【作者】 董玉梅；

【导师】 于贤昌；

【作者基本信息】 山东农业大学 ， 蔬菜学， 2011， 博士

【摘要】 抗坏血酸(Ascorbic acid, AsA)即维生素C (Vitamin C, Vc),是植物和大多数动物体内合成的一类含量丰富的抗氧化剂和多种酶的辅因子,在生物体的生长发育过程中具有重要的代谢功能和调节作用。人类自身无法合成而又必需AsA,只能从膳食中获得,因此,提高膳食中AsA含量对人类维持正常的生命活动至关重要。植物AsA生物合成主要是通过半乳糖途径,L-半乳糖酸-1,4-内酯脱氢酶(GLDH)是催化AsA合成最后一步的关键酶。本研究从马铃薯叶片中克隆到编码GLDH的基因StGLDH,构建了GLDH的正、反义表达载体,转入马铃薯中,获得转正义、反义GLDH基因的马铃薯株系,研究了该基因在马铃薯叶片和块茎中的表达水平、抗坏血酸含量及相关酶的活性；并研究了NaCl胁迫条件下,转基因试管苗和野生试管苗GLDH表达、抗坏血酸含量及抗坏血酸代谢系统部分酶活性的变化,初步明确了GLDH基因在马铃薯AsA代谢中的功能及超表达效果。主要结果如下：1.从马铃薯叶片中成功获得了L-半乳糖酸-1,4-内酯脱氢酶(L-galactono-1,4-lactone dehydrogenase, GLDH, EC1.3.2.3)基因,命名为StGLDH, GeneBank注册号为FJ755844。该基因全长2563 bp,其cDNA包含完整的开放阅读框(551-2323),编码590个氨基酸,推导的氨基酸序列分子量为67119 Da,等电点为8.5。在其5’端具有一段推定的线粒体靶信号肽(putative mitochondrial),其氨基酸序列的82FR/YA85间存在该信号肽的切割位点(cleavage site)。StGLDH具有3个跨膜区(transmembrane region),含有一个典型的黄素腺嘌呤二核苷酸结合域(FAD-binding region)和一个ALO结构域。StGLDH编码的氨基酸序列与番茄、辣椒、烟草同源性在90.6%-95.9%之间。2.将获得的StGLDH基因与含有35S启动子的pBI 121载体重组,构建真核表达载体,利用农杆菌介导法转化马铃薯。通过PCR、高浓度卡那霉素、荧光定量PCR等方法对带卡那霉素抗性的转基因植株进一步检测,证明获得了4个转正义、2个转反义GLDH的马铃薯株系。3. GLDH的反义表达抑制了马铃薯株系anti-GLDH 13的生长和产量,使植株表观发生了较大改变,表现出植株矮小,节间较短,叶片小、厚,表皮多毛,块茎小,产量低,出芽早,抗性差等性状。而GLDH的正义表达对植株表观影响较小,主要表现在使叶色变绿,部分株系(GLDH 155、GLDH 262)叶片偏圆,前期长势较快等方面。4.两个转正义GLDH的株系GLDH 516、GLDH 518 GLDH超表达效果显著,酶活性提高,AsA, DHA, AsA+DHA含量均相应增加。GLDH 516、GLDH 518两株系叶片AsA含量比野生型株系分别提高了47.16%、13.1%,块茎中AsA含量分别提高了10.3%、6.8%；转反义基因株系anti-GLDH 13表现相反,叶片和块茎中AsA含量分别比野生型株系减少了28.82%、10.3%。转基因株系叶片中AsA水平变化幅度高于块茎中AsA的变化幅度。5.用L-半乳糖内酯饲喂马铃薯叶片,所有株系GLDH活性与AsA、DHA、AsA+DHA含量都大幅度提高。其中DHA、AsA+DHA含量,比饲喂前提高了约2倍,株系间AsA含量饲喂后差异减小。饲喂后,GLDH 516、GLDH 518株系AsA含量仍保持最高,而anti-GLDH 13含量最低。6.0～250 mmol·L-1NaCl处理5天时,GLDH mRNA表达水平显著提高,随处理时间延长(15天),GLDH表达水平显著降低。AsA含量、GLDH活性与GLDH表达变化趋势一致,但远不如GLDH变化幅度大。7. GLDH的正义表达提高了GLDH 516、GLDH 518两个株系的耐盐性,而反义表达降低了anti-GLDH 13株系的耐盐性,各株系耐盐性与正常条件下试管苗AsA含量相关。250 mmol·L-1 NaCl胁迫5天后,所有株系APX、MDHAR、DHAR活性升高,AO活性降低,MDA含量升高,AsA氧化加速。耐盐株系胁迫后AsA含量的提高是AsA合成、再生加速共同作用的结果,而不耐盐株系AsA含量的减少是因为AsA合成的减少和氧化的加速,而并非其再生限制造成的。不耐盐株系膜脂过氧化较严重,anti-GLDH13出现明显胁迫症状。因此,GLDH表达水平和GLDH活性对马铃薯植株体内的AsA水平及植株的耐盐性具有重要的调节作用。更多还原

【Abstract】 L-Ascorbic acid (AsA), also known as vitamin C, is one of the most abundant antioxidants and cofactor for several enzymes. It has been reported that AsA is necessary for plant growth and development, and it has multiple roles in metabolism and in plant responses to abiotic stresses and pathogens. Moreover, Plant-derived ascorbate also provides the major source of vitamin C for the human, because humans are incapable of synthesizing AsA and must secure it by means of dietary uptake to be healthy. Increasing the plant vitamin C content is important for peoples’health.It had been well confirmed that L-galactose or Smirnoff-Wheeler pathway was the major biosynthetic pathway of ascorbate in plants, and GLDH was the key enzyme which catalyses the last step of this pathway, in the control of AA content under optimal and stress conditions. In the previous study,we isolated a full-length cDNA clone encoding L-galactono-1,4-lactone dehydrogenase (GLDH) from the leaf of potato (Solanum tuberosum L.) cv. Favorita and subcloned into the binary vector pBI121 to construct sense and antisense recombinant