因捕获水稻基因启动子

构建了嵌合质粒p13DGUTs,它是在Ds转座子中插入了无启动子的B．葡萄糖醛酸酶报告基因(GUS),用于分离水稻基因启动子。将p13DGUTs转化粳稻品种中花11的胚性愈伤组织,获得了496个转基因植株。抗性愈伤组织与转基因植株的GUS染色与PCR分析表明整合在水稻染色体上的Ds因子都发生了随机跳跃。转基因植株T0代与部分T1代的GUS染色结果表明,M92转基因植株中Ds转座子整合位置上游的水稻基因启动子指导GUS基因的表达及表达的特性是可遗传的。文章对此方法在分离水稻基因启动子与基因上的应用进行了讨论。     

胸腺肽基因多元植物表达载体的构建

Gus基因是最为常用的报告基因之一,为了便于对胸腺肽目的基因进行检测,在该研究中,胸腺肽目的基因、PRIB分泌基因和GUS报告基因DNA片段用DNA纯化回收试剂盒回收,然后用T4 DNA连接酶连接,构建成几个基因相融合的多元植物表达载体,并通过酶切进行鉴定。用基因枪转化法对胡萝卜愈伤组织进行了遗传转化,转化的胡萝卜愈伤组织经Southern杂交检测和X-Gluc染色,结果表明,多元表达载体成功地被导入胡萝卜愈伤组织。     

红树林耐盐相关基因转化水稻的研究

运用农杆菌介导法将红树林耐盐相关基因mangrin转入粳稻品种‘日本晴’中,通过GUS基因检测愈伤组织转化率,确定农杆菌菌液浓度OD600为0.5,浸染时间30min,共培养时间3d为最佳转化体系;经潮霉菌筛选,获得抗性再生植株。通过PCR扩增检测、Southern blot分析和GUS基因活性检测,结果表明,mangrin基因整合到再生水稻的染色体DNA上。耐盐性测定结果表明,转基因植株在200mmol/L NaCl胁迫下,成活率保持在83.3%,株高增长20%~40%,mangrin基因能提高转基因水稻对盐胁迫的抗性。     

根癌农杆菌介导的金发草遗传转化条件的优化

金发草(Pogonatherum paniceum)是一种多年生岩生草本植物,在生态恢复和景观建设中起着重要的作用。利用根癌农杆菌介导转化金发草胚性愈伤组织,通过GUS(β-葡萄糖苷酸酶)瞬时表达率研究菌液浓度、浸染时间、乙酰丁香酮(AS)浓度、葡萄糖浓度、共培养时间等因素对金发草转化的影响,并利用确定的最佳条件将GUS基因转入金发草,获得稳定表达转化植株。结果表明:菌液浓度(OD₆₀₀)为0.6,浸染时间为10min,添加20mg/L的乙酰丁香酮(AS)和10g/L的葡萄糖,共培养时间5d为最佳条件,GUS瞬时表达率最高。经过抗性筛选后最终获得阳性转基因植株频率为57%。再生植株经GUS染色和PCR检测证明,GUS基因已成功整合到金发草基因组中。此转化体系的建立为金发草的遗传改良及相关功能基因的研究奠定了基础。     

高羊茅组织培养再生体系及GUS基因瞬间表达研究

以成熟种子为外值体,对高羊茅纰织培养和植株再生体系进行了优化,分析了不同浓度2．4-D、6-BA和激动素对高羊茅愈伤组织诱导和愈伤组织分化成苗的影响．结果表明：9．0mg／L 2．4-L)对愈伤组织的诱导效果最佳．0．2mg／L激动素是愈伤组织分化成苗的最适浓度．二者的诱导率和分化率分别达到68．08%和45．83％。在愈伤组织继代培养基中附加1．0mg／L 2．4-D、0．5mg／L 6-BA和1．25mg／L CuSO4;有利于胚性愈伤组织的形成,可以明显促进愈伤组织分化。同时．采用基因枪法将GUS基因导入高羊茅愈伤组织中,通过组织化学染色检测到了GUS瞬间表达活性;并对影响CUS基因瞬间表达的因素进行了分析．以期为提高基因枪法遗传转化效率提供参考。     

