【作者】 王贵平；

【导师】 王玮；

【作者基本信息】 山东农业大学 ， 植物学， 2009， 博士

【摘要】 在自然生长环境中,植物经常处于多种胁迫并存的逆境条件下,如干旱和高温共胁迫、干旱与低温共胁迫等。人们对于干旱、高温单一胁迫对植物生长发育的影响已经研究了很多,但是有关干旱与高温共胁迫对植物影响方面的研究却很少。甜菜碱(GB)在提高植物对单一逆境(例如干旱或高温胁迫)的抗性方面有重要作用,但是在干旱和高温共胁迫条件下,甜菜碱改善植物抗性方面的研究资料还很少。本文利用超表达甜菜碱的转基因小麦99T6(T6)及其野生型小麦石4185(WT)(Triticum aestivum L.)为试材,研究了小麦对干旱、高温单一胁迫以及干旱高温共胁迫响应,甜菜碱提高小麦抗干旱、高温单因子胁迫以及干旱高温共胁迫的生理机制,分析了单一逆境与多因子综合逆境对小麦生理状态影响的差异以及甜菜碱作用的差异。转基因小麦99T6是通过基因工程转入山菠菜(Prunella asiatica Nakai)BADH基因产生的,BADH基因编码甜菜碱醛脱氢酶,此酶是甜菜碱合成的关键酶之一。转基因系中,BADH基因的超表达诱导了甜菜碱的过量积累。本文采用室内研究、网室盆栽和田间试验相结合的方法。室内和盆栽实验设干旱、高温胁迫和干旱高温共胁迫三个处理;大田试验在5月下旬利用夏季小麦收获前田间大气干旱、高温、高光强并存,即“干热风”发生的自然环境条件。检测了不同胁迫条件下,小麦叶片光合气体交换参数、水分状况和膜脂过氧化水平的变化差异。主要结果和结论如下:1.过量积累GB对不同胁迫下小麦幼苗叶片光合作用的影响砂培小麦幼苗生长到三叶一心时进行胁迫处理。用30%(w/v)PEG-6000(渗透势大约为-1.88Mpa)诱导干旱胁迫处理3天;高温胁迫处理是在光照培养箱中,温度40℃处理3小时;干旱和高温共胁迫处理是对已经进行干旱胁迫处理(30%(w/v)PEG-6000)3天的小麦幼苗放到40℃高温条件下胁迫处理3小时。进行胁迫处理后,对小麦的光合作用相关参数、水分状况和膜脂过氧化指标进行测定。结果如下:(1)不同胁迫方式对小麦幼苗叶片光合作用的影响不同。干旱与高温胁迫下,小麦叶绿素含量、光合能力(Pn)、羧化效率(CE)和表观量子效率(AQY)均明显降低(P<0.05),共胁迫下降低程度更大;干旱与高温胁迫比较,高温胁迫造成的降低程度高于干旱胁迫,但是干旱对蒸腾速率(Tr)、气孔导度(Gs)以及胞间CO2浓度(Ci)的影响程度高于高温胁迫;干旱胁迫下气孔关闭,高温胁迫下气孔张开,干旱高温共胁迫下气孔关闭。干旱、高温单一胁迫下光合作用降低。外源BADH基因的导入提高了小麦叶片中甜菜碱的积累水平,同时小麦叶片甜菜碱的积累还受干旱、高温以及干旱高温共胁迫的诱导。甜菜碱的过量积累不仅可以提高单一干旱或高温胁迫条件下小麦的光合能力,而且可以提高干旱高温共胁迫条件下小麦的光合作用。甜菜碱促进光合能力的提高与气孔状况和叶肉光合能力的改善有关。(2)GB的过量积累能维持较好的水分状况。干旱单一胁迫与干旱高温共胁迫造成小麦叶片相对含水量降低,但是高温单一胁迫对叶片水分含量的影响不明显。过量积累的甜菜碱除了本身是一种有效的渗透调节物质以外,还通过促进其它渗透调节物质的积累,如脯氨酸和可溶性糖,维持了小麦叶片在不同胁迫条件下较强的渗透调节能力,增强了吸水,从而维持转基因小麦株系较好的水分状况,水分状况的维持可能与水孔蛋白有关。(3)GB的过量积累提高了小麦叶片的抗氧化能力。胁迫导致活性氧(超氧阴离子和过氧化氢)含量提高,膜脂过氧化产物丙二醛(MDA)含量以及膜透性增加,最终破坏了生物膜的完整性和有序性。共胁迫下膜脂过氧化程度较单一胁迫下更为严重。过量积累甜菜碱的转基因植株在同样胁迫下膜系统的破坏小,膜脂过氧化水平低。测定结果表明,过量积累甜菜碱的转基因植株主要抗氧化酶(SOD、CAT、POD、APX)的活性相对较高,抗氧化物质(抗坏血酸AsA和还原型谷胱甘肽GSH)含量明显高于野生型,能间接清除活性氧,由于甜菜碱不能直接清除活性氧,这些间接机制可能是最终降低膜脂过氧化水平和胁迫对转基因小麦株系生物膜损伤的主要原因。(4)过量积累GB能维持类脂组分和功能来保护类囊体膜上功能蛋白和类囊体膜的完整性。胁迫改变类囊体膜中各类脂的相对比例,造成膜脂不饱和指数升高。过量积累甜菜碱的转基因植株类囊体膜脂组分和脂肪酸不饱和度相对稳定。胁迫同样影响了类囊体膜上各种蛋白的丰度,并有新多肽的出现。过量积累甜菜碱的植株中类囊体多肽受胁迫影响小。(5)过量积累GB对胁迫下小麦幼苗叶片叶绿体超微结构有保护作用。干旱胁迫导致小麦叶片的基粒片层肿胀,变得模糊不清;高温胁迫下,基粒片层解体;干旱高温共胁迫条件下,损伤现象更为严重;野生型小麦的叶绿体超微结构受不同胁迫的损伤较转基因株系T6更为明显;甜菜碱的过量积累对不同胁迫下小麦叶片的类囊体片层结构具有一定的保护作用,可以减轻不同胁迫条件下对叶绿体的超微结构的破坏。2. GB的过量积累对盆栽小麦旗叶光合能力的影响盆栽小麦于开花期进行胁迫处理。干旱胁迫的材料从开花期停止浇水,雨天用遮雨棚遮雨,对照必要时浇水。干旱和高温共胁迫通过对处于干旱胁迫状态的植株进行高温胁迫处理,高温胁迫处理是在光照培养箱中进行,温度40℃,处理时间为4小时。检测开花期旗叶相关指标,结果如下:(1)过量积累GB提高了小麦旗叶的光合作用能力。BADH基因的导入增加了小麦旗叶甜菜碱的积累,略高于幼苗小麦叶甜菜碱含量,这可能与生长阶段和生长环境有关。甜菜碱的过量积累不仅可以提高干旱或高温单一胁迫条件下旗叶的光合能力,而且可以提高干旱高温共胁迫条件下小麦旗叶的光合能力,这与幼苗期的结果一致。(2)胁迫下转基因小麦有较强的氮代谢能力。高温和干旱高温共胁迫显著降低了亚硝酸还原酶(NR)和谷氨酰胺合成酶(GS)活性(P<0.05),表明亚硝酸还原酶和谷氨酰胺合成酶对高温敏感。在三种胁迫下,T6中的NR和GS活性均高于WT,这表明逆境条件下,T6中GB积累诱导的游离脯氨酸及氨基酸含量的增加可能与氮代谢较为旺盛有关。(3)GB的过量积累增加了胁迫下小麦旗叶PSII的活性。干旱胁迫下Fv/Fm变化不明显;高温和干旱高温共胁迫条件下Fv/Fm和ФPSII明显降低,但T6中下降程度低于WT。说明T6中PSII的反应中心比较稳定,对胁迫具有较强的耐性,即BADH基因的过量积累可以增强PSII的耐逆性。(4)适当的干旱胁迫增强了小麦旗叶PSII的热稳定性。PSII的光化学活性在干旱高温共胁迫条件下明显高于高温胁迫下的PSII活性,表明干旱胁迫提高了PSII的抗热性。从qP的变化也可以看出,在高温胁迫下其降低的程度高于干旱高温共胁迫条件下,这也说明了干旱提高了PSII反应中心的活性。干旱提高PSII的热稳定性也与放氧复合体(OEC)的抗热性提高有关,在干旱高温共胁迫条件下出现的K相较高温胁迫下低。(5)过量积累GB提高了小麦旗叶热耗散能力。干旱、高温以及共胁迫下,T6和WT的旗叶NPQ明显增加。叶黄素循环的脱环氧化状态(A+Z)/(V+A+Z)的变化和NPQ的变化趋势一致。叶黄素循环的脱环氧化状态(A+Z)/(V+A+Z)与热耗散能力直接相关。这说明三种胁迫条件下,小麦的热耗散能力增强,干旱及共胁迫条件下明显高于高温胁迫下。过量积累甜菜碱提高了小麦的热耗散能力。3. GB的过量积累对田间灌浆期小麦旗叶光合日变化的影响在田间自然条件下,选择晴天对灌浆期小麦旗叶的光合日变化进行跟踪测定。主要结果如下:(1)GB的过量积累影响灌浆期小麦的光合日变化。田间栽培条件下,在小麦灌浆期,转基因小麦旗叶和野生型小麦旗叶的光合日变化呈双峰曲线,两者皆在10:00左右和16:00左右各有一个峰值。在12:00出现最低值,但T6中的峰值和谷值都明显高于野生型,其它气体参数的日变化与光合速率基本一致。GB的过量积累明显降低了峰值与谷值的差值。(2)GB的过量积累能减轻田间栽培条件下小麦的光抑制。PSII的最大光化学效率(Fv/Fm)和实际光化学效率(ФPSII)的日变化和净光合速率的日变化结果相似。GB的过量积累能减轻午间PSII光化学活性的降低,减轻小麦的光抑制,提高小麦的光合速率。这有利于提高小麦的产量,特别是在逆境条件下或者“干热风”发生的年份更有利。(3)GB的过量积累对小麦产量及其构成因子有影响。GB的过量积累对小麦产量没有明显的影响(P>0.05),但是转基因T6小麦的穗籽粒数明显(P<0.05)高于野生型石4185。推测GB的过量积累可能对小麦的发育有影响。(4)在灌浆期与灌浆后期,与野生型相比转基因小麦T6保持了较高的非光化学猝灭(NPQ)。灌浆后期“光合午休”加重,小麦旗叶PSII反应中心的受抑制程度增加,而此时两种基因型小麦的热耗散能力均大大降低,两基因型的NPQ在12点都降到最低。但是一天中T6的NPQ值总是高于野生型。以上结果表明,过量积累甜菜碱可以减轻小麦生育后期的光合“午休”,降低光抑制。更多还原

