果实成熟的基因工程研究

乙烯是催化果实成熟的内源植物激素。本文简要介绍用植物基因工程的手段分离和鉴定出乙烯合成和果实成熟有关的多聚半乳糖醛酸酶、ＡＣＣ氧化酶、ＡＣＣ合酶及ＡＣＣ脱氨酶的基因。并利用反意ＲＮＡ技术将它们的反意ＲＮＡ转入番茄中,得到了相应的反意转基因植株和果实,实现了在基因水平上对果实成熟的调控,开辟了植物育种的新途径。     

硬肉桃果实成熟前后几种与果实软化相关的生理指标的变化

比较桃品种'川中岛白桃'(一般常规桃)和'双久红'(硬肉桃)成熟前后20 d内果肉硬度、乙烯释放量和活性氧代谢有关指标的变化结果表明:两品种桃成熟前后20 d内O2产生速率、H2O2含量、丙二醛(MDA)含量和脂氧合酶(LOX)活性均持续上升,'双久红'果实明显低于'川中岛白桃';在成熟前20 d内两者的超氧化物歧化酶(SOD)和过氧化物~(POD)活性均呈下降趋势,果实成熟后20 d内SOD活性先上升后下降,而POD活性则持续上升;过氧化氢酶(CAT)活性在成熟前20～10 d内上升.以后呈下降趋势.果实成熟后'双久红'的SOD、POD和CAT活性均明显的高于'川中岛白桃'.     

番茄果实中乙烯与多聚半乳糖醛酸酶的关系

乙烯与多聚半乳糖醛酸酶(PG)都是果实成熟过程中关键的调节因子.一方面,在有乙烯合成缺陷的转反义ACS番茄和乙烯感受缺陷的Nr突变体番茄果实中PG基因表达量都明显下降,PG酶活性明显降低;用外源乙烯(100 μL/L)处理绿熟期番茄果实使PG基因的表达明显增强,而1-甲基环丙烯(1-MCP,1 μL/L)处理转色期番茄果实明显抑制PG基因表达.另一方面,转反义PG基因番茄果实乙烯释放量在授粉后低于其野生型,番茄乙烯受体基因LeETR4和乙烯反应因子LeERF2基因表达量比野生种低.PG降解果胶的产物D-GA(100 mg/L)促进未熟期番茄果实中的乙烯生成和LeETR4、LeERF2基因的表达.     

园艺作物成熟和衰老的分子生物学

对园艺作物乙烯和果实成熟、乙烯生物合成途径中二个关键酶ACC合成酶和ACC氧化酶的分子特性,基因克隆和表达及转基因研究等方面问题进行了评述。     

桃ACC合酶基因的分离及其差异表达

利用5'/3'RACE FCR技术,从桃（Prunus persica(L.)Batsch）果实中克隆了植物乙烯生物合成的关键酶-ACC合酶的全长cDNA pacs,对pacs基因进行全序列测定表明,该基因全长1848个碱基,编码区1449个碱基,5'端有177个碱基的非编码区序列,3'端有219个碱基的非编码区序列（不包括终止密码子TAA）。pacs基因编码区共编码483个氨基酸,蛋白质大小为54kd,等电点为6.43。pacs与番茄（S19677）、梅（AB031026)、番木瓜（U68216）、苹果（AB034993）等其他植物ACC合酶cDNA氨基酸序列同源性分别为65%、70%、90%,并存在与这些ACC合酶氨pacs12(af467782)在叶片和花中基因表达模式基本一致,伤处理和IAA均能诱导叶片pacs和pacs12基因的表达,但pacs在伤处理叶片的表达水平比pacs12高;pacs和pacs12基因在果实表达有所不同,pacs在绿熟和成熟果实中均有表达,而pacs12在绿熟果实中基本检测不同,在成熟果实中才有表达,两在果实中的表达水平比伤处理和IAA处理叶片和花中要低。     

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。     

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基因则主要抑制是ＰＧ酶活性。     

果实成熟过程相关调控基因研究进展

果实成熟过程中,多聚半乳糖醛酸酶（ＰＧ）参与果胶的分解,从而在果实软化中起作用,新近发现,果实软化过程中,协同展蛋白具有一定的作用：ＡＣＣ合成酶（ＡＣＳ）、ＡＣＣ氧化酶（ＡＣＯ）和ＡＣＣ脱氨酶与乙烯合成直接有关,ＡＣＳ是乙烯形成的关键酶,由多基因家族编码,各个基因协同表达,每一基因都有自己的转录特性,新近不断发现果实中ＡＣＳ基因家族中的新成员;ＡＣＯ是一种与膜结合的酶,这种酶具有结构上的立体专一性     

