一氧化氮处理对香蕉多聚半乳糖醛酸酶及MaPGs基因表达的影响

为了解多聚半乳糖醛酸酶（PG）在香蕉采后软化中的分子调控机制,采用40 μL/L NO熏蒸处理绿熟期的‘巴西’香蕉果实3 h后,在20℃和相对湿度为85%的条件下贮藏,研究NO对香蕉果实乙烯释放量、硬度、PG活性及MaPGs基因表达的影响。结果表明：NO处理降低了果实乙烯释放量,延缓了果实硬度的下降,抑制了PG的活性;降低了MaPG2、MaPG3和MaPG4基因的表达,延缓了香蕉果实的软化。     

番茄果实特异性启动子的克隆与遗传转化研究

为了实现外源基因在番茄果实中的高效和特异表达,克隆了番茄果实特异基因多聚半乳糖醛酸酶基因( Polygalacturonase,PG)的启动子.以中蔬四号番茄为材料,建立并优化了以子叶为外植体的番茄高效再生和遗传转化体系;以GUS为报告基因,构建PG:GUS植物表达载体,转化番茄.结果表明,在1.0 mg/L ZT的MS分化培养中,番茄子叶的发芽率最高,芽的诱导率高达91％,且发生畸态芽和褐化的外植体最少;通过抗生素浓度对农杆菌的抑制效果试验发现,当头孢霉素的浓度为200 mg/L时,抑制农杆菌的效果最好;成功克隆了番茄PG启动子,将PG启动子驱动的GUS基因转入番茄,对转基因后代果实的GUS染色表明,PG启动子驱动的外源基因在果实中特异表达.     

番茄多聚半乳糖醛酸酶cDNA的克隆及其对番茄中PG表达的反义抑制

通过PCR扩增获得了包含多聚半乳糖醛酸酶(PG)全部阅读框架的1.5kb cDNA,经限制酶酶谱和部分序列分析鉴定无误后,将其以反方向插入含两个增强子的35s启动子和Nos3＇端之间,构建成表达PG反义RNA的双元载体,经农杆菌途径转化番茄品种“丽春”,获得了60株抗卡那霉素再生植株,经PCR检测,证明有2／3的再生植株有外源PG基因导入,成熟果实的PG粗提液的SDS—PAGE分析表明：若干株系中PG蛋白量较对照有不同程度的下降。PG活性亦同步下降,其中一个株系3^＃,PG酶活下降了93%。这些结果表明外源PG基因的反方向导入有效地抑制了内源PG基因的表达。     

SlCBL1基因调控番茄果实成熟发育的分子机理

以番茄(Solanum lycopersicum L.)品种‘Micro Tom’为试材,从其果实中克隆得到番茄类钙调磷酸酶B基因(Tomato Calcineurin B-Like gene,SlCBL1),构建其带有报告基因的e-GFP植物表达载体,分析番茄果实中SlCBL1基因超表达与成熟发育进程的相互关系。结果显示:(1)与对照非转基因植株以及转空载植株相比,转SlCBL1基因番茄中SlCBL1基因过量表达,而且能够使番茄果实成熟期提前3~5d,表明SlCBL1基因可促进番茄果实成熟。(2)番茄果实成熟相关基因的表达量也受到不同程度调控,其中番茄成熟过程中的色素合成基因、乙烯路径基因以及果实成熟相关转录因子都受到强烈的调控,与对照相比表达量分别上调5~10倍。研究表明,SlCBL1基因能够促进番茄果实成熟,而且通过影响色素合成基因以及果实成熟相关转录因子来调控番茄果实成熟。     

两个反义基因在番茄工程植株中的生理抑制效应分析

用番茄乙烯形成酶（ＥＦＥ）和多聚半乳糖醛酸酶（ＰＧ）反义ｃＤＮＡ转化番茄子叶,获得两个转基因系统。分别比较了两个基因系统果实和叶片的乙烯生成速率、果实中ＥＦＥ酶活性和果胶酶活性,表明反义ＥＦＥ基因在番茄工程植株中能显著抑制ＥＦＥ酶活性和乙烯生成;反义ＰＧ基因则主要是抑制其ＰＧ酶活性。     

