黄瓜单性花决定基因M的表达分析

植物成花性型决定过程涉及多种机制, 而单一基因位点控制非完全花(单性花)的发育为葫芦科植物所特有, 其中以黄瓜(Cucumis sativus L.)和甜瓜(C. melo L.)研究最深入。文章利用两对黄瓜近等基因系材料, 结合定量PCR技术, 研究黄瓜单性花决定基因M(CsACS2, GenBank登陆号: FJ529216)的表达情况。利用药剂(AgNO3和艾维激素)处理不同基因型黄瓜材料, 研究在内源乙烯作用被干扰的情况下CsACS2基因的表达变化。结果表明: 内源乙烯可以诱导CsACS2基因的表达。在正常生长条件下, 这种内源乙烯可能来自CsACS1G基因(F基因)或CsACS2基因(M基因)本身, 而后一种情况可能涉及一种正回馈激活机制。     

Molecular characterization and isolation of the <Emphasis Type="Italic">F/f</Emphasis> gene for femaleness in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

The biological processes leading to sex expression in plants are of tremendous practical significance for fruit production of many agricultural and horticultural crops. Sex-expression studies in cucumber showed that the different sex types are determined by three major genes: M/m, F/f and A/a. The M/m gene in the dominant condition suppresses stamina development and thus leads to female flowers. The F/f gene in the dominant condition shifts the monoecious sex pattern downwards and promotes femaleness by causing a higher level of ethylene in the plant. To investigate the molecular character of the gene F/f, we used nearly isogenic gynoecious (MMFF) and monoecious (MMff) lines (NIL) produced by our own backcross programme. Our investigations confirmed the result of other groups that an additional genomic ACC synthase (key enzyme of ethylene biosynthesis) sequence (CsACS1G) should exist in gynoecious genotypes. A linkage was also verified between the F/f locus and the CsACS1G sequence with our plant material. After the exploration of different Southern hybridization patterns originating from different CsACS1 probes, a restriction map of the CsACS1 locus was constructed. By using this restriction map, the duplication of the CsACS1 gene and following mutation of the CsACS1G gene could be explained. The promoter regions of the genes CsACS1G and CsACS1 were amplified in a splinkerette PCR and sequenced. An exclusive amplification of the new isolated sequence (CsACS1G) in gynoecious (MMFF) and sub-gynoecious (MMFf) genotypes confirmed that the isolated gene is the dominant F allele.     

A positive feedback loop mediated by CsERF31 initiates female cucumber flower development

Sex determination is a crucially important developmental event that is pervasive throughout nature and enhances the adaptation of species. Among plants, cucumber (Cucumis sativus L.) can generate both unisexual and bisexual flowers, and the sex type is mainly controlled by several 1-aminocyclopropane-1-carboxylic acid synthases (CsACSs). However, the regulatory mechanism of these synthases remains elusive. Here, we used gene expression analysis, protein–DNA interaction assays, and transgenic plants to study the function of a gynoecium-specific gene, ETHYLENE RESPONSE FACTOR31 (CsERF31), in female flower differentiation. We found that in a predetermined female flower, ethylene signaling activates CsERF31 by CsEIN3, and then CsERF31 stimulates CsACS2, which triggers a positive feedback loop to ensure female rather than bisexual flower development. A similar interplay is functionally conserved in melon (Cucumis melo L.). Knockdown of CsERF31 by RNAi causes defective bisexual flowers to replace female flowers. Ectopic expression of CsERF31 suppresses stamen development and promotes pistil development in male flowers, demonstrating that CsERF31 functions as a sex switch. Taken together, our data confirm that CsERF31 represents the molecular link between female–male determination and female–bisexual determination, and provide mechanistic insight into how ethylene promotes female flowers, rather than bisexual flowers, in cucumber sex determination.

A key regulator promotes female flower development by triggering a positive feedback loop during cucumber sex determination.     

