The Expression of GUS Gene Driven by T-cyt Promoter in Transgenic Tobacco andPotato

The location of GUS gene expression under control of T-cyt gene (gene 4 of T- DNA coding isopenteryl transferase) 5′ region in transgenic tobacco (Nicotiana tabacum cv. W38) and potato (Solanum tuberosum L, cv. Desiree) plants was examined with biochemical assays. The results showed differential distribution in various organs and different cell types. The highest levels of GUS activity were found in tobacco stem where axillary bud was initiated and potato buds on tubers. Moreover, the expression of T-cyt promoter/GUS was found to be inducible in transgenic tobacco stem with cytokinin rather than auxin treatment. Additionally, the level of expression was high in the wounded leaf of transgenic potato. It was suggested that T-cyt promoter may be selectively induced by some exogenous plant hormones.     

Bt转基因抗虫植物研究进展

目前Bt成为世界上植物抗虫基因工程中研究和应用最多的基因。已经有多种转单一Bt和复合Bt的转基因玉米、棉花、马铃薯等作物得到大面积种植。还有许多新的具有良好抗虫性的Bt转基因植物,如水稻、大豆、油菜、苜蓿、花椰菜、蓖麻等已经试验成功,并逐渐推广种植。Bt转基因抗虫植物的培育为提高产量,减少杀虫剂的使用和保护环境做出了举足轻重的贡献。就Bt的分类、杀虫机理、Bt转基因抗虫植物的发展状况以及种植Bt抗虫植物对环境的影响进行了概述。     

Glyphosate-Tolerant Crops: Genes and Enzymes

This review focuses on the genes for the enzymes 5-enolpyruvyl-3-phosphoshikimlc acid synthase (EPSPS) and the glyphosate oxidoreductase (GOX). These genes have been used to genetically engineer plants that are resistant to the herbicide glyphosate. Overproduction of glyphosate-insensitive.EPSPS in transgenic crops has been used to overcome the deleterious effuts of this herbicide. The introduction into plants of GOX also confers glyphosate tolerance to plants and augments the tolerance of transgenic plants already expressing a glyphosate tolerant EPSPS. These genes also provide a method for selecting transformed plant tissue using the glyphosate tolerance as the selectable marker in the presence of inhibitory concentrations of glypllosate. Glyphosate tolerant transgenic plants of beet, corn, cotton, lettuce, poplar, potato, rapeseed. soybean, tobacco, tomato, and wheat have already been field tested and are entering agriculture.     

Genetically engineered resistance to potato virus X in four commercial potato cultivars

The genes for the capsid protein (CP) and the 8K movement protein of PVX were introduced into potato (Solanum tuberosum L.) and expressed under the control of CaMV 35S promoter using a binary vector andAgrobacterium tumefaciens. Four commercial potato cultivars (Russet Burbank, Shepody, Desirée and Bintje) have been efficiently transformed. Eleven independent transgenic clones, with CP expression levels higher than 0.05% of the soluble leaf proteins, were analyzed for resistance to inoculation with PVX (5 and 50µg/ml). The resistance of the transgenic plants to PVX was observed with the lower titer of virus inoculation (5 µg/ml) but not with higher titer (50 µg/ml). A significant reduction in the accumulation of virus in the inoculated transgenic potato plants has been observed under greenhouse and field conditions. Furthermore, the CP gene is very stable and is transferred to new plants originated from stem cuttings or from tubers. The transgenic plants appeared to be phenotypically identical to the nontransformed controls.Abbreviations BAP benzyl-aminopurine - BCIP 5-bromo-4-chloro-3-indolylphosphate p-Toluidine salt - CaMV cauliflower mosaic virus - CP capsid protein - GA₃ gibberellic acid - Kbp kilobase pair - NAA naphthalene acetic acid - NBT nitroblue tetrazolium chloride - NOS nopaline synthase - NPT II neomycin phosphotransferase II - PMSF phenyl methyl sulfonyl fluoride - PVX potato virus X - PVY potato virus Y     

Transgenic Potato with PVY Coat Protein Gene and Its Small-Scale Field Test

Potato virus Y (PVY) infection may cause a severe yield depression up to 80%. To develop the potato (Solanum tuberosum L. ) cultivars that resist PVY infection is very crucial in potato production. The authors have been cloned the coat protein gene of PVY from its Chinese isolate. A chimaeric gene containing the cauliflower mosaic virus 35S promoter and PVY coat protein coding region was introduced into the potato cultivars “Favorita”, “Tiger head” and “K4” via Agrobacterium tumefaciens. Results from PCR and Southern blot analysis confirmed that the foreign gene has integrated into the potato chromosomes. These transgenic potato plants were mechanically inoculated with PVY virus (20 mg/L). The presence of the virus in the potato plants was determined by ELISA and method of back inoculation into tobacco. The authors observed a drastic reduction in the accumulation of virus in some transgenic potato lines. Furthermore, some transgenic potato lines produced more tubers per plant than the untransformed potato did, and the average weight of these transgenic plant tubers was also increased. In the field test, the morphology and development of these transgenic potato plants were normal, 3 transgenic lines of “Favorita” exhibited a higher yield than the untrasformed virus-free potato with an increase ranged from 20% to 30%. From these transgenic lines, it will be very hopeful to develop a potato cultivar which not only has a significant resistance to PVY infection, but also a good harvest in potato production.     

