Isolation and functional analysis of MxNAS3 involved in enhanced iron stress tolerance and abnormal flower in transgenic Arabidopsis

Metal trace elements, such as Fe, Zn, and Mn, are necessary micronutrients required by all plants. In this study, the MxNAS3 gene was cloned from Malus xiaojinensis and MxNAS3 was localized in the cytoplasmic membrane. The expression level of MxNAS3 in root and new leaf was higher than in mature leaf and phloem, which was greatly influenced by high and low Fe stresses, IAA and ABA treatments in M. xiaojinensis. Over-expression of MxNAS3 in transgenic Arabidopsis thaliana contributed to enhanced Fe stress tolerance, as well as higher levels of root length, fresh weight, concentrations of chlorophyll, nicotianamine, Fe, Zn, and Mn, especially under high and low Fe stresses. More importantly, it was the first time for us to find that higher expression of MxNAS3 in transgenic A. thaliana contributed to misshappen flowers. Moreover, the MxNAS5-OE A. thaliana had increased expression levels of flowering-related genes (AtYSL1, AtYSL3, AtAFDL, AtAP1, ATMYB21, and AtSAP).     

Ectopic expression of nicotianamine synthase genes results in improved iron accumulation and increased nickel tolerance in transgenic tobacco

Heavy metals are essential for basic cellular processes but toxic in higher concentrations. This requires the precise control of their intracellular concentrations, a process known as homeostasis. The metal-chelating, non-proteinogenous amino acid nicotianamine (NA) is a key component of plant metal assimilation and homeostasis. Its precise function is still unknown. Therefore, this article aims to contribute new information on the in vivo function of NA and to evaluate its potential use for plant nutrition and crop fortification. For this purpose, a nicotianamine synthase gene of Arabidopsis thaliana was ectopically expressed in transgenic tobacco plants. The presence of extra copies of the nicotianamine synthase gene co-segregated with up to 10-fold elevated levels of NA in comparison with wild type. The increased NA level led to: (a) a significantly increased iron level in leaves of adult plants; (b) the accumulation of zinc and manganese, but not copper; (c) an improvement of the iron use efficiency in adult plants grown under iron limitation; and (d) an enhanced tolerance against up to 1 m m nickel. Taken together, the data predict that NA may be a useful tool for improved plant nutrition on adverse soils and possibly for enhanced nutritional value of leaf and seed crops.     

A novel tomato SUMO E3 ligase,SlSIZ1, confers drought tolerance in transgenic tobacco

SUMOylation is an important post‐translational modification process that regulates different cellular functions in eukaryotes. SIZ/PIAS‐type SAP and Miz1 (SIZ1) proteins exhibit SUMO E3 ligase activity, which modulates SUMOylation. However, SIZ1 in tomato has been rarely investigated. In this study, a tomato SIZ1 gene (SlSIZ1) was isolated and its molecular characteristics and role in tolerance to drought stress are described. SlSIZ1 was up‐regulated by cold, sodium chloride (NaCl), polyethylene glycol (PEG), hydrogen peroxide (H₂O₂) and abscisic acid (ABA), and the corresponding proteins were localized in the nucleus. The expression of SlSIZ1 in Arabidopsis thaliana (Arabidopsis) siz1‐2 mutants partially complemented the phenotypes of dwarf, cold sensitivity and ABA hypersensitivity. SlSIZ1 also exhibited the activity of SUMO E3 ligase to promote the accumulation of SUMO conjugates. Under drought stress, the ectopic expression of SlSIZ1 in transgenic tobacco lines enhanced seed germination and reduced the accumulation of reactive oxygen species. SlSIZ1 overexpression conferred the plants with improved growth, high free proline content, minimal malondialdehyde accumulation and increased accumulation of SUMO conjugates. SlSIZ1 is a functional homolog of Arabidopsis SIZ1 with SUMO E3 ligase activity. Therefore, overexpression of SlSIZ1 enhanced the tolerance of transgenic tobacco to drought stress.     

