【作者】 隋娜；

【导师】 孟庆伟；

【作者基本信息】 山东农业大学 ， 植物学， 2007， 博士

【摘要】 低温是限制冷敏感植物产量和地理分布的重要因素。生物膜是低温伤害的初始位点。植物的抗冷性与膜脂中脂肪酸的不饱和程度密切相关。由于叶绿体中磷脂酰甘油(PG)的sn-2位主要被饱和脂肪酸或反式不饱和脂肪酸所占据,因而PG在sn-1位的顺式不饱和脂肪酸水平决定了植物的抗冷性。决定PG中sn-1位顺式不饱和脂肪酸含量的是叶绿体甘油-3-磷酸酰基转移酶(GPAT: EC2.3.1.15)对底物的选择性。GPAT是PG生物合成过程中的第一个酰基脂化酶,它将脂肪酰转移到3-磷酸甘油的sn-1位上合成1-酰基-Sn-甘油-3-磷酸(溶血磷脂酸)。来源于抗冷性不同的植物的GPAT对底物酰基具有不同的选择性。一般来说,抗冷植物中GPAT优先选择C18:1-ACP作为底物,因此在这些植物中PG的sn-1位上就含有较高比例的18:1脂肪酸,这些脂肪酸可以在酰基脂肪酸去饱和酶的作用下进一步去饱和化成为顺式多聚不饱和脂肪酸;然而在冷敏感植物中,GPAT很难区分C18:1-ACP和C16:0-ACP,由于16:0脂肪酸不能被进一步去饱和形成顺式不饱和脂肪酸,结果这些植物中PG的sn-1位顺式不饱和脂肪酸含量较低,从而表现为冷敏感。本研究从番茄叶片中分离到叶绿体甘油-3-磷酸酰基转移酶基因,并对该基因的表达和功能进行了分析。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甘油-3-磷酸酰基转移酶基因的中间片段,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LeGPAT (DQ459433)。该基因全长为1770 bp,ORF为1314 bp,编码437个氨基酸,分子量约为48 kDa。同源序列比较发现,番茄甘油-3-磷酸酰基转移酶基因的序列与甜椒、红花、豌豆、菠菜的甘油-3-磷酸酰基转移酶基因的序列同源性较高。结构同源性分析表明LeGPAT有四个序列保守的结构域,block I的组氨酸和天冬氨酸残基,block III的甘氨酸残基和block IV的脯氨酸残基都是绝对保守的,它们组成了一个重要的催化位点。2.将p35S-LeGPAT-GFP融合蛋白在豇豆原生质体中瞬时表达。通过Confocal观察到GFP激发的绿色荧光和叶绿素的红色自发荧光完全重合,说明LeGPAT的基因产物定位于叶绿体。3. Northern杂交分析显示,LeGPAT在不同器官中呈非特异性表达,在叶绿素含量高的组织中表达量较高。同时,该基因在4-40℃的温度范围内均有表达,且受低温诱导,高温胁迫抑制其表达。4.将获得的LeGPAT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和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,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LeGPAT的番茄叶片类囊体膜PG中18:2和18:3含量明显增加,脂肪酸不饱和度升高,从而导致低温胁迫下转基因植株类囊体膜的流动性高于野生型植株。LeGPAT表达发生沉默的番茄植株中PG的18:2和18:3含量下降,而16:0,16:1和18:0含量增加,脂肪酸饱和度升高。5.构建了原核表达载体pET-LeGPAT,并在大肠杆菌BL21中表达融合蛋白,免疫小白鼠,制备抗体,其抗血清效价为1:500。Western杂交表明,转正义植株中LeGPAT已在蛋白水平过量表达。6.将野生型和转正义基因番茄的酶提取液,以及原核表达后纯化的蛋白分别与[1-14C]18:1-CoA,[1-14C]16:0-CoA,甘油-3-磷酸,HEPES-NaOH缓冲液和BSA反应测定甘油-3-磷酸酰基转移酶的选择性和该酶的活性。结果表明,尽管番茄是冷敏感植物,但甘油-3-磷酸酰基转移酶对18:1的选择性明显高于16:0,且转正义基因番茄的酶含量和总活性高于野生型。7.在低温弱光(4℃,100μmol m-2 s-1)胁迫条件下,野生型和转正义基因株系T1-5和T1-19的光合速率(Pn)都降低,但野生型的降低较明显,并且T1-5和T1-19的Pn可以在12 h内恢复,而野生型的Pn在12 h时仅恢复了73.2%,在24 h时恢复了86.4%。在低温胁迫过程中,T1-5和T1-19的光系统II最大光化学效率(Fv/Fm)降低的程度比野生型小,而且恢复较快,恢复8 h时T1-5和T1-19的Fv/Fm完全恢复,但此时野生型的Fv/Fm只恢复了95.2%。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,且区别不大,而转正义基因植株的氧化态P700恢复较快。经过24 h的恢复,T1-5的氧化态P700恢复了98.5%,T1-19的恢复了99.4%,而野生型的只恢复了85.3%;经过12 h的低温胁迫,T1-5和T1-19的相对电导率分别增加到21.3%和19.3%,而野生型的增加到24.4%。转正义基因植株和野生型的NPQ及(A+Z)/(V+A+Z)都增加,但转正义基因植株的NPQ和(A+Z)/(V+A+Z)增加的较多。野生型番茄的叶绿体SOD和APX活性在胁迫的最初6 h升高,随后降低,而转正义基因植株的叶绿体SOD和APX活性在处理9 h后才轻微的降低。低温处理6 h后,转正义基因植株的SOD和APX活性高于野生型。野生型番茄的O2和H2O2的含量在低温处理6 h后开始增加,而转正基因植株的O2和H2O2含量在处理9 h后才增加,而且野生型的O2和H2O2含量增加的程度明显高于转基因植株。胁迫结束时,T1-5,T1-19和野生型的O2含量分别增加了15.7%,14.8%和63.0%,而H2O2含量分别增加了26.0%,15.8%和77.6%。8.反义介导的LeGPAT的缺失能够影响番茄的育性。从形态上看,转反义基因株系(-)12的花粉粒大部败育。(-)12株系从小孢子母细胞时期绒毡层开始败育,在单核小孢子液泡期败育的花粉粒明显高于野生型。反义介导的LeGPAT的缺失能够减少内质网的合成,改变油脂的大小。当花粉粒在培养基上萌发60 min后,野生型的花粉有65%的萌发,而转反义基因株系(-)12的花粉仅有8%的萌发。当花粉粒在培养基上萌发120 min后,野生型花粉的萌发率几乎为100%,而(-)12株系的仅为20%。转反义基因株系(-)12中53.8%(14/26)的花败育,而野生型植株中仅有7.8%(4/51)的花败育。转反义基因植株的种子败育,失去再生能力,而野生型的种子发育正常。9.在高温弱光胁迫(45℃,100μmol m-2 s-1)下处理6 h和12 h,野生型和转反义基因番茄的放氧速率都降低,但野生型降低的比较明显。45℃处理6 h后,野生型,(-)7和(-)12株系的放氧速率分别降低到初始值的30.5%,47.4%和50.9%;12 h时分别降低到7.2%,18.5%和19.4%。高温胁迫过程中,野生型和转反义基因番茄的Fv/Fm都降低,但野生型降低的比较明显。45℃下处理12 h,野生型,(-)7和(-)12株系的Fv/Fm分别降低了42.5%,30.9%和26.8%。上述结果表明,LeGPAT的表达受低温诱导,被高温胁迫抑制。番茄叶绿体甘油-3-磷酸酰基转移酶对18:1的选择性明显高于16:0,且转正义基因番茄中该酶的含量和总活性高于野生型番茄。过量表达该基因可提高番茄植株的耐冷性,而抑制该基因表达可增强番茄植株的耐热性,影响番茄的育性。更多还原