Fructan as a New Carbohydrate Sink in Transgenic Potato Plants

Fructans are polyfructose molecules that function as nonstructural storage carbohydrates in several plant species that are important crops. We have been studying plants for their ability to synthesize and degrade fructans to determine if this ability is advantageous. We have also been analyzing the ability to synthesize fructan in relation to other nonstructural carbohydrate storage forms like starch. To study this, we induced fructan accumulation in normally non-fructan-storing plants and analyzed the metabolic and physiological properties of such plants. The normally non-fructan-storing potato plant was modified by introducing the microbial fructosyltransferase genes so that it could accumulate fructans. Constructs were created so that the fructosyltransferase genes of either Bacillus subtilis (sacB) or Streptococcus mutans (ftf) were fused to the vacuolar targeting sequence of the yeast carboxypeptidase Y (cpy) gene. These constructs were placed under the control of the constitutive cauliflower mosaic virus 35S promoter and introduced into potato tissue. The regenerated potato plants accumulated high molecular mass (>5 [times] 106 D) fructan molecules in which the degree of polymerization of fructose units exceeded 25,000. Fructan accumulation was detected in every plant tissue tested. The fructan content in the transgenic potato plants tested varied between 1 and 30% of dry weight in leaves and 1 and 7% of dry weight in microtubers. Total nonstructural neutral carbohydrate content in leaves of soil-grown plants increased dramatically from 7% in the wild type to 35% in transgenic plants. Our results demonstrated that potato plants can be manipulated to store a foreign carbohydrate by introducing bacterial fructosyltransferase genes. This modification affected photosynthate partitioning in microtubers and leaves and increased nonstructural carbohydrate content in leaves.     

A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop

It is generally thought that transformation of plant cells using Agrobacterium tumefaciens occurs at a very low frequency. Therefore, selection marker genes are used to identify the rare plants that have taken up foreign DNA. Genes encoding antibiotic and herbicide resistance are widely used for this purpose in plant transformation. Over the past several years, consumer and environmental groups have expressed concern about the use of antibiotic- and herbicide-resistance genes from an ecological and food safety perspective. Although no scientific basis has been determined for these concerns, generating marker-free plants would certainly contribute to the public acceptance of transgenic crops. Several methods have been reported to create marker gene-free transformed plants, for example co-transformation, transposable elements, site-specific recombination, or intrachromosomal recombination. Not only are most of these systems time-consuming and inefficient, but they are also employed on the assumption that isolation of transformants without a selective marker gene is not feasible. Here we present a method that permits the identification of transgenic plants without the use of selectable markers. This strategy relies on the transformation of tissue explants or cells with a virulent A. tumefaciens strain and selection of transformed cells or shoots after PCR analysis. Incubation of potato explants with A. tumefaciens strain AGL0 resulted in transformed shoots at an efficiency of 1-5% of the harvested shoots, depending on the potato genotype used. Because this system does not require genetic segregation or site-specific DNA-deletion systems to remove marker genes, it may provide a reliable and efficient tool for generating transgenic plants for commercial use, especially in vegetatively propagated species like potato and cassava.     

Molecular biology and application of plant peroxidase genes

Peroxidases are a family of isozymes found in all plants; they are heme-containing monomeric glycoproteins that utilize either H(2)O(2) or O(2) to oxidize a wide variety of molecules. These important enzymes are used in enzyme immunoassays, diagnostic assays and industrial enzymatic reactions. Peroxidase genes and their promoters can be used for molecular breeding of useful plants. Transgenic techniques have also been used to investigate the physiological and molecular functions of peroxidase genes in plants. Here, we review transgenic studies of peroxidase genes, including the functional analyses of the enzymes and their promoters. Regarding application of peroxidase genes, it has been reported that overexpression of the tomato TPX2 gene or the sweet potato swpa1 gene conferred increased salt-tolerance or oxidative-stress tolerance, respectively. The growth stimulation effect in transgenic tobacco and hybrid aspen upon overexpression of horseradish peroxidase gene is also discussed.     

