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。     

PG在番茄果实成熟中的作用及二价金属离子与乙烯对PG活性的影响

对采后番茄果实的电镜观察表明:当果实成熟衰老时,叶绿体数量减少,多数基粒结构丧失;成熟果实胞壁中胶层水解成中空的电子透明区,初生壁的纤丝也发生一定程度的水解,相邻细胞分离;外源 PG(多聚半乳糖醛酸酶)提取物处理绿熟期果实组织,也可引起胞壁结构和叶绿体发生与正常衰老相同的变化。Ca~(2+)、Mg~(2+)、Co~(2+)二价金属离子处理果实,可明显降低番茄红素含量和 PG 活性,延缓果实软化。外源乙烯处理果实,可促进番茄红素的形成,提高 PG活性,并能解除钙对 PG 活性的抑制。本文也对 PG 在乙烯和 Ca~(2+)调节果实成熟中的作用进行了讨论。     

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.     

钙对不同成熟期番茄果实的PG活性及其合成的影响

本文研究了钙处理不同成熟期番茄果实对果壁组织中钙含量与转化、多聚半乳糖醛酸酶(PG)活性与 PG 合成的影响。结果表明,钙处理绿熟期的番茄果实可使总钙和可溶性钙含量明显增加,并较多转化为结合钙;后期处理,进入和转化的钙都减少。同样,钙处理愈早,对果实 PG 活性的抑制愈强,绿熟期处理可完全抑制 PG 活性。凝胶电泳结合钌红染色,证明绿熟期果实无 PG,PG 是在果实成熟过程中新合成的。钙处理愈早,对 PG 合成的抑制愈强,绿熟期钙处理可完全抑制 PG 合成。     

热处理对脱涩后火柿生理生化变化的影响

以CO2脱涩后的火柿为试材,研究了脱涩后火柿在5℃条件下贮藏过程中主要生理生化变化及35℃热处理对其软化生理的影响。结果表明,柿果可溶性单宁含量的继续下降是柿果软化的一个原因;乙烯在脱涩柿果的软化中起着主导作用,呼吸速率及PE、PG和纤维素酶活性的变化与乙烯存在着紧密的关系。35℃热处理抑制了脱涩火柿可溶性单宁含量的下降速率,使乙烯生成量降低和乙烯峰出现时间推迟,刺激了早期的呼吸速率,抑制了后期的呼吸速率,降低了PE、PG和纤维素酶的活性,从而达到了延缓脱涩柿果轮化的目的。     

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。     

赤霉素和萘乙酸对柿果实采后成熟软化生理指标的影响

以采后'富平尖柿'果实为试材,常温下用60 mg/L赤霉素(GA3)和20 mg/L α-萘乙酸(NAA)进行处理,考察柿果实成熟软化相关生理指标及果胶物质代谢在贮藏过程中的变化,以明确GA3和NAA处理对柿果实贮藏效果的影响.结果显示:GA3、NAA处理果实的贮藏时间分别比对照延长了4 d和10 d;GA3和NAA处理可显著延缓果实硬度的下降进程,有效降低果实呼吸强度和乙烯释放量,且呼吸高峰和乙烯高峰的出现明显迟于对照;果实多聚半乳糖醛酸酶(PG)活性的升高受到抑制,从而延缓了原果胶的降解以及水溶性果胶含量的增加,阻碍了果实的软化进程.试验表明,GA3和NAA处理可有效延缓柿果实的后熟软化,延长其贮藏期限, 并以GA3的效果尤为明显.     

荔枝果实的氰不敏感呼吸

荔枝果实果皮和果肉的匀浆或组织的呼吸受KCN,SHAM和PG的抑制。KCN对呼吸的抑制达12％～58％。有KCN时,受SHAM抑制的呼吸为4％～29％。氰不敏感呼吸比率与果实成熟度有关,未熟绿果比半熟果和成熟果的高。乙烯利诱导成熟前果实呼吸增高但减少氰不敏感呼吸比率。DNP对绿果的总呼吸及氰不敏感呼吸均有轻微刺激作用,但对成熟果只明显刺激总呼吸率。     

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。     

乙烯对苹果果实细胞壁降解效应初探

以陕西主栽苹果品种'秦冠'为试材,研究了不同浓度乙烯利以及加热处理下苹果果实中与细胞壁代谢相关酶的活性变化及其与细胞壁组分降解的关系.结果表明:乙烯对各细胞壁酶活性的促进效应因乙烯利施用浓度不同而异.乙烯利浓度由10 mg/L增至1 000 mg/L时,果胶甲酯酶(PME)、多聚半乳糖醛酸酶(PG)和纤维素酶(CS)的活性先逐渐增强,而后又被抑制;木聚糖(Xyl)没有受到明显影响.加热处理可增进乙烯利的作用,如在60℃时,PME、PG、CS、Xyl活性分别是对照的1.5、2.7、1.1和1.5倍.PG活性的显著增加同时引起了果实可溶性糖含量的显著升高,但其他酶活性变化与可溶性糖含量无直接相关.     

