【作者】 卢其能；

【导师】 杨清；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 马铃薯（Solanum tuberosum）是仅次于小麦、玉米和水稻的第四大粮食作物。其块茎是繁殖器官，也是产品器官。块茎有不同颜色，如白色、红色和紫色。花色苷是有色块茎颜色的决定因子，作为天然色素，花色苷可应用于食品、医药和化妆品等行业。同时，它被证明具有抗癌、抗氧化和预防心血管疾病的作用。因此，近年来有关花色苷的生化与分子生物学研究，引起了全世界科学家的广泛关注和浓厚兴趣。花色苷的生物合成途径是被最为广泛而深入研究的植物次生代谢途径，特别在主要模式植物中，已经有了清楚的认识。一些花色苷生物合成途径中的关键酶基因，已经从马铃薯栽培种（S．tuberosum L．）中克隆到，如CHS基因、F3H基因、DFR基因和F3’5’H基因，而在马铃薯野生种（S．pinnatisectum）中，还未见到花色苷生物合成酶基因克隆及其表达研究的报道。本研究以野生种为材料，克隆了CHS、F3H、DFR和3GT四个基因，分析了这四个基因的表达情况，通过转基因对3GT基因进行了功能验证；以马铃薯栽培种（S．tuberosum cv．Chieftain）为材料，研究了CHS、F3H、F3’5’H、DFR和3GT五个基因的组织表达和诱导表达。研究的主要结果如下：1．马铃薯花色苷的种类、含量和稳定性及影响其生物合成的因素用1％（v／v）盐酸甲醇溶液分别从红色和紫色马铃薯中提取色素，用正已烷除杂，再经薄层层析（TLC）纯化后，在紫外-可见分光光度计下扫描。根据提取液特性、Rf值和紫外-可见光谱特点，参考已有相关资料，初步判断马铃薯紫色色素主要为锦葵素的衍生物，而红色色素主要为天竺葵素衍生物。紫色马铃薯的花色苷含量是红色马铃薯花色苷含量的2.9倍。光、热和pH值对花色苷的稳定性有显著影响，但紫色马铃薯花色苷的稳定性好于红色马铃薯花色苷。愈伤组织来源于马铃薯品种Chieftain，在红色愈伤组织中，低浓度的2，4-D有利于花色苷的积累，高浓度的2，4-D促进愈伤组织的生长而不利于花色苷的积累；高浓度的6-BA能促进红色愈伤组织中花色苷的积累和诱导白色愈伤组织合成花色苷，但抑制生长；卡那霉素能使白色愈伤组织变红并积累花色苷，随着卡那霉素浓度的提高，愈伤组织生长受到严重抑制并最终变褐死亡；提高蔗糖浓度能促进马铃薯愈伤组织花色苷的产生和积累而抑制生长。2．马铃薯野生种花色苷生物合成相关基因的克隆与序列分析设计简并引物，通过RT-PCR方法从马铃薯野生种（S．pinnatisectum）紫色芽的cDNA中克隆到了CHS、F3H、DFR和3GT基因的全长cDNA。序列分析表明，以上四个基因分别编码389、358、382和448个氨基酸残基的多肽，在氨基酸水平上与茄科植物相应多肽的同源性最高，达到76-96％；多重比较和系统发育分析表明它们分别属于各自基因家族中的一员。3．马铃薯野生种3GT基因的功能验证为了验证马铃薯野生种3GT基因的功能，构建带CaMV 35S启动子的表达载体pG3GT，转化农杆菌GV3101，用茵液浸泡花序法对拟南芥进行遗传转化，在含50mg／LKan的1／2MS培养基上筛选，得到4个抗性幼苗，转化率为0.13％，对移栽成活的3株进行PCR检测为阳性，Southern blot分析2株表现为阳性并为单拷贝整合。其中一株的叶片和茎杆颜色变成紫红色，经花色苷含量的测定表明其含量比野生型植株高出7.01倍，从而初步验证了3GT基因的功能和活性。4．马铃薯花色苷生物合成相关基因的表达分析用RT-PCR分析了马铃薯野生种的CHS、F3H、DFR和3GT基因的空间表达情况，这些基因主要在花、匍匐茎和顶芽中表达，除3GT基因外，其它3个基因在根中的表达没有检测到，CHS在根和块茎中没有检测到。在马铃薯野生种的白色块茎中这些基因在光诱导之前表达量很低或不表达，但在光照之后表达量显著增加，随着光照时间的延长，白色块茎变为紫红色，花色苷大量积累。用RT-PCR分析了CHS，F3H，DFR、F3’5’H和3GT五个基因在马铃薯栽培种Chieftain植株中的表达情况，结果表明，这些基因在匍匐茎的表达量最高，这与其花色苷积累量也较高是相一致的。在块茎中五个基因均有表达，但表达量相对较低，这可能与花色苷仅在红色表皮组织中合成而在占大部分体积的白色薯肉中不合成有关。在附加6-BA为2mg／L、2，4-D为0.5 mg／L的MS培养基上，分离到绿色、白色和红色三种愈伤组织（细胞系），三种愈伤组织在叶绿素和花色苷的含量上显著不同，其中绿色和白色愈伤组织中几乎不含花色苷，而红色愈伤组织则大量积累花色苷。对CHS、F3H、DFR、F3’5’H和3GT五个花色苷生物合成相关基因分析表明，绿色和白色愈伤组织不合成花色苷主要是由于DFR基因不表达造成的。稀土元素铈对马铃薯悬浮培养的愈伤组织中花色苷积累及其生物合成基因的表达有显著影响。研究表明，低浓度Ce⁴⁺能促进细胞和愈伤组织的生长，而高浓度Ce⁴⁺则抑制生长并导致细胞死亡。不同浓度的Ce⁴⁺能诱导正常的愈伤组织变红并积累花色苷。RT-PCR分析表明，用0.1 mM Ce⁴处理悬浮培养的愈伤组织能显著诱导以上五个基因的表达，其表达量与花色的含量呈协同关系。更多还原

【Abstract】 Potato （Solanum tuberosum） is the forth food crop following wheat, maize and rice inthe world. Potato tubers are multiplicative organs as well as product organs, which there aredifferent colors, e.g. white, red and purple. Anthocyanins are key factors that determinetuber color. As natural pigments, they have been used in foods, pharmaceuticals andcosmetics due to their function of anti-cancer, cardioprotection and antioxidization. So inrecent years, scientists have showed a great interest in biochemical and molecularbiological investigation of anthocyanin biosynthesis all over the world.The biosynthetic pathway of anthocyanins is one of the most extensively studiedpathways