转SOD基因烟草中SOD酶活力对逆境的耐性及其遗传学特征

温度、pH、酶抑制剂H2O2和KCN均对转SOD基因烟草及其子代(S1和F1)的SOD活性有影响。在这些不利条件下,转基因SOD高表达烟草品系的SOD耐性明显优于对照品系,且其S1、F1能很好地保持亲本的这种优势。     

转popW基因烟草的构建及相关表型分析

PopW是克隆于青枯劳尔氏菌Ralstonia solanacearum ZJ3721中的一种新的编码harpin蛋白的基因,原核表达的PopW蛋白能够诱导烟草对TMV的抗性、促进烟草生长、提高烟草品质。将popW基因连接到植物表达载体pBI121上,构建成重组转基因载体pB-popW,通过冻融法转化根癌土壤杆菌EHA105,获得阳性转化子。再采用叶盘法转化三生烟Nicotiana tobacum cv.Xanthi nc.,经卡那霉素抗性筛选、PCR检测、RT-PCR分析获得21个株系的T3代阳性植株。PCR及RT-PCR检测结果表明popW基因已经整合到烟草基因组中,并在转录水平正常表达。GUS染色进一步证明popW基因在翻译水平上进行了表达,且不同株系之间表达存在差异。对烟草花叶病毒(TMV)的抗病性测定结果表明,转基因烟草对TMV的抗病性增强,防效最高达54.25%。转基因烟草在生长上也具有一定优势,生长15 d的根长最高为野生型的1.7倍,移栽后60 d的株高、鲜重、干重最高分别为野生型烟草的1.4、1.7和1.8倍。     

Overexpression of plant uncoupling mitochondrial protein in transgenic tobacco increases tolerance to oxidative stress

An Arabidopsis thaliana cDNA clone encoding a plant uncoupling mitochondrial protein (AtPUMP1) was overexpressed in transgenic tobacco plants. Analysis of the AtPUMP1 mRNA content in the transgenic lines, determined by Northernblot, revealed variable levels of transgene expression. Antibody probing ofWestern blots of mitochondrial proteins from three independent transgenic lines showed significant accumulation of AtPUMP1 in this organelle. Overproduction of AtPUMP1 in transgenic tobacco plants led to a significantincrease in tolerance to oxidative stress promoted by exogenous hydrogen peroxide as compared to wild-type control plants. These results provide thefirst biological evidence for a role of PUMP in protection of plant cells against oxidative stress damage.     

Expression of human papillomavirus type 16 L1 protein in transgenic tobacco plants

To develop a plant expression system for the production of the human papillomavirus type 16 (HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt Ⅱ gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by cocultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV 16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein, was able to form 55nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV 16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV16 L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.     

微尺度下烟田铁的空间变异性及其与烟叶铁的相关分析

铁是作物必需的营养元素之一,是血红蛋白和细胞色素的组成成分,也是细胞色素氧化酶、过氧化氢酶、过氧化物酶等的组成成分,与烟叶香气前体物的形成密切相关.精准农业通常研究的是微尺度下(米级或亚米级)土壤养分的空间变异性.为此,在米级尺度下(11 m×10 m)对烟田全铁在种烟前后的空间变异性进行比较,并分析相应网格上烤后烟叶样品的铁含量,探讨土壤在种烟前后全铁含量与烟叶铁含量间的相互关系.结果表明:收完烟后试验地全铁含量平均值与种烟前相比有所下降,由30.3 g kg-1降到了24.79 g kg-1.种烟前土壤全铁具有中等的空间相关性(块金效应为48.8%),变异函数的最佳理论模型为线性模型,决定系数达到0.999,Block-Kriging内插图显示试验地全铁具有较强的空间异质性,方差分析的结果也表明取样点间的差异达极显著水平(p=0.00008).种烟后土壤全铁含量以球状模型为变异函数的最佳理论模型,决定系数达到0.956,Block-Kriging内插图显示种烟后土壤全铁含量的最高值比种烟前往南移动了一些,最低值也往北移了一些,整个试验地土壤全铁在种烟后的空间变异性比种烟前有所升高.相关分析表明,烟叶铁含量只与种烟前土壤全铁含量呈极显著相关,与种烟后土壤全铁含量、种烟前后土壤全铁的相对变化量都没有显著相关.根据相关分析的结果,建立了以种烟前土壤全铁含量为自变量,以烟叶全铁含量为因变量的回归方程,并对方程进行验证,结果表明在自变量X的分布区间内预测效果比较好.     

