摘要
苹果(Malus domestica Borkh)是世界上最主要的鲜食果品之一,其果实色泽是重要的外观品质,直接决定其商品价值。‘国光’是20世纪80年代中国北方广泛种植的苹果品种之一,具有晚熟、酸甜适口及抗逆性强等特点,近年来再次受到消费者的青睐,但着色差一直是限制其发展的重要因素。2005年课题组在山东省五莲县发现了一株‘国光’的红色突变,其突出的特点是比国光着色早,着色好,成熟时果面片红。果皮的红色主要由花青苷类物质的含量和成分决定的,花青苷是由一系列的酶促反应生成的次生代谢物。前人以‘国光’为对照,从生理层面上对红色芽变花青苷积累及合成途径关键酶的活性进行了研究。本文在此基础上,比较了两试材中花青苷合成相关结构基因、调节基因的表达水平以及三个调节基因的编码区,找出了引发红色突变的关键基因;并通过对关键基因MdMYB1基因组和上、下游调控序列的扩增和比对,以及MdMYB1启动子区域的甲基化状态的比较,阐明了MdMYB1表达量差异的原因。主要研究结果如下:
1.果实发育后期正常光照条件下的果实MdCHS、MdF3H、和MdUFGT表达量逐渐上升,采收前达到最大值;MdCHI、MdDFR1及MdLDOX的表达量亦逐渐上升,但在采收前略有下降;套袋处理(盛花后30天套袋,采收前30天除袋)的果实除袋前花青苷合成结构基因表达量均很低,除袋后迅速上升;与正常光照条件下的果实相比,套袋处理明显提高了MdDFR1, MdLDOX及MdUFGT的表达水平,但两不同光照处理MdCHS, MdCHI和MdF3H的表达差异不大。两种试材相对比,不同光照处理下红色芽变6个结构基因表达水平均不同程度地高于‘国光’,且位于合成途径下游的结构基因表达量差异更加明显。类黄酮合成另两条分支途径中,黄酮醇合成酶基因MdFLS的表达水平明显低于‘国光’,而单宁生物合成途径中无色花色素还原酶基因MdLAR1和MdANR的表达量在两种试材中差异不明显,仅个别阶段芽变表达量略低。
2.根据MdMYB1, MdbHLH3及MdbHLH33的cDNA序列设计特异引物,分别从‘国光’及其红色芽变中克隆出以上三个花青苷合成调节基因的编码区并进行测序比对。结果表明两种试材中MdMYB1和MdbHLH3两种调节基因的编码序列完全一致,编码结构相同的蛋白质;MdbHLH33蛋白的结构略有差异:氨基酸序列中第304位的氨基酸不同,‘国光’为甲硫氨酸(Met),而芽变中为苏氨酸(Thr)。
3.正常光照条件下,MdMYB1的表达量随着果实成熟逐渐升高,后期达到最大值并略微呈现下降趋势;套袋处理果实除袋前MdMYB1表达量很低,除袋后迅速上升。与正常光照条件下的果实相比,套袋处理明显提高了MdMYB1的表达水平。两种试材相对比,不同光照处理下芽变MdMYB1的表达量均明显高于‘国光’。MdbHLH3的表达与花青苷积累相关性不明显,MdbHLH33的表达与MdMYB1显著相关;两试材MdbHLH3和MdbHLH33的表达量无明显差异。
4.对‘国光’及其芽变MdMYB1基因组及上、下游序列进行克隆比对,琼脂糖凝胶电泳检测显示对应序列的长度无差异,说明芽变MdMYB1内含子及上、下游调节序列中无基因片段如反转录转座子等插入或缺失;对基因片段进行测序比对,也未发现两种试材中有明显差异,说明芽变MdMYB1未发生DNA水平上的变化。对两种试材MdMYB1启动子区甲基化水平进行比较,发现红色芽变-965~-416区域甲基化水平明显低于‘国光’。
综上所述,红色芽变中花青苷合成结构基因表达水平协同上调;MdMYB1表达量的升高是引起结构基因上调的关键原因;MdMYB1启动子区甲基化水平的降低是其表达上升的根本原因。
Apple (Malus×domestica Borkh) is one of several main types of fresh fruits in the world. Skin color is an important component of fruit quality and significantly influences the market value of apple fruits.‘Ralls’apple is one of the apple cultivars that widely planted in northern China in 1980s. It has the characters of late maturing, good flavors and strong stress resistance and has regained popularity with people in recent years. But poor coloration is the main limiting factor of its development. In 2005, we found a mutation which had been proved to be a bud sport of‘Ralls’. Fruits of the bud sport colored eailier and better, and were blushed-red at ripening. The degree of red coloration in apple skin is determined by the content and composition of anthocyanins, which are secondary metabolites synthesized through a series of enzymatic reactions. Previous work had studied the accumulation of anthocyanins and the activity of key enzymes in the biosynthetic pathway compared with‘Ralls’apple. In this study, we compared the transcript levels of anthocyanin biosynthetic and regulatory genes during late developing stage, cloned and compared the coding regions of MdMYB1, MdbHLH3 and MdbHLH33 to find out the key gene that caused the mutation. We also compared the genomic sequences and upstream, downstream regions of MdMYB1 and methylation status of MdMYB1 promoter region to explain the reason for the increasing of MdMYB1expression in the bud sport. The results are shown as follows:
