【作者】 韩菡；

【导师】 孟庆伟；

【作者基本信息】 山东农业大学 ， 植物学， 2009， 博士

【摘要】 光作为植物光合作用的唯一能源是植物生命活动的基础。然而,当植物吸收的光能超过其电子传递所需时,过剩的光能便会造成光合效率和光合功能的降低,引起光抑制,严重时还会发生光氧化破坏。植物除了在强光下会产生光抑制外,在低温弱光、高温、干旱、盐碱等逆境下由于CO2的固定受到限制,使得植物对光能的利用减少而导致光抑制。高等植物体内存在着多种防御机制以避免或减少光抑制,其中叶黄素循环被认为是逆境下保护植物光合机构免受过剩光能破坏的一种重要的机制。叶黄素循环是指植物吸收的光能过剩时,双环氧的紫黄质（V）在紫黄质脱环氧化酶（VDE）的催化下,经过中间物单环氧的花药黄质（A）转化为无环氧的玉米黄质（Z）;在暗处,则在玉米黄质环氧化酶（ZE）的作用下朝相反的方向进行,将Z重新环氧化为V,形成一个循环。依赖于叶黄素循环的非光化学猝灭（NPQ）能够保护植物光合机构免受过剩激发能的破坏。有过剩光能存在时,由紫黄质（V）脱环氧化而积累的玉米黄质（Z）也被人们普遍认为对光合机构起到至关重要的保护作用。本研究从番茄叶片中分离到番茄紫黄质脱环氧化酶基因,并对该基因的表达和功能进行了分析。结果表明,该基因的表达并受光强、温度和昼夜节律的影响。过量表达该基因可减轻番茄植株对光抑制的敏感性,而抑制该基因表达可加重番茄植株在低温弱光胁迫下的光抑制程度。主要结果如下:1.利用同源序列设计简并引物,通过RT-PCR的方法从番茄叶片克隆到紫黄质脱环氧化酶基因的中间片段,通过5’-RACE和3’-RACE分别克隆到5’和3’片段,拼接后设计特异引物扩增到全长cDNA。命名为LeVDE（FJ648424）。该基因全长为1670bp,ORF为1437bp,编码478个氨基酸,分子量约为53 kDa。同源序列比较发现,番茄紫黄质脱环化酶基因的序列与拟南芥、烟草、菠菜、莴苣、水稻的紫黄质脱环化酶基因的序列同源性较高。结构同源性分析表明LeVDE有三个特征结构域,block I为-端半胱氨酸富集区,有活性的VDE中N-端含有11-13个半胱氨酸,是VDE的抑制剂DTT作用的区域,低浓度DTT只能部分抑制VDE的活性,说明N-端的半胱氨酸形成不止一个二硫键。block II为脂质运载蛋白特征区,结合和转运疏水小分子的区域,为疏水底物A和Z的结合区。block III是C-端谷氨酸富集区,含有大量的负电荷,是VDE与类囊体膜结合的部位。2. Northern杂交结果表明,LeVDE基因呈非特异性表达,在叶绿素含量高的组织中表达量较高。同时,该基因在弱光和强光处理的24小时昼夜周期中都存在着相似的双峰表达模式,并部分受低温的抑制。Southern杂交结果表明,LeVDE基因在番茄基因组中是单拷贝的。3.将获得的LeVDE基因与含有35S启动子的pBI121载体重组,分别构建了正义和反义表达载体,利用农杆菌介导的叶盘法转化番茄,用PCR及Northern杂交的方法对带卡那抗性的转基因番茄植株进一步检测,结果证明成功地获得了转正义和反义基因的番茄植株。与野生型植株相比,过量表达LeVDE基因的番茄叶片中A和Z的含量增加,而V的含量减少,脱环氧化状态较野生型高。LeVDE基因表达受到抑制的番茄植株中V量积累,而A和Z含量非常低,脱环氧化状态保持较低的水平4.构建了原核表达载体pET-LeVDE,并在大肠杆菌BL21中表达融合蛋白,将强诱导带切下,溶于PBS获得抗原,免疫小白鼠,其抗血清效价为1: 500。Western杂交表明,转正义植株中LeVDE基因已在蛋白水平过量表达。另外,分别对低温弱光和强光处理的野生型番茄进行了Western杂交分析。结果表明,LeVDE在蛋白水平的表达始终处在稳定的水平,基本不受光强和温度的影响。5.在低温弱光(4℃,100μmol m^-2 s^-1)和强光(1200μmol m^-2 s^-1)胁迫条件下,野生型和转正义基因株系T1-7和T1-10株系的NPQ及叶黄素循环脱环氧化状态（A+Z）/（V+A+Z）都增加,但野生型的增加更为明显。在低温弱光及强光胁迫条件下,依赖叶黄素循环的NPQ能够耗散过剩激发能,而转基因株系比野生型耗散能力加强。强光处理过程中,野生型比转正义基因株系的Fv/Fm降低更明显,且转基因株系的Fv/Fm恢复较快,野生型的Fv/Fm恢复较慢。与在强光下类似,T1-7和T1-10株系的Fv/Fm在低温胁迫过程中降低的程度比野生型小,而恢复较慢。这表明LeVDE的过量表达减轻了PSII的光抑制程度,与转基因番茄相比,野生型的PSII反应中心受伤害较严重。强光胁迫12 h,野生型和转基因番茄的净光合速率降低非常显著,但野生型下降程度明显大于转基因株系。这表明由于LeVDE的过量表达增强了低温弱光和强光胁迫下PSII反应中心的稳定性,使光合机构所受到的伤害减轻。在低温弱光处理过程中转正义基因植株与野生型的氧化态P700都降低,且野生型降低幅度大于转正义基因植株。经过12 h的处理,T1-7,T1-10和野生型的O₂^-|-含量分别增加了52.3 %,59.8%和81.1 %, H2O2含量分别增加了40.1%,42.3 %和61.3 %。野生型中O2和H2O2含量增加的程度明显高于转基因株系。另外,在低温处理过程中,转正义基因植株和野生型的MDA含量都增加,但转正义基因植株的MDA含量增加的较少。6.抑制LeVDE的表达使得过剩光能胁迫下的NPQ减少。在转反义基因植株中V大量积累而Z和A的含量非常低。在强光和低温弱光胁迫后,转反义基因植株脱环氧化状态始终保持较低的水平。在强光和低温胁迫过程中野生型和转反义基因植株的NPQ都上升,且胁迫12 h后转反义基因植株的NPQ低于野生型。这表明抑制Z的生成使得依赖于叶黄素循环的能量耗散减弱。7.在强光胁迫下野生型和转反义基因植株的Fv/Fm都降低,但是转反义基因植株降低幅度大于野生型。低温弱光胁迫下野生型与转反义基因植株的Fv/Fm均下降,转反义基因植株下降更为明显,低温处理后,（-）2,（-）9和野生型植株的Fv/Fm分别下降了20.1%,17.9%,12.1%。在低温弱光处理过程中转反义基因植株与野生型的氧化态P700都降低,转反义基因株系植株P700的下降更为显著。低温处理后,（-）2,（-）9和野生型植株中O₂^-|-含量分别增加了79.1%,76.4%和62.7%。经低温弱光处理后,野生型和转反义基因植株中MDA含量都增加,转反义基因植株中MDA含量增加更为显著,低温胁迫12小时后野生型,（-）2和（-）9中MDA含量分别增加到138.5 %,159.1 %和161.7%。综上所述,LeVDE的表达受温度、光强和昼夜节律的影响。过量表达该基因减轻了PSII和PSI对光抑制的敏感性。抑制该基因的表达加重了强光和低温弱光胁迫下的光抑制。更多还原

