摘要
抗坏血酸(AsA)即维生素C,是一种普遍存在于植物组织的高丰度小分子抗氧化物质,它在清除自由基、调控细胞生长和分裂以及抵抗逆境等方面起着非常重要的作用,也是人类维持健康所必需的一种微量营养元素。因此,AsA的含量已成为衡量农产品品质的重要指标,受到人们的广泛关注。分离、克隆与AsA积累和代谢有关的基因,探讨这些基因在植物体内的功能及其表达模式,对进一步认识AsA在植物体内的积累代谢机制,提高植物体内AsA含量以及增强植物抵抗环境胁迫等方面具有重要意义。
本试验以维生素C含量丰富的草莓栽培品种‘丰香’(Fragaria×ananassa cv. Toyonaka)为材料,分离、克隆了AsA代谢途径中两个关键酶基因Faapx-c和Fadhar,并利用生物信息学手段对这两个基因及其编码的蛋白质进行了结构分析和功能预测,同时分析了这两个基因在草莓不同组织及其果实不同发育阶段的表达模式。主要得到了以下研究结果：
1.通过对GenBank上已登录的几种植物apx和dhar同源基因进行比较,分别在其保守区设计一对引物,扩增得到了草莓‘丰香’apx和dhar基因的中间片段。在此基础上,通过RACE技术扩增得到了包括3’端全长的apx基因序列,命名为Faapx-c;该序列长1034 bp,包含一个长753 bp的完整ORF,编码1个由250个氨基酸残基组成的蛋白质,预测分子量为27.359 kDa,等电点为5.92；3’端非编码区长248 bp,含有加尾信号aaataa和长28bp的poly(A)尾巴。通过SON-PCR技术,从草莓‘丰香’中分离了dhar基因编码区全长DNA序列,命名为Fadhar;该序列长1374 bp,包含一个长887 bp的完整ORF,编码区被3个内含子阻断,该编码区编码1个由190个氨基酸残基组成的蛋白质,预测分子量为20.987 kDa,等电点为6.24。
2.对草莓Faapx-c和Fadhar基因与其它植物apx和dhar基因进行同源比较分析表明,草莓Faapx-c和Fadhar基因序列与其它植物apx和dhar基因的同源性较高,核苷酸序列同源性分别达到65%和62%以上,氨基酸序列同源性达到79%和57%以上,都是比较保守的基因。
3.采用Bioedit软件分析了草莓Faapx-c和Fadhar基因全CDS序列的碱基组成,结果表明：Faapx-c基因碱基A、C、G、T的数目分别是171、200、201、181,四种碱基分别占总数的22.71%、26.56%、26.69%和24.04%,A+T的含量(46.75%)低于G+C含量(53.25%)；Fadhar基因碱基A、C、G、T的数目分别是144、125、147、157,四种碱基分别占总数的25.13%、21.82%、25.65%和27.40%,A+T的含量(52.53%)高于G+C含量(47.47%)。草莓Faapx-c和Fadhar基因CDS编码区密码子使用偏爱性分析表明,两个基因都偏向使用以T结尾的密码子。Faapx-c基因含有5个大肠杆菌稀有密码子,而且它们是分散的；而Fadhar基因只含有1个大肠杆菌稀有密码子。
4.采用Bioedit软件及ExPASy在线蛋白质分析软件的ProtParam工具分析了FaAPX-c和FaDHAR蛋白的理化性质,表明FaAPX-c和FaDHAR都是亲水性强、结构稳定的蛋白质。应用ANTHERWIN5.0软件和PredictionProtein服务器预测了FaAPX-c和FaDHAR蛋白的二级结构,结果表明FaAPX-c和FaDHAR蛋白二级结构都是主要以a-螺旋和无规则卷曲两种形式出现。采用EsyPred3D和CPH models-3.0分别构建了FaAPX-c和FaDHAR蛋白的三维结构模型。由ESyPred3D构建的FaAPX-c肽链三维模型中只有12个α-螺旋；而由CPH models-3.0构建的FaAPX-c肽链三维模型中有12个α-螺旋和2个β-折叠。FaDHAR肽链用ESyPred3D构建的三维模型由7个α-螺旋、2个β-折叠和1个转角组成；而用CPH models-3.0构建的三维模型由8个α-螺旋、3个β-折叠和1个转角组成。采用Swiss-Pdb Viewer对FaAPX-C和FaDHAR蛋白的两种模建结果进行了检测,计算得出了它们对应的Ramachandran图,结果表明所得到的三维结构模型都是可靠的。
5.采用网络服务器对FaAPX-C和FaDHAR蛋白的功能进行了预测,结果表明,FaAPX-c属于植物过氧化物酶家族,它的氨基酸序列中存在两段保守的多肽序列,即过氧化物酶活性位点序列(APlmLRLaWHSA)和过氧化物酶近端血红素配体序列(DIVALSGGHTL); FaDHAR属于谷胱甘肽-S-转移酶家族,其氨基酸序列中也存在两段潜在谷胱甘肽S-转移酶的特征序列。FaAPX-c和FaDHAR蛋白都没有信号肽,都不属于分泌性蛋白,也不存在卷曲螺旋结构,而且都没有跨膜结构,都不是跨膜蛋白,没有N-糖基化位点,但都存在多个潜在的O-糖基化位点和磷酸化位点。对apx和dhar基因的电子表达谱分析表明,apx和dhar基因在所能检索到的植物不同部位都能够表达,说明它们在植物生长发育中具有重要功能。
6. Faapx-c和Fadhar基因的表达模式分析表明,这两个基因在草莓‘丰香’不同组织部位都能够表达,在果实中表达量最大。Faapx-c基因在草莓‘丰香’果实不同发育阶段的表达基本是从小绿期开始呈阶梯式的增大,可能与果实生长发育相关;Fadhar基因在草莓‘丰香’果实整个发育过程中高度表达,该基因的表达可能调控着草莓果实内AsA的积累水平。
Ascorbic acid or vitamin C, is a small molecule antioxidant with high abundance in plant tissue. It plays an important role in scavenging free radicals, regulating of cell growth and division, and resistance to environmental stress. In addition, it is also a necessary micronutrient for maintain healthy of human. AsA content has become an important indicator to measure the quality of agricultural products, and thus be payed close attention. Isolation, cloning genes which be involved in AsA accumulation and metabolism, and analyzing the functions and expression patterns of these genes, have a great significance for further understanding of AsA accumulation and metabolism in plants, increasing AsA content and enhancing plant stress resistance.
