【作者】 牛姗姗；

【导师】 陈昆松； 徐昌杰；

【作者基本信息】 浙江大学 ， 果树学， 2011， 博士

【摘要】 以‘荸荠’(Myrica rubra cv. Biqi)、‘东魁’(M. rubra cv. Dongkui)和‘水晶’(M.rubra cv. Shuijing)三种不同果实颜色的杨梅品种为试验材料,分析了花青素苷与果实色泽之间的关系,克隆得到花青素苷生物合成途径的结构基因和MYB转录因子,采用qPCR技术分析上述基因在不同品种杨梅果实、植株组织器官、果实发育阶段和果实套袋等处理中的表达模式,并开展了转录因子MYB与花青素苷生物合成途径相关编码基因的互作模式研究。通过烟草瞬时表达体系,验证了MYB转录因子的功能,明确了其对花青素苷生物合成途径相关基因的调控模式。主要结果如下：1、‘荸荠’杨梅果实中花青素苷总量最高,为85.40 mg/100g FW；‘东魁’杨梅果实中花青素苷总量为46.44 mg/100g FW；‘水晶’杨梅果实中检测不到花青素苷含量,三个品种对应的CIRG值分别为7.56、5.27和3.84。分析‘荸荠’杨梅不同组织器官中花青素苷含量,发现叶片中未检测到花青素苷,幼根和幼茎中含有少量的花青素苷,果实的花青素苷总量最高。‘东魁’和‘荸荠’杨梅64DAFB采收的果实中均未检测到花青素苷,随着果实的发育,CIRG值和花青素苷总量呈上升趋势。‘荸荠’杨梅套袋果实中的花青素苷总量仅为0.56 mg/100g FW,未套袋果实中花青素苷总量为108.99 mg/100g FW；未套袋果实CIRG值约为套袋果实的两倍。2、从成熟的‘荸荠’杨梅果实中克隆得到MrACT (227 bp)、MrCHS (440 bp)、MrCHI (301 bp)、MrF3H (436 bp)、2个DFRs (MrDFR1,221 bp; MrDFR2,221 bp)、MrANS (428 bp)、MrUFGT基因(647 bp)和2个R2R3 MYB基因(MrMYB1和MrMYB2)。从杨梅EST数据库中得到了MrF3’H基因(412 bp)和1个MrMYB3基因(514 bp)。MrCHS和MrF3H基因与其他物种中对应基因的序列同源性高达90%, MrDFR1基因与拟南芥中对应基因的同源性为83%,而MrUFGT基因与其他物种的对应基因的序列同源性介于51%和70%之间,其余基因与其他物种对应基因的序列同源性则介于68%和82%之间。3、采用RACE技术扩增非编码区序列,得到MrMYB1序列(966 bp)和MrMYB2序列(996 bp),其中MrMYBl为全长。MrMYB1和MrMYB2与拟南芥中R2R3 MYB基因家族成员的序列同源性较高,接近70%,而MrMYB3与拟南芥的MYB同源性仅为55%。MrMYB1和MrMYB2蛋白的序列同源性较高,均含有R2R3结构域。4. MrCHS和MrCHI在三个不同品种杨梅果实中表达相似,而其他基因在两个有色品种中的表达水平都高于‘水晶’杨梅,MrF3H、MrF3’H, MrDFR1和MrUFGT基因的转录水平与花青素苷含量呈正相关关系。在‘荸荠’杨梅的不同组织器官中,除MrCHI在幼根中表达水平最高之外,其余基因均在成熟果实中表达水平最高,其中MrUFGT基因仅在成熟果实中表达。在‘东魁’和‘荸荠’杨梅果实的不同生长发育阶段,除MrCHI和MrDFR2外,其他结构基因的表达水平和花青素苷总量成正比,且在‘荸荠’果实中的表达水平要高于‘东魁’果实。套袋处理明显抑制花青素苷生物合成途径中的结构基因表达。5、三个不同品种的杨梅果实中转录因子MrMYB1的表达水平与花青素苷总量呈正相关关系。转录因子MrMYB1只在成熟‘荸荠’果实中表达,而不在根茎叶中表达。‘东魁’和‘荸荠’杨梅的发育过程中,转录因子MrMYB1的表达逐渐增强,并在发育后期达到峰值。在套袋果实中转录因子MrMYB1的表达被显著抑制,表明光照可能通过影响MrMYB1表达进而影响果实花青素苷积累。6、烟草瞬时表达试验的结果表明,过量表达MrMYB1基因诱导烟草叶片中花青素苷积累。MrMYB1协同bHLH转录因子可激活AtDFR启动子,表明MrMYB1转录因子调控花青素苷生物合成途径中结构基因的表达,从而影响花青素苷合成。7、从‘水晶’、‘东魁’和‘荸荠’杨梅果实中分别克隆得到各自的MrMYBl全长序列。发现‘水晶’杨梅的MrMYB1(命名为MrMYB1d)在ATG起始密码子后第30位上缺失了一个胞嘧啶,导致移码突变,不能合成正常的MYB1蛋白；‘荸荠’杨梅仅含正常功能的MrMYB1基因,‘东魁’杨梅则同时含有MrMYB1和MrMYB1d基因。上述结果表明,MrMYB1调控杨梅花青素苷合成途径基因的协同表达。更多还原