挥发性香气物质和乙烯生产在"湖景蜜露"桃果实发育过程中的变化

以"湖景蜜露"水蜜桃(Prunus persica L.)为试材,检测了果实从未成熟到成熟发育过程中乙烯生成、呼吸速率及挥发性香气性物质的变化;同时对果实大小、果皮色泽、果肉硬度、可溶性固形物、可滴定酸进行了测定;对与果实乙烯产生密切相关的1-氨基环丙烷-1-羧酸(ACC)含量、ACC合成酶活性、ACC氧化酶活性也进行了测定.结果表明,随果实成熟度的增加,果实大小、果皮L*值、可溶性固形物含量增加,而果实硬度、果皮h°值、可滴定酸含量减少.在未成熟的果实中,C6的醛类(反式-2-己烯醛)和醇类(顺式-3-己烯醇)是主要的成分;乙烯生成量很低;呼吸速率较高.到跃变阶段C6～C12的内酯类物质明显增加,尤其是γ和δ-内酯类成为果实主要的香气挥发性物质.推测果实乙烯、呼吸作用等基本的生理变化可能调节着内酯类物质的生成.在乙烯跃变上升时果肉中ACC氧化酶的活性下降,ACC含量和ACC合成酶活力的变化与乙烯生成量变化的趋势一致.根据以上结果可以认为桃果实主要的香气挥发性物质的形成与乙烯、呼吸跃变的开始密切相关.香气物质形成速率动态变化可能是桃果实发育过程中成熟度的另一个生理学指标.     

用反义技术解决番茄保鲜的研究进展

目前发现20多种与果实发育有关的酶和蛋白,其中14种mRNA在果实成熟过程中增加,8种受乙烯诱导,相关的基因已被克隆、测序并在染色体上定位．利用反义技术抑制这些酶和蛋白的表达,一方面可研究它们在果实成熟过程中的作用,另一方面从分子水平上解决了番茄保鲜问题．已将7种反义基因导入番茄,反义果实在离体条件下可保鲜90～120d．     

桃ACC合酶基因的分离及其差异表达

利用5′/3′RACE PCR技术,从桃(Prunus persica (L.) Batsch)果实中克隆了植物乙烯生物合成的关键酶--ACC合酶的全长cDNA pacs,对pacs基因进行全序列测定表明,该基因全长1 848个碱基,编码区为1 449个碱基,5′端有177个碱基的非编码区序列,3′端有219个碱基的非编码区序列(不包括终止密码子TAA).pacs基因编码区共编码483个氨基酸,蛋白质大小为54 kD,等电点为6.43.pacs与番茄(S19677)、梅(AB031026)、番木瓜(U68216)、苹果(AB034993)等其他植物ACC合酶cDNA氨基酸序列同源性分别为65%、70%、75%、90%,并存在与这些ACC合酶氨基酸的活性位点保守序列SLSKDMGFPGFR.RT-PCR结合杂交分析表明,pacs和我们以前克隆的桃ACC合酶cDNA pacs12(AF467782)在叶片和花中基因表达模式基本一致,伤处理和IAA均能诱导叶片pacs 和pacs12基因的表达,但pacs在伤处理叶片的表达水平比pacs12高;pacs 和pacs12基因在果实表达有所不同,pacs在绿熟和成熟果实中均有表达,而pacs12在绿熟果实中基本检测不到,在成熟果实中才有表达,两者在果实中的表达水平比伤处理和IAA处理叶片和花中要低.     

Wound ethylene and 1-aminocyclopropane-1-carboxylate synthase in ripening tomato fruit

Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ACC-synthase activity were compared in intact and wounded tomato fruits (Lycopersicon esculentum Mill.) at different ripening stages. Freshly cut and wounded pericarp discs produced relatively little ethylene and had low levels of ACC and of ACC-synthase activity. The rate of ethylene synthesis, the level of ACC and the activity of ACC synthase all increased manyfold within 2 h after wounding. The rate of wound-ethylene formation and the activity of wound-induced ACC synthase were positively correlated with the rate of ethylene production in the intact fruit. When pericarp discs were incubated overnight, wound ethylene synthesis subsided, but the activity of ACC synthase remained high, and ACC accumulated, especially in discs from ripe fruits. In freshly harvested tomato fruits, the level of ACC and the activity of ACC synthase were higher in the inside parts of the fruit than in the pericarp. When wounded pericarp tissue of green tomato fruits was treated with cycloheximide, the activity of ACC synthase declined with an apparent half life of 30–40 in. The activity of ACC synthase in cycloheximide-treated, wounded pericarp of ripening tomatoes declined more slowly.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

Expression of ACC synthase is enhanced earlier than that of ACC oxidase during fruit ripening of mume (Prunus mume)