Molecular cloning and characterization of betaine aldehyde dehydrogenase gene from Suaeda liaotungensis and its use in improved tolerance to salinity in transgenic tobacco

cDNA encoding betaine aldehyde dehydrogenase (BADH) from the halophyte Suaeda liaotungensis has been cloned, sequenced and expressed in tobacco (Nictiana tabacum 89). The full-length cDNA is 1506 base pairs (bp) long and encodes a 502 amino-acid polypeptide. The cDNA fragment coding for the mature enzyme was cloned into vector pCAMBIA-1301 for expression in tobacco. Southern blotting analysis showed that that the Badh gene was integrated into the genome of tobacco. Tobacco expressing BADH survived on MS medium containing 200 mM NaCl, whereas the untransformed plants turned yellow after about 20 d and died.     

Expression of Arabidopsis CBF1 regulated by an ABA/stress inducible promoter in transgenic tomato confers stress tolerance without affecting yield

Modern‐day plants are subjected to various biotic and abiotic stresses thereby limiting plant productivity and quality. It has previously been reported that the use of a strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive the expression of Arabidopsis CBF1 in tomato improved tolerance to cold, drought and salt loading, at the expense of growth and yield under normal growth conditions. Hence in the present study, the suitability of expressing the Arabidopsis CBF1 driven by three copies of an ABA‐responsive complex (ABRC1) from the barley HAV22 gene in order to improve the agronomic performance of the transgenic tomato plants was investigated. Northern blot analysis indicated that CBF1 gene expression was induced by chilling, water‐deficit and salt treatment in the transgenic tomato plants. Under these tested stress conditions, transgenic tomato plants exhibited enhanced tolerance to chilling, water‐deficit, and salt stress in comparison with untransformed plants. Under normal growing conditions the ABRC1‐CBF1 tomato plants maintained normal growth and yield similar to the untransformed plants. The results demonstrate the promise of using ABRC1‐CBF1 tomato plants in highly stressed conditions which will in turn benefit agriculture.     

Constitutive over-expression of AtGSK1 induces NaCl stress responses in the absence of NaCl stress and results in enhanced NaCl tolerance in Arabidopsis

GSK3/shaggy-like protein kinases have been shown to play diverse roles in development and signal transduction pathways in various organisms. An Arabidopsis homologue of GSK3/shaggy-like kinase, AtGSK1, has been shown to be involved in NaCl stress responses. In order to further clarify the role of AtGSK1 in NaCl stress responses in plants, we generated transgenic Arabidopsis plants that over-expressed AtGSK1 mRNA. These plants showed enhanced resistance to NaCl stress when assayed either as whole plants or by measurement of root growth on NaCl plates. In addition, AtGSK1 transgenic plants in the absence of NaCl stress showed phenotypic changes, such as accumulation of anthocyanin, that were similar to those observed in wild-type plants under NaCl stress. Transgenic plants accumulated 30-50% more Na+ than did wild-type plants when subjected to NaCl stress, and Ca2+ content was increased by 15-30% in the transgenic plants regardless of the NaCl stress level. Northern blotting revealed that AtGSK1 over-expression induced expression of the NaCl stress-responsive genes AtCP1, RD29A and CHS1 in the absence of NaCl stress. In addition, AtCBL1 and AtCP1 were super-induced in the NaCl-stressed transgenic plants. Taken together, these results suggest that AtGSK1 is involved in the signal transduction pathway(s) of NaCl stress responses in Arabidopsis.     

Reduced sedoheptulose-1,7-bisphosphatase levels in transgenic tobacco lead to decreased photosynthetic capacity and altered carbohydrate accumulation

Transgenic tobacco (Nicotiana tabacum L. cv. Samsun) plants with reduced levels of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37) were produced using an antisense construct in which the expression of a tobacco SBPase cDNA clone was driven by the cauliflower mosaic virus (CaMV) promoter. The reduction in SBPase protein levels observed in the primary transformants correlated with the presence of the antisense construct and lower levels of the endogenous SBPase mRNA. No changes in the amounts of other Calvin cycle enzymes were detected using Western blot analysis. The SBPase antisense plants with less than 20% of wild-type SBPase activity were observed to display a range of phenotypes, including chlorosis and reduced growth rates. Measurements of photosynthesis, using both light-dosage response and CO₂ response curves, of T1 plants revealed a reduction in carbon assimilation rates, which was apparent in plants retaining 57% of wild-type SBPase activity. Reductions were also observed in the quantum efficiency of photosystem II. This decrease in photosynthetic capacity was reflected in a reduction in the carbohydrate content of leaves. Analysis of carbohydrate status in fully expanded source leaves showed a shift in carbon allocation away from starch, whilst sucrose levels were maintained in all but the most severely affected plants. Plants with less than 15% of wild-type SBPase activity were found to contain less than 5% of wild-type starch levels. The results of this preliminary analysis indicate that SBPase activity may limit the rate of carbon assimilation. Received: 23 February 1997 / Accepted: 2 May 1997     