The climacteric-like behaviour of young, mature and wounded citrus leaves

Although leaves and other vegetative tissues are generally considered as non-climacteric, citrus leaves show a climacteric system II behaviour after detachment. Upon harvest, young, fully expanded 'Valencia' orange (Citrus sinensis) leaves ( approximately 60-d-old) exhibited two phases of ethylene production. The first phase, up to 6 d after detachment, was characterized by a low and constant ethylene production (system I pathway), associated with a constitutive expression of ACC synthase 2 (CsACS2), CsERS1, and CsETR1. ACC synthase 1 (CsACS1) was not expressed during this phase and autoinhibition of ethylene production was apparent following treatment with exogenous ethylene or propylene. The second phase, 7-12 d after detachment, was characterized by a climacteric rise in ethylene production, preceded by the induction of CsACS1 and ACC oxidase 1 (CsACO1) gene expression in the system II pathway. This induction was accelerated and augmented by exogenous ethylene or propylene, indicating an autocatalytic system II ethylene biosynthesis. Mature leaves (6-8-months-old) behaved similarly, except that the climacteric peak in ethylene production occurred earlier (day 5). Young and mature leaves varied in the timing of the climacteric ethylene rise and CsACS1 and CsACO1 induction. The two phases of ethylene production, system I and system II, were also detected in wounded leaf discs of both young and mature leaves. The first phase peaked 15 min after excision and the second phase peaked after 6 h.     

转基因烟草中Bt毒蛋白基因的表达行为

Bt toxin genes were the insecticidal genes most widely used in genetic engineering of pest resistant plant, were of important significance to study their expression behavior in transgenic plants. In this work, a plant expression vector, pBinMoBc, was constructed. It contained the Cry IA(c) gene under control of chimeric OM promoter and the Ω factor. The vector was transferred into tobacco (Nicotiana tabacum L.) plant via Agrobacterium-mediated transformation. ELISA assay showed that the expression levels of the Cry IA(c) gene in transgenic tobacco plants were significantly higher than that in wild-type tobacco plants. The highest could be up to 0.255% of total soluble proteins; the expression level of CryIA(c) gene in transgenic tobacco plant was changeable during the development stages of tobacco plant. Bioassay showed that pBinMoBc transgenic tobacco plants had more notable insecticidal activity than the wild-type tobacco plants. The above results indicated that pBinMoBc was an effective pest-resistent plant expression vector. This study would be very helpful in screening transgenic cotton with high resistance to cotton bollworm (Heliothis armigeva Hubner).     

A Conserved Ethylene Biosynthesis Enzyme Leads to Andromonoecy in Two Cucumis Species

Andromonoecy is a widespread sexual system in angiosperms, characterized by plants carrying both male and bisexual flowers. Monoecy is characterized by the presence of both male and female flowers on the same plant. In cucumber, these sexual forms are controlled by the identity of the alleles at the M locus. In melon, we recently showed that the transition from monoecy to andromonoecy result from a mutation in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, CmACS-7. To isolate the andromonoecy gene in cucumber we used a candidate gene approach in combination with genetical and biochemical analysis. We demonstrated co-segregation of CsACS2, a close homolog of CmACS-7, with the M locus. Sequence analysis of CsACS2 in cucumber accessions identified four CsACS2 isoforms, three in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed the four isoforms in Escherichia coli and assayed their activity in vitro. Like in melon, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active CsACS2 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. Consistent with this, CsACS2, like CmACS-7 in melon, is expressed specifically in carpel primordia of buds determined to develop carpels. Following ACS expression, inter-organ communication is likely responsible for the inhibition of stamina development. In both melon and cucumber, flower unisexuality seems to be the ancestral situation, as the majority of Cucumis species are monoecious. Thus, the ancestor gene of CmACS-7/CsACS2 likely have controlled the stamen development before speciation of Cucumis sativus (cucumber) and Cucumis melo (melon) that have diverged over 40 My ago. The isolation of the genes for andromonoecy in Cucumis species provides a molecular basis for understanding how sexual systems arise and are maintained within and between species.     

Assessment of the utility of the tomato fruit-specific E8 promoter for driving vaccine antigen expression

To assess the utility of the tomato fruit-specific E8 gene's promoter for driving vaccine antigen expression in plant, the 2.2 kb and 1.1 kb E8 promoters were isolated and sequenced from Lycopersicon esculentum cv. Jinfeng #1. The 1.1 kb promoter was fused to vaccine antigen HBsAg M gene for the transfer to Nicotiana tabacum, and the CaMV 35S promoter was used for comparison. Cholera toxin B (ctb) gene under the control of the 1.1 kb promoter was transformed into both N. tabacum and L. esculentum. Southern blot hybridization confirmed the stable integration of the target genes into the tomato and tobacco genomes. ELISA assay showed that the expression product of HBsAg M gene under the control of the 1.1 kb E8 promoter could not be detected in transgenic tobacco tissues such as leaves, flowers, and seeds. In contrast, the expression of HBsAg M gene driven by CaMV 35S promoter could be detected in transgenic tobacco. ELISA assay for CTB proved that the 1.1 kb E8 promoter was able to direct the expression of exotic gene in ripe fruits of transgenic tomato, but expression was absent in leaf, flower, and unripe fruit of tomato, and CTB protein was not detected in transgenic tobacco tissues such as leaves, flowers, and seeds when the gene was under the control of the 1.1 kb E8 promoter. The results indicated that the E8 promoter acted not only in an organ-specific, but also in a species-specific fashion in plant transformation.     