of plant secondary products, and it is clearly elucidated in some model organism.A few genes of anthocyanin synthesis have been cloned from potato cultivars （S. tuberosumL.）, e.g., chalcone synthase （CHS）, dihydroflavonol 4-reductase （DFR）, flavanone3-hydroxylase （F3H）, and flavonoid 3’, 5’-hydroxylase （F3’5’H） genes. However, noreports in this aspect have been seen until now in wild potato species. In this research,cDNAs encoding CHS, F3H, DFR and 3GT were isolated from S. pinnatisectum, and theirexpression was analyzed. The function of 3GT was basely confirmed by transgenic plant.The expression of CHS, F3H, F3’5’H, DFR and 3GT genes from S. tuberosum cv. Chieftainwas investigated. The main results were showed as following:1. Preliminary studies on the categories, contents of skin color pigments and effectsof growth regulators, antibiotic and sucrose on anthocyanin synthesis in potatoes.The skin color pigments of potatoes were extracted in 1％HC1 （v/v） in methanol fromred and purple tubers, respectively. The extract was separated with hexane, purified by ThinLayer Chromatography （TLC）. The Rf and UV-Vis spectra analysis indicated that the redpigments were primarily anthocyanin pelargonins and the purple pigments were primarilyanthocyanin petunins. The content of anthocyanins in purple tubers were 2.9 times of thatin red tubers. The stability of anthocyanins was significantly influenced by light, heat andpH value, but the anthocyanins from purple tubers were more stable than that from red tubers.The callus originated from the explants of S. tuberosum cv. Chieftain. The lowercontents of 2,4-D promote accumulation of anthocyanins in red callus, the higher contentsof 2,4-D stimulated callus growth and inhibited anthocyanin production in red callus. Thehigher contents of 6-BA promoted accumulation of anthocyanins in red callus, inducedanthocyanin production in white callus and inhibited callus growth. The white callus couldbe induced to turn red and accumulated anthocyanins by different concentrations ofkanamycin, while the higher contents of kanamycin inhibited callus growth and turnedbrown to lead callus dead eventually. The elevated contents of sucrose could stimulateanthocyanin production and inhibite callus growth.2. Cloning and sequence analysis of anthocyanin biosynthetic genes in wild potatospecies （S. pinnatisectum）.Four complete-length cDNAs encoding CHS, F3H, DFR and 3GT were isolated fromthe sprouts by RT-PCR with the degenerated primers. The sequence analysis indicated thatCHS, F3H, DFR and 3GT cDNAs encoded the polypeptide of 389, 358, 382 and 448 aminoacid residues, respectively. Sequences of the encoded polypeptide comparison showed thatthey shared 76-96％identities with each corresponding solanaceous proteins reportedpreviously. The multiple alignment and phylogenetic analysis demonstrated that each genewas a member of a multigene family.3. The function of 