NWIPB OpenIR
小麦品种高原115紫色籽粒中花青素合成调控机理研究
其他题名Genetic and molecular analysis of seed anthocyanin accumulation in the puple wheat variety Gaoyuan 115
刘宝龙
学位类型博士
导师张怀刚
2009-01-09
学位授予单位中国科学院西北高原生物研究所
学位授予地点西北高原生物研究所
关键词小麦 紫色籽粒 花青素 Myb Bhlh Wd40
摘要花青素是一种重要的植物次生代谢产物。在小麦中,育种家们已经选育了不同颜色的小麦(红、紫、黑、蓝),但是其色素形成的遗传机理还不完全清楚。本论文的研究目的是以紫色籽粒小麦品种高原115为主要试验材料比较系统地分析控制种子花青素积累的生理机制和遗传机理。 采用徒手切片和冰冻切片,研究了紫色籽粒小麦品种高原115花青素组织化学定位,发现高原115籽粒的花青素定位于果皮和种皮。利用HPLC技术,从高原115籽粒中分离得到五种不同的花青素类化合物。测定高原115与Opata(白粒小麦)开花后籽粒中花青素含量的变化。结果发现高原115籽粒中花青素含量高于Opata。高原115籽粒中花青素合成开始于开花后14天,到开花后28天籽粒中花青素含量达到最大。通过研究光对开花后不同时期籽粒花青素合成的影响,揭示了光在高原115紫色籽粒花青素合成中的作用。光对高原115籽粒的花青素合成具有诱导作用,并影响成熟籽粒的花青素含量。 以高原115与白粒小麦品种Opata为亲本,建立F2代分离群体,研究了紫色籽粒性状的遗传规律。结果表明高原115籽粒的紫色籽粒性状是由种子的母本基因型决定的,控制紫色花青素积累的基因呈现不完全显性遗传。但在F2/F3遗传分离群体中,籽粒(包括深紫色和浅紫色)与白粒的分离比例符合3 :1,推测紫粒性状由一个主效基因决定。 用EMS和离子束对高原115种子进行诱变,获得了10732个M2突变家系,从中鉴定出190份白粒M2突变家系,40份农艺性状优良M2突变家系,31份无芒M2突变家系,9份紧颖壳M2突变家系。EMS和离子束诱变具有剂量效应,0.3%、0.4%和0.5% EMS 诱变效率为1.2%、2.2%和2.5%;3 × 1017和4 × 1017 ions/cm2诱变率分别为1.0%和1.7%。白粒突变M2突变家系的分离规律表明,花青素合成通路上的基因的拷贝数、染色体分布和作用方式可能是多样的。 以高原115为材料,分离并鉴定了高原115紫色籽粒发育过程中调控黄酮类化合物合成的转录因子MYB、bHLH和WD40。从高原115中克隆到MYB类调控因子基因TaMYB3,定位于4B染色体,蛋白产物定位于细胞核。进化分析表明其从属于需要bHLH基因协作的花青素合成MYB基因的分支,与ZmC1亲缘关系较近,其蛋白产物具有DNA结合域和转录调控结构域。瞬时表达功能验证表明,单一的TaMYB3或玉米的ZmR(bHLH)均不能使得暗处理高原115胚芽鞘细胞产生红色的花青素。TaMYB3能与玉米的ZmR协同作用使得暗处理的高原115胚芽鞘细胞产生花青素,并且在功能上可与ZmC1相互替代。从高原115的籽粒中克隆到bHLH基因的6种剪切变异体cDNA序列,分别命名为TaMYC1-1,TaMYC1-2,TaMYC1-3,TaMYC1-4,TaMYC1-5和TaMYC1-6,相对表达丰度分别为2.0%、7.5%、86.6%、1.0%、1.0%和2.0%。除TaMYC1-4外,其它变异体的蛋白产物都具有完整的bHLH结构域并定位于细胞核。瞬时表达功能验证表明,TaMYC1-3在TaMYB3存在的前提下能够诱导Opata白色胚芽鞘细胞产生红色花青素,功能上与玉米的ZmR基因可以相互替换。单一的TaMYB3和TaMYC1-3不能诱导花青素的合成。从高原115的籽粒中克隆到TaWD401基因,其蛋白产物具有WD40结构域。半定量PCR结果表明,TaMYB3与TaMYC1的表达与开花后籽粒中花青素的合成呈正相关,并且具有受光诱导的特性。利用大麦条纹花叶病毒(BSMV)介导的基因沉默试验发现,降低TaMYB3与TaMYC1的表达显著减少了高原115籽粒花青素的合成。因此推断TaMYB3和TaMYC1参与了高原115籽粒花青素的合成,光可能是通过诱导TaMYB3和TaMYC1的表达来促进高原115籽粒花青素的合成。 白粒品种Opata中具有TaMYB3和TaMYC1基因,并且与高原115的碱基序列高度相似。TaMYB3和TaMYC1的编码区不是紫粒种子形成色素的原因。瞬时表达TaMYB3和ZmR能够使得Opata白色的胚芽鞘细胞产生红色花青素,表明白粒小麦胚芽鞘中并不缺少花青素合成的结构基因。
其他摘要Anthocyanins are important secondary plant metabolites, which can be stimulated by different environmental factors. Wheat varieties bearing colored (i.e., purple, blue, black) grains, which are caused by anthocyanin accumulation in seed tissues, have been bred and cultivated in many countries. However, the knowledge on the physiological and genetic mechanisms regulating seed anthocyanin accumulation is still not well understood. The objectives of my PhD project are to study the physiological and genetic mechanisms regulating seed anthocyanin accumulation in Gaoyuan 115, a common wheat variety accumulating purple anthocyanin pigments in its seeds. Under field conditions, seed anthocyanin accumulation started approximately 14 days after flowering (DAF) and reached maximum at 28 DAF. Seed anthocyanin pigments were predominantly located in the coat and pericarp tissues. High performance liquid chromatography (HPLC) assays showed the presence of at least five major anthocyanins in Gaoyuan 115 seeds, none of