苹果GPP和IMP基因的cDNA克隆、原核表达及抗体制备
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摘要
抗坏血酸(AsA)是生物体内重要的抗氧化剂和很多酶的辅因子,植物体内的AsA是人类天然AsA的来源。作为大众水果的苹果,其AsA含量并不高,严重影响了其品质。研究植物AsA的合成代谢途径进而改善植物组织中AsA的含量对人类健康和植物自身都有重大意义。研究苹果AsA合成关键酶基因的功能是研究苹果果实AsA合成积累、机制的基础。本实验主要对植物AsA生物合成途径中的两个关键酶——L-半乳糖-1-磷酸磷酸酶(GPP)和肌醇-1-磷酸磷酸酶(肌醇单磷酸酶,IMP)基因进行了以下研究,以期为进一步研究GPP、IMP基因的功能奠定基础,进而从分子水上为苹果果实AsA合成、积累机理的研究提供理论依据。
用提取的苹果幼果总RNA做反转录,通过RT-PCR扩增GPP和IMP基因的cDNA全长序列,并分别对其序列进行生物信息学分析。对亚克隆得到的GPP、IMP基因和pET-32a载体同时进行双酶切,经连接、转化构建pET-GPP和pET-IMP表达载体,并对二者的重组菌液进行IPTG诱导,研究其融合蛋白的特性。利用His-GPP和His-IMP融合蛋白的包涵体制备抗原免疫家兔,制备抗血清并检测其特异性。用丙酮-三氯乙酸法提取苹果不同组织的总蛋白,采用Western杂交方法探索苹果GPP和IMP基因编码蛋白在苹果不同组织中的表达情况。实验结果如下:
1.克隆得到了全长为1121 bp的苹果GPP基因序列和全长为1201bp的IMP基因序列,开放阅读框分别为813 bp和1008 bp,分别编码270和335个氨基酸。通过序列分析获得了预测的GPP和IMP基因编码蛋白的理化性质、二级结构、三级结构等结果,发现二者存在一定的相似度。
2.根据克隆得到的GPP、IMP基因序列和pET-32a的序列重新设计引物引入合适的酶切位点,从pMD-GPP和pMD-IMP中亚克隆得到目的片段,经双酶切、连接和转化成功的构建了pET-GPP和pET-IMP表达载体并在E.coli BL21中得到高效表达,其表达产物分子量分别49 KD和57 KD左右。经过原核表达分析可知,His-GPP最适的IPTG诱导浓度和时间分别是0.1 mmol?L-1和5 h,而His-IMP则为0.01 mmol?L-1和4 h,且二者的融合蛋白均主要以包涵体的形式存在。
3.用佐剂乳化His-GPP和His-IMP重组蛋白的包涵体免疫家兔,获得的抗血清分别经纯化的His-GPP和His-IMP可溶性蛋白检测,Western结果显示均具较高的特异性。
4.丙酮-三氯乙酸法提取得到了高质量的苹果组织总蛋白,经Western杂交实验在苹果各组织中均检测到了大小分别约为33 KD和39 KD的不同强度的GPP和IMP蛋白表达信号。
L-Ascorbic acid (AsA) is one of the most important antioxidants and cofactor for many enzymes, it is the main source of natural AsA to people. Apple fruit is a kind of favorite fruit, but it contains lower AsA than other fruit, which seriously affected its quality. Studying the AsA biosynthesis of plant and improving the content of AsA in plant tissues are significant for plant themselves and human health. Studying the function of AsA biosynthesis and metabolism genes which involved in apple fruit is the basis of apple fruit AsA accumulation mechanism. This experiment focused on the L-galactose-1-P phosphatase(GPP) and Myo-inositol-1-P phosphatase(IMP) which involved in AsA biosynthesis and the results are useful for further study of function of GPP and IMP gene, and they also provide the theoretical basis of the molecular mechanism of synthesis and accumulation of AsA in apple fruit.