In this study, two key enzymes genes Faapx-c and Fadhar, which involved in AsA metabolic pathway were cloned from Fragaria×ananassa cv. Toyonaka rich in vitamin C. And the structures and functions of them were predicted by means of bioinformatics. At the same time, the expression patterns of the two genes be analyzed in different tissues and different developmental stages of fruit in strawberry. The following findings were obtained:
1. The primers were respectively designed by comparing homologous genes from GenBank, and the conserved regions of apx and dhar were obtained from Fragaria×ananassa cv. Toyonaka. On this basis, Faapx-c gene sequence including the full of 3' end was obtained by RACE, named Faapx-c. The sequence was 1034 bp in length, containing a ORF of 753 bp, which encodes a polypeptide of 250 amino acid residues a molecular mass of 27.359kDa and a pI of 5.92. The non-coding region of 3'end was 248 bp, containing the potential polyadenylation signal aaataa and poly (A) of 28bp. A full length of open reading frame of dhar gene was isolated by SON-PCR, named Fadhar. The sequence was 1374 bp in length interrupted with three introns, including a full ORF of 887 bp, which encoding a deduced protein of 190 amino acids with a molecular weight of 20.987 kDa and a pI of 6.24.
2. Homology analysis of Faapx-c and Fadhar genes showed that they shared high homology with other plants.The nucleotide sequence homology were respectively over 65% and 62%, and amino acid sequence homology were respectively over 79% and 57%. This suggested they are all conservative genes.
3. The base compositions of Faapx-c and Fadhar CDS were analyzed by Bioedit. The results showed that the numbers and percents of A, C, G, T in Faapx-c are 171 (22.71%),200(26.56%),201(26.69%) and 181(24.04%), respectively. The content of A +T is (46.75%) lower than G+C(53.25%); The numbers and percents of A, C, G, T in Fadhar are 144(25.13%),125(21.82%),147(25.65%),157(27.40%), respectively. The content of A+T is (52.53%) higher than G+C(47.47%). The analysis of codon preferences of Faapx-c and Fadhar CDS showed that they all preferred to use of T ending codons. Faapx-c contains five E. coli rare codon, and they are scattered in the suquences; and Fadhar contains only one E. coli rare codon.
4. The analysis of physicochemical properties of FaAPX-c and FaDHAR indicated that they were all acidic, hydrophilic and structural stable protein by Bioedit and ProtParam tools in ExPASy. Secondary structures analysis showed that FaAPX-c and FaDHAR contained mainly a-helix and random coil predicted by ANTHERWIN5.0 and PredictionProtein.3D structures of FaAPX-c and FaDHAR were predicted using the ESyPred3D and CPH models-3.0. FaAPX-c only contains 12α-helices predicted by ESyPred3D, but it contains 12α-helices and twoβ-sheets predicted by CPH models-3.0. FaDHAR contains sevenα-helix, twoβ-sheet and one turn predicted by ESyPred3D, but it contains eightα-helix, threeβ-sheet and one turn predicted by CPH models-3.0.3D structures of FaAPX-C and FaDHAR were reliable by analysis of corresponding Ramachandran blots using Swiss-Pdb Viewer.
5. Function prediction was carried out in net server. The results showed that FaAPX-C was a member of plant peroxidase superfamily, containing peroxidases active site signature (APlmLRLaWHSA) and peroxidases proximal heme-ligand signature (DIVALSGGHTL). And FaDHAR was a member of GST-S-family superfamily, containing two soluble glutathione S-transferase terminal domain. FaAPX-c and FaDHAR had no signal peptides, coils, transmenbrane domains, thus they were not secreted proteins or transmembrane proteins. In addition, they had no N-glycosylation sites, but with some O-glycosylation sites and phosphorylation sites. Electronic expression profiles revealed that apx and dhar expressed in different tissues of plants, which suggested that they played an important function in plant development.
6. The expression profile analysis showed that Faapx-c and Fadhar can be expressed in different tissues of Fragaria×ananassa cv. Toyonaka, especially in fruits. Faapx-c may be associated with fruit growth and development, because the amounts of expression were increased gradually from small green; Fadhar is highly expressed during fruit development, which regulates the level of AsA accumulation in strawberry fruits.
引文
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