【Abstract】 Studies of anthocyanins biosynthesis pathway were carried out using Chinese bayberry (Myrica rubra Sieb. et. Zucc.) cultivars’Biqi’,’Dongkui’and’Shuijing’. The relationship between the anthocyanins and the colour of bayberry fruit was analyzed. Structural genes in anthocyanins biosynthesis and MYB transcription factors (TFs) were cloned. Their expression modes in the fruit and pulp of the different cultivars, in different tissues and organs, in different developmental stages and in bagged treatment were analysed by qPCR. Research on reciprocal action mode between MYB and the coding genes in anthocyanins biosynthesis was carried on. The function of MYB transcription factors and their regulation modes on the genes in anthocyanins biosynthesis were confirmed by transient tobacco expression system. The main results are listed as follows:1. The highest anthocyanins content was found in the fruit of’BQ’, which was 85.40 mg/100g FW, and the content in the fruit of’DK’was 46.44 mg/100g FW, while no anthocyanin was tested in the fruit of’SJ’. The CIRG values were 7.56,5.27 and 3.84 in ’BQ’,’DK’and’SJ’, respectively. In the analysis of anthocyanin contents in different tissues and organs in’BQ’, little anthocyanin was tested in root and young stem, on the contrary, anthocyanin content in the fruits was the highest. In the fruits of the three cultivars on 64d DAFB, no anthocyanin was detected in the fruit of’DK’and’BQ’. The anthocyanin contents and CIRG values increased with the development of fruits. For ’BQ’, the anthocyanin content in bagged fruit was only 0.56 mg/100g FW, however, the anthocyanin content in non-bagged fruit was 108.99 mg/100g FW. The CIRG values of the non-bagged fruits were about two fold of those bagged ones.2. From the mature fruit of’BQ’, we cloned ten genes, i.e. Mr ACT (227 bp), MrCHS (440 bp), MrCHI (301 bp), MrF3H (436 bp), two DFR members (MrDFR1 221 bp and MrDFR2 221 bp), MrANS (428 bp), MrUFGT (647 bp)and two R2R3 MYB genes (MrMYB1 and MrMYB2). From EST database of bayberry, we obtained two genes, MrF3’H (412 bp) and MrMYB3 (514 bp). The homology of MrCHS and MrF3H with the counterpart genes in other species was as high as 90%. MrDFR1 showed a similarity of 83% with the counterpart genes in Arabidopsis thaliana. However, the similarities between MrUFGT and the counterpart genes in other species ranged from 51% to 70%. The rest genes had similarities from 68% to 82% compared with counterpart genes in other species.3. By 3’RACE (for both members)and 5’RACE (for Mr MYB1 only),966 bp and 996 bp fragments were obtained and named MrMYB1 and MrMYB2. MrMYB1 was a full length fragment. MrMYB1 and MrMYB2 showed a similarity up to 70% with the gene family of R2R3 MYB in Arabidopsis thaliana. Wheras, MrMYB3 showed only a similarity of 55% to AtMYB. MrMYB1 and MrMYB2, all of which contained structural domain of R2R3, showed a high identity of amino acid sequence.4. The expression of MrCHS and MrCHI were similar in fruits of the three cultivars, while that of other genes was higher in the two colored cultivars than those in’SJ’. The transcriptional levels of MrF3H, MrF3’H, MrDFRl and MrUFGT were directly associated with anthocyanin content. In different tissues and organs of’BQ’, except for MrCHI whose highest expression level was found in root, the highest expression levels of all other genes were found in ripe fruits. Besides, MrUFGT was expressed exclusively in ripe fruits. In fruits of different developmental stages of’DK’and’BQ’, except for MrCHI and MrDFR2, expressions of all other structural genes directly associated with the total content of anthocyanin, and the expression levels in’BQ’fruits were higher than those of’DK’fruits. Bag treatment significantly inhibited the expression of structural genes in anthocyanin biosynthesis.5. The transcriptional level of MrMYB1 directly associated with the total content of anthocyanin. MrMYB1 was exclusively expressed in ripe fruits of’BQ’, not in root, stem and leaf. The expressions of MrMYB1 in’DK’and’BQ’increased along with the fruit development and reached the summit in the late developmental stage. The expression of MrMYB1 in bagged fruits was significantly inhibited, suggesting that light could affect the anthocyanin accumulation by MrMYBl expression.6. The results of tobacco transient expression showed that over-expression of MrMYBl resulted in the accumulation of anthocyanin. MrMYBl coordinated with bHLH could activated AtDFR promoters, indicating that MrMYB1 regulated the expressions of structural genes in the pathway of anthocyanin biosynthesis, which further affected the biosynthesis of anthocyanin.7. The full length sequences of MrMYB1 were separately cloned from the fruits of ’BQ’,’DK’and’SJ’. A cytosine was lost from the+30 position after the start codon of ATG in MrMYB1 (named MrMYB1d) of’SJ’, resulting in frameshift mutation, which leaded to failing in normal protein synthesis of MYB1. MrMYB1 gene was normal in’BQ’, otherwise, MrMYB1 and MrMYB1d genes were all normal simultaneously in’DK’. Probably, the sequence mutation resulted in which anthocyanin was not able to biosynthesis in fruits of’SJ’.In conclusion, MrMYB1 regulated the expressions of structural genes in the pathway of anthocyanin biosynthesis in Chinese bayberry.更多还原