【作者】 张春平；

【导师】 何平；

【作者基本信息】 西南大学 ， 植物学， 2012， 博士

【摘要】 土壤盐渍化是影响农业生产与生态环境的一个重要因素,由于工业的发展和人口的不断增加,农业用耕地面积越来越少,因此提高作物的单产及抗逆性成为未来农业可持续发展及环境治理所面临的重要课题。土壤盐渍化是由于长期使用含有某些难溶性盐类的水灌溉或化肥的过度使用造成的,原始土壤和生产用水对设施土壤次生盐渍化形成有重要影响,不合理的施肥、特殊的水分运行方式、不合理的种植方式都是造成设施土壤次生盐渍化的重要因素。土壤盐渍化越来越成为影响作物生长、分布和产量的非生物因素。如何能使现有的作物正常生长在盐渍生境中并能获得一定的产量,这是长期以来科研工作者要努力解决的问题。黄连(Coptis chinensis Franch.)又名味连、川黄连、鸡爪连,为毛茛科黄连属多年生草本植物,三级渐危种,为我国著名的传统中药。黄连在四川、重庆、湖南、湖北等省均有栽培,重庆石柱是黄连的主产区之一。黄连根部有效成份小檗碱的独特药效使得黄连极具药用价值,但在长期的栽培种植过程中,由于施肥方式的单一和栽培地的局限,使得黄连栽培地土壤的理化性质发生了较大的改变,土壤的日渐盐化便是其中之一。近年来发现多种外源化学物质能够诱导植物产生抗盐性,为了深入研究外源性物质对盐胁迫下植物的具体作用,本研究中以黄连种子和幼苗为材料,选择了5-氨基乙酰丙酸(ALA)、一氧化氮(NO)供体硝普钠(SNP)、一氧化碳(CO)供体高铁血红蛋白(Hematin)、亚精胺(Spd)及水杨酸(SA)共5种外源性化学物质对NaCl模拟盐胁迫下的黄连种子萌发生理、幼苗光合特性及叶绿素荧光特性、抗氧化酶体系及保护性渗透物质、次生代谢产物等的影响做了研究,根据实验结果了解了黄连的耐盐机理,并提出了缓解盐胁迫的方法,为黄连在栽培生产中遇到的盐胁迫问题提供理论依据和解决方法,并通过一系列问题的探讨,为黄连的大面积优质推广,规范黄连的种植提供理论依据。本研究的主要结果如下：1.不同浓度的NaCl处理对黄连种子的萌发均有不同程度的抑制作用,较低浓度的NaCl (25mmol-L-1)处理黄连种子后,各萌发指标(发芽势、发芽率、发芽指数及活力指数)的变化并不明显。但是当NaCl浓度达到50mmol·L-1时,黄连种子的各萌发指标便显著降低,随着浓度的进一步升高,各指标变化趋势相似且不断降低,当浓度为400mmol·L-1时,黄连种子的4个萌发指标均降为零,这意味着在此浓度下,黄连种子的萌发完全被抑制。当用外源性物质进行处理后,各指标均有不同程度的提高,并且每种外源物质在所设置的4个浓度梯度中,均存在着相对效果最佳的处理浓度。2.在不同浓度NaCl模拟的盐胁迫下,黄连植株叶片的Chla、Chlb、Chl a+b、Car含量均发生了不同程度的降低,且随着胁迫程度的增大和处理时间的延长降低幅度逐渐增大。Chla/b在盐胁迫初期呈现出先升高后降低的趋势,随着胁迫时间的进一步延长,呈现出规律性的降低现象。Car/Chl(a+b)的变化则自始至终都呈现出升高的趋势,这说明盐胁迫对Chl a的伤害较Chl b更大,对总叶绿素的伤害比对Car的伤害作用更加明显。不同外源物质处理均提高了各种色素的含量,提高了Chl a/b比值,即对Chl a的含量的促进作用大于对Chl b的作用；Car/Chl a+b的比值则随着处理时间的延长呈现出降低趋势,此结果表明外源物质处理显著提高了总叶绿素含量,且提高幅度高于Car。3.在不同浓度的NaCl胁迫下,黄连植株的的净光合速率(Pn),气孔导度(Gs)及叶片蒸腾速率(Tr)均发生下降,并且随着胁迫时间和胁迫浓度的增加,下降幅度逐渐增大,胞间CO2浓度(Ci)则呈上升趋势。这说明在盐胁迫下,黄连净光合速率降低的主要影响因素是非气孔因素。在应用外源性物质进行处理后,黄连的Pn、Gs及Tr均有不同程度的提高,Ci也有不同程度的降低,并且不同的浓度梯度存在着显著的效果差异,这说明外源物质对维持黄连幼苗叶片正常的光合作用,维持光系统的较高活性具有积极的作用。4.黄连植株在经过盐胁迫后,黄连叶片的最大荧光(Fm)、最大光化学效率(Fv/Fm)、PSⅡ潜在活性(Fv/Fo). PSⅡ有效光化学效率(Fv’/Fm’)、PSⅡ实际光化学效率(ΦPSⅡ)、光化学淬灭系数(qP)、电子传递率(ETR)和光化学速率(PCR)和光化学反应能(P)均有不同程度的降低,而初始荧光(Fo)、非光化学淬灭系数(NPQ)、天线色素热耗散能(D)和非光化学反应耗散能(E)则呈升高趋势。上述结果说明盐胁迫显著地抑制了PS Ⅱ的功能,造成了PS Ⅱ反应中心的破坏。PS Ⅱ天线色素吸收的能量流向光化学的部分减少,以荧光形式等散失的能量增加,并且随着时间的延长,破坏程度越高。并降低了PSⅡ反应中心QA的氧化态数量,使QA→QB传递电子的能力下降,从而形成较多的QB,导致PSⅡ反应中心放氧活性降低。在经过外源性物质处理后,显著提高了黄连叶片Fm、Fv/Fm、Fv/Fo、ΦPSⅡ、 Fv’/Fm、qP、ETR、PCR和P的水平,降低了Fo、NPQ、E和D的值,提高了ΦPS Ⅱ的水平,减少PSⅡ和电子传递链的过分还原,可能还增强了假循环式光合磷酸化过程,用来消耗多余的能量,保护光合器官免受过多光能而造成伤害,减轻了膜脂过氧化作用,进而缓解了盐胁迫对黄连造成的伤害。5.盐胁迫提高了黄连幼苗叶片的相对电导率及质膜过氧化指标(H202含量、02产生速率、LOX酶活性、MDA含量)。经过5种外源物质的处理后,均显著的降低了黄连叶片膜氧化的水平,叶片的相对电导率、H202含量、02产生速率及MDA含量均显著降低,并且ALA、SNP及Hematin显著地降低了LOX的活性。Spd和SA处理对LOX的活性的影响并不明显,但是其它指标均有显著降低,这可能是两种物质缓解胁迫及膜氧化的方式与其它3种物质有所差异。6.低浓度和中等浓度的盐处理使黄连叶片SOD酶活性在前期和中期呈显著的上升趋势,而在处理后期则呈现逐渐下降的趋势,高浓度盐处理则呈现持续下降的变化趋势,这可能是由于高浓度下SOD的防御功能已经达到极限,可能会通过其它方式进行调节。POD活性在盐处理初期随处理浓度的升高逐渐升高,在处理的中后期,中低浓度处理仍然使得活性升高,而高浓度处理则呈现出降低趋势。中低浓度的盐处理使得CAT活性呈现出逐渐升高的趋势,而高盐处理则使得CAT呈现出先升高后下降的趋势,并且在盐处理后期,CAT活性降到最低。APX活性在胁迫初期显著升高,并且随着NaCl浓度的增大,活性升高的趋势逐渐加大,中低浓度的NaCl处理呈现出先增加后降低的趋势,高浓度NaCl处理则呈现出持续下降的趋势。GR活性在低浓度NaCl处理的整个过程出现不断升高的趋势,高浓度处理在整个过程呈现出不断降低的变化趋势,而中等浓度处理则呈现出先升高后降低的变化趋势。外源物质的应用在不同的处理时期有效的提高了盐胁迫下黄连幼苗叶片SOD、POD、CAT、APX及GR活性,从而形成了较为有效的抗氧化体系,有效地清除因为膜质过氧化积累下来的活性氧,以实现缓解过氧化的目标。