【作者】 李群睿；

【导师】 朱长甫；

【作者基本信息】 东北师范大学 ， 植物学， 2010， 博士

【摘要】 类胡萝卜素对人类营养和健康具有重要价值。类胡萝卜素主要作为人体的抗氧化剂和维生素A的前体。较高的类胡萝卜素饮食摄入量与各种类胡萝卜素缺乏相关疾病的降低密切相关[1]。动物和人体无法直接合成类胡萝卜素,必须从其食物中获得类胡萝卜素。谷物里一般缺乏这些化合物,导致一些以谷物为主食国家的人们患有与类胡萝卜素缺乏相关的许多疾病。由于这些原因人们对植物类胡萝卜素合成途径和应用遗传工程技术改良禾谷类作物以及提高类胡萝卜素含量的研究产生了极大的兴趣。近年来通过对类胡萝卜素生物合成途径的遗传操作在提高主要农作物可食用器官的类胡萝卜素含量方面已取得了可喜的进展[2-4]。但在应用遗传工程技术提高植物靶器官中某一特定类胡萝卜素含量时,存在很大的盲目性,这主要是由于目前人们对植物体内类胡萝卜素合成和积累的调控机制等还不甚清楚。为了深入了解玉米(Zea mays L.)种子胚乳中类胡萝卜素生物合成和积累分子调节机制,我们从玉米自交系B73种子胚乳中克隆了编码类胡萝卜素异构酶(CRTISO)和番茄红素ε-环化酶(LYCE)的基因(cDNAs)。我们克隆到两个截然不同的玉米(Zea mays L.)crtiso cDNAs(Zmcrtiso1和Zmcrtiso2)。这两个crtiso基因分别位于玉米基因组的不同染色体上。与拟南芥和番茄crtiso基因相似,Zmcrtiso1和Zmcrtiso2基因在玉米基因组中也都具有12个内含子。Zmcrtiso1 cDNA核苷酸序列编码一个含587个氨基酸残基的蛋白质,分子量为63.7 kDa,等电点为8.24。用ChloroP 1.1信号肽预测工具预测ZmCRTISO1蛋白存在一个含有43个氨基酸残基的质体信号肽。在玉米自交系B73中,Zmcrtiso2 cDNA在第143个核苷酸序列(与Zmcrtiso1 cDNA对比,Zmcrtiso2 cDNA推测的翻译起始密码子第一个核苷酸序列为1)位置的碱基从胞嘧啶(C)颠换(transversion)为腺嘌呤(A)。但重要的是,这种碱基颠换(transversion)并不发生在白玉米M37W,黄玉米EP42和A632以及登录在国际基因数据库(GenBank)中的其他玉米品系中(登录号:FL133727和EU957482)。玉米自交系B73Zmcrtiso2 cDNA中的这种C到A的单碱基颠换,导致翻译提前终止而产生无义突变。因此,预测玉米自交系B73Zmcrtiso2 cDNA翻译成一个缩短(truncated,或截短)了127个氨基酸残基的蛋白质。研究表明,ZmCRTISO1能将7,9,7’,9’-四顺式-番茄红素(前番茄红素)转换为全反式番茄红素,但不能异构化含有15-顺式双键的9,15,9’-三顺式-ζ-胡萝卜素。在玉米自交系B73,Zmcrtiso2 cDNA由于含有提前终止密码子,编码的缩短了的ZmCRTISO2没有类胡萝卜素异构酶活性,但在其它的玉米品种中由于缺乏这种DNA单碱基颠换,他们的Zmcrtiso2基因编码的蛋白质(酶)具有与ZmCRTISO1相同的类胡萝卜素异构酶活性的功能。玉米自交系B73Zmlyce的最长开放阅读框(ORF,open reading frame)预测可编码537个氨基酸残基的蛋白质;对其编码蛋白的预测,分子量为59.7 kDa,等电点为6.33。Zmlyce cDNA推导的氨基酸序列与拟南芥、番茄和黄花龙胆lyce cDNA推导的氨基酸序列有72.1%到73.8%的相似性(similarity)以及63.2%到63.9%的同一性(identity)。通过在大肠杆菌中的异源互补实验(complementation experiment)证实,Zmlyce cDNA编码的LYCE能经过含有一个ε-紫罗兰酮(ionone)环的δ-胡萝卜素,在δ-胡萝卜素的另一个末端添加第二个ε-紫罗兰酮环而生成含有两个ε-紫罗兰酮环的ε-胡萝卜素。这与拟南芥,番茄和黄花龙胆LYCE只能催化全反式番茄红素生成含有一个ε-紫罗兰酮环的δ-胡萝卜素的酶活性明显不同。为了探讨在玉米种子胚乳中类胡萝卜素生物合成和积累的分子调控机制,我们研究了Zmcrtiso1和Zmcrtiso2 mRNA表达与玉米种子胚乳中类胡萝卜素的积累之间的关系。随玉米种子胚乳发育,直到授粉后第25天,Zmcrtiso1和Zmcrtiso2 mRNA在胚乳中的积累量逐渐增加,这与玉米种子胚乳类胡萝卜素的积累(尤其是β-胡萝卜素,玉米黄质和叶黄体素组分的积累)相一致。Zmcrtiso1 mRNA积累量随后逐渐降低,而Zmcrtiso2 mRNA积累量一直保持到授粉后第30天。我们深入讨论了这两个Zmcrtiso平行同源基因(paralogs)在玉米种子胚乳类胡萝卜素合成和积累中的功能。更多还原