Utilization of PVX-Cre expression vector in potato

Trait genes are usually introduced into the plant genome together with a marker gene. The last one becomes unnecessary after transgene selection and characterization. One of the strategies to produce transgenic plants free from the selectable marker is based on site-specific recombination. The present study employed the transient Cre-lox system to remove the nptII marker gene from potato. Transient marker gene excision involves introduction of Cre protein in lox-target plants by PVX virus vector followed by plant regeneration. Using optimized experimental conditions, such as particle bombardment infection method and application of P19 silencing suppressor protein, 20-27% of regenerated plants were identified by PCR analysis as marker-free. Based on our comparison of the recombination frequencies observed in this study to the efficiency of other methods to avoid or eliminate marker genes in potato, we suggest that PVX-Cre mediated site-specific excisional recombination is a useful tool to generate potato plants without superfluous transgenic sequences.     

Cloning and characterization of a cathepsin D inhibitor gene from Solanum tuberosum L.

A DNA clone encoding a cathepsin D inhibitor CathInh was isolated from a potato genomic library using a CathInh cDNA as hybridization probe. The amino acid sequence of the coding region is nearly identical with a CathInh cDNA and CathInh proteins previously isolated from a tuber-specific cDNA library and from tubers, respectively. Analysis of GUS activity resulting from expression of chimeric CathInh promoter-GUS genes in transgenic potato plants revealed expression exclusively confined to potato tubers. No GUS activity could be detected in any other organ of the transgenic plants either constitutively or after wounding or treatment with abscisic and jasmonic acid (JA). Interestingly, part of the promoter region of the CathInh gene, essential for GUS activity in tubers, shows striking similarity to promoter regions of tuber-specific class I patatin genes.     

Overexpression of jasmonic acid carboxyl methyltransferase increases tuber yield and size in transgenic potato

Jasmonates control diverse plant developmental processes, such as seed germination, flower, fruit and seed development, senescence and tuberization in potato. To understand the role of methyl jasmonate (MeJA) in potato tuberization, the Arabidopsis JMT gene encoding jasmonic acid carboxyl methyltransferase was constitutively overexpressed in transgenic potato plants. Increases in tuber yield and size as well as in vitro tuberization frequency were observed in transgenic plants. These were correlated with JMT mRNA level––the higher expression level, the higher the tuber yield and size. The levels of jasmonic acid (JA), MeJA and tuberonic acid (TA) were also higher than those in control plants. Transgenic plants also exhibited higher expression of jasmonate-responsive genes such as those for allene oxide cyclase (AOC) and proteinase inhibitor II (PINII). These results indicate that JMT overexpression induces jasmonate biosynthesis genes and thus JA and TA pools in transgenic potatoes. This results in enhanced tuber yield and size in transgenic potato plants.     

转铜/锌超氧化物歧化酶和抗坏血酸过氧化物酶基因马铃薯的耐氧化和耐盐性研究

高盐等逆境可以加剧植物体内活性氧的产生,进而引起植物细胞死亡。为开发抗逆境作物,以置于氧化诱导型启动子下定位于叶绿体的转铜/锌超氧化物歧化酶（Cu/ZnSOD）和抗坏血酸过氧化物酶基因(APX)马铃薯为材料,研究了其对MV和 NaCl所引起的氧化胁迫的耐受性。结果表明, MV胁迫下,转基因马铃薯叶片膜的相对电导率明显低于对照; NaCl胁迫下,其叶绿素含量高于对照。 在含NaCl 的培养基上,转基因幼苗生根率明显大于对照。另外,NaCl胁迫下转基因马铃薯叶片的SOD和APX酶活性显著高于对照,与其耐盐性的提高相一致。这些研究表明,转入Cu/ZnSOD和APX基因的马铃薯清除活性氧的能力增强,抗逆性得到提高。本实验采用氧化诱导型启动子调控下的SOD和APX两个基因协同作用,使外源基因只有在逆境胁迫时才特异性表达,增强转基因植株的抗逆效果,为培育抗逆经济作物开阔了思路。     

Expression of a Flax Allene Oxide Synthase cDNA Leads to Increased Endogenous Jasmonic Acid (JA) Levels in Transgenic Potato Plants but Not to a Corresponding Activation of JA-Responding Genes