7.盐胁迫下,可溶性糖在处理前期和中期均呈上升趋势,但是在处理后期呈现出下降趋势,游离脯氨酸含量在整个过程一直呈现上升趋势,可溶性蛋白在处理初期随着NaCl浓度的增大呈现出不断升高的趋势,但是随着处理时间的延长,含量均有不同程度的降低。总黄酮含量整体呈上升趋势,并且随着时间的延长呈上升趋势,处理中期和后期的差异并不显著。盐酸小檗碱在低浓度NaCl处理下含量升高,而在高浓度下(>100mmol·L-1)含量呈逐渐下降的趋势。当用外源物质进行处理后,可溶性蛋白、可溶性糖和游离脯氨酸含量均有显著的增加,并存在着浓度梯度效应。总黄酮含量也有增加,但是各处理浓度之间差异并不显著,盐酸小檗碱含量在经过外源物质处理后也有显著的升高。8.在用外源物质进行处理盐胁迫下黄连种子及种苗的过程中,各指标出现最佳效果的时间虽略有差异,但是最佳处理浓度基本一致。种子在进行外源物质处理时,筛选出基本的最佳浓度分别是：ALA (10mg·L-I)、SNP (0.1mmol·L-1)、Hematin (0.5μmol·L-1)、Spd (0.25mmol·L-1)、SA(10mg-L-1)。黄连植株在进行处理时,最佳处理浓度分别是：ALA(50mg·L-1)、SNP (0.25mmol·L-1)、Hematin (2.0μmol·L-1)、Spd (0.50mmol·L-1)、SA(50mg·L-1)。综上所述,5种外源物质通过提高盐胁迫下黄连种子萌发指标、植株叶片光合色素含量、抗氧化酶活性、光合参数(Pn、Gs、Tr, Fm、Fv/Fm、Fv/Fo、ΦPSⅡ、Fv’/Fm、qP、ETR、PCR和P)的水平,代谢产物(可溶性糖、游离脯氨酸、可溶性蛋白质、黄酮)的含量,降低Ci、Fo、NPQ、E、D H2O2含量、O2产生速率、LOX酶活性、MDA含量等的方式缓解了盐胁迫对黄连的伤害,提高了黄连的抗盐性。更多还原

【Abstract】 Soil salinity is an important factor affecting agricultural production and ecological environment, the proportion of arable land becoming less and less with the development of industry and the increase of population. So improving the crop resistance to adversity has become an important problem to the future sustainable development of agriculture and environmental management. Given the erosion of large areas of land and increased salinity stress experienced by numerous plants worldwide, soil salinity has become a global problem. Soil salinity, a major abiotic stress that affects germination, crop growth, and productivity, is a common adverse environmental factor. Soil salinity affects plant growth, the global geographic distribution of vegetation, and medicinal plant yields. Soil salinity is a long-term using of water for irrigation contains some insoluble salts or excessive use of fertilizers. The original soil and water for production formation of the facilities secondary soil salinity, unreasonable fertilization, unconscionable cropping patterns are the important factors in the soil salinity. It is a significant problem to the scientists to let the crops growth normally in the salinity habitats and obtain a certain yield.Coptis chinensis Franch. is a well-known traditional Chinese medicinal plant belonging to the medically and economically important of genus Coptis family Ranunculaceae with other name like "weilian","chuanhuanglian","jizhualian", and so on. It is widely distributed in the province of Sichuan, Chongqing, Hunan, Hubei. Shizhu is the main producing areas in Chongqing. The active ingredient berberine in the root of C. chinensis with distinctive therapeutic effectiveness make it of great medicinal value. In the long-term cultivation process, due to the limitations of single fertilization methods and the areas of cultivated land, the physical and chemical properties of soil has large changes. In recent years, more and more research had fund that variety of exogenous chemicals can induce plants to improve the salt tolerance under salt stress. In this study, seeds