马铃薯GBSS基因5′侧翼区调控作用的研究

将０．４、０．８、１．６、２．９ｋｂＧＢＳＳ基因的５′侧翼区与ＧＵＳ基因融合,构建了双元表达载体。０．８ｋｂＧＢＳＳＧＵＳ通过基因枪介导在块茎切片中获得了瞬间表达。以上建构物通过农杆菌介导转入了马铃薯（ＳｏｌａｎｕｍｔｕｂｅｒｏｓｕｍＬ．ｃｖ．Ｄｅｓｉｒｅｅ）。ＸＧｌｕｃ染色及ＰＣＲ结果证实已获得转基因植株。利用离体块茎诱导系统,ＧＵＳ表达用荧光进行定量检测,结果显示,２．９、１．６、０．４ｋｂＧＢＳＳＧＵＳ的表达均以块茎明显高于茎段,达２～１０倍。０．８、１．６、２．９ｋｂＧＢＳＳＧＵＳ表达高于０．４ｋｂＧＢＳＳＧＵＳ。蔗糖浓度的升高可诱导ＧＢＳＳＧＵＳ的表达,而光照抑制了ＧＢＳＳＧＵＳ的表达。     

马铃薯GBSS基因5‘侧翼区调控作用的研究

将０．４、０．８、１．６、２．９ｋｂＧＢＳＳ基因的５＇侧翼区与ＧＵＳ基因融合,构建了双元表达载体。０．８ｋｂＧＢＳＳ－ＧＵＳ通过基因枪介导在块茎切片中获得了瞬间表达。以上建构物通过农杆菌介导转入了马铃薯。Ｘ－Ｇｌｕｃ染色及ＰＣＲ结果证实已获得转基因植株。利用离体块茎诱导系统,ＧＵＳ表达用荧光进行定量检测,结果显示,２．９、１．６、０．４ｋｂＧＢＳＳ－ＧＵＳ的表达均以块茎明显高于茎段,达２￣１０倍。     

细胞分裂素基因（T—cyt）启动子驱动下的GUS基因在烟草和马铃薯中的表达

构建了来自根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍ ｅｆａｃｉｅｎｓ） Ｔ－ＤＮＡ 的细胞分裂素基因（Ｔ－ｃｙｔ）启动子驱动下的ＧＵＳ基因的表达质粒,并用以转化烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ ｃｖ． Ｗ ３８）和马铃薯（Ｓｏｌａｎｕｍ ｔｕｂｅｒｏ－ｓｕｍ Ｌ． ｃｖ． Ｄｅｓｉｒｅｅ）,研究其在转基因植物中表达的定位。结果表明,Ｔ－ｃｙｔ启动子在转基因植株的根、茎、叶、块茎和萌发的种子中均可表达。其中在茎和块茎中的表达是不均一的：在维管束部分表达较强,在侧芽或叶柄的生长点及块茎的芽生长点表达活性较高。此外,在培养基中加入０．１ ｍ ｇ／ＬＢＡＰ,转基因烟草茎中ＧＵＳ基因的表达活性增强,而对ＮＡＡ 没有明显的反应。看来某些外源植物激素对Ｔ－ｃｙｔ启动子的活性有一定的诱导作用     

烟草黄矮双生病毒中双向启动子的活性及其调节控制

将烟草黄矮双生病毒（ＴｏｂＹＤＶ）中双向启动子的区域,以不同长度的片段和方向插入启动子分析载体ｐＧ１,与ＧＵＳ报道基因和ＮＯＳ终止子融合。同时,将各个ＴｏｂＹＤＶ读码框区域插入表达载体ｐＡＲＴ７中,置于ＣａＭＶ３５Ｓ启动子和ＯＣＳ终止子之间。用电穿孔法将各种启动子构建物个别地或者与读码框构建物成对地导入烟草和玉米原生质体,以考察ＴｏｂＹＤＶ启动子控制下ＧＵＳ基因瞬间表达的活性,以及ＴｏｂＹＤＶ的读     

