Co-transformation to tobacco of Cre/lox site-specific recombination system and its precise recombination

For the temporally and spatially regulated expression of the barnase gene in plant,two kinds of plasmids with cre gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure.The transgenic plants were conformed by PCR analysis.The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome.Cloning and sequencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre/lox site-specific recombination.     

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.     

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。     

Kas基因启动子区的克隆及功能初步分析

利用TAIL-PCR技术,克隆到了与辣椒素合成有关的胎座特异表达基因——3-酮酯酰．ACP合成酶基因（Kas）上游400bp的调控区域．将其全长片段与GUS基因连接构建植物表达载体并转化烟草．GUS组织化学染色表明,克隆到的440bp片段具有启动子活性．对该片段进行序列分析发现,在起始密码子ATG上游存在2个TATA-box,分别为-316～-311位的TATAAA和-224～-219位的TATAAA;在TATA-box上游还存在1个位于-378～-374处的CAAT-box,序列为CCAAT．该研究旨在为利用基因调控辣椒素的生物合成,提高辣椒果实中的辣椒素含量奠定基础．     

Isolation of a strong matrix attachment region (MAR) and identification of its function in vitro and in vivo

Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspensioncultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene TM1, TM2, TM3 and AM1 increased uidA GUS gene expression level approximately 1.5-fold, 5-fold, 1.35-fold, 1.3-fold respectively and AM2 has no effect on gene expression. TM2 was found to be a strong MAR that could effectively increase gene expression level and could be used as an effective enhancing element to construct high efficient expression vectors. In this note the relations among the sequence features, binding strength in vitro and function in vivo of the five MARs were analyzed, and the potential significance of TM2 in plant genetic engineering was discussed.     

水稻基因整合平台系统供体质粒pURKMT的构建

提高水稻产量,改良稻米品质是育种学家广泛研究的课题．随着现代生物技术的发展,水稻已成为植物基因工程的重要研究对象．许多实验室已成功地建立了一系列供外源基因转化水稻的系统．但是这些转化系统主要应用Ｔｉ质粒衍生的载体,通过Ｔ－ＤＮＡ左右两端的序列将目的基...     

Evaluation of transgenic tobacco expressing two insecticidal genes to delay resistance development ofHelicoverpa armigera

The resistance ratio ofHelicoverpa armigera to Cry1 Ac insecticidal protein fromBacillus thuringiensis (Bt) is 13.1- and 3.02-fold after 18 generations of selection by transgenic tobacco expressing Bt or two (Bt and CpTI) insecticidal protein genes, in which the average corrected mortality for each selection treatments is about 60%. The mortality of selected population by transgenic Bt gene tobacco is significantly lower than the control strain when fed on transgenic tobacco plants. The mortaltty of the selected population by transgenic two genes tobacco was not significantly different from the control strain. This is the first experiment under laboratory condition which has proved that transgenic two genes tobacco could significantly delay resistance development ofH. armigera compared with one gene.     

Activity of the truncated promoter of pollen-specific G9 gene of cotton and the transgenic recessive nuclei-sterile tobacco

A 557 bp fragment from the translation initiation site of the G9 gene expressed in maturing pollens of cotton was isolated from genomic DNA of upland cotton (Gossypium hirsutum L.) cv. “Zhongkang 17”, and two expression vectors for plant transformation were constructed via fusing this fragment with β-glucuronidase gene (Gus) and cytotoxin gene Barnase. The promoter activity of this fragment was demonstrated via transient expression of Gus gene in cotton and by the integrated expression of Barnase gene in tobacco. This promoter can initiate the expression of exogenous gene specifically and efficiently in plant pollen. The transgenic tobacco plant containing G9-Barnase fusion gene showed the characteristics of recessive nuclei-sterility.     

Functional analysis of cis-acting sequences regulating root-specific expression in transgenic tobacco

Two different length fragments, RSF1 and RSF2 which contained the cis-acting sequences of root-specific gene TobRB7, were isolated from tobacco genome. The abilities of these fragments to direct root-specific expression were studied by fusing them to the β-glucuronidase (GUS) report gene with different directions. After the recombined vectors were transformed into tobacco, the expression pattern was performed by histochemical staining and the quantitative analysis of GUS activity. The data suggested that the cis-acting element of TobRB7 gene direct GUS expression not only as root-specific but also as bidirectional. In our studies, the short fragment, RSF2, performed stronger activity than RSF1 with any direction. The stronger activity of GUS expression was determined by reverse inserting of RSF1 or RSF2 than positive inserting.     

