【作者】 吕双双；

【导师】 李天来；

【作者基本信息】 沈阳农业大学 ， 蔬菜学， 2009， 博士

【摘要】 果实的成熟衰老是一个与生俱来的过程,它涉及了许多生化和生理过程,从而使果实的硬度、颜色、风味、香气和质地发生明显的变化。果实软化是决定果实品质和后熟长短的重要因素。甜瓜作为一种典型的呼吸跃变型果实,在成熟过程中果肉硬度迅速降低,从而限制了运输、贮藏和货架寿命。在果实成熟和品质形成的诸多因素中,果实中的钙含量及乙烯释放具有极为重要的作用。许多研究表明,钙素具有抑制许多植物果实成熟衰老的作用,而乙烯具有促进许多植物果实成熟衰老的作用。然而,有关钙对乙烯促进植物果实成熟衰老影响方面尚缺乏系统研究报道,而这一研究不仅对于深入探讨乙烯促进植物果实成熟衰老的机理具有重要意义,而且对于钙素防止植物果实成熟衰老的应用也具有重要意义。本研究以网纹甜瓜为试材,采用采前植株喷钙处理和采后果实浸钙处理、不同成熟度果实采后外源乙烯处理以及先用乙烯处理再用钙素处理和先用钙处理再用乙烯处理等试验设计方法,系统研究了果实成熟衰老的相关生理因素变化、乙烯的生物合成过程、果肉细胞形态学变化、果实品质变化,以及衰老相关酶-多聚半乳糖醛酸酶基因表达等,探明了外源钙和乙烯在网纹甜瓜果实衰老和软化过程中的作用,为研制防止网纹甜瓜衰老、延长货架期的调控技术措施提供依据。1.通过采前喷钙和采后浸钙两种方式处理网纹甜瓜,筛选出了钙合理的处理浓度为0.5%。通过研究采前喷钙和采后浸钙对果实软化过程中硬度、呼吸速率和果实品质变化,明确了采前喷钙和采后浸钙均能有效地延缓果实硬度下降的速度,降低呼吸速率;同时钙提高了果糖、葡萄糖、蔗糖、可溶性总糖和淀粉含量,降低了可溶性固形物含量,证实了钙在减缓网纹甜瓜果实的软化速度同时还在一定程度上改善了果实的品质。2.通过研究采前喷钙和采后浸钙对果实软化过程中细胞壁水解酶和衰老相关酶活性的影响,明确了采前喷钙和采后浸钙均能降低细胞壁水解酶的活性包括:多聚半乳糖醛酸酶（PG）、果胶甲酯酶（PE）和羧甲基纤维素钠酶（CMC-Na）,并提高过氧化物酶（POD）、超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性,同时降低果肉中可溶性蛋白含量、外渗电导率的升高速度和脂氧合酶（LOX）活性、pH值的作用,说明钙是通过调节果实内细胞壁水解酶和衰老相关酶的活性而延缓果实衰老软化的速度。3.通过研究采前喷钙和采后浸钙对果实软化过程中乙烯的生物合成的影响,明确了采前喷钙和采后浸钙均能有效地推迟乙烯释放高峰出现的时间,还降低了乙烯释放量。这主要是通过调节乙烯的生物合成来实现的,钙推迟了ACC含量和ACS达到高峰时出现的时间,并降低其峰度值;同时钙明显降低了ACO催化的ACC向乙烯的转化,从而降低了乙烯的释放量。4.通过研究采前喷钙和采后浸钙对果实衰老软化过程果肉组织结构的变化,明确了钙可以推迟网纹甜瓜果肉细胞“水浸状”现象出现的时间,尤其以采后浸钙处理更好。通过果肉组织结构观察发现,钙处理后的果实细胞壁结构保持得较为完整,维持了细胞之间的致密性,抑制了细胞壁区域的膨胀,从而延缓了果肉的软化速度。5.采用浓度为10μL·L^-1的外源乙烯处理不同成熟度果实,验证了乙烯能够明显加快果实的软化速度。通过研究果实衰老软化过程中果实硬度、呼吸速率和品质的变化,明确了外源乙烯处理能够显著提高成熟和完熟网纹甜瓜果实呼吸速率,并且降低了果实中可溶性固形物、果糖、葡萄糖和可溶性淀粉含量,略微提高了果实中蔗糖和可溶性总糖含量。证实了乙烯可以加速网纹甜瓜果实的软化并在一定程度上降低了果实的品质。6.通过研究外源乙烯对不同成熟度果实软化过程中细胞壁水解酶和衰老相关酶活性的影响,明确了乙烯能够提高细胞壁水解酶活性,并降低果实中POD、SOD和CAT活性、可溶性蛋白质的含量,并提高PAL和LOX活性、外渗电导率和pH值,表明乙烯是通过调节果实内细胞壁水解酶和衰老相关酶的活性而加速果实衰老和软化。7.通过研究外源乙烯对不同成熟度网纹甜瓜果实乙烯的生物合成的影响。外源乙烯处理后使甜瓜果实内源乙烯的含量迅速增加,这一过程是通过提高果肉中ACC含量及ACS和ACO的活性来实现的。8.通过研究外源乙烯促进不同成熟度网纹甜瓜软化过程中果肉组织结构的变化,说明外源乙烯处理可加快网纹甜瓜果肉细胞“水浸状”现象出现的时间并增加了其发生程度。这可能是由于外源乙烯提高了内源乙烯的释放量,而乙烯的释放促进了细胞壁的解体和/或质膜透性的增加进而提高了细胞间隙的大小导致果肉水浸状出现。通过光学显微镜观察,说明外源乙烯促进了果肉细胞壁的解体,破化了质膜的结构。9.通过研究钙调控乙烯诱导网纹甜瓜软化过程中果实硬度、呼吸速率和果实品质的变化。明确了而与单用钙处理相比,先钙后乙烯处理也可在一定程度上提高呼吸速率;先乙烯处理后增施钙比单用乙烯处理可进一步降低了果实的硬度并提高了呼吸速率。先先钙后乙烯处理和乙烯处理后施钙,比单独使用乙烯和钙处理,降低了果实中可溶性固形物、果糖、葡萄糖和可溶性淀粉含量,提高了果实中蔗糖和可溶性总糖含量。10.通过研究钙调控乙烯诱导网纹甜瓜软化过程中细胞壁水解酶活性和衰老相关酶活性的变化。结果表明而与单用钙处理相比,先钙后乙烯处理也可在一定程度上提高细胞壁水解酶的活性和衰老相关指标:先乙烯处理后增施钙比单用乙烯处理可进一步提高了细胞壁水解酶的活性和衰老相关指标。11.通过研究钙调控乙烯诱导网纹甜瓜软化过程中乙烯的生物合成的影响。结果表明先钙后乙烯处理比单用钙处理也提高了乙烯释放量,但是仍旧低于对照,先乙烯处理后增施钙比单用乙烯处理可进一步提高乙烯释放量,这一过程中均是通过调控乙烯生物合成的进程来实现的。可见作为果实成熟衰老抑制物质的钙素与作为果实成熟衰老促进物质的乙烯之间存在某种互作关系,钙可促进乙烯诱导条件下网纹甜瓜果实衰老的作用。12.通过研究钙调控乙烯诱导网纹甜瓜软化过程中果肉组织结构的变化发现,通过观察果肉细胞组织结构,除了钙处理果实外,其余各个处理果实各部位均有“水浸状”现象出现,并且流汁量很大,就发生“水浸状”现象的程度来看,Eth-Ca>Eth>CK>Ca-Eth。通过光学显微镜观察,说明尽管钙有效地稳定了细胞结构保护了细胞的完整性,但是乙烯的作用位置在膜上导致膜质过氧化作用,使先钙后乙烯处理的果肉细胞在一定程度上受到了破坏,削弱了钙对细胞膜的保护作用,最终表现为细胞的部分融合。另外乙烯处理破坏了网纹甜瓜果实质膜结构的完整性,而再用钙处理进一步放大了乙烯的作用,使细胞壁几乎完全被破坏。13.通过Northern blot证明,采用钙素和乙烯处理甜瓜,在贮藏过程前、中、后期果实不同部位的MPGs的表达变化是不同的。两种PG基因在果实贮藏过程中的表达相似。随着贮藏时间的延长,表达水平逐渐加强。钙处理有效地抑制了甜瓜果实中MPG1和MPG2基因的表达,但是不同钙处理的抑制效果存在明显差异。我们还发现甜瓜果实各个部位中MPG1和MPG2基因的表达也存在差异。MPG1和MPG2基因的表达均从胎座组织开始,由果实内部向外逐渐表达减弱。在贮藏过程中,乙烯处理有效地促进了MPG1基因的表达,完熟果实中的MPG1的表达量明显高于成熟果实,而对MPG2的刺激作用轻微。另外,不同成熟度果实的不同部位对外源乙烯的相应也存在差异,乙烯处理后成熟果实中果皮和外果皮MPG1基因的表达量增加明显,而在胎座和中果皮中MPG2基因的表达明显升高。乙烯处理后完熟果实各个部位MPG1和MPG2基因的表达均明显升高。外源钙素和乙烯共同处理时,以先乙烯后钙处理果实中MPG1和MPG2基因表达量最高。更多还原