矮牵牛花同源异型基因fbp2的克隆及其对烟草花形态的影响

以矮牵牛（Ｐｅｔｕｎｉａ　ｈｙｂｒｉｄａ　Ｌ．）栽培品种为材料,取开放前的花蕾分离ｍＲＮＡ,反转录合成ｃＤＮＡ,以ｃＤＮＡ为模板,通过ＰＣＲ扩增,对获得的目的片段进行序列分析。结果表明,分离的目的片段含有６８６个核苷酸（含有起始密码和终止密码）。核苷酸序列与文献报道相比,同源率为９９．６％,只有３个碱基发生改变,５’端的ＭＡＤＳ盒区域完全相同。将得到的矮牵牛花同源异型基因ｆｂｐ２的ｃＤＮＡ（ｙｆｂｐ２）与ＣａＭＶ３５５启动子和ＮＯＳ３’终止子融合,构建了表达载体ｐＢＢＰ２。表达载体通过农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ　ｔｕｍｅｆａｃｉｅｎｓ）ＬＢＡ４４０４（ｐＡＬ４４０４）介导转化烟草（ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ．）叶片,在含有１００ｍｇ／Ｌ卡那霉素的抗性培养基上再生成株。对抗性株进行总ＤＮＡＳｏｕｔｈｅｒｎ杂交和总ＲＮＡ的点杂交,证明目的基因已导入烟草细胞中,整合到烟草基因组上,并且在烟草细胞中转录。同源异型基因ｆｂｐ２导入烟草后导致烟草花型改变,在雄蕊上产生了花瓣。     

乙型肝炎病毒前S2抗原决定簇（HBVPreS2epitope）与乙型肝炎病毒核心抗原（HBcAg）的基因融合与表达

乙肝前Ｓ２（ＨＢＶＰｒｅＳ２）肽段由５５个氨基酸组成,其Ｎ端肽段含Ｔｈ和Ｂ细胞抗原决定簇。我们将化学合成的ＰｒｅＳ２ｅｐｉｔｏｐｅ（１２０－１４５）基因与ＨＢｃＡｇ基因不同位点进行融合,融合基因在大肠杆菌中获得表达,并对融合蛋白进行了纯化。经ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ实验表明,融合蛋白具有ＰｒｅＳ２和ＨＢｃＡｇ两者的抗原性。此外,研究还表明,强启动子能使表达水平有一定提高。     

种子特异表达ipt转基因棉花根和纤维的改变

将种子特异表达的菜豆蛋白启动子（Ｐｈ／Ｐ）与ｉｐｔ基因融合,构建了植物表达载体。该载体含有由３５Ｓ启动子驱动的ｇｕｓ报告基因。应用该载体通过花粉管通道法转化棉花（Ｇｏｓｓｙｐｉｕｍ ｈｉｒｓｕｔｕｍ Ｌ．）,种子萌发后剪取幼根进行ＧＵＳ染色,获得ＧＵＳ阳性植株２３棵。ＰＣＲ检测证明有３棵ＧＵＳ阳性植株中含有Ｐｈ／Ｐ－ｉｐｔ基因,并进一步用地高辛标记的ＤＮＡ探针作杂交验证了上述结果。分析表明２棵转基     

禾谷类作物雄性不育及育性恢复表达载体的构建

通过ＰＣＲ反应扩增出了玉米花药特异启动子ＣＡ５５,将其分别与Ｂａｒｎａｓｅ和Ｂａｒｓｔａｒ基因融合,构建成了植物雄性不育基因ＣＡ５５ＢＮＮＯＳ和其育性恢复基因ＣＡ５５ＢＳＮＯＳ,再将它们分别插入到ｐＣＡＭＢＩＡ３３００中,获得了应用于禾谷类作物的基因工程雄性不育及育性恢复的表达载体。     

