摘要
赤霉素(gibberellin, GA)是一类四环二萜羧酸,是种类最多、生理功能最广的植物内源激素之一。目前,已发现的赤霉素类物质有136种,其中只有少数具有生物活性,影响着高等植物生活史的各个阶段。随着研究手段和技术的飞速发展,各种突变体材料的获得和利用,在GA生物合成及其调控方面,特别是GA合成中关键酶基因的克隆成功及对其功能的研究取得了较大的进展。GA2-氧化酶是由多基因编码的双加氧酶,调节赤霉素代谢,作用于有生物活性的GA1和GA4,将其转变成无活性的GA8和GA34,使植物体内GAs的活性降低,呈现矮化、节间缩短的表型。此外,GA2-氧化酶还参与植物光形态的建成。光通过诱导GA2-氧化酶基因的表达,使植物体内有生物活性的GA4降低而促进光形态建成。但目前仍没有关于GA2-氧化酶抗体的报道。因此,本论文通过生物信息学预测分析GA2ox1基因编码蛋白质的基本性质,从拟南芥基因组中克隆GA2ox1基因,构建高效原核表达载体pDEST17-GA2ox1,使其在宿主菌E.coli BL(21)star中表达,并优化表达条件。另外采用亲和层析方法纯化融合蛋白GA2ox1, Western Blot鉴定是否为目的蛋白。论文的具体结果如下：
(1)通过生物信息学分析GA2ox1基因编码蛋白。结果显示,GA2ox1基因的ORF编码蛋白质全长329个氨基酸,分子量为36.7314kD,等电点为8.5793,没有明显疏水性,含一个潜在的跨膜区,N端没有信号肽序列,二级结构丰富,含有磷酸化位点、N-糖基化位点、N-酰基化位点、ATP/GTP结合位点等蛋白修饰及活化位点,三级结构显示该蛋白空间构象不对称,折叠成松散的球状蛋白。
(2)以哥伦比亚野生型拟南芥为实验材料,拟南芥总RNA为模板,采用RT-PCR扩增得到GA2ox1基因的ORF,大小约为990bp,与预期相符。
(3)成功地构建了原核表达载体pDEST17-GA2ox1。将其转化宿主菌E.coli BL(21)star,得到原核表达工程菌,命名为BL(21)star-pDEST17-GA2oxl。
(4)使用诱导剂IPTG诱导GA2ox1基因在宿主菌E.coli BL(21)star中成功表达融合蛋白GA2ox1, SDS-PAGE分析该蛋白是以包涵体形式存在,分子量约40kD与预期相符。并确定最优表达条件为：IPTG浓度0.2 mmol/L、温度30℃、诱导时间6 h。
(5)探索表达蛋白GA2ox1的纯化条件。得到了大量、高纯度的GA2ox1融合蛋白,经Western Blot鉴定,纯化的蛋白为我们所需的目的蛋白。以上结果为下一步研制GA2ox1多克隆抗体、对GA2ox1基因进行蛋白水平的分析和研究GA2ox1蛋白的功能,及赤霉素调控植物生长的相关研究奠定了基础。
GA (gibberellin) is a class of tetracyclic diterpenoid carboxylic acid, is one of endogenous hormones in plants, which has the largest species and the most extensive physiological function. People have discovered 136 kinds of GAs up to now, and a small number of them are biologically active, which affects all stages of higher plants life cycle. With the rapid development of technology and access to a variety of mutant materials, greater progress had been made in studying biosynthesis and regulation of GA, especially cloning key enzyme genes in GA synthesis. GA2-oxidase is encoded by multiple genes dioxygenase. It regulates GA metabolism, changes bioactive GA1 and GA4 into inactive GA8 and GA34 and reduces the activity of GAs in plants, so plants showed dwarfing, internode shortening phenotype. GA2-oxidase is also involved in plant photomor-phogenesis. Light induces GA2-oxidase gene expression, and plants reduce the biological activity of GA4, so that promote the completion of photomorphogenesis. However, there is no report about GA2-oxidase antibody currently. Therefore, this thesis predicted and analysed GA2ox1 gene encoding protein by bioinformatics, then cloned GA2ox1 from the Arabidopsis thaliana genome, constructed prokaryotic expression vector pDEST17-GA2ox1, expressed it in host strain E.coli BL (21) star, and optimized the expression condition; we also purified fusion protein GA2ox1 by means of affinity chromatography, Western Blot identificated whether is the target protein or not. The specific results of this study are as follows:
(1) Bioinformatics analysis showed GA2ox1 encoded 329 amino acids. This protein had no obvious hydrophobic and its molecular weight was 36.7314kD, isoelectric point was 8.5793. It was a soluble protein with a potential transmembrane domain. It had not N-terminal signal peptide sequence. Its secondary structure enriched as well as contained a number of protein modification and activation of sites, such as the phosphorylation sites, N-glycosylation sites, N-myristoylation sites, ATP/GTP binding site and so on.The tertiary structure prediction showed that the protein was asymmetric and was an loosely folded globular protein.
(2) We took columbia wild-type Arabidopsis thaliana as experimental material, Arabidopsis thaliana total RNA as template, and amplified GA2ox1 gene ORF by RT-PCR, which was about 990bp, as expected.
(3) We successfully constructed prokaryotic expression vector pDEST17-GA2ox1, and confirmed by sequencing and PCR, it turned out the target gene was correctly inserted into the vector cloning site.Then we transformed it into host bacteria E. coli BL(21)star, we got prokaryotic-expressed engineering strain BL(21)star-pDEST17-GA2ox1.
(4) We successfully inducted GA2ox1 to express in the host bacteria E.coli BL(21)star with IPTG. SDS-APGE analysis reviewed its expression mainly in the form of inclusion bodies and the molecular weight was about 40kD as we expected. The optimal conditions as follows:IPTG concentration was 0.2mmol/L, temperature was 30℃, the inducted time was 6 h.
(5) We explored the expression of protein purification conditions, and obtained high purity GA2oxl fusion protein. Western Blot further analysis confirmed that the fusion protein was our target protein. This study establishes a basis for GA2oxl polyclonal antibody preparation, further studying GA2ox1 protein's function and researches on gibberellin regulation of plant growth.
引文
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