and seedlings of C. chinensis were used to research the specific role of exogenous substances under salinity stress. The5-aminolevulinic acid (ALA), nitric oxide (NO) donor sodium nitroprusside (SNP), carbon monoxide (CO) donor hematin, spermidine (Spd) and salicylic acid (SA) were applied to study the effects to the seed germination, photosynthetic characteristics of seedlings, chlorophyll fluorescence characteristics, lipid peroxidation, antioxidant enzyme machinery, permeability protection substances, secondary metabolites of C. chinensis seeds and seedlings. The main results of this study as follows:1. Seed germination indices, such as Gv, Gr, Gi, and Vi, of C. chinensis were inhibited with different degrees under different concentrations of NaCl. The treatment with lower NaCl concentration dose not show an obviously change compared with the control. When the concentration of NaCl got50mmol·L-1, the germination indices reflected the same trend and showed an obvious inhibition with the improvement of the concentration. When the concentration got400mmol·L-1, every indices became to zero and this result means the germination was inhibited completely. With the treatments of different exogenous substances, every index improved with different degrees. Every exogenous substance with different concentrations got an appropriate concentration finally.2. The Chi content of C. chinensis seedling leaves treated under different concentrations of NaCl stress were inhibited with different degrees and yielded lower concentrations than the control. Chi a, Chi b, and total Chi concentrations decreased with similar tendencies following treatment days. Chi a/b has the trend of increased first and then decreased in the early days of treatment, and decreased with the treatment days. The change of Car/Chl (a+b) shown a tendency of decrease in the whole process of treatment. This result shown that the injury of salinity stress to Chi a was more severe compared with Chi b, the stress to total Chi was more obvious to Car. The content of different pigments, the level of Chi a/b were all improved and the level was Car/Chl a+b decreased with the treatments of different exogenous substances. The result indicated that exogenous substances increased the content of total Chi and the level improved more significant to Car.3. The level of Pn, Gs, and Tr of C. chinensis seedlings were all decreased with the treatment days and the increased concentrations of NaCl under salinity stress, and the value of Ci increased with the opposite tendency. These results indicate that the main influencing factor to the decreasing net photosynthetic rate of C. chinensis is non-stomatal factors. After treated with different exogenous substances, the Pn, Gs, and Tr were all increased with different degrees, and the level of Ci was decreased. Among the different concentrations in the same exogenous substance has a appropriate concentration. It indicated that exogenous substances have a positive effect of to maintain normal photosynthesis and the higher activity of photosynthetical system of C. chinensis. 