Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl Transferase Gene

Cytokinins, a class of phytohormones, appear to play an important role in the processes of plant development. We genetically engineered the Agrobacterium tumefaciens isopentenyl transferase gene, placing it under control of a heat-inducible promoter (maize hsp70). The chimeric hsp70 isopentenyl transferase gene was transferred to tobacco and Arabidopsis plants. Heat induction of transgenic plants caused the isopentenyl transferase mRNA to accumulate and increased the level of zeatin 52-fold, zeatin riboside 23-fold, and zeatin riboside 5[prime]-monophosphate twofold. At the control temperature zeatin riboside and zeatin riboside 5[prime]-monophosphate in transgenic plants accumulated to levels 3 and 7 times, respectively, over levels in wild-type plants. This uninduced cytokinin increase affected various aspects of development. In tobacco, these effects included release of axillary buds, reduced stem and leaf area, and an underdeveloped root system. In Arabidopsis, reduction of root growth was also found. However, neither tobacco nor Arabidopsis transgenic plants showed any differences relative to wild-type plants in time of flowering. Unexpectedly, heat induction of cytokinins in transgenic plants produced no changes beyond those seen in the uninduced state. The lack of effect from heat-induced increases could be a result of the transient increases in cytokinin levels, direct or indirect induction of negating factor(s), or lack of a corresponding level of competent cellular factors. Overall, the effects of the increased levels of endogenous cytokinins in non-heat-shocked transgenic plants seemed to be confined to aspects of growth rather than differentiation. Since no alterations in the programmed differentiation pattern were found with increased cytokinin levels, this process may be controlled by components other than absolute cytokinin levels.     

Expression of Isopentenyl Transferase Gene Controlled by Seed-Specific Lectin Promoter in Transgenic Tobacco Influences Seed Development

The bacterial isopentenyl transferase (ipt) gene involved in cytokinin biosynthesis was fused with a seed-specific lectin promoter from soybean and introduced into tobacco. Under the control of the lectin promoter, the expression of the ipt gene increased cytokinin levels and promoted cell division in the embryo in transgenic tobacco seeds. Compared with controls, the number of plerome cell layers and the cell number of cotyledons and pleromes were significantly increased from 16 DAF (days after flowering); the embryo diameter of transgenic tobacco was enlarged at 16, 19, and 21 DAF (16.1%, 12.7%, and 13.9% increase, respectively). Furthermore, the soluble protein content of the transgenic mature seeds was increased by 9.8–22.2% and the dry weight of transgenic tobacco seeds was increased by 8.8–21.8% compared with that of controls. The transgenic tobacco seedlings also grew quickly and a greater increase in fresh weight compared with controls was observed at 20 and 35 days after germination (average 14% and 8% increase above controls, respectively).     

黄瓜导入异戊烯基转移酶基因后对内源激素的影响

用根癌农杆菌C58Cl_PHZ6111)转化黄瓜外植体,在无激素的MS_0培养基上获得转化植株。经胭脂碱测定和氯霉素抗性实验,所测16株均为转化型。气相色谱分析表表,转化植株2—ip和IPA的含量分别为非转化植株的10.7倍和66.7倍,基因4的引入不仅提高了转化植株1AA的会量,也促进了ABA和赤霉素的合成和积累。     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens I. Auxin and Cytokinin Contents of Cultured Tissues Transformed with Wild-Type and Mutant Ti Plasmids

Carrot and tobacco plants were transformed with Agrobacteriumtumefaciens harboring wild-type, aux^– or cyt^– Tiplasmids. In tobacco, these wild and mutant Ti plasmids inducedthe formation of non-morphogenic galls, galls with shoots, andgalls with roots, respectively. In carrot, however, transformationwith any of these plasmids resulted in only the formation ofamorphous tumors. Determination of IAA and cytokinin contentsshowed that in tobacco, significantly high amounts of cytokininsor IAA are present in the cells transformed with Ti plasmidspossessing cytokinin or IAA biosynthetic genes, respectively.In carrot, cytokinin contents were also high in the cells transformedwith Ti plasmids having cytokinin biosynthetic genes, whereasIAA contents of the cells were similar regardless of the plasmidsused for transformation. These results suggest that the mechanism regulating IAA metabolismmay be different in tobacco and carrot. (Received June 25, 1987; Accepted February 1, 1988)     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens II. IAA Biosynthetic Activity of Cultured Carrot Tissues Transformed with Wild-Type and Mutant Ti Plasmids