【Abstract】 Carotenoids have fundamental roles in human nutrition as antioxidants and vitamin A precursors, and a high dietary intake of carotenoids is associated with a reduced risk for a range of diseases[1]. Animals are unable to synthesize carotenoids directly and must obtain them from their diets. Cereal grains are generally deficient in these compounds, leading to deficiency diseases in countries where cereals are the staple diet. For these reasons there is much interest in studying the carotenoid biosynthetic pathway in plants and in modifying crop plants to enhance their carotenoid content. The limited data concerning endogenous regulation of carotenogenic genes has made the precise engineering of crop plants to enhance carotenoid content and composition difficult despite recent progress in cereal crops, particularly in corn[2-4]. In order to gain further insight into the partly-characterized carotenoid biosynthetic pathway in corn (Zea mays L.), we cloned cDNAs encoding the enzymes carotenoid isomerase (CRTISO) and lycopeneε-cyclase (LYCE) using endosperm mRNA isolated from inbred corn line B73. For CRTISO enzyme, two different cDNAs (Zmcrtiso1 and Zmcrtiso2), encoding full-length carotenoid isomerase (CRTISO) enzymes, were amplified from 25 DAP (days after pollination) B73 corn endosperm mRNA. The full-length Zmcrtiso1 cDNA encoded a 587-amino-acid protein with a molecular weight of 63.7 kDa, a pI of 8.24, and a putative 43-residue transit peptide for chloroplast targeting. The B73 Zmcrtiso2 cDNA contained a C-to-A transversion at position 143 (the putative translation start codon of the Zmcrtiso2 cDNA is referred to as 1) which was not present in homologous EST and cDNA sequences from white maize (M37W), yellow maize (EP42 and A632), and sequences from other cultivars (accession nos. FL133727 and EU957482). The resulting nonsense mutation prematurely terminates protein synthesis and yields a truncated 127-amino-acid protein. Zmcrtiso1 and Zmcrtiso2 were identified mapping to unlinked genes each containing 12 introns, a feature conserved among all crtiso genes studied thus far. ZmCRTISO1 was able to convert tetra-cis prolycopene to all-trans lycopene but could not isomerize the 15-cis double bond of 9,15,9′‐tri‐cis-ζ-carotene. ZmCRTISO2 is inactivated by the premature termination codon in B73 corn as described above, but importantly the mutation is absent in other corn cultivars and the active enzyme showed the same activity as ZmCRTISO1.ZmLYCE from maize B73 was predicted to be 59.7 kDa (537 residues) and a pI of 6.33. The full-length amino acid residues of Zmlyce have high similarities (72.1% to 73.8%) and identities (63.2% to 63.9%) with functional LYCE proteins from Arabidopsis, tomato and gentian. However, by heterologous complementation experiment in Escherichia coli, ZmLYCE can add twoε-ionone rings at the both ends of lycopene to generate the bicyclicε,ε-carotene through mono-cyclicδ-carotene, which is significant different from that from Arabidopsis, tomato and gentian where LYCE can add only oneε-ionone ring at one end of lycopene to give rise to mono-cyclicδ-carotene. Furthermore, in order to understand how carotenoid accumulation is regulated in maize endosperm development, we investigated the correlation between carotenoid accumulation in endosperm and mRNA expression of Zmcrtiso1 and Zmcrtiso2. Both of Zmcrtiso1 and Zmcrtiso2 genes were expressed during endosperm development, with mRNA levels rising in line with carotenoid accumulation (especiallyβ-carotene, zeaxanthin and lutein) until 25 DAP. Thereafter, expression declined for Zmcrtiso1 gene, whereas Zmcrtiso2 mRNA levels maintained by 30 DAP. We discuss the impact of paralogs with different expression profiles and functions on the regulation of carotenoid synthesis in corn.更多还原