农杆菌介导的玉米遗传转化

用带有质粒pGIH(35S-intron-GUS/ Hptr)的根癌农杆菌EHA101转化玉米愈伤组织,获得潮霉素抗性植株,再生性好的苏玉1 图7 Southern blot分析HpaII消化的质粒pGIH 和gusA基因沉默的愈伤组织的总DNA Fig.7 Southern blot analysis of plasmid pGIH(P) and plant genomic DNA of gusA gene si- lence callus(T)digested with HpaII号转化率可以达到8.1%。对转化植株进行GUS染色分析及PCR和Southern杂交检测证明外源基因已经整合,并能够稳定表达。反向PCR分析的三个转化植株,T-DNA插入片段均为单拷贝。部分失去分化能力的抗性愈伤组织,Southern blot分析发现其基因组中有gusA基因插入,但X-gluc染色呈阴性,经HpaII酶切分析发现整合的gusA基因发生了高度甲基化。     

高赖氨酸蛋白基因导入水稻及可育转基因植株的获得

构建了一个植物高效表达质粒,使来源于四棱豆（Psophocarpus tetragonolobus(L.)DC)的高赖氨酸蛋白基因（lys）受控于单子叶植物ubiqutin强启动子下表达。用基因枪法将其导入水稻(Oryza sativa L.)幼胚诱导的愈伤组织,经潮霉素抗性筛选,得到可育的再生植株。经PCR和Southem blotting检测,表明该基因已整合到水稻的基因组织。GUS组织化学染色表明转基因水稻植株的叶、茎和根中均有gus基因的表达。测定112株转基因水稻叶片中赖氨酸叶量,大部分植株有不同程度的提高,最高幅度为16．04％。     

Agrobacterium tumefaciens–mediatated transformation of shoot-buds of chicory

Vegetative propagation of chicory via axillary shoot proliferation is one of the best ways to obtain an offspring with complete genetic stability. These shoot buds were used in transformation experiments using Agrobacterium tumefaciens strains containing binary plasmids carrying the neomycin phosphotransferase gene (nptII) and the β-glucuronidase gene (uidA). Selection was carried out on basal medium containing 100 mg l⁻¹ kanamycin. Transformed plantlets were recovered at a frequency of about 10% within four weeks after co-cultivation. The presence of the uidA gene was demonstrated by transient gene expression experiments using the histochemical GUS staining procedure. Evidence for stable transformation was shown by subculturing leaf discs on kanamycin selection medium, and Southern blot analysis confirmed the integration of the nptII and the uidA genes in the plant genome. Analysis of the progenies showed that kanamycin resistance was inherited as a single dominant trait. This method for obtaining transgenic chicory plants represents an alternative to leaf disc transformation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Promoter analysis in transient assays using a GUS reporter gene construct in creeping bentgrass (Agrostis palustris)

Transient expression profiles for several chimeric beta-glucuronidase (GUS) gene constructs were determined in tissues (young leaves, mature leaves and roots) of creeping bentgrass (Agrostis palustris, cv. Penn A4) following microprojectile bombardment. The constructs analyzed consisted of the uidA (GUS) reporter gene driven by four different promoters (ubiquitin 3-potato, ubiquitin corn, ubiquitin rice and CaMV 35S). The total number of GUS hits (or transient expression units; TEUs) were determined manually under a dissecting scope after histochemical staining for GUS. Results suggest that the ubiquitin rice promoter is most active in cells of turfgrass, regardless of the developmental stage or tissue-type. The ubiquitin corn promoter was the next best. Of the four promoter used, except for ubiquitin 3-potato, reporter gene activity was dramatically higher in mature leaves compared to young leaves. The relative efficiency of each promoter was about the same in roots and leaves. We have also analyzed uidA (GUS) reporter gene activity following microprojectile bombardment in transient expression assays with callus from two cultivars (Providence or Penn A4) of creeping bentgrass. Differences in the frequency of GUS positive hits were observed between cultivars up to 72 hours post-bombardment. However, this difference between cultivars disappeared after 72 hours post-bombardment. This information describing promoter functionality in bentgrass will be important when designing gene constructs for trait modification and when choosing appropriate cultivars for improvement through gene transfer experiments. This is the first in depth report on organ-specific and developmental gene expression profiles for transgenes in a turfgrass species.     