Production of HIV-1 p24 protein in transgenic tobacco plants

The production of antigens for vaccines in plants has the potential as a safe and cost-effective alternative to traditional production systems. Toward the development of a plant-based expression system for the production of human immunodeficiency virus type I (HIV-1) p24 capsid protein, the p24 gene was introduced into the genome of tobacco plants using Agrobacterium tumefaciens-mediated gene transfer. Southern blot analyses confirmed the presence of the p24 coding sequence within the genome of transgenic lines. Western blot analysis of protein extracts from transgenic plants identified plant-expressed p24 protein that cross-reacted with a p24-specific monoclonal antibody, thus confirming the maintenance of antigenicity. Quantification of the p24 protein using enzyme-linked immunosorbent assay (ELISA) estimated yields of approx 3.5 mg per g of soluble leaf protein. Similar accumulation levels of p24 were also detected in T1 plants, confirming that the p24 gene is transmitted stably. Our results indicate that plant-based transgenic expression represents a viable means of producing p24 for the development of HIV vaccine and for use in HIV diagnostic procedures.     

Functional expression and subcellular localization of the Nectria haematococca Mak1 phytoalexin detoxification enzyme in transgenic tobacco

Medicarpin and maackiain are antifungal pterocarpan phytoalexins produced by many legumes, and are thought to be important components of the defense response of these legumes to certain fungal pathogens. The Mak1 gene from the fungal pathogen Nectria haematococca encodes an FAD-dependent mono-oxygenase, known to specifically hydroxylate the phytoalexins medicarpin and maackiain, converting them to less fungitoxic derivatives. Two binary vector constructs were made containing the coding regions from two fungal clones, a Mak1 cDNA (intronless) and a genomic (including three fungal introns) clone, regulated by an enhanced cauliflower mosaic virus 35S promoter. The constructs were introduced into tobacco to check for expression of active fungal enzyme in plant cells and for splicing of fungal introns. Leaves of tobacco plants transformed with the Mak1 cDNA construct readily metabolized infiltrated medicarpin to 1a-hydroxymedicarpin, indicating high levels of active enzyme. RT-PCR analysis of tobacco plants transformed with the Mak1 genomic construct indicated no processing of Mak1 introns, and no Mak1 activity was detected in these plants. When using plants containing the Mak1 cDNA construct, immunolocalization with a Mak1-specific antibody together with cellular fractionation indicated that Mak1 protein accumulated in the plant cytoplasm, associated with endoplasmic reticulum membranes; medicarpin biosynthetic enzymes have been localized to the same subcellular region. The Mak1 cDNA construct is therefore suitable for use in studies to selectively eliminate medicarpin accumulation to assess the relative importance of medicarpin in the antifungal defense mechanisms of alfalfa and other legumes.     

Developmental specific expression and organelle targeting of the Escherichia coli fabD gene,encoding malonyl coenzyme A-acyl carrier protein transacylase in transgenic rape and tobacco seeds

In both plants and bacteria, de novo fatty acid biosynthesis is catalysed by a type II fatty acid synthetase (FAS) system which consists of a group of eight discrete enzyme components. The introduction of heterologous, i.e. bacterial, FAS genes in plants could provide an alternative way of modifying the plant lipid composition. In this study the Escherichia coli fabD gene, encoding malonyl CoA-ACP transacylase (MCAT), was used as a model gene to investigate the effects of over-producing a bacterial FAS component in the seeds of transgenic plants. Chimeric genes were designed, so as not to interfere with the household activities of fatty acid biosynthesis in the earlier stages of seed development, and introduced into tobacco and rapeseed using the Agrobacterium tumefaciens binary vector system. A napin promoter was used to express the E. coli MCAT in a seed-specific and developmentally specific manner. The rapeseed enoyl-ACP reductase transit peptide was used successfully, as confirmed by immunogold labelling studies, for plastid targeting of the bacterial protein. The activity of the bacterial enzyme reached its maximum (up to 55 times the maximum endogenous MCAT activity) at the end of seed development, and remained stable in mature transgenic seeds. Significant changes in fatty acid profiles of storage lipids and total seed lipid content of the transgenic plants were not found. These results are in support of the notion that MCAT does not catalyse a rate-limiting step in plant fatty acid biosynthesis.