1. Under normal sunlight condition, the transcript levels of MdCHS, MdF3H and MdUFGT in the skin tissues of fruits increased gradually during late developmental stage till harvesting, but that of MdCHI, MdDFR1 and MdLDOX peaked earlier and then declined to a lower level. The transcript levels of six anthocyanin biosynthetic genes in the fruits covered in bags were very low, but induced greatly after bag removal. Compared with those under normal sunlight, the transcript levels of MdDFR1, MdLDOX and MdUFGT were significantly improved in bagging treated fruits; but MdCHS, MdCHI and MdF3H showed little differece. Take a comparison between the two varieties, transcript levels of the biosynthetic genes in the bud sport were significantly higher than that in‘Ralls’; the differences were more obvious in late genes of the pathway. In the other two branches of flavonoid biosynthesis, the transcript levels of MdFLS in the bud sport were significantly lower than that in‘Ralls’. The transcription of MdLAR1and MdANR showed little difference between the two materials. They were a little lower in the bud sport only at one stage.
2. Specific primers were designed according to the cDNA regions of MdMYB1, MdbHLH3 and MdbHLH33. The coding regions of the three common regulatory genes were isolated from both‘Ralls’and its bud sport. Sequence comparison revealed that the coding regions of MdMYB1 and MdbHLH3 were identical between the two varieties. So the transcription factors they encode were of the same structure. The amino acid residues at the 304 position of transcription factor MdbHLH33 were different: it was Met in‘Ralls’while Thr in the bud sport.
3. Analysis of transcription of MdMYB1, MdbHLH3 and MdbHLH33 suggested that: under normal sunlight, the transcript level of MdMYB1 increased progressively during late developing stage, reached its maximum level before harvesting and then declined to a lower level. MdMYB1 transcript level in the fruits covered in bags was very low, but induced greatly after bag removal. Compared with those fruits under normal sunlight, the transcript level of MdMYB1 was greatly improved in those bagging treated fruits. Take a comparison between the two varieties, the transcript level of MdMYB1 in the bud sport was significantly higher than‘Ralls’under both treatments. The transcription profile of MdbHLH3 did not show any link with the accumulation of anthocyanin. Whereas MdbHLH33 shared the similar transcription profile with that of MdMYB1. However, both their transcript levels showed no significant differences between the two apple varieties.
4. The genomic sequences and upstream, downstream regions of MdMYB1 were isolated and compared. Analysis by agarose gel electrophoresis showed no size differences between the corresponding fragments of the two cultivars; sequence alignment also showed no significant differences between different varieties. Therefore, there were no large insertions or deletions like transposable element occurred in the DNA sequence of MdMYB1 in the bud sport. Besides,the methylation status of MdMYB1 promoter region was discovered much lower in the bud sport.
5. From the findings above, it can be concluded that the transcription of all the biosynthetic genes was coordinately up-regulated; elevation of MdMYB1 transcription was the main reason that caused the up-regulation of biosynthetic genes; the lowered methylation level of MdMYB1 promoter region resulted in the elevation of MdMYB1 transcription.
引文
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