Molecular and physiological evidence suggests the existence of a system II-like pathway of ethylene production in non-climacteric<Emphasis Type="Italic"> Citrus</Emphasis> fruit

Mature citrus fruits, which are classified as non-climacteric, evolve very low amounts of ethylene during ripening but respond to exogenous ethylene by ripening-related pigment changes and accelerated respiration. In the present study we show that young citrus fruitlets attached to the tree produce high levels of ethylene, which decrease dramatically towards maturation. Upon harvest, fruitlets exhibited a climacteric-like rise in ethylene production, preceded by induction of the genes for 1-aminocyclopropane-1-carboxylate (ACC) synthase 1 (CsACS1), ACC oxidase 1 (CsACO1) and the ethylene receptor CsERS1. This induction was advanced and augmented by exogenous ethylene or propylene, indicating an autocatalytic system II-like ethylene biosynthesis. In mature, detached fruit, very low rates of ethylene production were associated with constitutive expression of the ACC synthase 2 (CsACS2) and ethylene receptor CsETR1 genes (system I). CsACS1 gene expression was undetectable at this stage, even following ethylene or propylene treatment, and CsERS1 gene expression remained constant, indicating that no autocatalytic response had occurred. The transition from system II-like behavior of young fruitlets to system I behavior appears to be under developmental control.Abbreviations ACC 1-Aminocyclopropane-1-carboxylate - CsACS1, CsACS2 ACC synthase - CsACO1 ACC oxidase - CsERS1, CsETR1 Ethylene receptors - DAFB Days after full bloom - 1-MCP 1-Methylcyclopropene     

The M locus and ethylene-controlled sex determination in andromonoecious cucumber plants.

Sex determination in cucumber (Cucumis sativus L.) plants is genetically controlled by the F and M loci. These loci interact to produce three different sexual phenotypes: gynoecious (M-F-), monoecious (M-ff), and andromonoecious (mmff). Gynoecious cucumber plants produce more ethylene than do monoecious plants. We found that the levels of ethylene production and the accumulation of CS-ACS2 mRNA in andromonoecious cucumber plants did not differ from those in monoecious plants and were lower than the levels measured in gynoecious plants. Ethylene inhibited stamen development in gynoecious cucumbers but not in andromonoecious ones. Furthermore, ethylene caused substantial increases in the accumulation of CS-ETR2, CS-ERS, and CS-ACS2 mRNA in monoecious and gynoecious cucumber plants, but not in andromonoecious one. In addition, the inhibitory effect of ethylene on hypocotyl elongation in andromonoecious cucumber plants was less than that in monoecious and gynoecious plants. These results suggest that ethylene responses in andromonoecious cucumber plants are reduced from those in monoecious and gynoecious plants. This is the first evidence that ethylene signals may influence the product of the M locus and thus inhibit stamen development in cucumber. The andromonoecious line provides novel material for studying the function of the M locus during sex determination in flowering cucumbers.     

Ethylene formation and phenotypic analysis of transgenic tobacco plants expressing a bacterial ethylene-forming enzyme

A bacterial ethylene-forming enzyme (EFE) catalyzes oxygenation of 2-oxoglutarate to produce ethylene and carbon dioxide in contrast to a plant enzyme which uses 1-aminocyclopropane-1-carboxylic acid as a substrate. We constructed several lines of transgenic tobacco plants which expressed an EFE from Pseudomonas syringae pv. phaseolicola PK2. The gene encoding a chimeric protein consisting of EFE and beta-glucuronidase (GUS) was introduced into the tobacco genome using a binary vector which directs expression of the EFE-GUS fusion protein under the control of constitutive promoter of cauliflower mosaic virus 35S RNA. Two lines of transgenic plants produced ethylene at consistently higher rates than the untransformed plant, and their GUS activities were expressed in different tissues. A significant dwarf morphology observed in the transgenic tobacco displaying the highest ethylene production resembled the phenotype of a wild-type plant exposed to excess ethylene. These results demonstrate a potential use of bacterial EFE to supply ethylene as a hormonal signal via an alternative route using an ubiquitous substrate 2-oxoglutarate in plant tissues.     