【Abstract】 Low temperature is the major factor limiting the productivity and geographical distribution of chilling-sensitive plant species. It was suggested that membrane was the primary position to be damaged under chilling stress. The tolerance of plants to chilling stress was closely connected with the fatty acid unsaturation of plant membrane lipids. Sn-2 position is occupied mainly by saturated and trans-unsaturated fatty acids, so the content of cis-unsaturated fatty acids at the sn-1 position of Phosphatidylglycerol (PG) determines chilling resistance. The dominant factor that determines the level of cis-unsaturated fatty acids in PG is the substrate selectivity of glycerol-3-phosphate acyltransferase(GPAT: EC2.3.1.15)in chloroplasts, which catalyzes the first step of glycerolipid biosynthesis by transferring the acyl group of acyl-(acyl-carrier protein) (ACP) to the sn-1 position of glycerol-3-phosphate to yield 1-acylglycerol-3-phosphate (lysophosphatidate; LPA). GPAT from chilling-resistant plants prefers oleoyl-ACP (18:1-ACP) to palmitoyl-ACP (16:0-ACP) as a substrate. Thus, a large proportion of oleic acid (18:1) occurs at the sn-1 position of PG in chilling-resistant plants. Under chilling stress oleic acid (18:1) of sn-1 position desaturates further into cis-polyunsaturated fatty acids of linoleic acid (18:2) and linolenic acid (18:3) by acyl-fatty acid desaturase in chloroplast membranes. The enzyme from chilling-sensitive plants hardly distinguishes 18:1-ACP from 16:0-ACP. Fatty acid of sn-1 position remains unchanged, resulting in a low level of cis-unsaturated fatty acids at the sn-1 position of PG, which increase sensitivity of plants to chilling stress. In this study, we isolated and characterized chloroplast glycerol-3-phosphate acyltransferase gene from tomato. 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, which named LeGPAT (Acession Numeber:DQ459433), contains 1770 bp nucleotides with an open reading frame (ORF) of 1314 bp comprising 437 amino acid residues with the predicted molecular mass of 48 kDa. The deduced amino acid sequence showed high identities with GPAT from Capsicum annuum, Carthamus tinctorius, Pisum sativum, Spinacia oleracea. Amino acid sequence alignment revealed that the plant members contained four acyltransferase domains. The His and Asp residues in block I, the Gly residue in block III, and the Pro residue in block IV, all of which have shown to form a catalytically important site in this family of acyltransferases, are absolutely conserved.2. p35S-LeGPAT-GFP fusion protein was constructed and transiently expressed in cowpea protoplasts derived from leaf tissue. It was observed with confocal microscopy that the green fluorescence was clearly associated with chloroplasts and colocalized with the red autofluorescence of chloroplasts, demonstrating that LeGPAT subcellular localization on chloroplast.3. Northern hybridization shows that LeGPAT constitutively expressed in stems, petals, fruits and leaves of wild type plants. The transcripts were high in the tissues abundant of chlorophyll. LeGPAT expressed extensively from 4 to 40℃in leaves and the expression of