plant expression vectors respectively. The recombinant were introduced into potato, and 4 sense transgenic potato lines and 2 anti-sense ones were obtained. Both the StGLDH expression level, StGLDH activity and AsA content in leaf and tuber of potato were investigated. Because of the postulated role of AsA as a stress-related Antioxidants, the expression of NaCl stress-responsive were examined in transformed and nontransformed potato plantlets grown in vitro. From which we get some information about the founction of GLDH in AsA metabolism system in Potato, as well as the overexpression of StGLDH. The main results were as follows:1. A full-length cDNA clone encoding L-galactono-1,4-lactone dehydrogenase (GLDH), named as StGLDH (GenBank:FJ755844), was isolated from the leaf of potato (Solanum tuberosum L.) cv. Favorita StGLDH transcript is 2563 nt long with an open reading frame of 1773 bp and encodes a polypeptide of 590 amino acids. The StGLDH amino acid sequence predicted a molecular weight of 67 119 Da and a pI of 8.5, which contained a putative mitochondrial-targeting domain in its first 83 amino acids and a cleavage site (FR/YA) similar to other known GLDHs. Three possible transmembrane regions were predicted between residues 50 and 68,247 and 269,479 and 503. Analysis of StGLDH amino acid sequence identified a putative FAD binding domain between residues 105 and 240, wherein was located a region (134VGSGLSP140) common to all GLDHs characterized to date. Sequence analysis showed that deduced StGLDH protein was highly homologous to other GLDH proteins from different species, especially with Solanum lycopersicum, Capsicum annuum, Nicotiana tabacum, about 90.6%-95.9% identity in amino acid sequence.2. The full-length cDNA, as well as partial cDNA of GLDH, were subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form a sense construct. Then the constructs were introduced into potato via Agrobacterium fumeracens-mediated procedure. PCR and Real time PCR results indicated that the StGLDH had been recombined into potato genome,4 sense transgenic potato lines and 2 anti-sense ones were obtained.3. Among the six transgenic potato lines, anti-GLDH 13 lines exhibited an obviously phenotypic difference from others. Its aerial parts was reduced and displayed very severe growth defects such as stunted plants with deformed leaves. Detailed characterization revealed that the plant growth rate, plant height, node height, leaf size, stem diameter and fruit weight were all reduced, and the epidermal hair density increased compared to the wild type lines, and the To tuber germinate more earlier than the others. Accordingly, the ability of anti-GLDH 13 line against stress was also decreased.The sense transgenic lines showed a slightly phenotypic difference from wild type lines, the node height and leaf thickness were increased compared to the wild type lines. Sense transgenic lines turn slightly round in leaf shape and To Tuber. Furthermore, the leaf color of sense transgenic lines was greener than that of the wild type and the anti-sense ones. But there was no obviously differerce in plant