转反义PG基因番茄果实细胞结构变化的研究

经细胞学观察发现,转反义PG基因番茄果实在不同成熟期及存放前后,其果皮外面几层细胞的厚度都比未转基因的厚1～5 μm,细胞结构、细胞质和细胞核等的状态都有明显区别。尤以贮存后更为明显,未转基因果实的果皮细胞结构解体、细胞质凝聚、细胞核变的模糊程度都比转基因的严重。经外源乙烯处理后,转基因和未转基因果实的细胞结构也有相似的变化。结果表明：反义PG基因的转入降低了PG活性,并且减弱了外源乙烯的作用,延缓了果实的衰老,提高了耐贮性能,从而起到果实保鲜作用。     

转反义PG基因番茄果实细胞结构变化的研究

经细胞学观察发现 ,转反义PG基因番茄果实在不同成熟期及存放前后 ,其果皮外面几层细胞的厚度都比未转基因的厚 1～ 5 μm ,细胞结构、细胞质和细胞核等的状态都有明显区别。尤以贮存后更为明显 ,未转基因果实的果皮细胞结构解体、细胞质凝聚、细胞核变的模糊程度都比转基因的严重。经外源乙烯处理后 ,转基因和未转基因果实的细胞结构也有相似的变化。结果表明 :反义PG基因的转入降低了PG活性 ,并且减弱了外源乙烯的作用 ,延缓了果实的衰老 ,提高了耐贮性能 ,从而起到果实保鲜作用     

PG与番茄果实成熟的关系

系统比较了转多聚半乳糖醛酸酶(PG)反义基因和对照番茄果实成熟过程中绿熟、转色、粉顶、粉红、全红5个时期的PG活性和与其相关的生理、生化组分的动态变化.实验表明,转基因果与对照果相比,PG活性始终处于较低水平,PG活性强烈被抑制是在全红期;果实的硬度、贮藏寿命指数都高于对照果;番茄红素合成积累进程被延缓;可溶性果胶含量、电解质外渗百分率均低于对照果.外源乙烯刺激引起对照果PG活性剧增,而转基因果表现钝化.讨论了PG活性与果实成熟、耐贮性及软化的关系,及对外源乙烯刺激的敏感性.首次明确提出PG活性在对照果中极大地表达,在转基因果中强烈被抑制是在全红期,而不是在整个成熟期;PG活性在果实软化中起直接和重要作用;PG活性的高低与番茄红素的合成与积累有关.     

SlCBL1基因在番茄抗灰霉病中的作用

以番茄(Solanum lycopersicum L.)品种‘Micro Tom’为试材,分析番茄叶片和果实的灰霉病发病规律,及番茄类钙调磷酸酶B基因(tomato calcineurin B-like gene,SlCBL1)在叶片和果实中的表达变化;比较转SlCBL1基因番茄与对照的叶片和果实的灰霉病发病过程,分析转基因番茄抗病相关转录因子表达变化。结果表明:(1)非转基因番茄中,不同叶龄的叶片均在接种灰霉病4d开始发病;不同发育阶段的果实接种灰霉病后发病时间也不同,其中绿果(花后16~18d)接种5d还未发病,白果(花后34~36d)接种11d开始发病,红果(花后40~42d)接种5d开始发病;SlCBL1基因表达量在番茄叶片中较低,在绿果期和白果期的果实中表达量最高,红果期果实中表达量最低。(2)转SlCBL1基因后,SlCBL1基因的过量表达能够抑制番茄叶片和果实灰霉病发生;同时番茄叶片和果实中几乎所有的抗病转录因子的表达量都上调,其中WRKY转录因子家族基因SlWRKY33和SlWRKY70受到强烈调控。研究说明,SlCBL1基因过量表达能够提高番茄的抗灰霉能力,其主要机理是通过影响抗病相关转录因子进而调控番茄抗灰霉病的能力。     

辣椒多聚半乳糖醛酸酶基因CaPG的克隆与序列分析

以耐贮辣椒品系P98为材料,采用RACE方法,首次获得辣椒果实多聚半乳糖醛酶(PG)基因的全长cDNA,命名为CaPG,登录号为FJ596175.序列分析结果表明,该基因cDNA长1 668 bp,5′非编码区为119 bp,3′非编码区为442 bp,CDS长1 107 bp,编码368个氨基酸.Blast比对发现,该基因核苷酸序列与已报道的番茄和番木瓜PG基因具有84%和85%的相似性.聚类分析表明,该基因与番茄和番木瓜的亲缘关系较近,与拟南芥PG基因的亲缘关系较远.     