【Abstract】 Fruit ripening is a genetically programmed event that is characterized by a number of biochemical and physiological processes that alter fruit firmness,fruit colour,flavour,aroma, and texture.Fruit softening is a major factor that determines fruit quality and post-harvest life. In melon（Cucumis melo L.）,a typical climacteric fruit,some cultivars show an extremely rapid decrease in flesh firmness during ripening,limiting their transport,storage,and shelf-life.In the factors influencing fruit ripe and quality,both the calcium content and ethylene production have an important role.A number of studies have suggested that calcium is able to inhibit the maturation and senescence of many fruit and the ethylene have promotion this process.However,the report about effect of calcium on fruit maturation and senescence of ethylene-promoted has little systematic research.Moreover,this research is vitally important on both the mechanism of ethylene promoting and the application of calcium suppressing during the maturation and senescence of fruits.The muskmelon was studied by following treatments:pre-harvest spraying plants, post-harvest immersing fruit,exo-ethylene treating different maturity of fruit,and the treating sequence of calcium and ethylene.We investigated the ripe-related physiological parameters, the process of ethylene biosynthesis,the morphology of flesh cell,quality,and the gene expression of soften-related enzyme-polygalacturonase,and so on.The results have determined that both calcium and ethylene regulated the maturity and senescence of muskmelon which provided a reliable base in theory for the regulation technology of delaying senescence and extending shelf life.The more details are the following.1.The suitable concentration of calcium is 0.5 percent base on both pre-harvest and post-harvest.We investigated the flesh firmness,respiratory rate and quality related parameters via two ways of applying calcium.The results evaluated calcium availably delayed the softening of flesh,dropped respiratory rate,improved the content fructose, glucose,sucrose and soluble total sugar and starch,and as well reduced the content of soluble solids.Above results confirmed the calcium played an important role on delaying soften and improving the quality of fruit.2.The effect of pre-harvest and post-harvest calcium on the cell wall hydrolases and senescence-related enzymes was investigated during the process of fruit soften.The results showed that calcium decreased the activity of cell wall hydrolases（PG,PE,CMC-Na）, increased the activity of POD,SOD and CAT,and reduced the content of soluble solids, electrical conductivity,the activity of LOX and pH ratio.It turned out that calcium has ability of delaying the fruit soften by regulating the activity of cell wall hydrolases and senescence-related enzymes.3.The ethylene biosynthesis during the softening and senescence of fruit were studied in view of two ways of calcium treatment.The results suggested that calcium postponed the peak of ethylene and reduced the production.This process was implemented by the regulation of ethylene biosynthesis:postponing the peak and production of ACC content and ACS activity,decelerating the