砧用南瓜提高黄瓜嫁接苗耐盐性的生理机制及蛋白质组学研究
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摘要
随着我国设施园艺的迅速发展,设施栽培中的土壤次生盐渍化问题日趋突出,已成为限制设施蔬菜产业发展的主要障碍。黄瓜(Cucumis sativus L.)是设施栽培的主要蔬菜作物,其耐盐性较弱。研究表明,利用耐盐砧木进行嫁接栽培是提高蔬菜作物耐盐性的一条有效途径。尽管在黄瓜嫁接栽培技术和耐盐生理方面已有报道,但主要集中在NaCl胁迫方面,嫁接黄瓜对Ca(NO3)2胁迫和NaCl胁迫响应的生理机制差异等研究,国内外尚鲜有报道。本研究以筛选出的耐盐白籽南瓜‘青砧1号’(Cucurbita maxima×C. moschata)为砧木,日光温室主栽黄瓜品种‘津优3号’为接穗,在营养液栽培条件下,对等渗的60mmo1·L-1Ca(N03)2和90mmol·L-1NaCl胁迫下砧木嫁接株和自根嫁接株生理机制和蛋白质组学等进行了比较,主要研究结果如下:
     1.等渗Ca(NO3)2和NaCl胁迫下,各砧用南瓜幼苗生长和抗氧化酶系统均受到不同程度的抑制,其中,‘青砧1号’的盐害指数最小,生物量及超氧化物歧化酶SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性的下降幅度以及相对电导率的上升幅度均小于其他砧木。Ca(NO3)2胁迫下,各砧木SOD、POD和CAT酶活性均高于等渗的NaCl,而盐害指数和相对电导率低于NaCl,表明Ca(NO3)2对砧木南瓜幼苗生长的危害小于NaCl.白籽南瓜‘青砧1号’具有较强的生长势和有效清除体内活性氧能力,降低膜质过氧化伤害程度,抗盐性较强。
     2.等渗Ca(NO3)2和NaCl胁迫下砧木嫁接和自根嫁接幼苗的生长和抗氧化酶系统均受到不同程度的抑制;与自根嫁接株相比,砧木嫁接株的生物量和叶片SOD、POD、CAT、APX、GR、DHAR和MDAR的活性及GSH、ASA、氧化还原力GSH/GSSG值均较高,而盐害指数、膜相对透性、MDA含量、O2-产生速率则明显降低,并可调节SOD、POD、CAT和APX同工酶的差异表达。等渗Ca(NO3)2和NaCl对黄瓜幼苗的盐胁迫效应不同,Ca(NO3)2处理各幼苗酶活性及氧化还原力均显著高于NaCl处理。表明耐盐砧木嫁接株具有抗氧化系统和AsA-GSH循环效率,能有效清除体内活性氧能力,降低膜质过氧化伤害程度,表现出较强的耐盐性。
     3.等渗Ca(NO3)2和NaCl胁迫下,与自根嫁接株相比,砧木嫁接株显著降低了Na和Cl-含量,较强地限制根系内皮层Na+、Cl-向中柱导管中的装载,进而将Na+、Cl-较多的截流在根部,阻止其向地上部分运输;增强了对K+、Ca2+、Mg2+的选择性吸收和向地上部的运输,可明显提高叶片和根系中硝酸还原酶(NR)活性及N03--N、可溶性糖、脯氨酸和可溶性蛋白含量,抑制NH4+-N升高,并提高叶片质膜H+-ATPase.液泡膜H+-ATPase、H+-PPase活性和相关基因表达,从而保持砧木嫁接株较强的耐盐性。NaCl胁迫下积累较多的Na+、Cl-,以离子胁迫为主,Ca(NO3)2胁迫以积累C1-为主,主要通过渗透胁迫影响植株生长,NaCl胁迫对植株的伤害程度大于等渗的Ca(NO3)2处理。
     4. Ca(NO3)2胁迫下黄瓜植株光合色素含量、净光合速率(Pn)、气孔导度(GS)、水分利用率(WUE)、光系统Ⅱ实际光化学效率(ΦPSⅡ)、光化学淬灭系数(qP)、Rubisco活性和含量、碳同化关键酶基因表达水平均显著高于等渗的NaCl胁迫;Rubisco活性显著下降是导致NaCl胁迫下Pn下降的主要原因。不同盐胁迫下,砧木嫁接株光合色素含量、Pn、Gs、Ci、WUE、ΦPSⅡ、Rubisco活性和含量均显著高于自根嫁接株,且可上调RbcL (Rubisco大亚基)、RCA (Rubisco活化酶)和PRK(5-磷酸核酮糖激酶)的基因表达,下调RbcS (Rubisco小亚基)的基因表达。因此认为,等渗Ca(NO3)2和NaCl胁迫下砧木嫁接株能维持较高的光系统II活性和碳同化能力。
     5. NaCl胁迫下砧木嫁接和自根嫁接株叶片2-DE图谱发现58个差异表达的蛋白点,成功鉴定55个蛋白点。与自根嫁接株目比,30个蛋白点在砧木嫁接中上调,14个蛋白点下调,45.45%的差异蛋白参与光合、20.00%参与蛋白质生物合成、14.55%参与胁迫防御,9.09%参与代谢、其它为能量和转运及功能未知等相关蛋白。除pc外,其它9个候选转录子与对应的蛋白丰度表现出一致的转录积累特性。Ca(NO3)2胁迫下发现48个差异表达的蛋白点,成功鉴定45个蛋白点,33个蛋白点在砧木嫁接中上调,10个蛋白点下调,46.67%的差异蛋白参与光合、24.44%参与代谢、15.56%参与胁迫防御反应,其它为能量和转运及蛋白质生物合成和功能未知等相关蛋白。10个候选转录于apx、pod、adh、mdh、pgam、rca、gs、rbcl、rbcs、pc与对应的蛋白丰度表现出一致的转录积累特性。表明砧木嫁接改变了植株NaCl和Ca(NO3)2胁迫下差异蛋白质的积累特性,并从转录水平上调节了盐胁迫下差异蛋白的表达,增强黄瓜植株对NaCl和Ca(NO3)2的胁迫耐性。