IAA biosynthetic activity was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens harboring wild-type,aux^– or cyt^– Ti plasmids. In vitro IAAM hydrolaseactivities in tissues transformed with wild-type, and cyt^–Ti plasmids were 3.09 and 19.82 nmol/g proteins/30 min, respectively,but not detectable when aux^– Ti plasmids were used. Theactivity of IAA biosynthesis, determined by the incorporationof radioactivity into IAA in tissues fed with [¹⁴C]-tryptophan,was 34.13, 10.92 and 32.47 pmol/g fr wt/30 min in tissues transformedwith wild type, aux^– and cyt^– Ti plasmids, respectively.The incorporation of radioactivity into the IAAM fraction wasdetected only in the tissues transformed with wild type andcyt^– Ti plasmids. These results showed that the T-DNAencoded pathway of IAA biosynthesis was active in tissues transformedwith wild-type and cyt^h Ti plasmids, and that the activity ofIAA biosynthesis in those tissues was higher than that in tissuestransformed with the aux^– Ti plasmid. (Received March 16, 1988; Accepted July 31, 1988)     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens III. Formation of IAA Conjugates in Cultured Carrot Tissues Transformed with Wild-type and Mutant Ti Plasmids

Inactivation of IAA was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens that harbored wild-type,aux^– or cyt^– Ti plasmids. IAA oxidase activities were similar among the three types oftransformed tissue. Metabolites of IAA were examined by followingthe fate of 3-indolyl-[l-^l4C]IAA. IAA conjugates were detectedin all transformed tissues as well as in hypocotyl segmentsof non-transformed carrot seedlings. The rate of formation ofIAA conjugates was ten times higher in the tissues transformedwith wild-type or cyt^– Ti plasmids than in the tissuestransformed with aux^– Ti plasmids. When the tissues transformedwith aux^– Ti plasmids were cultured on medium that containedIAA for 6 h, the rate of formation of IAA conjugates in thesetissues became as high as that in tissues transformed with wild-typeor cyt^– Ti plasmids. The tissues transformed with wild-type or cyt^– Ti plasmidsnot only synthesize larger amounts of IAA but also convert alarger amount of free IAA to conjugated IAA than do non-transformedand aux^– transformed tissues. Presumably, in carrot, theformation of IAA conjugates decreases the amount of free IAAin the transformed tissues that synthesize large amounts ofIAA and, consequently, the level of free IAA can be maintainedfairly constant. (Received June 2, 1989; Accepted May 23, 1990)     

Relationship of Pruning and Growth Morphology with Hormone Ratios in Shoots of Pillar and Standard Peach Trees

Genotype and cultural management determine the shape of peach [Prunus persica (L.) Batch] tree canopies in orchards. Not well understood, however, is the relationship between terminal growth, lateral branching, and shoot hormone levels that can fundamentally affect tree canopy development. In this experiment, two peach cultivars with widely differing growth habits (Pillar, KV930479 and Standard, ‘Harrow Beauty’) were budded on ‘Lovell’ rootstock, planted in the field in 1998, and characterized for shoot morphology and hormone concentrations in 2002 and 2003 (the fourth and fifth leaf, respectively). Auxin (indole-3-acetic acid) and cytokinins (largely trans-zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine) were measured in shoot tips (2002) and current-year shoots (2003) using mass spectrometry. In 2002, Pillar trees had less sylleptic branching, more upright growth, and higher auxin and auxin-to-cytokinin ratios than Standard trees. In Pillar trees in 2003, auxin concentrations and shoot growth were highest in current year shoots; in pruned trees, only auxin levels increased. Peach tree growth habits may be the result of altered hormone metabolism. Growth forms leading to superior production efficiency may be developed by selection based on specific target hormone concentrations and ratios.     

Senescence-Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

Cassava (Manihot esculenta Crantz) sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-IPT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.     