4. The indices, such as maximum fluorescence (Fm), potential maximum photochemical efficiency (Fv/Fm), potential photochemical efficiency (Fv/Fo), photochemical efficiency (Fv’/Fm’), actual photochemical efficiency ((DPSII), and photochemical quench coefficient (qP), electronic transfer rate (PCR), photochemistry rate (ETR), and absorbed light in photochemistry energy (P) were all significantly decreased with NaCl stress compared with the control, the values of these parameters increased with the number of treatment days, while the minimal fluorescence (Fo), non-photochemical quenching coefficient (NPQ), antenna heat dissipation (D), excess energy (E) were all increased. These results indicate that the primary light energy conversion efficiency of PSII was significantly inhibited under salinity stress and the PSII response centre is damaged by photooxidation and photoinhibition. The energy absorbed by the PS II antenna pigments to the photochemical energy reduced and the part that dissipated with the form of fluorescent improved. The number of PSII open centres and the original electronic receptor QAs decreased. With the treatments of different exogenous substances, the level of Fm、Fv/Fm、Fv/Fo、ΦPSⅡ、Fv’/Fm、 qP、ETR、PCR, and P were all improved significantly, the level of Fo、NPQ、E和1D were all decreased. The photosystem, by increasing non-radiative heat dissipation, could consume the excessive light energy absorbed by PSII. Consequently, the PSII response centre is protected from damage by photooxidation and photoinhibition for absorbing excess light energy and the salinity stress-induced damage to C. chinensis had been alleviated.5. An age-dependent increase in TBARS concentration, H2O2content, LOX activity, production rate of O2-, and the relative conductivity were all observed in leaves subjected to NaCl-only treatment. The level of plasma membrane peroxidation was decreased with five different exogenous substances, the TBARS concentration, H2O2content, the production rate of O2-, and the relative conductivity were all decreased significantly. ALA, SNP, and Hematin treatments decreased the LOX activity significantly. The effects of Spd and SA treatments to the LOX activity were not significantly. This might due to the form alleviating the membrane peroxidation was different to other three substances.6. NaCl treatments with lower and medium concentrations made the SOD activity of C. chinensis leaves increased significantly in the early and mid, and decreased in the late period of treatment. The SOD activity decreased with higher concentration of NaCl treatments has a continuing trend. This may be the defense function of the SOD has reached its limitation and adjusted by other modes. POD activity gradually increased with the increase of NaCl concentration