The protected cultivation of horticultural crops has expanded rapidly in recent years in China. Meanwhile, the secondary salinization of the protected horticulture is becoming increasingly severe, and has become an obstacle to limit vegetables production. Cucumber (Cucumis sativus L.) is one of the most popular cultivation conditions, and is sensitive to salt stress. In the present study, the effectiveness of grafting on salt tolerant rootstock can increase cucumber salt tolerance. However, a few researches on grafting techniques and salinity tolerance physiology of grafted cucumber have been carried out, which were mainly focused on NaCl stress, and few studies on physiological mechanisms of grafted cucumber in response to calcium nitrate stress have been reported. In this study, we use 'Qingzhen Nol'(Cucurbita maxima×Cucurbita. moschata) a salt tolerant cultivar as rootstock, and'Jinyou No3'a major cultivar in greenhouse as scion, grafting was made to compare the differences physiological mechanisms and proteomics between self-grafted and rootstock-grafted cucumber seedlings under60mmol·L-1Ca(NO3)2or90mmol·L-1NaCl stress. The main results were as follows:
     1. Under hight salt concentration60mmol·L-1Ca(NO3)2or90mmol·L-1NaCl treatment, the antioxidative systems of rootstock-Seedling were inhibited to varying degrees. The biomass and SOD, POD, CAT activity of'Qingzhen No.1' were higher than other rootstock varieties, whereas its salt injury index, plasma membrane electrolytic leakage, MDA content and O2production rate were significantly lower than others. At the same time, Enzyme activity of the various rootstock under Ca(NO3)2stress were higher then ones of rootstock under NaCl stress, but its salt injury index, plasma membrane electrolytic leakage, MDA content and O2production rate were significantly lower than ones under NaCl stress. The antioxidative ability varied with different genotypes of rootstock under salt stress.'Qingzhen No.l'was less affected by salt stress and held high resistance to salt stress; Both salt inhibited the plant growth, the oxidative damage was much heavier under NaCl stress.