【Abstract】 Within their natural habitat, plants are often subjected to a combination of various abiotic stress conditions such as drought and high temperature. The effects of individual drought or high temperature stress on plant have been extensively studied. However, only a little is known about how their combination impacts on plant. Glycine betaine (GB) plays an important role in protecting plant cell from damage by various single stresses, but what does it under the combination of drought and high temperature stresses? In the present study, to investigate the different responses of wheat to drought, high temperature stress and their combination, and analyze the physiological mechanisms of GB improvement involved in the wheat tolerance to stress conditions, a transgenic wheat line T6 and the wild-type (WT) line Shi4185 were used. The transgenic line was generated by introducing a BADH gene into wheat by microprojectile bombardment, the BADH gene encoded a betaine aldehyde dehydrogenase, which was cloned from Atriplex hortensis L. in the plasmid pABH9 containing maize ubiquitin promoter and bar gene, the transgenic wheat plants T6 exhibits higher BADH activity and GB accumulation than WT. The preparation of experimental treatments in this study was conducted by three ways: laboratory cultivation, pot cultivation outroom and field cultivation. There were three treatments in laboratory test and pot experiment, including single drought (DS), heat stress (HS) and their combination (DS+HS), Field investigation was conducted on 20 and 28 may 2009 under natural condition. In this time, air dry, high temperature and high light stresses occurred simultaneously. Photosynthetic gas exchange, water status, and lipid peroxidation of the wheat leaves were examined in all experiments.1. The effects of over-accumulated GB on photosynthesis of wheat seedlings leaveWhen the 3 rd leaf was expanded fully, all treatments were performed with at least three parallels. Drought was imposed by 30% (w/v) PEG-6000 (osmotic potential -1.88 MPa) until the relative water content (RWC) of leaves reached to 83% to 86% (continual 3 days typically). A combination of drought and high temperature stress was performed by subjecting the drought-stressed plants to a high temperature of 40°C for 3 h. High temperature stress was applied by raising the temperature in artificial chamber to 40°C for 3 h at the same time when stress combination was executed. The results are as follows:(1) The effects of different stresses on the photosynthesis of wheat seedlings leaves were different. Chlorophyll content, net photosynthesis rates (Pn), the apparent quantum yield (AQY) and the carboxylation efficiency of photosynthesis (CE) were decreased significantly in both wheat lines (P<0.05) under both drought and heat stress, and they were greatly aggravated by a combination of drought and heat stress. Compared the results under drought stress to high temperature stress; the decrease of these parameters was greater under high temperature than that under drought stress. But the effect of drought stress on transpiration rate (Tr), stomatal conductance (Gs) and intercellular CO2 concentration (Ci) were more serious than high temperature stress. Individual drought stress resulted in opening of stomata, but under individual high temperature stress, that was opened as non-stressed control. However, after drought stress treatment, the succedent high temperature stress can not lead to opening stomata. Introducing foreign BADH gene into wheat induced visibly the overaccumulation of GB, and GB accumulation level was also induced by drought,high temperature stress and their combination. Overexpression of GB in T6 increased the tolerance of photosynthesis not only to individual drought or high temperature stress but also to their combination.(2) Over accumulated GB can help the transgenic wheat leaves to maintaining well water status compared to WT. Both drought and a combination of drought and heat stresses resulted in a significant (P<0.05) decrease in RWC of leaves, but heat stress can not gave significant effect on it. Besides as an osmolyte, overaccumulated GB could increase the level of osmotic adjustment (OA) via accumulating some other compatible solutes such as proline and soluble sugar, all them can decrease osmotic potential of the cell and enhance water-absorbing, and consequently maintain a better water status in wheat leaves. Otherwise, the improvement of GB on water status may be related to protecting the aquaporin (AQP) in cell membrane.(3) Overaccumulated GB enhanced the antioxidant ability of the transgenic wheat. Stresses caused the increases of reactive oxygen species (O2 and H2O2) content, malondialdehyde (MDA) content, and ultimately resulted in electrolyte leakage of plant cell in wheat leaves. Both drought and high temperature stress increased ion leakage of the cell significantly (p<0.05), but the increase was slightly greater under heat than drought stress, and the most increase was observed under stress combination. The activity observations of several main antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) suggested that overaccumulated GB can maintain higher enzyme activity in transgenic wheat leaves under stresses, and the increased accumulations of two non-enzyme antioxidants, namelyascirbic acid (AsA) and glutathione (GSH) were also higher in T6 than that in WT. The increased antioxidant enzymes activity and non-enzyme antioxidant accumulation may be the major cause that GB can help to scavenge O2 and H2O2 indirectly, because GB is ineffective in scavenging O2 and H2O2 directly.(4) Overaccumulated GB alleviates the damage of functional protein and the thylakoid membrane integrity under stresses through maintaining lipid components and polypeptides. The index of unsaturated fatty acids (IUFA) of thylakoid membrane lipids decreased under almost all stresses. Transgenic line T6 with overaccumulated GB had a relatively stable status under stresses in components and levels of thylakoid membrane lipids. Similarly, the abundance of 20~60 kDa polypeptides was affected by stresses and some new polypeptide appeared under stresses. However, the influences were smaller in T6 than that in WT.