Effect of the Calcium on Polygalacturonase (PG) Activity and Synthesis at Different Ripening Stages of Tomato Fruits

It has been reported that PG is a key enzyme related to the tomato fruit ripening and that the application of calcium can dramatically decrease the PG activity and delay the ripening of fruits. In this paper the effects of calcium treament at various ripening stages on the transformation of absorbed calcium, PG activity and PG synthesis in tomato fruits were studicd. According to the analysis of calcium by atomic absorption spectroscopy, it was shown that the soluble and total calcium contents in pericarp of fruits treated with calcium at mature-green stage were increased significantly, and that more soluble calcium was transformed into bound calcium. Both the absorption and transformation of calcium decreased in fruits treated with calcium at later stage of ripening. The inhibition of calcium on PG activity was most effective by treatment at mature-green stage, but less effective at later stage of ripening. One reason for the decrease of calcium inhibition was probably due to the decline of calcium absorption as fruit ripening. The polyacrylamide gel electrophoresis of PG showed that PG with a molecular weight of 46.7 kD was absent in mature-green fruits, and PG synthesis occurred only at the later stage of ripening. It seems that the earlier the treatment was done the more effective of the calcium inhibition of PG synthesis. Based on the above results, it was concluded that the PG plays a major role in ripening and senescence of tomato fruits, and both PG synthesis and its activity were inhibited by calcium. In order to delay the ripening and senescence of tomato fruits, the treatment with calcium should be done at mature-green stage.     

Analysis of Transgenic Tomatoes with Expression of Antisense Polygalacturonase Gene

Authors have been transfered antisense polygalacturonase (PG, E, C, 3, 2, 1, 15) gene into two tomato cultivars (Lycopersicon esculentum cv. “Lichun” and “Qingfeng”) by Agrobacterium-mediated transformation and obtained homozygotes with expression of antisense PG gene. Analysis of Northern blot and PG activity showed that antisense PG gene had stably expressed in T0～T2 generations of transgenic tomatoes, and this expression could be inherited and enhanced in the progeny. For example, PG activity of To, T1 and T2 generation fruits in line T2-19 of"Lichun” was dropped to 27%, 20% and 4% respectively. But the remarkable decrement of PG activity did not have obvious inhibitory effect on normal fruit softening. The transgenic plants of T0～T2 generations grew and developed normally. Average yield per plant and average weight per fruit in these transgenic plants were a little more than those in nontransgenic control. The content determination of vitamin C, soluble sugar and soluble solid substances indicated that transgenic fruits kept the original texture and taste. The contents of vitamin C and soluble solid substances of “Lichun” T2-19 and soluble sugar of “Qingfeng” T2-46 were higher than those of the control. Stored in 28～32℃ for 30 days, all of nontransgenic fruits and about 50% of the transgenic ones were infected by pathologcal bacteria, a few of remainders could be perserved for 50 days. These evidences suggested that the resistance of the transgenic fruits to pathological bacteria during preservation has been strengthened. The approach which the authors used to study regulation of tomato fruit ripening will be desirable to improve the storability of certain famous and precious fruits.     

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。     

The Role of Polygalacturonase (PG) in Tomato Fruit Ripening and Effects of Divalent Metal Ions and Ethylene on PG Activity

It has been reported that PG is a key enzyme related to the tomato fruit ripening. In this study tomato fruits were harvested at the mature-green stage and stored at room temperature. The cell ultrastructure of pericarp tissue was observed at different ripening stages, and the effects of treatments with ethylene and calcium on PG activity and fruit ripening were examined. The object of this study is to elucidate the role of PG in regulation of tomato fruit ripening by ethylene and calcium. PG activity, was undetectable at mature-green stage, but it rose rapidly as fruif ripening. The rise in PG activity was coincided with the dechnmg of fruit firmness during ripening of tomato fruits. The observation of cell ultrastructure showed that the most of grana in chloroplast were lost and the mitochondrial cristae decreased as fruit ripening. Striking changes of cell wall structure was most noted, beginning with dissolution of the middle lamella and eventual disruption of primary cell wall. A similar pattern of changes of cell wall and chloroplast have been observed in pericarp tissue treated with PG extract. In fruits treated with calcium and other divalent metal ions atmature-green stage, the lycopene content and PG activity decreased dramatically. Ethylene application enhanced the formation of lycopene and PG activity. The inhibition of Ca2+ on PG ac ivity was removed by ethylene. Based on the above results, it was demonstrated that PG played a major role in ripening of tomato fruits, and suggested that the regulation of fruit ripening by ethylene and Ca2+ was all mediated by PG. PG induced the hydrolysis of cell wall and released the other hydrolytic enzymes, then effected the ripening processes follow up.     

草莓果实发育成熟过程中糖苷酶和纤维素酶活性及细胞壁组成变化

以丰香和红丰草莓为试材,对果实发育成熟过程中细胞壁水解酶活性和细胞壁成份变化进行了研究．结果表明：半乳糖苷酶和α-甘露糖苷酶活性随草莓果实成熟而提高,葡萄糖苷酶活性不随草莓果实成熟而提高．随着果实发育成熟,纤维素酶活性、果胶酶活性不断提高．果实中未检测到内切多聚半乳糖醛酸酶活性,外切多聚半乳糖醛酸酶活性变化不随果实成熟软化而提高．随果实发育成熟,细胞壁中可溶性果胶和半纤维素增加,而离子结合果胶和共价结合果胶及纤维素减少．     

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。