in the early period and the treatments with lower and medium concentrations in the mid and late period were also increased, while the higher concentration showed a decreasing trend. The lower and medium concentrations of NaCl treatments increased the CAT activity and the higher concentration showed a trend that increased first and then decreased. In the late period of the NaCl treatment, CAT activity got the minimum. APX activity in the initial period of salinity stress was significantly increased, and the low and mid concentrations of NaCl treatment showing a trend of increasing first and then decreasing. The higher concentration of NaCl treatment shown a continuous decline trend. GR activity in the whole process of the low concentration of NaCl treatment has a rising trend, and showing a continuous decrease trend with the higher concentration. Medium concentrations treatment made a trend that increasing first and then decreasing. The application of exogenous substances in different treatment periods to C. chinensis improved the activities of SOD, POD, CAT, and APX and GR effectively. This can form a more effective antioxidant system and remove the accumulated lipid peroxidation reactive oxygen species in order to ease the oxidation of the target.7. The content of soluble sugars has a increasing trend in the early and mid period of treatment, while in the late period, the content of soluble sugars decreased. The content of free proline increased in the whole process of the treatment. The soluble protein has the increasing tendency in the early period of treatment with the increase of NaCl concentrations, and the content has a decreasing trend with different degrees with the treatment time. Total flavones content increased with the treatment times and has no significant in the mid and late period of the treatment. The content of berberine hydrochloride increased with the lower concentration of NaCl, while the content decreased in the higher NaCl concentration. The application of exogenous substances in different treatment periods to C. chinensis increased the content of soluble sugars, free proline, soluble protein, and berberine hydrochloride. Total flavones content also has a improvement, but there is no significant difference between different concentrations.8. In the process of C. chinensis treated with different exogenous substance under salinity stress, the time that got the optimum result of every indices has a slightly different, while the optimum concentration to every indices was the same. The seeds of C. chinensis treated with different exogenous substance got the optimum concentration were as follows:ALA (10mg-L-1)、 SNP (0.1mmol·L-1)、Hematin(0.5μmol·L-1)、Spd (0.25mmol·L-1)、SA (10mg·L-1).The seedlings of C. chinensis treated with different exogenous substance got the optimum concentration were as follows:ALA (50mg·L-1)、SNP (0.25mmol·L-1)、Hematin (2.0μmol·L-1)、Spd (0.50mmol·L-1)、 SA (50mg·L-1)更多还原