转Cry1Ac活性杀虫蛋白及慈菇蛋白酶抑制剂B基因的棉花

根据植物基因的结构特征。合成了Cry1Ac活性杀虫蛋白的编码序列并与内质网定位肽编码序列组成嵌合杀虫蛋白基因Bt29K。构建了含Bt29K基因及慈菇蛋白酶抑制剂B（API-B）基因表达框的双抗虫基因植物表达载体。通过根癌土壤杆菌（Agrobacterium trmefaciens(Smith et TOwnsend)ConnLBA4404)介导转化了棉花（Gossypium hirsu-tunL.)的两个生产品种（系）。根据抗棉铃虫（Heliothis armigera)试验及农艺性状的观察调查结果。经6代筛选,获得了抗棉铃虫90.0%_99.7%且农艺性状优良的9个双价抗虫棉纯合品系。分子生物学分析结果表明,两个抗虫基因在棉花基因组中的插入拷贝数为1个或2个,活性Cry1Ac和API-B蛋白在转基因抗虫棉株系中的表达量分别约占总可溶性蛋白的0.17%和0.09%。对双抗纯合系植株及仅转Bt基因的棉花纯合系抗虫性检测结果表明前者的抗虫性明显高于后者,因此推断本研究采用的双抗虫基因表达载体构建策略是合理的。     

玉米异戊烯基焦磷酸异构酶基因的克隆及其功能分析

以玉米嫩叶为材料,采用RT-PCR技术克隆了植物萜类物质前体生物合成过程最后一个关键酶--异戊烯基焦磷酸异构酶(IPI)基因的全长cDNA,命名为ZmlPI(GenBank登录号为AY738148),该基因cDNA全长为1 382 bp,包含1 104 bp的开放阅读框,编码367个氨基酸残基.颜色互补分析表明ZmlPI能推动工程菌XLI-Blue pTreZmlPI pAC-BETA超量表达β-胡萝卜素,证实ZmIPI具有典型的IPI基因的功能.     

非组培转化法获得转基因植株及其Basta耐受性研究

以玉米‘昌7-2’、大豆‘晋遗19’和高粱‘晋粱5号’的种子为受体,质粒pCAMBAR.CHI.11为供体,采用非组培转化法导入bar基因。PCR扩增和Southern杂交分析结果证明,‘昌7-2’、‘晋遗19’和‘晋粱5号’的划胚种子都获得了转化植株,转化率均在5%以上,未划胚种子的转化率为0。共培养液中加入浓度为200μmol/L的乙酰丁香酮(AS)能使玉米种子转化率比对照提高8倍,赋予300W强度的超声波处理,转化率可再提高60%。划胚处理对种子的出苗率影响较大,为17%左右。划胚种子于实验室条件下的Basta耐受试验结果表明,不同作物对Basta的耐受性存在差异,玉米、大豆和高粱转bar基因植株的Basta耐受临界值分别为0.15%、0.1%和0.05%,Basta处理后的幼苗成活率在各作物间差异不大。田间喷雾Basta溶液结果证明,按Basta耐受性大小排列顺序为高粱<大豆<玉米,但按对Basta敏感性的排序为大豆<高粱<玉米。不同生育期喷雾除草剂的实验结果(T2)证明,在生育期后期作物植株对除草剂的敏感性比前期有所降低,生长前期喷雾后玉米、大豆和高粱的苗存活率分别为57%、49%和52%,生长后期喷雾后苗存活率分别为77%、70%和73%,但各个群体的PCR扩增阳性结果率均在80%以上,证明存活率差异是由植株对除草剂耐受性和敏感性的差异引起的。多代转基因植株的检测结果证明目的基因可稳定遗传。     

转金属硫蛋白αα突变体基因的矮牵牛对铅的抗性及积累的研究

利用农杆菌介导的方法将MT及αα基因导入矮牵牛中.转基因植株对铅(Pb)的抗性及吸收能力明显高于对照.转MT及αα基因的植株分别能在150及200 μmol/L Pb中正常生长，而对照只能在50 μmol/L Pb中正常生长.转MT及αα基因植株后代种子萌发与对照相比表现出明显的抗Pb优势.Pb的吸收实验表明转MT、αα基因的植株对Pb的吸收分别比对照提高了28%和35%.     