transformation from ACC to ethylene by ACO catalysis,and thereby reducing the ethylene production.4.The textural structure of flesh was observed with two ways of calcium treatment.As it turned out,calcium could delay the appearance of water-core,especially for the post-harvest calcium.Moreover,calcium maintained the integrity of cell wall,kept the density among cells, and inhibited regional expansion of cell wall,and thus delayed the softening of fruit.5.The different maturity of fruits was treated by 10μL·L^-1 exo-ethylene,which demonstrated ethylene accelerating the softening of fruit.The survey of firmness,respiratory rate and quality revealed that ethylene significantly elevated respiratory rate,dropped the content of soluble solids,fructose,sucrose and starch,and slightly increased the content of sucrose and soluble total sugar.The results of the tests indicated that ethylene accelerated the softening of different maturity fruits and deteriorated in the quality of fruit to the extent.6.The changes of cell wall hydrolases and senescence-related enzymes were investigated after the different maturity of muskmelon being treated by ethylene.We found out that the ethylene were able to increase the activity of cell wall hydrolases,dropped the activity of POD,SOD and CAT,and the content of soluble protein,at the same time elevated the activity of PAL and LOX,electrical conductivity and Ph ratio.Base on these results ethylene could promote the maturity and senescence of fruit by ways of regulation the activity of cell wall hydrolasases and senescence-related enzymes.7.The effect of muskmelon of different maturity on ethylene biosynthesis was studied after the ethylene treatment.The exo-ethylene significantly increased the endo-ethylene production by means of heightening the content of ACC and the activities of ACS and ACO.8.The change of flesh structure was observed during the softening process of ethylene promoting different maturity of muskmelon.Ethylene could induce the phenomenon of water-core and make it even worse.This is probably the result that exo-ethylene has capability of promoting the production of endo-ethylene,stimulated the disassembly of cell wall and/or maintained better cell membrane integrity,and then enlarged intercellular space which lead to the appearance of water-core.In terms of light microscope,the ethylene was able to accelerate the deterioration of cell wall and destroy of structure of cell membrane.9.The flesh firmness,respiratory rate and fruit quality in relation to the reduction of calcium regulating ethylene were investigated during the process of muskmelon soften.The results were as follows:exogenous applied ethylene in calcium condition has higher respiratory ratio than single calcium treatment.Meanwhile,compared to single ethylene treatment,the time sequence of calcium and ethylene further reduced the flesh firmness and elevated the respiratory ratio.In addition,the treatments of time sequence of calcium and ethylene have lower contents of soluble solids,fructose,glucose and soluble starch,as well higher sucrose and soluble total sugar than the single ethylene and calcium.10.The effect of calcium on activities of cell wall hydrolases