The complete GPP and IMP gene sequences were amplificated by RT-PCR method, and the bioinformatic tools were used to analyze the sequences. In order to further understand the function of the two genes, expression plasmids of GPP and IMP gene were constructed with pET-32a and expressed in E.coli BL21, IPTG was added to induce the fusion protein expression,then analyzed the characteristics of the fusion proteins. His-GPP and His-IMP fusion proteins were used to immunolize rabbits to prepare Polyclonal antibody, then the specific recognition were detected by Western blot experiment, and the expression of GPP, IMP gene in apple different tissues were analysised by the same method. The main results were as follows:
1. The full-length cDNA of GPP is 1121 bp and contains a complete open reading frame of 813 bp, which encoding 270 amino acids. And the full-length cDNA of IMP is 1201 bp and contains a complete open reading frame of 813 bp, which encoding 335 amino acids. Homology analysis of their sequences showed high homology with the cDNA from other plants, respectively. Through the bioinformatic analyze, we found the secondary structure and tertiary structure of GPP are similar to IMP’s, maybe it is the explanation of the similar function between GPP and IMP gene.
2. We signed other two pairs of specific primers based on the sequences of GPP, IMP gene and pET-32a, then GPP and IMP gene were subcloned into the expression vector pET-32a. The recombinant expression vectors pET-GPP and pET-IMP were digested by BamHI and SalI and the results show that GPP and IMP were correctly linked to the pET-32a. The recombinant expression vectors pET-GPP and pET-IMP were transformed into E.coli BL21 cells and then induced by IPTG, the molecular weights of the cells specifically expressed proteins are 49 KD and 57 KD based on SDS-PAGE gel results, respectively. They are identical to the known His-GPP and His-IMP fusion proteins, so the GPP and IMP gene were expressed in E.coli BL21. We also fixed the best expression condition by compare the expression result of different IPTG density and cell induced culture times. The result shows that the best expression condition for His-GPP induced is 0.1 mmol?L-1 IPTG at 28℃for 5 h, and the best expression condition for His-IMP induced is 0.01 mmol?L-1 IPTG at 37℃for 4 h. Through solublity analysis we found His-GPP and His-IMP fusion proteins were almost expressed in inclusion body.
3. Inclusion body of His-GPP and His-IMP from E.coli induced by IPTG were used as antigens, then injected into rabbit to prepare the antibodies. The validity of polyclonal antibodies were confirmed by western blot and the results show that they had specific antigen-antibody recognition to the His-GPP and His-IMP.
4. Total Proteins were extracted from different tissues of apple and separated by SDS-PAGE,then transferred to a PVDF membrane. Western blot was Performed using antibodies against GPP and IMP, respectively. Specific bands about 33 KD and 39 KD were found in Western blot immune images. The reason of the appearance of these bands are the expression signals of GPP and IMP gene in apple tissues were detected.
用提取的苹果幼果总RNA做反转录,通过RT-PCR扩增GPP和IMP基因的cDNA全长序列,并分别对其序列进行生物信息学分析。对亚克隆得到的GPP、IMP基因和pET-32a载体同时进行双酶切,经连接、转化构建pET-GPP和pET-IMP表达载体,并对二者的重组菌液进行IPTG诱导,研究其融合蛋白的特性。利用His-GPP和His-IMP融合蛋白的包涵体制备抗原免疫家兔,制备抗血清并检测其特异性。用丙酮-三氯乙酸法提取苹果不同组织的总蛋白,采用Western杂交方法探索苹果GPP和IMP基因编码蛋白在苹果不同组织中的表达情况。实验结果如下:
1.克隆得到了全长为1121 bp的苹果GPP基因序列和全长为1201bp的IMP基因序列,开放阅读框分别为813 bp和1008 bp,分别编码270和335个氨基酸。通过序列分析获得了预测的GPP和IMP基因编码蛋白的理化性质、二级结构、三级结构等结果,发现二者存在一定的相似度。
2.根据克隆得到的GPP、IMP基因序列和pET-32a的序列重新设计引物引入合适的酶切位点,从pMD-GPP和pMD-IMP中亚克隆得到目的片段,经双酶切、连接和转化成功的构建了pET-GPP和pET-IMP表达载体并在E.coli BL21中得到高效表达,其表达产物分子量分别49 KD和57 KD左右。经过原核表达分析可知,His-GPP最适的IPTG诱导浓度和时间分别是0.1 mmol?L-1和5 h,而His-IMP则为0.01 mmol?L-1和4 h,且二者的融合蛋白均主要以包涵体的形式存在。
3.用佐剂乳化His-GPP和His-IMP重组蛋白的包涵体免疫家兔,获得的抗血清分别经纯化的His-GPP和His-IMP可溶性蛋白检测,Western结果显示均具较高的特异性。
4.丙酮-三氯乙酸法提取得到了高质量的苹果组织总蛋白,经Western杂交实验在苹果各组织中均检测到了大小分别约为33 KD和39 KD的不同强度的GPP和IMP蛋白表达信号。
L-Ascorbic acid (AsA) is one of the most important antioxidants and cofactor for many enzymes, it is the main source of natural AsA to people. Apple fruit is a kind of favorite fruit, but it contains lower AsA than other fruit, which seriously affected its quality. Studying the AsA biosynthesis of plant and improving the content of AsA in plant tissues are significant for plant themselves and human health. Studying the function of AsA biosynthesis and metabolism genes which involved in apple fruit is the basis of apple fruit AsA accumulation mechanism. This experiment focused on the L-galactose-1-P phosphatase(GPP) and Myo-inositol-1-P phosphatase(IMP) which involved in AsA biosynthesis and the results are useful for further study of function of GPP and IMP gene, and they also provide the theoretical basis of the molecular mechanism of synthesis and accumulation of AsA in apple fruit.