Mume (Japanese apricot: Prunus mume Sieb. et Zucc.) is a climacteric fruit that produces large amounts of ethylene as it ripens. Ripening is accompanied by marked increases in the activities of two ethylene-biosynthetic enzymes, namely, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. To study the molecular aspects of ripening of mume, we isolated cDNA clones for proteins that we considered likely to be involved in the biosynthesis and perception of ethylene during ripening, namely, ACC synthase, ACC oxidase and the ethylene receptor. Northern blotting analysis revealed the markedly increased expression of ACC synthase prior to that of ACC oxidase and the increase in ethylene production during ripening. Overall, the levels of the mRNAs for the genes corresponded closely to the levels of activity of the ethylene-biosynthetic enzymes. Exposure of mature green mume fruit to ethylene for 12 h induced strong expression of ACC synthase, as well as of ACC oxidase. Wounding of the pericarp of mume fruit induced the expression of ACC synthase but not of ACC oxidase. The rate of ethylene production increased only slightly after wounding. These results suggest that expression of the genes for ACC synthase and ACC oxidase must be activated sequentially for maximum production of ethylene during ripening of mume fruit and that several mechanisms regulate the expression of ethylene-biosynthetic genes during ripening.     

Expression of MdCAS1 and MdCAS2, encoding apple beta-cyanoalanine synthase homologs, is concomitantly induced during ripening and implicates MdCASs in the possible role of the cyanide detoxification in Fuji apple (Malus domestica Borkh.) fruits

Fruit ripening involves complex biochemical and physiological changes. Ethylene is an essential hormone for the ripening of climacteric fruits. In the process of ethylene biosynthesis, cyanide (HCN), an extremely toxic compound, is produced as a co-product. Thus, most cyanide produced during fruit ripening should be detoxified rapidly by fruit cells. In higher plants, the key enzyme involved in the detoxification of HCN is β-cyanoalanine synthase (β-CAS). As little is known about the molecular function of β-CAS genes in climacteric fruits, we identified two homologous genes, MdCAS1 and MdCAS2, encoding Fuji apple β-CAS homologs. The structural features of the predicted polypeptides as well as an in vitro enzyme activity assay with bacterially expressed recombinant proteins indicated that MdCAS1 and MdCAS2 may indeed function as β-CAS isozymes in apple fruits. RNA gel-blot studies revealed that both MdCAS1 and MdCAS2 mRNAs were coordinately induced during the ripening process of apple fruits in an expression pattern comparable with that of ACC oxidase and ethylene production. The MdCAS genes were also activated effectively by exogenous ethylene treatment and mechanical wounding. Thus, it seems like that, in ripening apple fruits, expression of MdCAS1 and MdCAS2 genes is intimately correlated with a climacteric ethylene production and ACC oxidase activity. In addition, β-CAS enzyme activity was also enhanced as the fruit ripened, although this increase was not as dramatic as the mRNA induction pattern. Overall, these results suggest that MdCAS may play a role in cyanide detoxification in ripening apple fruits.     

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.     

Regulation of ascorbate oxidase expression in pumpkin by auxin and copper

Ascorbate oxidase expression in pumpkin (Cucurbita spp.) tissues was studied. Specific ascorbate oxidase activities in pumpkin leaf and stem tissues were about 2 and 1.5 times that in the fruit tissues, respectively. In seeds, little ascorbate oxidase activity was detected. Northern blot analyses showed an abundant ascorbate oxidase mRNA in leaf and stem tissues. Fruit tissues had lower levels of ascorbate oxidase mRNA than leaf and stem tissues. Ascorbate oxidase mRNA was not detected in seeds. Specific ascorbate oxidase activity gradually increased during early seedling growth of pumpkin seeds. The increase was accompanied by an increase in ascorbate oxidase mRNA. When ascorbate oxidase activity in developing pumpkin fruits was investigated, the activities in immature fruits that are rapidly growing at 0, 2, 4, and 7 d after anthesis were much higher than those in mature fruits at 14 and 30 d after anthesis. The specific activity and mRNA of ascorbate oxidase markedly increased after inoculation of pumpkin fruit tissues into Murashige and Skoog's culture medium in the presence of an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D) but not in the absence of 2,4-D. In the presence of 10 mg/L of 2,4-D, ascorbate oxidase mRNA was the most abundant. Thus, ascorbate oxidase is induced by 2,4-D. These results indicate that ascorbate oxidase is involved in cell growth. In pumpkin callus, ascorbate oxidase activity could be markedly increased by adding copper. Furthermore, immunological blotting showed that the amount of ascorbate oxidase protein was also increased by adding copper. However, northern blot analyses showed that ascorbate oxidase mRNA was not increased by adding copper. We suggest that copper may control ascorbate oxidase expression at translation or at a site after translation.