Both jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are thought to be significant components of the signaling pathway regulating the expression of plant defense genes in response to various stresses. JA and MeJA are plant lipid derivatives synthesized from [alpha]-linolenic acid by a lipoxygenase-mediated oxygenation leading to 13-hydroperoxylinolenic acid, which is subsequently transformed by the action of allene oxide synthase (AOS) and additional modification steps. AOS converts lipoxygenase-derived fatty acid hydroperoxide to allene epoxide, which is the precursor for JA formation. Overexpression of flax AOS cDNA under the regulation of the cauliflower mosaic virus 35S promoter in transgenic potato plants led to an increase in the endogenous level of JA. Transgenic plants had six- to 12-fold higher levels of JA than the nontransformed plants. Increased levels of JA have been observed when potato and tomato plants are mechanically wounded. Under these conditions, the proteinase inhibitor II (pin2) genes are expressed in the leaves. Despite the fact that the transgenic plants had levels of JA similar to those found in nontransgenic wounded plants, pin2 genes were not constitutively expressed in the leaves of these plants. Transgenic plants with increased levels of JA did not show changes in water state or in the expression of water stress-responsive genes. Furthermore, the transgenic plants overexpressing the flax AOS gene, and containing elevated levels of JA, responded to wounding or water stress by a further increase in JA and by activating the expression of either wound- or water stress-inducible genes. Protein gel blot analysis demonstrated that the flax-derived AOS protein accumulated in the chloroplasts of the transgenic plants.     

A sucrose non‐fermenting‐1‐related protein kinase‐1 gene,IbSnRK1, improves starch content,composition, granule size,degree of crystallinity and gelatinization in transgenic sweet potato

Sucrose non‐fermenting‐1‐related protein kinase‐1 (SnRK1) is an essential energy‐sensing regulator and plays a key role in the global control of carbohydrate metabolism. The SnRK1 gene has been found to increase starch accumulation in several plant species. However, its roles in improving starch quality have not been reported to date. In this study, we found that the IbSnRK1 gene was highly expressed in the storage roots of sweet potato and strongly induced by exogenous sucrose. Its expression followed the circandian rhythm. Its overexpression not only increased starch content, but also decreased proportion of amylose, enlarged granule size and improved degree of crystallinity and gelatinization in transgenic sweet potato, which revealed, for the first time, the important roles of SnRK1 in improving starch quality of plants. The genes involved in starch biosynthesis pathway were systematically up‐regulated, and the content of ADP‐glucose as an important precursor for starch biosynthesis and the activities of key enzymes were significantly increased in transgenic sweet potato. These findings indicate that IbSnRK1 improves starch content and quality through systematical up‐regulation of the genes and the increase in key enzyme activities involved in starch biosynthesis pathway in transgenic sweet potato. This gene has the potential to improve starch content and quality in sweet potato and other plants.     

Transgenic potato expressing Abeta reduce Abeta burden in Alzheimer's disease mouse model

Beta amyloid (Abeta) is believed one of the major pathogens of Alzheimer's disease (AD), and the reduction of Abeta is considered a primary therapeutic target. Immunization with Abeta can reduce Abeta burden and pathological features in transgenic AD model mice. Transgenic potato plants were made using genes encoding 5 tandem repeats of Abeta1-42 peptides with an ER retention signal. Amyloid precursor protein transgenic mice (Tg2576) fed with transgenic potato tubers with adjuvant showed a primary immune response and a partial reduction of Abeta burden in the brain. Thus, Abeta tandem repeats can be expressed in transgenic potato plants to form immunologically functional Abeta, and these potatoes has a potential to be used for the prevention and treatment of AD.     

The catecholamine potentiates starch mobilization in transgenic potato tubers.

In human and animal cells, the catecholamines are involved in glycogen mobilization. Since the compounds are found in a potato, their function in starch mobilization was hypothesized. In order to verify this hypothesis, the transgenic potato plants Solanum tuberosum L. cv. Desiree overexpressing tyrosine decarboxylase (TD EC 4.1.1.25) cDNA from parsley has been generated. The cDNA expression was judged by the northern blot analysis and the enzyme activity measurements. Four independent transgenic lines with the highest TD mRNA expression were selected and analyzed. The expected substantial decrease in tyrosine content was followed by significant increase in tyramine and dramatic enhancement of norepinephrine synthesis was detected. The level of L-3,4-dihydroxyphenylalanin (L-Dopa) was only slightly increased and dopamine significantly decreased in most cases in these plants. The increase in norepinephrine was accompanied by changes in carbohydrate metabolism. The significant increase in glucose and sucrose and the decrease in starch content were characteristic features of TD overexpressed transgenic potato tubers. The features mentioned above indicate that catecholamines potentiate starch mobilization in potato plants in common with animal cells. The decrease in tyrosine content in transgenic plants is also compensated by significant increase in chlorogenic acid synthesis thus potentially increasing the antioxidant capacity of transgenic tubers. The glycoalkaloids content is changed in the transformants. This may originate from glucose accumulation and glycolysis activation. The obtained transgenic potato provides material for further detailed studies of the physiological function of catecholamines in plants.