(5) Overaccumulated GB protected chloroplast ultrastructure in wheat seedling leaves. The shape of the chloroplast was spherical, the thylakoid lamellae was swollen under drought stress, and thylakoid lamellae was scattered under high temperature; the most severe damage was found under combined stress. However, over accumulated GB can protect chloroplast ultrastructure from damage by stresses. The chloroplast and thylakoid lamella ultrastructure were damaged by all kinds of stresses, but the demolition in WT was more serious than that in T6 under stresses, suggesting the protection of overexpressed GB to photosynthetic apparatus of wheat leaves.2. The effects of overaccumulated GB on photosynthesis of wheat flag leaves at flowering stage.Seeds of both WT and T6 were sown in earthenware pots (high, 18cm; diameter, 24 cm) filled with 3 kg soil composed of loam and organic fertilizer by the ratio in 7:3 and 250 g complete fertilizer (NH4NO3:K3PO4:KNO3 = 20:10:20). Seven seeds were sown in each pot initially, and five plants per pot were left after germination. Then, all plants were grown in a greenhouse with conventionality cultivation until flowering stage. All treatments were performed in parallel. Drought stress was imposed by withdrawing water from plants until the flag leaves reached relative water content (RWC) of 76% to 84% (typically 6–7 d). High temperature was applied by raising the temperature in the growth chamber to 40°C for 4 h. A combination of drought and high temperature was performed by subjecting drought-stressed plants (RWC of 76%–84%) to a high temperature treatment (40°C for 4 h). After treatments, the relative indexes were examined. The results are as follows:(1) Over accumulated GB increased photosynthesis of the wheat flag leaves. Introducing of foreign BADH gene visibly enhanced the accumulation of GB in wheat flag leaves, and the contents were higher than that in wheat seedling leaves, this may be related to the developmental stages and environment of wheat growth. Similarly, overaccumulated GB in T6 increased the tolerance of wheat flag leaves photosynthesis not only to individual drought or high temperature stress but also to their combination.(2) Under stresses, the T6 plants maitained the higher nitrogen assimilation than WT. Drought stress induced the increase of the activities of nitrate reductase (NR) and glutamine synthetase (GS), but high temperature and combined stress significantly decreased them (P < 0.05), indicating the sensitivity of two enzymes to high temperature. The activities of NR and GS were significantly higher in T6 than that in WT under different stresses. The higher proline and free amino acid level in T6 may be involved in the higher nitrogen (N) metabolism under stresses. (3) Under stresses, overaccumulated GB increased the wheat flag leaves PSII photochemistry activity. Drought had no significant effect on the maximal efficiency (Fv/Fm), however, the high temperature and the combined stress significantly decreased the Fv/Fm and actual efficiency (ФPSII). Overaccumulated GB in T6 alleviated the decrease of both Fv/Fm andФPSII under stress conditions, suggesting the higher activity of PSII reaction center in T6 than WT. The results showed that the PSII complex in T6 is better in with standing photoinduced inactivation than WT under stress conditions.(4) Drought stress increases thermostability of PSII. An increased resistance of PSII to high temperature in drought-stressed leaves was observed, which was associated with an improvement of the resistance of PSII reaction centre to high temperature, as indicated by a smaller increase in the proportion of QB-non-reducing PSII reaction centre during high temperature in drought-stressed leaves than in well-watered plants. In addition, these results demonstrate that increased thermostability of PSII in drought-stressed plants was also associated with an improvement in thermostability of the O2-evolving complex (OEC), as shown by a less pronounced phase K in the polyphasic fluorescence transients in combination-stressed plants than in the high temperature -stressed plants.