【作者】 宿福园；

【导师】 罗正荣；

【作者基本信息】 华中农业大学 ， 果树学， 2012， 博士

【摘要】 柿(Diospyros kaki Thunb.)原产于中国,栽培面积和产量均居世界第一,但传统产区多为涩柿,人工脱涩处理耗费大量人力、物力和财力。柿果呈现涩感是因其果肉中单宁细胞的液胞内含有大量的原花青素(Proanthocyanidins, PAs;也称缩合单宁,condensed tannin)之故。单宁作为类黄酮代谢途径的终产物之一,其生成途径有众多结构基因参与,而结构基因的表达又受转录因子调控。已有研究表明,MYB、basic Helix-Loop-Helix (bHLH)和WD40三类转录因子构成MBW复合体,共同调控单宁的代谢途径。但中国原产完全甜柿自然脱涩是否与上述转录因子有关目前尚不清楚。本研究以中国原产完全甜柿‘罗田甜柿’为主要研究试材,分离克隆了MYB、bHLH和WD40三类转录因子,开展了柿单宁代谢调控相关研究工作。具体研究结果如下。1.发育期柿果实单宁含量测定。在花后5周,供试材料中单位果重内可溶性与不溶性单宁含量均最高,并随果实发育而逐渐减少,到花后13周,‘前川次郎’(日本原产完全甜柿)和‘西村早生’(日本原产不完全甜柿)果实已完成脱涩,而‘罗田甜柿’(中国原产完全甜柿)果实在花后21周后脱涩,‘磨盘柿’(中国原产完全涩柿)不能在树上完成脱涩。‘前川次郎’和‘西村早生’单果的单宁含量在果实发育早期停止增长,而‘罗田甜柿’和‘磨盘柿’的单宁累积持续到果实发育后期。2.‘罗田甜柿’乙醇脱涩处理后单宁含量变化。花后17周,对‘罗田甜柿’进行离体35%乙醇脱涩处理,处理两天后,果实脱涩,可以观察到单宁细胞颜色明显加深,果实中可溶性单宁含量显著减少,同时不溶性单宁含量相应增加,可溶性单宁转变为不溶性单宁,果实通过“凝固效应”而脱涩。3.柿MYB基因克隆及分析。以拟南芥和葡萄MYB转录因子序列为信息探针,在柿EST数据库中进行BLAST分析,找到一个候选片段DC587740,随后在‘罗田甜柿’中扩增出其片段,通过3’RACE和5’Hi-TAIL PCR扩增得到其全长序列1152bp,其中ORF长843bp,编码280个氨基酸,将该基因命名为DkPAl,序列已提交GenBank。DkPA1具有MYB转录因子的典型结构域,属于R2R3类MYB转录因子,通过邻接法构建进化树,发现其与已报道的其他物种中参与单宁调控MYB转录因子具有极高的序列相似性。DkPA1在‘罗田甜柿’与‘磨盘柿’中的表达模式与两个材料中单宁的累积模式一致,与结构基因DkDHD、DkSCPL、DkF3’H、DkF3’5’H、DkANR的表达模式也一致。4.柿bHLH基因克隆及分析。以拟南芥bHLH转录因子AtTT8为信息探针,检索柿EST数据库,找到一个候选片段DC587220,在‘罗田甜柿’中扩增出其片段,通过3’RACE和5’Hi-TAILPCR扩增得到其全长序列2687bp,ORF长2160bp,编码719个氨基酸,将该基因命名为DkMYC1,序列已提交GenBank。DkMYC1且有bHLH家族典型的碱性螺旋-环-螺旋结构,属于bHLH转录因子Ⅲf家族成员,与葡萄和拟南芥中参与单宁代谢调控的bHLH转录因子具有极高的序列相似性。在‘罗田甜柿’、‘前川次郎’和‘磨盘柿’中,DkMYC1的表达模式与三个材料中单宁的累积模式一致,也与DkANR、DkF3’5’H等结构基因的表达模式一致。在‘罗田甜柿’中分离得到单宁代谢途径两个特异基因DkLAR和DkANR启动子序列,发现在DkANR的启动子区具有多个bHLH转录因子的关键调控元件MYCATERD1及MYCCONSENSUSAT,进一步确定其受DkMYC1调控。5.柿WD40基因克隆及分析。通过同源克隆法设计简并引物,在‘罗田甜柿’中扩增出WD40基因片段,通过3’RACE和5’Hi-TAIL PCR扩增到全长序列1640bp,ORF全长1014bp,编码337个氨基酸,将该基因命名为DkWD40,序列已提交GenBank。DkWD40具有4个典型WD40结构域,与葡萄VvWDR1相似性最高。在发育期的果实中,DkWD40的表达与单宁代谢途径中结构基因(DkDHD、DkSCPL、 DkF3’H、DkF3’5’H、DkANR)的表达并不十分相符,而与单宁凝固基因CDkADH1)相符。在乙醇脱涩处理果实中,DkWD40也与单宁凝固基因表达一致。将DkWD40构建超表达载体打入农杆菌,并侵染拟南芥花序,转基因后的拟南芥种皮颜色为黑褐色。6.组织特异性表达分析。DkPA1、DkMYC1和DkWD40在四种供试材料茎、叶、花、萼片、果皮、果肉和种子中均有表达,并非果实特异表达基因,在不同品种的不同部位,其丰度不同,在完全涩柿中表达量高于完全甜柿,且在幼嫩的叶和萼片中表达量较高,在果实中的表达量高低与果实中单宁含量成正相关。7.‘华柿1号’种质鉴定。通过测定‘华柿1号’果实单宁含量及单宁细胞大小,观察与统计果实及种子情况,结合RAPD和ISSR等多种分子标记鉴定结果,推测‘华柿1号’是一个起源于日本的不完全涩柿种质,其在柿的遗传改良中可能具有重要价值。本研究从中国原产完全甜柿‘罗田甜柿’中分离到MYB转录因子基因DkPAl, bHLH转录因子基因DkMYCl以及WD40转录因子基因DkWD40,通过在不同脱涩类型的柿品种果实发育期自然脱涩过程及乙醇脱涩处理前后果实中进行实时定量表达分析,结合单宁含量测定及单宁代谢结构基因表达情况,推测DkPAl和DkMYCl直接参与柿果实发育期单宁代谢调控,而DkWD40参与可溶性单宁向不溶性单宁转化的脱涩过程。更多还原

【Abstract】 Persimmon(Diospyros kaki Thunb.) originates in China, and China is the largest cultivator and producer worldwide. But an overwhelming amount of persimmons cultivated in China are astringent which take a lot of money and labors to de-astringency and process before eating every year. Persimmon fruit is astringent because it accumulates abundant proanthocyanidins (PAs, also called condensed tannin) in its fruit cells. Tannin, as one of the final products of flavonoid biosynthesis pathway, its biosynthesis pathway was controlled by a series of structural genes, and expressions of these structural genes were controlled by transcription factors (TFs). As it was reported in many researches, regulation of the structural gene expression in the flavonoid pathway was orchestrated