which was detected in the white grains of a control variety. Analysis of segregating genetic populations indicated that the purple grain color was controlled maternally and by a single gene. Mutational analysis, based on M2 mutant populations generated with chemical and radiation mutagenesis, suggested the potential involvement of multiple genes in synthesizing the purple pigments in Gaoyuan 115 kernels. Further examinations of the obtained mutants reveal significant difference in the genetic controls of anthocyanin accumulation in the seed and coleoptile tissues of Gaoyuan 115. Despite such difference, light was essential for anthocyanin biosynthesis in both organs. Attempts were also made to isolate MYB、bHLH and WD40 genes that are known to control anthocyanin accumulation in plant tissues using a homologous cloning strategy. TaMYB3, showing high nucleotide similarity with the C1 gene involved in controlling anthocyanin accumulation in maize seeds, was isolated and mapped to wheat chromosome 4B. Using transient expression assays, TaMYB3 was found to direct anthocyanin biosynthesis in plant cells in the presence of maize R gene, which has been shown to cooperate with the C1 gene to control anthocyanin accumulation in purple colored maize seeds. A bHLH gene showing significant nucleotide sequence similarity the maize R gene was isolated, which produced multiple types of splice variants (tentatively named as TaMYC1-1, TaMYC1-2, TaMYC1-3, TaMYC1-4, TaMYC1-5 and TaMYC1-6) in Gaoyuan 115 seeds. Except for TaMYC1-4, the remaining splice forms were all capable of encoding putative proteins with intact bHLH domain. Random sequencing of cloned PCR products indicated that TaMYC1-3 was the main species, whose protein product was located in the nucleus in protoplasts. In transient expression experiments, TaMYC1-3 stimulated anthocyanin biosynthesis in the presence of TaMYB3 or ZmR, indicating that the protein product of TaMYC1-3 is likely to be functional in Gaoyuan 115 seed tissues. Light increased the transcript levels of TaMYB3 and TaMYC1-3, which correlated with enhanced anthocyanin accumulation in Gaoyuan 115 seeds. Reducing the transcript levels of TaMYB3 or TaMYC1 through virus induced gene silencing (VIGS) based on barley stripe mosaic virus vector decreased seed anthocyanin accumulation in Gaoyuan 115 developing seeds, confirming the important roles of the two genes in directing anthocyanin biosynthesis in the purple grains of Gaoyuan 115. Interesingly, TaMYB3 and TaMYC1 genes were also present in the seed tissues of the white grained wheat variety Opata, suggesting that the lack of anthocyanin accumulation in white grained wheat varieties is not due to the absence of MYB and MYC regulatory genes and those involved in the bisosynthetic pathway of anthocyanins.
页数95
语种中文
文献类型学位论文
条目标识符http://210.75.249.4/handle/363003/3148
专题中国科学院西北高原生物研究所
推荐引用方式
GB/T 7714
刘宝龙. 小麦品种高原115紫色籽粒中花青素合成调控机理研究[D]. 西北高原生物研究所. 中国科学院西北高原生物研究所,2009.
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