Regulation of Tomato Fruit Polygalacturonase mRNA Accumulation by Ethylene: A Re-Examination

Polygalacturonase (PG) is the major enzyme responsible for pectin disassembly in ripening fruit. Despite extensive research on the factors regulating PG gene expression in fruit, there is conflicting evidence regarding the role of ethylene in mediating its expression. Transgenic tomato (Lycopersicon esculentum) fruits in which endogenous ethylene production was suppressed by the expression of an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene were used to re-examine the role of ethylene in regulating the accumulation of PG mRNA, enzyme activity, and protein during fruit ripening. Treatment of transgenic antisense ACC synthase mature green fruit with ethylene at concentrations as low as 0.1 to 1 μL/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 μL/L. Neither PG enzyme activity nor PG protein accumulated during this 24-h period of ethylene treatment, indicating that translation lags at least 24 h behind the accumulation of PG mRNA, even at high ethylene concentrations. When examined at concentrations of 10 μL/L, PG mRNA accumulated within 6 h of ethylene treatment, indicating that the PG gene responds rapidly to ethylene. Treatment of transgenic tomato fruit with a low level of ethylene (0.1 μL/L) for up to 6 d induced levels of PG mRNA, enzyme activity, and protein after 6 d, which were comparable to levels observed in ripening wild-type fruit. A similar level of internal ethylene (0.15 μL/L) was measured in transgenic antisense ACC synthase fruit that were held for 28 d after harvest. In these fruit PG mRNA, enzyme activity, and protein were detected. Collectively, these results suggest that PG mRNA accumulation is ethylene regulated, and that the low threshold levels of ethylene required to promote PG mRNA accumulation may be exceeded, even in transgenic antisense ACC synthase tomato fruit.     

多聚半乳糖醛酸酶反义基因在转基因番茄中的表达

番茄的多聚半乳糖醛酸是一种在果实成熟阶段特异性表达的酶。为了研究它在果实成熟中的作用,将其ｃＤＮＡ与花椰菜花叶病毒３５Ｓ启动子嵌合后,以反义基因的形式经农杆菌介导导入番茄植株,进一步分析了反义基因的整合与表达。结果表明,在转基因番茄中,反义基因的表达能明显抑制果实内源多聚半乳糖醛酸酶的活性。     

Reduced expression of the tomato ethylene receptor gene LeETR4 enhances the hypersensitive response to Xanthomonas campestris pv. vesicatoria

The hypersensitive response (HR) involves rapid death of cells at the site of pathogen infection and is thought to limit pathogen growth through the plant. Ethylene regulates senescence and developmental programmed cell death, but its role in hypersensitive cell death is less clear. Expression of two ethylene receptor genes, NR and LeETR4, is induced in tomato (Lycopersicon esculentum cv. Mill) leaves during an HR to Xanthomonas campestris pv. vesicatoria, with the greatest increase observed in LeETR4. LeETR4 antisense plants previously were shown to exhibit increased sensitivity to ethylene. These plants also exhibit greatly reduced induction of LeETR4 expression during infection and an accelerated HR at inoculum concentrations ranging from 10(5) to 10(7) CFU/ml. Increases in ethylene synthesis and pathogenesis-related gene expression are greater and more rapid in infected LeETR4 antisense plants, indicating an enhanced defense response. Populations of avirulent X. campestris pv. vesicatoria decrease more quickly and to a lower level in the transgenic plants, indicating a greater resistance to this pathogen. Because the ethylene action inhibitor 1-methylcyclopropene alleviates the enhanced HR phenotype in LeETR4 antisense plants, these changes in pathogen response are a result of increased ethylene sensitivity.     

Differential regulation of the tomato ETR gene family throughout plant development