LeGPAT was obviously induced by low temperature and inhibited by high temperature.4. The full-length LeGPAT 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 hybridization. It was indicated that the LeGPAT had been recombined into tomato genome and both sense and antisense transgenic tomato plants were obtained. A higher content of 18:2 and 18:3 in PG was detected in sense transgenic plants compared with the wild type (WT) tomato plants. The fluidity of thylakoid membrane of sense transgenic plants was higher than WT under low temperature. Depletion of LeGPAT in tomato decreased the content of unsaturated fatty acids (18:2 and 18:3) in PG. But the contents of 16:0, 16:1 (△3- trans, sometimes referred to as high-melting-point fatty acids) and 18:0 increased in antisense transgenic plants compared to that of WT plants.5. A recombinant of prokaryotic expression vector pET-LeGPAT was constructed and transformed to E.Coli. BL21. 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 hybridization revealed the presence of the strong positive protein signals corresponding to LeGPAT in sense transgenic plants.6. Substrate selectivity and enzyme activity of LeGPAT were measured by using purified enzyme fractions of wild type and sense transgenic tomato leaves and protein from the E. coli. cells expressing LeGPAT. Each reaction mixture contained [1-14C]18:1-CoA and [1-14C]16:0-CoA, glycerol-3-phosphate, HEPES-NaOH buffer and BSA. Results showed that LeGPAT exhibited 18:1-selectivity over 16:0 and transgenic plants had higher selectivity (18:1) than wild type plants.7. Although Pn of WT and sense transgenic plants decreased markedly under chilling stress in the low irradiance (4℃, 100μmol m-2 s-1), the decrease of Pn was more obvious in WT than in sense transgenic plants. After tomato plants were transferred to a condition of 25℃and a PFD of 600μmol m-2 s-1, Pn of T1-5 and T1-19 recovered completely in 12 h, whereas Pn of wild type plants recovered only 73.2% in 12 h and 86.4% in 24 h. Fv/Fm decreased obviously during chilling stress (4℃) and recovered slowly in wild type plants relative to in sense transgenic plants. Fv/Fm of T1-5 and T1-19 recovered completely in 8 h, while Fv/Fm of WT only recovered 95.2%. The oxidizable P700 decreased significantly both in WT and sense transgenic plants under chilling stress in the low irradiance and there were no evident differences. When tomato plants were transferred to a suitable condition of 25℃and a PFD of 100μmol m-2 s-1, the oxidizable P700 of sense transgenic plants recovered more quickly than WT. After 24 h recovery, the oxidizable P700 could recover 98.5%, 99.4% and 85.3% in T1-5, T1-19 and WT, respectively. After treatment at 4℃for 12 h, the relative electrolytic leakage of T1-5 and T1-19 increased to 21.3% and 19.3%, whereas 24.4% in WT. Both NPQ and the de-epoxidized ratio of the xanthophylls cycle, (A+Z)/(V+A+Z), increased in WT as well as in sense transgenic plants at chilling temperature. NPQ and (A+Z)/(V+A+Z) of sense transgenic plants markedly increased relative to that of WT during chilling stress. The chloroplastic SOD and APX activities of WT plants increased during first 6 h of chilling stress and then