Expression of two insect-resistant genescryIA (b&c)/GNA in transgenic tobacco plants results in added protection against both cotton bollworm and aphids

The synthesizedBacillus thuringiensis insecticidal protein gene cryIA(b&c) and the synthesized geneGNA, (the mannose specific lectin from snowdrop (Galanthus nivalis)), tumefaciens have been inserted into plant expression vector pGW4BAI. Leave stripes ofNicotiana tabacum var. K326 have been transformed withAgrobacterium tumefaciens strain LBA4404 harboring the plant expression vector. 28 kanamycin resistant tobacco plants have been obtained. PCR and Southern blot analyses show that the foreigncryIA andGNA genes have been inserted into the genome of transformed tobacco plants. Haemagglutination assays show thatGNA has a functional activity. Leaf disc bioassays against cotton bollworm (H. armigera) show that the transgenic tobacco plants have a high insecticidal activity. The inhibition of aphid population in leaf disc bioassays againstMyzus persicae shows that the fecundity of aphid on transgenic plants is lower than that on untransformed plants; the aphid population on the transgenic tobacco plants is 25%–70% that on untransformed tobacco plants. ELISA analysis of ClyIA protein in tobcco leaves provides similar data to bioassay results. Through the two bioassays againstH. armigera andM. persicae, several transgenic tobacco plants showing high insect-resistant activities to both pests have been obtained.     

Overexpression of rice OsLOL2 gene confers disease resistance in tobacco to Pseudomonas syringae pv. Tabaci

LSD1-related proteins of Arabidopsis with LSDl-like zinc finger domains regulate disease resistance and programmed cell death （PCD）. We cloned a rice OsLOL2 gene, orthologous to LSD1 of Arabidopsis and expressed it in a tobacco plant. Transgenic tobacco lines displayed enhanced disease resistance to a virulent bacterium Pseudomonas syringae pv. tabaci （Pst）. RT-PCR analysis showed that overexpression of OsLOL2 in transgenic tobacco lines resulted in upregulation of two pathogenesis-related （PR） protein genes, PR2 and PR5. Our results suggest that overexpression of OsLOL2 in transgenic tobacco enhances the resistance through the induction of PR proteins and hypersensitive response-like reaction.     

Introduction of antisensewaxy gene into main parent lines ofindica hybrid rice

Amylose content in rice endosperm is one of the key determinants of rice eating and cooking quality, and the poor quality ofindica hybrid rice is closely related to the high amylose level in rice grains. In order to improve the grain quality of theindica hybrid rice by genetic engineering, an antisense fragment of ricewaxy gene, driven by the 5′-franking sequences of the ricewaxy gene, was successfully introduced into three major parent lines ofindica hybrid rice, all contain a high amylose level in the grains, viaAgrobacterium, and more than 100 hygromycinresistant plants were regenerated. The analysis of PCR amplification and Southern blots indicated that the T-DNA containing the antisensewaxy gene had been integrated into the genome of transgenic rice plants. Most of the primary transgenic rice plants grew normally, and the mature seeds from these transgenic plants were performed for analysis of the amylose content. The results showed that the amylose content in the endosperm of some grains was reduced and the lowest reached 7.02% in one homozygous transgenic line, 72.4% lower than that of the wild type. The influence of the altered amylose content on the gelatinization temperature and gel consistency was also observed in several homozygous transgenic rice plants. The two authors contributed equally to this work.     

Synthesis of medium-chain-length-polyhydroxyalkanoates in tobacco via chloroplast genetic engineering

Medium-chain-length-polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters containing monomers ranging from 6 to 14 carbons in length. The key enzymes of their biosynthesis are PHA-polymerase (product of phaC gene) and 3-hydroxyacyl-acyl carrier protein-CoA transferase (product of phaG gene). With aadA (aminoglycoside 3‘-adenylyltransferase) gene as screening marker, two chloroplast transformation vectors of pTC2 harboring phaC2 gene only and pTGC harboring both phaC and phaG genes were constructed and introduced into tobacco chloroplast genome through particle bombardment. PCR and Southern blot analysis confirmed the insertion of the introduced genes into chloroplast genome. The content of mcl-PHAs accumulated in transgenic plants was analyzed by gas chromatography, mcl-PHAs accumulated up to 4.8 mg/g dry weight (dw) in transgenic line $4-3; their monomers were 3-hydroxyoctanoate and 3-hydroxydecanoate. Accumulation of mcl-PHAs polymers in the tobacco chloroplast was also observed by transmission electron microscopy. To our knowledge, this is the first report on the synthesis of mclPHAs in tobacco via chloroplast genetic engineering.     