by a ternary complex MYB-bHLH-WD40(MBW), composing of transcription factors from R2R3-MYB, MYC Like basic helix-loop-helix (bHLH), and WD40classes. In this research, we have isolated MYB, bHLH and WD40transcription factor genes from sweet persimmon’Luotiantianshi’which originated in China and done some tannin biosynthesis regulation studies.1. Tannin content analysis in developing persimmon fruits. The materials used in the research was’Luotiantianshi’(pollination-constant and nonastringent originated in China, C-PCNA),’Maekawa-Jiro’(PCNA originated in Japan, J-PCNA),’Nishimura-wase’(pollination-variant and nonastringent originated in Japan, J-PVNA) and’Mopanshi’(pollination-constant and astringent originated in China, C-PCA). In the four materials included, soluble tannin contents were at the highest level at5weeks after full bloom (WAB), and reduced with fruits development. At13WAB’Maekawa-Jiro’and ’Nishimura-wase’almost lost their astringency.’Luotiantianshi’lost its astringency after21WAB, and’Mopanshi’could not lose astringency naturally on the tree. The tannin stopped accumulation in’Maekawa-Jiro’and ’Nishimura-wase’at early stage of fruits development, but in’Luotiantianshi’and’Mopanshi’tannin accumulation lasted to the late stage of fruits development. 2. Tannin content analysis in fruits of’Luotiantianshi’treated with ethanol.’Luotiantianshi’fruits were treated with35%ethanol in vitro at17WAB. On the second day after treatment, the fruits lost astringency and the tannin cell color darkened. Soluble tannin content decreased dramatically; meanwhile insoluble tannin content increased correspondingly. The soluble tannin changed to insoluble tannin which was a sign that the fruits lost astringency by "coagulation effect"3. Molecular cloning and analysis of persimmon MYB gene. The sequences of Arabidopsis and grape MYB transcription factors were used as query sequences against D. kaki ESTs database using BLAST analysis. One significant match DC587440was found. The sequence was subsequently amplified from’Luotiantianshi’. After3’RACE and5’Hi-TAIL PCR amplification, the full length1152bp was obtained which had an ORF of843bp, encoding280amino acid residues. It was named as DkPA1and the sequence was submitted to GenBank. DkPA1had two conserved MYB domains and belonged to R2R3MYB transcription factors. Phylogenetic analysis showed DkPA1shared high similarity with MYB transcription factors from other species which were reported to be involved in tannin biosynthesis regulation. In quantitative real-time PCR analysis (qRT-PCR), expression pattern of DkPAl was correlated with tannin accumulation in’Luotiantianshi’ and’Mopanshi’; also coincided with expression of flavonoid biosynthesis structural genes DkDHD, DkSCPL, DkF3’H, DkF3’5’Hand DkANR.4. Molecular cloning and analysis of persimmon bHLH gene. The sequence of Arabidopsis bHLH transcription factor AtTT8was used as a query sequence to probe the D. kaki ESTs database. One significant match DC587220was found. The sequence was subsequently amplified from’Luotiantianshi’. After3’RACE and5’Hi-TAIL PCR amplification, the full length2687bp was obtained which had an ORF