A robust genetic transformation protocol to obtain transgenic shoots of Solanum tuberosum L. cultivar ‘Kufri Chipsona 1’

The genetic transformation of plants is an important biotechnological tool used for crop improvement for many decades. The present study was focussed to investigate various factors affecting genetic transformation of potato cultivar ‘Kufri Chipsona 1’. It was observed that explants pre-cultured for 2 days on MS2 medium (MS medium containing 10 µM silver nitrate, 10 µM BA, 15 µM GA3), injured with a surgical blade and co-cultivated with Agrobacterium tumefaciens strain EHA105 [O.D600 (0.6)] for 2 days results in maximum transient β-glucuronidase (GUS) expression. The addition of 100 µM acetosyringone in MS2 medium also increased rate of transient GUS expression in both the explants. Clumps of putative transgenic shoots were regenerated using the optimised culture conditions from leaf and internodal explants. The stable integration of T-DNA was established using histochemical staining for GUS and amplification of DNA fragment specific to nptII and uidA genes. Within the clumps, around 67.85% of shoots showed uniform GUS expression in all the tissues and about 32.15% shoots show intermittent GUS expression establishing chimeric nature. Uniform GUS staining of the tissue was used as initial marker of non-chimeric transgenic shoots. Quantitative expression of nptII transgene was found to be directly proportional to uniformity of GUS staining in transgenic shoots. The present investigation indicated that manipulation of culture conditions and the medium composition may help to get transgenic shoots with uniform expression of transgene in all the tissues of potato cultivar ‘Kufri Chipsona 1’.     

Stable genetic transformation of<Emphasis Type="Italic"> Vigna mungo</Emphasis> L. Hepper via<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>

Vigna mungo is one of the large-seeded grain legumes that has not yet been transformed. We report here for the first time the production of morphologically normal and fertile transgenic plants from cotyledonary-node explants inoculated with Agrobacterium tumefaciens carrying binary vector pCAMBIA2301, the latter of which contains a neomycin phosphotransferase ( nptII) gene and a beta-glucuronidase (GUS) gene ( uidA) interrupted with an intron. The transformed green shoots, selected and rooted on medium containing kanamycin, tested positive for nptII and uidA genes by polymerase chain reaction (PCR) analysis. These shoots were established in soil and grown to maturity to collect the seeds. Mechanical wounding of the explants prior to inoculation with Agrobacterium, time lag in regeneration due to removal of the cotyledons from explants and a second round of selection at the rooting stage were found to be critical for transformation. Analysis of T(0) plants showed the expression and integration of uidA into the plant genome. GUS activity in leaves, roots, flowers, anthers and pollen grains was detected by histochemical assay. PCR analysis of T(1) progeny revealed a Mendelian transgene inheritance pattern. The transformation frequency was 1%, and 6-8 weeks were required for the generation of transgenics.     

Agrobacterium-mediated transformation and plant regeneration of Brassica oleracea var. capitata

An efficient protocol for Agrobacterium tumefaciens-mediated transformation of four commercial cultivars of Brassica oleracea var. capitata is described. A strain of A. tumefaciens LBA4404 with the neomycin phosphotransferase gene (nptII) and a CaMV 35S-peroxidase gene cassette were used for co-cultivation. Preliminary selection of regenerated transgenic plants was performed on kanamycin-containing medium. The frequency of transgenic plants was calculated on the basis of GUS (β-glucuronidase) activity detected by the histochemical X-gluc test. Tissue-specific GUS expression driven by the peroxidase gene promoter in transgenic plants was analysed by GUS staining. The transformation rates of the commercial cultivars of B. oleracea was higher than in previous reports. Southern blot analysis revealed that integration of marker genes occurred in single and multiple loci in the genome. All transgenic plants grew normally after a brief vernalization period and showed stable inheritance of the marker gene. The present study demonstrates that morphologically normal, fertile transgenic plants of B. oleracea can be obtained. Received: 24 August 1999 / Revision received: 23 November 1999 / Accepted: 3 December     