growth rate, except GLDH 155 lines showed a rapid growth in earlier period.4. Two sense transgenic lines GLDH 516, GLDH 518 overexpress StGLDH. Overexpression of StGLDH resulted in enhanced GLDH activity. AsA content, DHA content, AsA+DHA content increased in both leaves and tubers correspondly. AsA content in leaves of GLDH 516 and GLDH 518 lines increased 47.16% and 13.1% respectively compared to wild type line, and AsA content in tubers increased 10.3% and 6.8% respectively. An adverse result occurred to the anti-sense transgenic lines’anti-GLDH 13’, which had a decrease in AsA content in both leaves (28.82%) and tubers (10.3%). AsA content in leaves showed large variations in amplitude than that of in tubers.5. Feeding potato leaves with AsA precursor was very effective at increasing AsA. Fed with L-GalL, the GLDH activity, AsA content, DHA content and AsA+DHA content in either transgenic or wild potato leaves were all significantly higher than those of treated with distilled water. L-GalL feeding leaves were approximately 2-fold DHA content and AsA+DHA content more than their corresponding controls.The difference in AsA content among 7 potato lines decreased after L-GalL feeding. AsA content in GLDH 516 and GLDH 518 lines were still higher than that of others, while anti-GLDH 13 lines had the lowlest AsA content in both leaves and tubers as yet.6. NaCl-stress could regulate GLDH mRNA expression in vitro potato plantlets. Under 0～250 mmol·L-1NaCl concentration and within 5 days, NaCl stress could significantly up regulated the expression of GLDH mRNA in the planetlets grown in vitro, then down regulate it continuously with the prolongation of time. The AsA level was positively correlated with the mRNA transcript expression and also GLDH activity. But there existed great difference of variation amplitude between GLDH mRNA expression level and AsA content, as well as GLDH activity.7. Sense expression of StGLDH in GLDH 516, GLDH 518 lines conferred better tolerance to NaCl stresses, while anti expression of StGLDH in GLDH 13 had observed adverse effects. The NaCl tolerance of potato plantlets was associated with AsA level before stress. After 5 days treatment, APX activity, MDHAR activity, DHAR activity and MDA content increased while the AO activity decreased. The increase of AsA level in better tolerance potato planets, such as GLDH 516, GLDH 518 lines, was resulted from the cooperation of rapid AsA synthesis and regeneration. The decrease of AsA level in the salt sensitive lines, such as anti-GLDH 13 lines, was resulted from the cooperation of decreased AsA synthesise and rapid oxidation, not for the reason of AsA regeneration. Anti-GLDH 13 line showed evident injured symptom due to high level of membrane lipid peroxidation initiated by NaCl stress.Consequently, the level of AsA accumulation in potato plantlets was directly correlated with their ability to withstand NaCl stresses. These results further demonstrated that the expression of GLDH and GLDH activity had played an important role on regulating AsA level as well as tolerance of potato plantlets to NaCl stresses.更多还原