Sequence modification of merB gene and high organomercurial resistance of transgenic tobacco plants

Mercury pollution has caused severe damage to environment and great attention has been paid to its control. Phytoremediation may become one of the most efficient measures to recover the polluted soil since it is economical, highly efficient and friendly to environment. In this report, plant genetic engineering methods were employed to modify the DNA sequence of merB genes that catalyze the conversion of organomercurals into ionic mercury. The modified merBhe genes were introduced into tobacco by Agrobacterium, and the resultant transgenic plants were verified by Southern and Northern hybridization. High level of organomercurial resistance was detected on progenies of transgenic plants, some of which were resistant to PMA (phenyl mercury acetate) of 2.5 μmol/L whereas 0.1 μmol/L PMA killed the seedlings of wild-type tobacco in soiless culrure. With the increase of PMA concentration, the inhibition of the seedling growth became apparent. This result makes it possible to breed mercury-resistant tobacco for phytoremediation of mercury-polluted soil.     

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 ubiqutinl 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 CrylAh protein in the construct containing the ubil 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 ubil intron was higher than that of the intronless construct. These results indicated that the maize ubil intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently     

A novel tobacco gene coding for a product similar to bacterial two-component regulators

The two-component signaling system has been studied in bacteria. It takes part in signal transduction of adaptive behavior. Recent studies have shown that a similar two-component system is also present in eukaryotes. Examples of this areETRl andCKLl genes which may involve the signal transduction of plant hormone ethylene and cytokinin respectively. The cloning and characterization of a novel gene (NTHKl) fragment from tobacco are presented. Its partial sequence codes for a product which shows similarity to many two-component signaling proteins. Southern blot analysis indicated that there are 2 to 3 copies ofNTHKl gene in tobacco genome (allotetraploid). Homologous genes may also exist in other plants such as Arabidopsis, soybean and spinach. The expression ofNTHKl gene has also been analyzed in tobacco. Further studies on the isolation of full-length cDNA ofNTHKl gene will elucidate more clearly its function in signal perception and transduction.     

Transformation of japonica rice with<Emphasis Type="Italic">RHL</Emphasis> gene and salt tolerance of the transgenic rice plant

Overexpression of the yeastHAL2 gene increases salt tolerance of yeast and plant. RiceHAL2-like (RHL) gene was introduced into ajaponica rice cultivar HJ19 withAgrobacterium tumefaciens-mediated transformation. Transgenic plants in R₀ generation were selected on the principle of GUS-positive,RHL gene PCR-positive and normal growth. Hygromycin-resistant plants of some transgenic lines in R₁ generation increased salt tolerance during the seedling and booting stage, being less damaged in the cytomembrane and stronger in leaf tissue viability under salt stress during booting period. Southern analysis of transgenic lines tolerant to salt in R₁ generation showed that theRHL gene expression cassette had been successfully integrated into rice genome. Moreover, gene engineering breeding methodology and really salt-tolerant rice cultivar were discussed.     

Transgenic tobacco plants expressing synthetic Cry1Ac and Cry1Ie genes are more toxic to cotton bollworm than those containing one gene

Transgenic tobacco plants carrying CrylAc, Crylle or both genes were obtained. In the leaves of transgenic plants carrying both genes, the contents of CrylAc and Crylle proteins were 0.173% and 0.131% of the total proteins, respectively. CrylAc protein content was 0.182 % and Cry1 le protein content was 0.124% of the total proteins in the leaves of transgenic plants containing only one Bt gene. Fresh leaves of transgenic tobacco and wild-type plants were used for the insect bioassay against wild-type and Cry1Ac-resistant cotton bollworm （Helicoverpa armigera）. The bioassay results showed that transgenic plants carrying both genes were significantly more toxic to wild-type and CrylAc-resistant cotton bollworm than those carrying CrylAc or Crylle alone. This study indicates that the higher toxicity of transgenic tobacco plants carrying both genes is caused by the cooperative function of both Bt proteins, thus providing a potential way to delay the development of insect resistance to transgenic crops.