     2. That growth and antioxidant system were inhibited in rootstock-grafted and self-grafted seedlings under either Ca(NO3)2or NaCl stress. The activities of SOD, POD and CAT in rootstock-grafted seedlings were higher than self-grafted seedlings, while the index of salt injury, relative permeability of membrane, content of MDA and the production rate of Of were significantly lower. The response of iso-osmotic Ca(NO3)2or NaCl stress in cucumber seedling was different, compared to NaCl stress, the relative permeability of membrane, the content of MDA and the production rate of O2were lower in Ca(NO3)2stress, while the activities of SOD, POD, CAT and APX were higher in Ca(NO3)2stress. It is conclude that grafting with salt-tolerant pumpkin could removal the reactive oxygen species and reduce the injury of lipid peroxidation, which was beneficial for plant growth in iso-osmotic Ca(NO3)2or NaCl stress.
     The activities of APX, GR, DHAR, MDAR and the content of GSH and ASA, as did the ratio of GSH/GSSG in rootstock-grafted seedlings were significantly higher than self-grafted seedlings. The isozymes expression of SOD, POD, CAT and APX were significantly induced by either Ca (NO3)2or NaCl stress,
     3. Compared with self-grafted seedlings, the rootstock-grafted reduced content of Na+and Cl-and inhibited the loading from root endodermis to column catheter, which could maintain much Na+, Cl-in roots to prevent transport to shoots. The selective absorption and transport to shoots of K+, Ca2+, Mg2+was enhanced by grafting with salt-tolerant pumpkin, and increased the activities of plasma membrane H+-ATPase and tonoplast H+-PPase and corresponding gene expression to enhance the salt tolerance. NaCl stress cause ion stress by accumulation of Na+and Cl-in plant, while Ca (NO3)2stress mainly affect plant growth through osmotic stress by accumulation of Cl". The damage under NaCl stress was much heavier than those of under Ca (NO3)2stress.
     4. The photosynthetic pigment content, net photosynthetic rate (Pn), the stomatal conductance (Gs), water use efficiency (WUE), the actual photochemical efficiency of photosystem Ⅱ (ΦPS Ⅱ), the photochemical quenching (qP), the content and activity of rubisco, the expression of key genes of carbon assimilation in leaves of both rootstock-grafted and self-grafted seedlings were higher under Ca(NO3)2stress than those of under NaCl stress. Intercellular CO2concentration (Ci) was decreased by Ca(NO3)2 treatment, while was increased by NaCl treatment. The decline of Pn in seedlings was due to the decreased of Rubisco activity under NaCl stress.The photosynthetic pigment content, Pn, Gs, Ci, WUE, ΦPS II, qP, rubisco activity and content in rootstock-grafted seedlings were more higher than that of self-grafted seedlings under iso-osmotic Ca(NO3)2or NaCl stress. And the genes of RbcL (Rubisco large subunit), RCA (Rubisco activase) and PRK(5-phosphoribulokinase) were also up-regulated while RbcS (Rubisco small subunit) was down regulated by rootstock-grafted under salt stress. We concluded that grafting with salt-tolerant pumpkin could maintain higher activity of PS Ⅱ and carbon assimilation capacity.
     5. We compared the high resolution two-dimensional gel electrophoresis (2-DE) protein profiles of self-grafed and rootstock-grafted, and the intensity of58protein spots varied. Of these spots, the identities of55were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and liquid chromatography electro-spray ionization tandem mass spectrometry.29protein spots were up-regulated in rootstock-grafted seedlings, and14protein spots were down-regulated. The intensity of48protein spots varied under Ca(NOs)2stress. Of these spots, the identities of45were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and liquid chromatography electro-spray ionization tandem mass spectrometry.31protein spots were up-regulated in rootstock-grafted seedlings, and13protein spots were down-regulated. These proteins were related to photosynthesis, metabolism, defending against stress, protein biosynthesis and energy and transpor, respectively. Other proteins were related to unknown proteins. Moreover, that the transcript accumulation patterns of majority candidate proteins were consistent with protein accumulation patterns. These results imply that rootstock-grafting mediated leaves protein different accumulation patterns under salt stress. The identified proteins may play an important role in regulating adaptation activities following exposure to NaCl or Ca(NO3)2stress.
引文
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