Interspecific Gene Transfer (Implications for Broadening Temperature Characteristics of Plant Metabolic Processes)

We report here an approach to metabolic engineering to alter the temperature characteristics of an enzyme pool based on the concept of thermal kinetics windows (TKWs), a useful indicator of enzyme performance. A chimeric cucumber NADH-hydroxypyruvate reductase (HPR) gene under the control of a cauliflower mosaic virus 35S promoter was constructed and introduced into the genome of tobacco (Tobacum tobacum). The root system of the R1 generation of the resultant transgenic plants expresses only the cucumber enzyme (the native tobacco HPR gene is light regulated and only found in the aerial portions of the plant). Enzyme isolated from the transgenic root tissues exhibits a TKW centered at 32.5[deg]C, characteristic of cucumber. The pool of HPR in the shoots, containing both tobacco and cucumber enzymes, exhibits a broad TKW consistent with an equal mix of the two forms. These data do not simply demonstrate that an introduced gene can be expressed in a transgenic plant but that the kinetics properties of the resultant enzyme are unaltered and when sufficient enzyme is produced the temperature characteristics of the total pool are altered. This suggests that the temperature characteristics of plant biochemical pathways can be broadened to suit changing thermal environments.     

在转基因烟草中表达番茄反义ACC合酶基因及其对芽再生的影响

利用从番茄(Lycopersicum esculentum Mill.)果实中分离到的ACC合酶cDNA,反向置于CaMV 35S启动子的控制之下,并转入烟草(Nicotiana tabacum L.)。PCR扩增证明此反义基因已整合到烟草的基因组上。Northern杂交及逆转录PCR分析表明,这种异源反义基因能在转基因烟草组织中表达,并抑制了烟草内源乙烯的合成,对乙烯合成的抑制在芽再生过程中更为明显,同时这也导致了转基因烟草在组织培养过程中芽再生能力的增强。这些结果从基因水平证明,乙烯在芽形成过程中具有重要的调控功能。     

Spatial expression and characterization of a putative ethylene receptor protein NTHK1 in tobacco

A putative ethylene receptor gene NTHK1 encodes a protein with a putative signal peptide, three transmembrane segments, a putative histidine kinase domain and a putative receiver domain. The receiver domain was expressed in an Escherichia coli expression system, purified and used to generate polyclonal antibodies for immunohistochemistry analysis. The spatial expression of the NTHK1 protein was then investigated. We found that NTHK1 was abundant during flower and ovule development. It was also expressed in glandular hairs, stem, and in leaves that had been wounded. The NTHK1 gene was further introduced into the tobacco plant and we found that, in different transgenic lines, the NTHK1 gene was transcribed to various degrees. Upon ACC treatment, the etiolated transgenic seedlings showed reduced ethylene sensitivity when compared with the control, indicating that NTHK1 is a functional ethylene receptor in plants.     

Tests of a mechanistic model of one hormone regulating both sexes in Cucumis sativus (Cucurbitaceae)

A mechanistic model of sex determination in flowering plants, which assumes that one hormone has male and female cell receptors to inhibit one sex and induce the other independently, was tested in cucumbers (Cucumis sativus) by applications of hormones and their inhibitors. Applications of gibberellic acid (GA) and an inhibitor of its synthesis (paclobutrazol) showed that GA had dual effects on sex expression of promoting maleness and inhibiting femaleness. Conversely, applications of Ethrel (an ethylene release agent) and AgNO₃ (an ethylene action inhibitor) indicated that ethylene induced femaleness and inhibited maleness. Results of various combined applications of the two hormones and their inhibitors suggested that ethylene had overriding effects on GA and acted more directly on sex expression in cucumber. These experiments indicated that there is only one hormone, not two, regulating sex expression in cucumber, and that the sex hormone is likely to be ethylene. Results thus supported the assumption of the model that one hormone can regulate both sexes by inducing one and inhibiting the other independently. Model predictions were confirmed that from a monoecious line, female plants could be induced by increasing the ethylene level, and male plants induced by decreasing the ethylene level. On the other hand, as the model predicts, applications of ethylene or its inhibitor induced gynomonoecious, female, and trimonoecious plants from a hermaphroditic line. In conclusion, all assumptions and predictions of the one-hormone model were confirmed in the experiments.