Gene Manipulation of a Heavy Metal Hyperaccumulator Species Thlaspi caerulescens L. via Agrobacterium-mediated Transformation

Thlaspi caerulescens L. is well known as a Zn/Cd hyperaccumulator. The genetic manipulation of T. caerulescens through transgenic technology can modify plant features for use in phytoremediation. Here, we describe the efficient transformation of T. caerulescens using Agrobacterium tumefaciens strain EHA105 harboring a binary vector pBI121 with the nptII gene as a selectable marker, the gus gene as a reporter and a foreign catalase gene. Based on the optimal concentration of growth regulators, the shoot cluster regeneration system via callus phase provided the basis of the genetic transformation in T. caerulescens. The key variables in transformation were examined, such as co-cultivation period and bacterial suspension density. Optimizing factors for T-DNA delivery resulted in kanamycin-resistant transgenic shoots with transformation efficiency more than 20%, proven by histochemical GUS assay and PCR analysis. Southern analysis of nptII and RT-PCR of catalase gene demonstrated that the foreign genes were integrated in the genome of transformed plantlets. Moreover, the activity of catalase enzyme in transgenic plants was obviously higher than in wild-type plants. This method offers new prospects for the genetic engineering of this important hyperaccumulator species.     

胡萝卜组织培养和高效遗传转化体系的建立

为了建立高效的胡萝卜遗传转化体系,本实验选用3个胡萝卜(Daucuscarotavar.sativa)栽培品种:‘金笋五寸’、‘Carol’和‘改良黑田七寸’,以它们的下胚轴和子叶为外植体,首先建立了高频愈伤诱导体系。在此基础上,以Carol的下胚轴和愈伤组织为受体材料,利用根癌农杆菌LBA4404介导转化质粒pBI121。经X-Gluc染色,证明GUS基因瞬间表达成功,经PCR方法鉴定,证明GUS基因已整合到胡萝卜的染色体中,从而建立了高效的胡萝卜遗传转化体系。     

用基因枪法介导OSISAP1基因遗传转化洋葱

以洋葱栽培品种‘HG400B’的鳞茎盘胚性愈伤组织为受体,利用基因枪介导法将水稻锌指蛋白基因OSISAP1导入洋葱中。组织化学染色检测到GUS基因在胚性愈伤组织中的瞬间表达活性,PCR、Southern杂交和RT-PCR分析,证实OSISAP1基因已整合到洋葱基因组中并实现高水平表达,转化率约为10%。对获得的转基因植株进行NaC1和NaHCO_3胁迫处理,当总浓度为200 mmol/L、处理1周后,未转基因植株会黄化、枯萎、死亡,而转基因植株却有很强的抗性,能耐受400mmol/L浓度的胁迫,表明OSISAP1基因的导入提高了转基因植株的耐盐碱性。     

影响农杆菌介导的麻疯树基因转化因素的研究(简报)

麻疯树(Jatropha curcas)为大戟科麻疯树属植物,是一种多年生木本油料植物。原产美洲,广泛分布于热带亚热带地区。因其种子含油量高达 60％,可作生物燃料之用,麻疯树是目前正在被开发利用的重要能源植物之一。除此之外,麻疯树的种子含有多种活性成分,有着重要的农药和医药价值;其生长能耐干旱贫瘠,可用于荒山造林。因此,麻疯树是一种具有多种用途、重要经济价值和学术研究价值的植物。目前对其研究多停留在植物组织培养、植物化学、毒理学、种植业方面等。