of2160bp, encoding719amino acid residues. It was named as DkMYC1and the sequence was submitted to GenBank. DkMYC1had the typical basic Helix-Loop-Helix domain and belonged to the bHLH subgroup Ⅲf. Phylogenetic analysis showed DkMYCl shared high similarity with bHLH transcription factors from Arabidopsis and grape which were reported to be involved in tannin biosynthesis regulation. In qRT-PCR analysis, expression pattern of DkMYC1correlated with tannin accumulation in’Luotiantianshi’,’Maekawa-Jiro’and’Mopanshi’, also coincided with expression of flavonoid biosynthesis structural genes DkANR and DkF3’5’H. Promoters of DkLAR and DkANR were isolated from’Luotiantianshi’, and several bHLH-binding cis-motifs (MYCATERD1and MYCCONSENSUSAT) were found in the promoter region of DkANR, which further supported that it was regulated by DkMYC1.5. Molecular cloning and analysis of persimmon WD40gene. Based on homology cloning method, we isolated a WD40fragments from’Luotiantianshi’. After3’RACE and5’Hi-TAIL PCR amplification, the full length1640bp was obtained which had an ORF of1014bp, encoding337amino acid residues. It was named as DkWD40and the sequence was submitted to GenBank. DkWD40had four conserved WD40repeat domains. Phylogenetic analysis showed DkWD40shared high similarity with WD40transcription factors from grape which was reported to be involved in tannin biosynthesis regulation. DkWD40showed coincident expression pattern with genes involved in tannin coagulation, but it didnot coincide very well with structural genes (DkDHD, DkSCPL, DkF3’H, DkF3’5’H and DkANR) in tannin biosynthesis during fruits development. In ethanol treatment, DkWD40also showed similar expression pattern with DkADHl which involved in tannin coagulation. When DkWD40overexpressed in Arabidopsis, seed coat where tannin concentrated was black brown.6. Tissue specific expression analysis. Expressions of DkPAl、DkMYCl and DkWD40were analysed in stem, leaf, flower, calyx, fruit peel, flesh and seed of four materials included. The results showed the three transcription factor genes expressed in almost all the organs and tissues included, rather than fruits specific expressed.7. Identification of’Huashi1’.’Huashi1’has been cultivated as a sweet persimmon. However its origin and astringent type was not clear. In this research, the tannin content, tannin cell size and fruits characters were measured. Combined with molecular markers amplification results, it was confirmed that’Huashi1’was a PVA type persimmon originated in Japan and it had big potential in persimmon breeding. In this research, MYB transcription factor gene DkPAl, bHLH gene DkMYC1and WD40gene DkWD40were isolated from’Luotiantianshi’(pollination-constant and nonastringent originated in China, C-PCNA). Expressions of the three genes were analyzed in developing persimmon fruits with different astringent type, and in ethanol treated persimmon fruits. Combined with tannin content analysis and expression of structural genes, it is inferred that DkPA1and DkMYC1were directly involved in tannin metabolism of developing persimmon fruits. However, DkWD40were involved in soluble tannin converting to insoluble tannin.更多还原