(5) Overaccumulated GB improved xanthophyll cycle-dependent energy dissipation. Under all three stress conditions, the NPQs of two wheat plants were significantly (P<0.05) increased, and the NPQ was relatively higher in T6 than WT. Meanwhile, the changes of the (A + Z) / (V + A + Z) in wheat leaves were consistent with the NPQ under different stresses. Because (A + Z) / (V + A + Z) is positive correlation to the energy dissipation ability, the results suggested that overaccumulated GB can enhance the thermal dissipation in the T6 leaves by increasing xanthophyll cycle.3. The effects of overaccumulated GB on diurnal variation of photosynthetic rate in wheat flag leaves during grain-filling period in the field condition.Under natural field condition, diurnal variation of photosynthesis in wheat flag leaves during grain-filling period was measured on a sunny day. The results are as follows:(1) Under the field condition, diurnal variation of stomatal conductance and net photosynthetic rate revealed two peaks at about 10:00 and 16:00 separately because of the strong intensity of sunlight, high temperature and relatively low humidity. The maximum at 10:00 was higher than that at 16:00 with a minimum at 12:00. The changes of other gas parameters were almost consistent with the net photosynthetic rate. Overaccumulated GB significantly decreased the gap between the maximum and minimum of net photosynthetic rate.(2) Overaccumulated GB can alleviate the photoinhibition of photosynthesis in wheat flag leaves in the filed. We compared the photoinhibition of photosynthesis of T6 leave to WT subjected to midday strong light stress under field conditions. This was done by measuring diurnal variation of Fv/Fm andФPSII of PSII. The diurnal changes of the Fv/Fm andФPSII were similar to that of net photosynthetic rate. The Fv/Fm andФPSII in WT were much lower than those of T6 leaves in both maxima and minimum level, showing a more severe photoinhibition in WT. Based on these results, we concluded that the overaccumulated GB may be benefit to increase the quality and yield of wheat under stress or in the years which occurre Dry-heat Wind.(3) The effects of overaccumulated GB on the yield parameter. Over accumulated GB had no significant effects on wheat yield (P>0.05); however, there were more seeds per spike in T6 than in WT, significantly (P<0.05), indicating that overaccumulated GB maybe affect on the spike development of the wheat.(4) At wheat filling stage, the non-photochemical quenching (NPQ) was higher in T6 than that in WT. During the wheat filling stage,“Midday Depression of wheat Photosynthesis”was serious in further, the depression of the PSII reaction center in the wheat flag leaves aggravated, and the energy dissipation in wheat flag leaves of two wheats decreased, too, with a minimum at 12:00. But the NPQ was higher in T6 than that in WT during a day. These results show that overaccumulated GB can alleviate“Midday Depression of wheat Photosynthesis”in the later growing period of wheat by decreasing the photoinhibition.更多还原