The complete GPP and IMP gene sequences were amplificated by RT-PCR method, and the bioinformatic tools were used to analyze the sequences. In order to further understand the function of the two genes, expression plasmids of GPP and IMP gene were constructed with pET-32a and expressed in E.coli BL21, IPTG was added to induce the fusion protein expression,then analyzed the characteristics of the fusion proteins. His-GPP and His-IMP fusion proteins were used to immunolize rabbits to prepare Polyclonal antibody, then the specific recognition were detected by Western blot experiment, and the expression of GPP, IMP gene in apple different tissues were analysised by the same method. The main results were as follows:
1. The full-length cDNA of GPP is 1121 bp and contains a complete open reading frame of 813 bp, which encoding 270 amino acids. And the full-length cDNA of IMP is 1201 bp and contains a complete open reading frame of 813 bp, which encoding 335 amino acids. Homology analysis of their sequences showed high homology with the cDNA from other plants, respectively. Through the bioinformatic analyze, we found the secondary structure and tertiary structure of GPP are similar to IMP’s, maybe it is the explanation of the similar function between GPP and IMP gene.
2. We signed other two pairs of specific primers based on the sequences of GPP, IMP gene and pET-32a, then GPP and IMP gene were subcloned into the expression vector pET-32a. The recombinant expression vectors pET-GPP and pET-IMP were digested by BamHI and SalI and the results show that GPP and IMP were correctly linked to the pET-32a. The recombinant expression vectors pET-GPP and pET-IMP were transformed into E.coli BL21 cells and then induced by IPTG, the molecular weights of the cells specifically expressed proteins are 49 KD and 57 KD based on SDS-PAGE gel results, respectively. They are identical to the known His-GPP and His-IMP fusion proteins, so the GPP and IMP gene were expressed in E.coli BL21. We also fixed the best expression condition by compare the expression result of different IPTG density and cell induced culture times. The result shows that the best expression condition for His-GPP induced is 0.1 mmol?L-1 IPTG at 28℃for 5 h, and the best expression condition for His-IMP induced is 0.01 mmol?L-1 IPTG at 37℃for 4 h. Through solublity analysis we found His-GPP and His-IMP fusion proteins were almost expressed in inclusion body.
3. Inclusion body of His-GPP and His-IMP from E.coli induced by IPTG were used as antigens, then injected into rabbit to prepare the antibodies. The validity of polyclonal antibodies were confirmed by western blot and the results show that they had specific antigen-antibody recognition to the His-GPP and His-IMP.
4. Total Proteins were extracted from different tissues of apple and separated by SDS-PAGE,then transferred to a PVDF membrane. Western blot was Performed using antibodies against GPP and IMP, respectively. Specific bands about 33 KD and 39 KD were found in Western blot immune images. The reason of the appearance of these bands are the expression signals of GPP and IMP gene in apple tissues were detected.
引文
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Szarka A, Horemans N, Banhegyi G. 2004. Facilitated glucose and dehydroascorbate transport in plant mitochondria. Arch Biochem Biophys, 428: 73～80
Tabata K, ?ba K, Suzuki K. 2001. Generation and properties of ascorbic acid-deficient transgenic tobacco cells expressing antisense RNA for L-galactono-1,4-lactone dehydrogenase. Plant J, 27: 139～148
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Wolucka B A, Van Montagu M. 2003. GDP-mannose 3,5-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants. J Biol Chem, 278: 47483～47490
Zhang W, Gruszewski HA, Chevone BI, Nessler CL. 2008. An Arabidopsis purple Acid phosphatase with phytase activity increases foliar ascorbate. Plant Physiol, 146: 431-440