【Abstract】 Photosynthesis is important for plant growth and survival. On a daily as well as seasonal basis most plants receive more sunlight than they could actually use for photosynthesis. If the excessive light energy which has been absorbed by photosynthetic apparatus can not be dissipated rapidly, it may reduce the photosynthetic efficiency and result in photoinhibition, even photooxidative damage to the photosynthetic reaction center. The environmental stresses such as low and high temperatures, drought and salinity enhance photoinhibition. Photoinhibition occurs in the field in plants exposed to conditions of high light. The combination of low temperature with irradiance also has the potential to induce chronic photoinhibition of PSII. This is partly because lower temperature generally reduces the rates of biological reactions particularly carbon dioxide reduction and photorespiration, and therefore limits the sinks for the absorbed excitation energy. Among the short-term mechanisms, the xanthophyll cycle has been conceived to play a key role in photoprotection. The xanthophyll cycle exists in the thylakoid membranes of all higher plants, ferns, mosses and algae. Its pigments have been demonstrated to be associated with all light-harvesting components, including LHCI. It comprises intercoversions of three carotenoid pigments, violaxanthin, antheraxanthin and zeaxanthin, which catalysed by two enzyme, violaxanthin de-epoxidase （VDE, EC:1.10.99.3） and zeaxanthin epoxidase （ZE, EC:1.14.13.90）. When light energy is excessive, violaxanthin is catalysed to zeaxanthin via antheraxanthin catalysed by violaxanthin de-epoxidase （VDE）. However, when light stress disappeared, epoxidation of zeaxanthin to antheraxanthin and violaxanthin is catalysed by another enzyme, zeaxanthin epoxidase （ZE）. The xanthophyll cycle and the associated non-photochemical quenching （NPQ） exert their protective role by thermal dissipation of excess light energy. Zeaxanthin is thought to be the main photoprotector in chloroplasts. Many studies show zeaxanthin can directly quench the 1Chl* state and some reactive oxygen species. So the photosynthetic apparatus can be protected from photooxidation. However, there are other researches which support that the xanthophyll pigments protect the thylakoid membrane by an indirect process. They can allosterically regulate the quenching process inside the LHCII. Zeaxanthin is a allosteric activator of this process and violaxanthin is a inhibitor.In this study, we isolated and characterized violaxanthin de-epoxidase gene from tomato using homological clone. The functional analysis showed that expression of the gene was induced by diurnal rhythm, light intensity and temperature. It is interesting that overexpression of LeVDE increased the level of de-epoxidation and thermal dissipation capacity. It is suggested that the overexpression of LeVDE could alleviate photoinhibition of PSII and PSI under high light and chilling stress. However, the suppression of LeVDE enhanced the photoinhibition of tomato plants under low temperature. The main results are as follows:1. Two degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from tomato leaves according to the homologous sequences from other plants. The middle fragment of interested cDNA was obtained by RT-PCR. The 5’and 3’fragment of the cDNA was isolated by 5’and 3’RACE. The clone was named LeVDE （Acession numeber: FJ648424）, contains 1670bp nucleotides with an open reading frame （ORF） of 1437bp, comprising 478 amino acid residues with the predicted molecular mass of 53 kDa. The deduced amino acid sequence showed high identities with VDE from Arabidopsis thaliana, Nicotiana tabacum,Oryza sativa, Spinacia oleracea, Lactuca sativa. Amino acid sequence alignment revealed that the plant members contained the