decreased, whereas the SOD and APX activities of sense transgenic plants increased during first 9 h of chilling stress and then slightly decreased. After 6 h chilling stress, Chloroplast SOD and APX activities of transgenic plants were higher than that of WT. The contents of O 2 and H2O2 in WT increased after 6 h chilling stress, while the contents of O 2 and H2O2 of sense transgenic plants increased only after 9 h chilling stress. Both O 2 and H2O2 contents increased more markedly in WT plants than in sense transgenic plants. At the end of chilling stress, O 2 content in leaves of T1-5, T1-19 and WT plants increased for about 15.7%, 14.8% and 63.0% of initial values, respectively, and H2O2 content of T1-5, T1-19 and WT increased for about 26.0%, 15.8% and 77.6% of initial values, respectively.8. Antisense-mediated depletion of LeGPAT severely affected tomato male fertility. Examination of scanning electron micrographs of pollen grains revealed that the majority of pollen grains of antisense line were collapsed in morphology. Clear evidence of tapetum developmental defects was detected beginning at the microspore mother cell stage. More arrested pollen grains were found at the vacuolated microspore stage in antisense line (-)12 than in wild type tomato plants. In addition, lipid bodies in antisene line (-)12 were more evident and ER was less than in WT. After germination on culture medium for 60 min, approximately 65% of WT pollen grains germinated, while only 8% of (-)12 germinated. After 120 min, the proportion of germinated pollen grains in WT was nearly 100%, compared to only 20% in (-)12 line. According to the statistical analysis, flower development was arrested in 53.8% (14/26) of the samples from antisense lines, compared to only 7.8% (4/51) in wild type plants. Seeds of wild type developed normally and could reproduce, whereas the progenitive ability of antisense seeds was lost.9. The O2 evolution rates of WT and antisense transgenic tomato plants significantly decreased at 45℃for 6 h and 12 h. The decrease was more obvious in the wild type than in antisense transgenic plants. After 6 h heat stress, the O2 evolution rates in wild type, antisense transgenic lines (-)7 and (-)12 decreased to about 30.5%, 47.4% and 50.9% of initial values, respectively. After 12 h heat stress the O2 evolution rates of wild type, (-)7 and (-)12 lines decreased to 7.2%, 18.5% and 19.4% of initial values, respectively. Fv/Fm decreased in both WT and transgenic plants at 45℃heat stress, with wild types showing the greater decrease. At the end of 12 h heat stress, Fv/Fm in wild type, (-)7 and (-)12 lines decreased about 42.5%, 30.9% and 26.8%, respectively.The functional analysis showed that expression of the gene was induced by low temperature, whereas it was inhibited by heat stress. LeGPAT exhibited 18:1-selectivity over 16:0. The content of LeGPAT was higher in transgenic plants than in wild type tomato. It is interesting that overexpression of chloroplast LeGPAT increased the resistance to chilling stress. However, the depletion of LeGPAT was helpful in improving the thermal tolerance of tomato plants to high temperature and caused a massive arrest in pollen development.更多还原