and senescence-related enzymes is different in ethylene condition.The treatments of time sequence of calcium and ethylene have further advanced above two enzymes than single calcium and ethylene treatments.11.During the process of muskmelon softening,the ethylene biosynthesis was investigated after calcium and ethylene treated in time sequence.The treatments of time sequence of calcium and ethylene elevated the ethylene produciton than single calcium and ethylene treatments by means of adjusting ethylene biosynthesis.This result indicated that there was a relation between the promoter of fruit senescence（Ca） and the inhibitor of fruit senescence（ethylene）.Namely,calcium can enlarge the reduction of ethylene during the fruit maturity and senescence.12.Light microscope analyzed the texture structure of flesh during the calcium regulation of ethylene-induced in fruit maturity and senescence.All other treatments of different parts appeared the phenomenon of water-core and visibly flowed juice besides the single calcium treatment.In terms of the extent of water-core,the results of this comparison are as follows: Eth-Ca＞Eth＞CK＞Ca-Eth.Moreover,light microscope analysis that the ethylene destroyed the integrity of cell membrane of muskmelon,and the calcium further enlarged the influence of ethylene.Exogenous applied ethylene in calcium condition almost completely deteriorated the cell wall.Furthermore,although calcium effectively stabilized the structure and maintained the integrity of cell,interacting sites of ethylene was the cell membrane leading to lipid peroxidation.Therefore,exogenous calcium applied in ethylene condition destroyed to the extent the flesh cell,weaken the protection of calcium on cell membrane,and then lead to partly cell mergence.13.Northern blot analysis showed that the expression of MPGs in the four segments of fruit of various storage stages（earlier,middle and final stage） was different after calcium and ethylene treatments.The expression pattern of two MPGs was similar during the storage.With the extension of storage life,the expression of levels was heightened.The expression of both MPG1 and MPG2 were significantly inhibited by calcium,but it was various to express in different calcium treatments.Moreover,there is different expression of both MPG1 and MPG2 in different parts of flesh.The expression of MPGs started in placenta,and frequently weakened from the seed cavity to outside.Furthermore,during the storage,the expression of MPG1 was promoted by ethylene.The expression level of MPG1 in ripe fruit was evidently higher than the maturity fruit,but the sitimulation of ethylene on MPG2 was less.In addition, the response of different segments in different maturity was also various.The expression of MPG1 in mesocarp and exocarp of fruit increased significantly,but the expression of MPG2 in placenta and endocarp was elevated obviously under the ethylene condition.Moreover,the expression of both MPG1 and MPG2 in four segments made an evident raise after ethylene treatment.When the exogeous calciuam and ethylene were applied at the same time,the expression of MPG1 and MPG2 were the hightest in the calcium treatment under ethylene condition.更多还原