previously defined regions. The cysteine-rich domain in block I, the lipocalin signature domain in block II and the C-terminal glutamate-rich domain in block III, all of which have been shown to form a catalytically essential site in VDE, are absolutely conserved. The cysteine-rich domain in block I was suggested to be the active site of VDE. The lipocalin signature domain could be a possible binding site for violaxanthin and MGDG which was the substrate for VDE. At the C-terminal region, the glutamate-rich domain in block III was thought to be involved in binding of VDE to the thylakoid membrane.2. Northern blot analysis showed that LeVDE constitutively expressed in roots, leaves, fruits, stems and calyxes of wild type （WT） plants. The transcripts were high in the tissues abundant of chlorophyll. LeVDE transcript level was similar for extracts from plants treated with high light and low light condition, and exhibited a diurnal rhythm expression pattern. Furthermore, the LeVDE transcript level was inhibited by both light intensity and low temperature. Southern blot analysis showed that LeVDE gene was a single copy in tomato genome.3. The full-length LeVDE cDNA was subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form sense and antisense constructs. The constructs were first introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method and verified by PCR and northern blot. It was indicated that the LeVDE gene had been recombined into tomato genome and both sense and antisense transgenic tomato plants were obtained. A lower content of V and higher content of A and Z were detected in sense transgenic plants compared with WT plants. The de-epoxidation ratio of xanthophyll cycle pigments （A+Z）/（V+A+Z） of sense transgenic plants was higher than that of WT. Suppression of LeVDE in tomato decreased the content of Z and A. But V accumulated in antisense transgenic plants compared to that of WT plants.4. A recombinant of prokaryotic expression vector pET-LeVDE was constructed and transformed to E.Coli BL21 to express. The strong induced fusion protein bands were collected into PBS solution and used to immunize white mice to obtain antiserum. The value of antibody reaches 1: 500. Western blot revealed the presence of the strong positive protein signals corresponding to LeVDE in sense transgenic plants. Western blot analysis was carried out over the diurnal cycle. Unexpectedly, the LeVDE protein level remained constant in leaves. Moreover, there was no difference between leaves exposed to high light and chilling stress.5. Although both NPQ and （A+Z）/（V+A+Z） of WT and sense transgenic plants increased markedly under chilling stress in the low irradiance (4℃, 100μmol m^-2 s^-1) and high light stress (1200μmol m^-2 s^-1), the increase of NPQ and （A+Z）/（V+A+Z） was more obvious in sense transgenic plants than in WT. Fv/Fm decreased in both WT and transgenic plants under high light stress, but the decrease of Fv/Fm in transgenic plants was more significant than that in WT. At the end of high light stress, Fv/Fm in WT, T1-10 and T1-7 lines decreased by 32.1%, 13.4 % and 11.2 %, respectively. When tomato plants were transferred to suitable condition of 25℃and a PFD of 100μmol m^-2 s^-1, Fv/Fm recovered in both WT and transgenic plants. However, the recovery of Fv/Fm in the transgenic plants was faster. Fv/Fm also