玉米19kD醇溶贮藏蛋白基因启动子种子特异性表达控制区段的分析

为研究玉米（Ｚｅａｍａｙｓ　Ｌ．）１９ｋＤ醇溶贮藏蛋白（ｚｅｉｎ）基因启动子种子特异性表达的控制区段,将全长６９４ｂｐ的启动子进行５’端缺失,共得到６个缺失突变体,长度分别为４８８ｂｐ、３７８ｂｐ、３０２ｂｐ、１５２ｂｐ、１２４ｂｐ和８５ｂｐ。将６个片段分别与报告基因ｇｕｓ连接构建成表达载体ｐＤＧＢ系列,经土壤农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ）介导转化,引入烟草。ＧＵＳ活性检测证明,４８８ｂｐ启动子片段能促使ｇｕｓ基因在种子中特异表达。３７８ｂｐ、３０２ｂｐ、１５２ｂｐ和１２４ｂｐ片段启动子引导的ｇｕｓ基因在烟草根、叶柄、种子中均可表达。     

小麦中外源基因瞬间表达调控研究及兔防御素（NP—1）基因的转化

将不同５上游调控序列驱动下的ＧＵＳ基因用基因枪法导入小麦幼胚和胚性愈伤组织,通过组织化学分析法和荧光分析法对ＧＵＳ基因的表达进行定量检测,比较了几种烟草花叶病毒（ＴＭＶ）Ω增强子序列对小麦中外源基因瞬间表达的调控作用;然后将其中效率最高的玉米Ｕｂｉｌ启动子与兔防御素（ＮＰ－１）连接起来,并加上Ｎｏｓ终止子,构民ＮＰ－１基因小麦表达载体,并转化小麦幼胚,经ＰＣＲ－Ｓｕｔｈｅｒｎ ｂｌｏｔ分析,初步确     

Tapetum-specific expression of theOsg6B promoter-β-glucuronidase gene in transgenic rice

The promoter of an anther tapetum-specific gene,Osg6B, was fused to a-glucuronidase (GUS) gene and introduced into rice byAgrobacterium-mediated gene transfer. Fluorometric and histochemical GUS assay showed that GUS was expressed exclusively within the tapetum of anthers from the uninucleate microspore stage (7 days before anthesis) to the tricellular pollen stage (3 days before anthesis). This is the first demonstration of an anther-specific promoter directing tapetum-specific expression in rice.Abbreviations GUS ßGlucuronidase     

Molecular characterization of rice genes specifically expressed in the anther tapetum

In situ localization of mRNA was carried out on two cDNAs (Osc4 and Osc6) that had been isolated from rice anthers at the microspore stage. The mRNA corresponding to each cDNA was shown to be localized only in the tapetal cells of the rice immature anthers, but not in the microspores or the mature pollen. The corresponding genomic clone, Osg6B, was isolated, and its 5-upstream region was found to regulate -glucuronidase expression in the tapetum of transgenic tobacco. A set of 5 deletions was also generated and a 1095 bp 5 region was revealed to be necessary for activation of the Osg6B promoter in transgenic tobacco.     

A promoter directing high level expression in pistils of transgenic plants

The promoter of the potato (Solanum tuberosum L.) SK2 gene, encoding a pistil-specific basic endochitinase, was cloned. Various fragments of the SK2-promoter, from 1 kb down to 0.23 kb in length, were fused to the GUS reporter gene. Chimaeric SK2 promoter-GUS fusion constructs were transformed into potato by Agrobacterium tumefaciens-mediated transformation. The SK2-GUS transgenic potato plants exhibited a highly specific GUS activity in the pistil. Expression in the pistil was shown to be developmentally regulated. In addition to the GUS activity in pistils, transgenic plants also showed a much weaker ectopic expression in anthers. In other tissues no systematic expression was detectable. All SK2 promoter fragments analysed conferred pistil-specific expression without significant qualitative or quantitative differences, demonstrating that the regulatory elements mediating this expression pattern are located within a 230 bp SK2 promoter fragment. The SK2 promoter may be used to engineer high levels of expression in pistils of transgenic plants.     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.