Ethylene perception in plants is co-ordinated by multiple hormone receptor candidates sharing sequence commonalties with prokaryotic environmental sensor proteins known as two-component regulators. Two tomato homologs of the Arabidopsis ethylene receptor ETR1 were cloned from a root cDNA library. Both cDNAs, termed LeETR1 and LeETR2, were highly homologous to ETR1, exhibiting ～ 90% deduced amino acid sequence similarity and 80% deduced amino acid sequence identity. LeETR1 and LeETR2 contained all the major structural elements of two-component regulators, including the response regulator motif absent in LeETR3, the gene encoding tomato NEVER RIPE (NR). Using RNase protection analysis, the mRNAs of LeETR1, LeETR2 and NR were quantified in tissues engaged in key processes of the plant life cycle, including seed germination, shoot elongation, leaf and flower senescence, floral abscission, fruit set and fruit ripening. LeETR1 was expressed constitutively in all plant tissues examined. LeETR2 mRNA was expressed at low levels throughout the plant but was induced in imbibing tomato seeds prior to germination and was down-regulated in elongating seedlings and senescing leaf petioles. NR expression was developmentally regulated in floral ovaries and ripening fruit. Notably, hormonal regulation of NR was highly tissue-specific. Ethylene biosynthesis induced NR mRNA accumulation in ripening fruit but not in elongating seedlings or in senescing leaves or flowers. Furthermore, the abundance of mRNAs for all three LeETR genes remained uniform in multiple plant tissues experiencing marked changes in ethylene sensitivity, including the cell separation layer throughout tomato flower abscission.     

Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes

The role of the cell wall hydrolase polygalacturonase (PG) during fruit ripening was investigated using novel mutant tomato lines in which expression of the PG gene has been down regulated by antisense RNA. Tomato plants were transformed with chimaeric genes designed to express anti-PG RNA constitutively. Thirteen transformed lines were obtained of which five were analysed in detail. All contained a single PG antisense gene, the expression of which led to a reduction in PG enzyme activity in ripe fruit to between 5% and 50% that of normal. One line, GR16, showed a reduction to 10% of normal PG activity. The reduction in activity segregated with the PG antisense gene in selfed progeny of GR16. Plants homozygous for the antisense gene showed a reduction of PG enzyme expression of greater than 99%. The PG antisense gene was inherited stably through two generations. In tomato fruit with a residual 1% PG enzyme activity pectin depolymerisation was inhibited, indicating that PG is involved in pectin degradation in vivo. Other ripening parameters, such as ethylene production, lycopene accumulation, polyuronide solubilisation, and invertase activity, together with pectinesterase activity were not affected by the expression of the antisense gene.     

Ethylene receptor degradation controls the timing of ripening in tomato fruit

Fruit ripening in tomato requires the coordination of both developmental cues and the phytohormone ethylene. The multigene ethylene receptor family has been shown to negatively regulate ethylene signal transduction and suppress ethylene responses. Here we demonstrate that reduction in the levels of either of two family members, LeETR4 or LeETR6, causes an early-ripening phenotype. We provide evidence that the receptors are rapidly degraded in the presence of ethylene, and that degradation probably occurs through the 26S proteasome-dependent pathway. Ethylene exposure of immature fruits causes a reduction in the amount of receptor protein and earlier ripening. The results are consistent with a model in which receptor levels modulate timing of the onset of fruit ripening by measuring cumulative ethylene exposure.     

Inhibition of the ethylene response by 1-MCP in tomato suggests that polyamines are not involved in delaying ripening, but may moderate the rate of ripening or over-ripening

Ethylene initiates the ripening and senescence of climacteric fruit, whereas polyamines have been considered as senescence inhibitors. Ethylene and polyamine biosynthetic pathways share S-adenosylmethionine as a common intermediate. The effects of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, on ethylene and polyamine metabolism and associated gene expression was investigated during ripening of the model climacteric fruit, tomato (Solanum lycopersicum L.), to determine whether its effect could be via polyamines as well as through a direct effect on ethylene. 1-MCP delayed ripening for 8 d compared with control fruit, similarly delaying ethylene production and the expression of 1-aminocyclopropane-1-carboxylic acid (ACC)-synthase and some ethylene receptor genes, but not that of ACC oxidase. The expression of ethylene receptor genes returned as ripening was reinitiated. Free putrescine contents remained low while ripening was inhibited by 1-MCP, but increased when the fruit started to ripen; bound putrescine contents were lower. The activity of the putrescine biosynthetic enzyme, arginine decarboxylase, was higher in 1-MCP-treated fruit. Activity of S-adenosylmethionine-decarboxylase peaked at the same time as putrescine levels in control and treated fruit. Gene expression for arginine decarboxylase peaked early in non-treated fruit and coincident with the delayed peak in putrescine in treated fruit. A coincident peak in the gene expression for arginase, S-adenosylmethionine-decarboxylase, and spermidine and spermine synthases was also seen in treated fruit. No effect of treatment on ornithine decarboxylase activity was detected. Polyamines are thus not directly associated with a delay in tomato fruit ripening, but may prolong the fully-ripe stage before the fruit tissues undergo senescence.