decreased significantly in transgenic plants during chilling stress （4℃） relative to that in WT plants. At the end of chilling stress, Fv/Fm in the the WT and transgenic plants of T1-10 and T1-7 decreased about 12.1 %, 8.3 % and 7.1 %, respectively. The recovery of Fv/Fm in transgenic plants was also faster than that in WT. Under high light stress for 12 h, the Pn of WT and transgenic tomato plants obviously decreased. This decrease was more significant in WT than in transgenic plants.The oxidizable P700 decreased significantly both in WT and sense transgenic plants under chilling stress in the low irradiance, and the decrease was more significant in WT than in transgenic plants. At the end of chilling stress, O₂^-|- content in leaves of T1-7, T1-10 and WT plants increased for about 52.3 %,59.8% and 81.1 % of initial values, respectively, and H2O2 content of T1-7, T1-10 and WT increased for about 40.1%,42.3 % and 61.3 % of initial values, respectively. The level of peroxide of membrane lipids was enlarged and the MDA contents of T1-7, T1-10 and WT plants increased to 115.1%, 121.7% and 140.5%, respectively.7. Antisense-mediated suppression of LeVDE affected NPQ under light stress. V accumulated in antisense transgenic tomato plants, but Z and A content was very low. The de-epoxidation ratio of xanthophyll cycle pigments （A+Z）/（V+A+Z） in antisense transgenic plants sustained a very low level before and after high light and low temperature stress. Although both NPQ of WT and antisense transgenic plants increased markedly under chilling stress in the low irradiance and high light stress, the increase was more significant in WT than in transgenic plants. It was suggested that the suppression of LeVDE decreased the energy dissipation in PSII. 8. Fv/Fm decreased in both WT and antisense transgenic plants under chilling stress in the low irradiance, and WT showed the greater decrease. At the end of low temperature stress for 12 h, Fv/Fm in （-）2, （-）9 and WT decreased about 20.1%,17.9%,12.1%, respectively. The oxidizable P700 decreased significantly both in WT and antisense transgenic plants under chilling stress in the low irradiance, and the decrease of P700 was more obvious in WT than in antisense transgenic plants. O₂^-|- contents increased more markedly in antisense transgenic plants than in WT plants. At the end of chilling stress, O^-|- content in （-）2, （-）9 and WT plant leaves increased for about 79.1%,76.4% and 62.7%, respectively. The MDA contents of WT and antisense transgenic tomato plants increased under chilling stress in the low irradiance. The increase was more obvious in antisense transgenic plants than in the wild type. After 12 h stress, the MDA contents in WT, antisense transgenic lines （-）2 and （-）9 increased to about 138.5 %, 159.8 % and 161.7%, respectively.In conclusion, we demonstrated that the expression of LeVDE was induced by light, temperature and diurnal rhythm and it was partially inhibited by chilling temperature. Overexpression of LeVDE increased the level of de-epoxidation and the thermal dissipation capacity under high light and chilling stress. The sensitivity of PSII and PSI photoinhibition to high light and chilling stress was therefore alleviated. The suppression of LeVDE affected NPQ under light stress and enchanced the photoinhibition of tomato plants under low temperature.更多还原