3GT gene was confirmed by Agrobacterium-mediatedtransformation of arabidopsis thaliana.Arabidopsis thaliana was transformed by floral dip method with AgrobacteriumGV3101 carrying expression vector pG3GT. Four transformants were selected for theirgrowth ability on 1/2MS medium containing 50mg/L kanamycin. The frequency oftransformation was 0.13％. PCR analysis confirmed that three survival transformants werepositive. Southern blot confirmed that two transformants were positive, in which only onecopy of 3GT gene was detected. Among the transformants, there was one whose stem andleaves turn purple. The analysis showed that the content of anthocyanin was 7.01 times ofwild type. These results indicated that 3GT gene normally expressed in the transformant.4. Expression analysis of anthocyanin biosynthetic genes in potatoes.The spatial expression analysis of CHS, F3H, DFR and 3GT genes in wild potatospecies （S. pinnatisectum） indicated that these genes were preferentially expressed inflowers, stolons and terminal buds. In roots, and their transcripts could not detected except 3GT. The expression of CHS was not detected in tubers. In white tubers the genes wereexpressed a little or not, but after receiving light all the genes were induced to express intuber skins and the expression level increased greatly. The color of tubers changed fromwhite into purple with the prolongation of lighting.The spatial expression patterns of CHS, F3H, DFR, F3’5’H and 3GT genes in potatocultivar （S. tuberosum cv. Chieftain） were examined by RT-PCR. The expression analysisindicated that the expression of the genes was higher in stolons, and lower in tubers androots.The green, white and red callus were isolated from the explants of S. tuberosum cv.Chieftain on the MS medium containing 6-BA 2mg/L and 2,4-D 0.5 mg/L. The contents ofchlorophyll and anthocyanins were significantly different in the three different callus. Therewere few anthocyanins in green and white callus, but a great deal of anthocyanins in redcallus. The expression analysis of CHS, F3H, DFR, F3’5’H and 3GT genes in threedifferent callus were performed by RT-PCR. The results showed that no DFR transcriptswere detected in green and white callus to lead no anthocyanin accumulated in them.The effects of cerium on callus growth, anthocyanin content and expression ofanthocyanin biosynthetic genes in callus suspension cultures of S. tuberosum cv. Chieftainwere studied. The results indicated that 0.1 mmol L^-1 Ce⁴⁺ could promote callus growth,increase accumulation of anthocyanins, and enhance expression of five anthocyaninbiosynthetic genes （CHS, F3H, F3’5’H, DFR and 3GT） most efficiently. But higherconcentration (1mmol·L^-1) of Ce⁴⁺ inhibited partially callus growth and 2 mmol·L^-1 of Ce⁴⁺caused cell death eventually. The results revealed that Ce4^could induce the expression ofanthocyanin biosynthetic genes and accumulation of anthocyanins.更多还原