摘要
膜脂不饱和度与植物抗冷性关系密切。高等植物抗冷能力与叶绿体膜脂中磷脂酰甘油(PG)的顺式不饱和脂肪酸水平密切相关,抗冷植物中PG 的sn-1 位具有较高比例的顺式不饱和脂肪酸,而冷敏感植物中顺式不饱和脂肪酸比例则较低。甘油-3-磷酸酰基转移酶(GPAT)是叶绿体中PG 生物合成过程中的第一个酰基酯化酶,它将脂肪酰转移到3-磷酸甘油的C-1 位上合成1-酰基-Sn-甘油-3-磷酸(溶血磷脂酸)。一般来说,抗冷植物中GPAT 优先选择C18:1-ACP 作为底物,因此在这些植物的叶绿体中PG 的sn-1 位置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,从而对低温胁迫敏感。
本文以甜椒为实验材料,利用反转录PCR(RT-PCR)克隆了甜椒甘油-3-磷酸酰基转移酶的基因,通过同源性比较证明所克隆的基因是目的基因。根据获得的基因序列制备了杂交探针,分析了该基因在甜椒中的表达特征。构建了GPAT 基因的正义和反义表达载体,并通过农杆菌介导的叶盘法转入烟草中,探讨了温度逆境条件下GPAT 基因的功能。主要结果如下:
1. 利用同源序列设计兼并引物,通过RT-PCR 的方法从甜椒茄门中克隆到了甘油-3-磷酸酰基转移酶基因的中间片段,然后通过3’-RACE 和5’-RACE 分别克隆到了甘油-3-磷酸酰基转移酶基因的3’片段和5’片段,拼接后设计特异引物扩增到甘油-3-磷酸酰基转移酶基因的全长cDNA,利用RT-PCR 方法首次从甜椒叶片中克隆了甘油-3-磷酸酰基转移酶基因的cDNA
The chilling resistance of higher plants is closely correlated with the level of cis-unsaturated fatty acids in phosphatidylglycerol(PG)of chloroplast membranes. Chilling-resistant plants contain a large proportion of cis-unsaturated fatty acids at the sn-1 position of PG. The dominant factor that determines the level of cis-unsaturated fatty acids in PG is the substrate selectivity of glycerol-3-phosphate acyltransferase of chloroplasts(GPAT: EC2.3.1.15), which catalyzes the first step of glycerolipid biosynthesis in chloroplasts by transferring the acyl group of acyl-(acyl-carrier protein) (ACP) to the sn-1 position of glycerol-3-phosphate. GPAT from chilling-resistant plants prefers 18:1-ACP to 16:0-ACP as a substrate. Thus, a large proportion of 18:1 occurs at the sn-1 position of PG in chilling-resistant plants, which is further desaturated into cis-polyunsaturated fatty acids by acyl-lipid desaturases in chloroplast membranes. By contrast, the enzyme from chilling-sensitive plants hardly distinguishes between 18:1-ACP and 16:0-ACP, resulting in a low level of cis-unsaturated fatty acids at the sn-1 position of PG, because no 16:0 is desaturated into cis-unsaturated fatty acids of PG in these plants.
In this reseach, a full length cDNA of GPAT gene was cloned from sweet pepper using RT-PCR approach with degenerate primers based on the conserved motifs found in a number of plant GPAT genes. The homology comparison indicated that the cloned gene is the expected one.
The hybridization probe was synthesized according to the sequence of the gene, and the expression properties of the gene were analyzed in sweet pepper. Results indicated that the gene was closely correlated with the chilling-resistance of plants. Then the sense-and antisense-expression vectors were constructed and transferred into the tobacco by Agrobacterium tumefaciens-mediated leaf disk transformation. The main results are as follows: 1. Two degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from the leaves of sweet pepper according to the homologous sequences from other plants. The middle fragment of interested cDNA was obtained by RT-PCR. The 3’and 5’fragments were further isolated by 3’-and 5’-RACE, then the total length was cloned using the special primers designed according to the 3’and 5’sequences. The gene contains 1791bp with a 5’-untranslated region (5’UTR) of 51 bp, an open reading frame (ORF) of 1392 bp comprising 463 amino acid residues, and a 3’-untranslated region (3’UTR) of 348 bp. The gene was named CaGPAT(AY318749). 2. The deduced amino acid sequence showed high identities with other plant GPAT from tomato, tangerine, cucumber, sawflower, spinach and rice, which were 85.5%, 60.7%, 58.3%, 58.1%, 57.2% and 51.7%, respectively. But it had low identities with E.coli, which was only 24.2%. 3. Northern blot analysis of different organs such as the leaves, petals, stems, roots and fruits in sweet pepper indicated that CaGPAT was mainly expressed in the leaves. Transcripts of the gene were less in the fruits and stems, but transcripts were hardly detected in the roots and petals. Northern blot analysis also indicated that the expression of CaGPAT was induced by low temperature, while high temperature (35℃) had no obvious influence on the expression of CaGPAT. When temperature was lower than 15℃, CaGPAT was expressed after treatment of 3 hours and then the expression level dropped
after 12 hours. When temperature was lower than 15℃, the gene expression level was distinctly higher than that at 25℃, but no difference was observed from 5℃to 15℃. 4. Recombining GPAT gene and the PBI121 plasmid containing 35S promoter, we constructed the sense-and antisense-expression vector. Then the gene was introduced into the tobacco of NC89 by A. tumefaciens-mediated leaf disk transformation. The transgenic plants were selected by 1% kanamycin. The kanamycin-resistant transgenic tobacco plants were identified by PCR. The result indicated that the GPAT gene had been recombined into the genome of tobacco in parts of the transgenic tobacco plants. The composition of membrane lipids was investigated in leaves of transgenic and wild type(WT) tobacco plants treated at 15℃for 24 hours. Comparing with the wild type tobacco, the change mainly occurred in PG of the transgenic plants. The proportion of PG in sense-transgenic tobaccos did not obviously change, but its composition of fatty acids changed greatly comparing with WT. The leaves of sense-transgenic tobacco contained more 16:0 and 16:1, but less 18:2 and 18:3 fatty acids. There are no obvious difference in the unsaturated degree of PG between antisense-transgenic and wild type tobacco plants, while contents of PG decreased greatly. As a result, the fluidity of its membrane also dropped under low temperature. 5. The photosynthetic rate (Pn) of transgenic plants was always lower than that of wild type tobacco plants under low temperature, and the recovery of Pn in the transgenic plants were also slow. However, the transgenic tobaccos had higher photosynthetic rate than wild type palnts under high temperature, which was due to lower unsaturated fatty acids in the transgenic tobacco plants. The results indicated that the unsaturation of PG had important influence on chilling resistance of plants. In addition, the maximum photochemical efficiency of PS Ⅱ(Fv/Fm) and actual photochemical efficiencyof PSⅡ(φPSⅡ) of transgenic tobacco were both lower than wild type tobacco plants, suggesting that the unsaturation degree and amount of
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