RNA/DNA嵌合分子介导的高效基因修复

本文介绍了RNA/DNA嵌合分子介导的高效基因修复技术。这一技术是1996年开始发展起来的全新技术,它通过人工合成的双链开环RNA/DNA嵌合分子转染细胞而使特定基因靶位点产生单碱基改变,从而修复突变基因。这一技术高效（目前最高可达50％以上）、特异性强、安全、无随机插入致变的危险、无免疫反应、无明显毒性,能够用于定点突变、基因敲除、动植物功能基因组学、药物遗传学等很多方面的研究,在不久的将来能够应用于人类基因治疗,具有很高的应用价值和医学前景。 Abstract：We introduce a new technique?targeted gene correction directed by chimeric RNA/DNA oligonucleotides which began at 1996.It uses synthetic double?stranded non?circular RNA/DNA chimeric oligonucleotides to transfect cells and make a single?based change at the targeted site of the target gene.It is highly efficient (the highest efficiency is more than 50％),highly special,safe,without danger of mutation caused by random insertion,without immune response,and without obvious toxicity.It can be used to make point mutation,or gene knock?out plants and animals,and is very likely to be used in human gene therapy in the near future.It is also valuable in the study of functional genomics,pharmacogenetics,and medicine.     

一个新的产黄青霉谷胱甘肽转移酶基因的克隆和鉴定

以产黄青霉(Penicillium chrysogenum Thom)cDNA为模板,克隆得到一个新的谷胱甘肽转移酶基因PcgstB,其开放阅读框长651bp,编码216个氨基酸的蛋白质。与已知序列进行BLASTp比较显示,该蛋白具有保守的GST结构域,与烟曲霉GstB的序列一致性最高,达65%。将PcgstB与原核表达载体pTrc99A连接得到表达质粒pTrc-gstB,转化大肠杆菌DH5α,经IPTG诱导后获得以可溶形式表达的重组PcGstB蛋白。以1-chloro-2,4-dinitrobenzene(CDNB)为底物检测,确认该蛋白具有GST活性。     

芪合酶基因转化番茄产生白藜芦醇的研究

为了获得含有白藜芦醇的转基因番茄,从葡萄雷司令中克隆到芪合酶基因,以之构建了含有组成型启动子的植物表达载体pBS2,用于农杆菌介导对番茄品种Tx00l4的遗传转化。通过对诱导愈伤、出芽、生根、再生植株的筛选,得到5株再生小苗,经PCR、Southem检测证实,有3株为真正的转基因植株。用HPLC对3株转基因植株叶片进行白藜芦醇含量鲜重分析,它们中白藜芦醇的含量分别为12．45μg/g,5.35μg/g,4.55μg/g。     

Correlated Tissue Expression of Genes of Cytoplasmic and Mitochondrial Nucleotide Metabolisms in Normal Tissues is Disrupted in Transformed Tissues

Cells maintain dual metabolic pathways to provide substrates for the replication of mitochondrial and nuclear DNA. These pathways involve two separate sets of genes in the nuclear DNA, with one set encoding proteins targeted to the mitochondrion. However, the cytoplasmic and mitochondrial metabolisms are capable of communication through the transport of deoxyribonucleosides and deoxyribonucleotides between the two subcellular compartments. Cytoplasmic and mitochondrial deoxyribonucleoside triphosphate concentrations are strongly correlated in normal cells but not in transformed cells. We were therefore interested in comparing the interactions in normal and transformed tissues between the corresponding cytoplasmic and mitochondrial metabolisms that produce deoxyribonucleoside triphosphates. We conducted an analysis of gene expression data in normal and transformed human tissues obtained from the UniGene database for a selected set of genes for proteins involved in nucleoside salvage in either the cytoplasm or mitochondria. We also included ribonucleotide reductase in our analysis due to its importance in generating deoxyribonucleoside triphosphates. This analysis revealed a large number of highly significant positive correlations between the tissue expression profiles of the genes of the mitochondrial and cytoplasmic pathways in normal tissues, indicating that in normal tissues, the two metabolisms coordinately generate deoxyribonucleoside triphosphates. In transformed tissues, this correlation structure was disrupted. Multiple correlations involving the mitochondrial nucleoside kinase gene DGUOK were statistically significantly different between normal and transformed tissues, suggesting that control of DGUOK expression relative to other cytoplasmic genes is important in transformed tissues.

[Supplementary materials are available for this article. Go to the publisher's online edition of (Nucleosides, Nucleotides & Nucleic Acids) for the following free supplemental resource(s): Supplementary figure 1, Supplementary figure 2, Gandhi_Samuels_Gene_Expression_Supplemental_file.xls (Vol. 31, No. 2)]