紫花椰菜MYB-TT8-TTG1转录因子调控烟草花青素积累的研究

发布时间：2018-03-22 14:12

本文选题：紫花椰菜　切入点：烟草　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：花青素是一类黄酮类化合物,对植物生长和动物健康都具有积极的意义。对于植物本身而言,花青素赋予植物营养器官、生殖器官等鲜艳的颜色,避免植物细胞受到病害、光照、氧化性的伤害,延长果品货架期及增加植物对逆境条件的适应能力等。花青素作为一种天然食用色素,安全、无毒、资源丰富,而且具有一定营养和药理作用,在食品、化妆、医药方面有着巨大应用潜力。近年来,调控植物花青素合成途径的分子机制越来越明晰。其中MYB、bHLH和WD40这三类转录因子所组成的MBW(MYB-bHLH-WD40)转录复合体被广泛证明卷入了花青素合成调控。鉴于来自不同物种的转录因子异源表达后常表现出与原植物中不同的花青素调控模式,且这三个转录因子如何在异源植物中协同调控花青素的合成机制仍不清楚。因此,本研究从紫色花椰菜中分离三个转录因子BoMYB2、BoTT8和BoTTG1在35S启动子的驱动下在烟草中单独或共表达,比较其形态学特征、花青素积累模式以及花青素合成结构基因的表达,揭示了三个基因在烟草花青素积累中的功能和作用。本研究中获得主要结果如下:1.提取紫花菜品种Graffiti花球总RNA,逆转录获得cDNA,根据Genbank公布的GU219986,GU219990以及GU21999序列信息,设计相应的特异引物。从cDNA中分别分离到744bp BoMYB2基因,1566bp BoTT8基因,1014bp BoTTG1基因。经核苷酸序列、氨基酸序列比较、进化树分析,显示获得的基因为调控花青素合成的三个重要转录因子。2.构建了以Bar基因为筛选标记的植物表达载体35Spro:MYB2、35Spro:TT8和35Spro:MYB2TT8以及Hpt基因为筛选标记的35Spro:TTG1的植物表达载体。通过农杆菌介导的单独叶盘转化或者共转化法将其导入普通烟草,分别获得35Spro:MYB2,35Spro:TT8和35Spro:TTG1三个单转录因子表达植株;35Spro:MYB2TT8,35Spro:MYB2TTG1和35Spro:TT8TTG1三个双转录因子共表达植株;一个35Spro:MYB2TT8TTG1三转录因子共表达植株。35Spro:TT8,35Spro:TTG1以及35Spro:TT8TTG1转基因在愈伤组织、根系及嫩叶中均未见花青素积累。而所有表达BoMYB2转录因子的转基因烟草,其愈伤组织、根系及嫩叶中均可见花青素积累,其中35Spro:MYB2TT8和35Spro:MYB2TT8TTG1的愈伤组织、根、茎、叶均呈紫色,花青素积累量更高。针对花青素合成途径的结构基因的表达分析显示,所有基因型的转基因烟草叶片中的EBGs表达量与野生型间无较大的差异,而积累花青素的35Spro:MYB2、35Spro:MYB2TTG1、35Spro:MYB2TT8基因型的转基因烟草叶片的LBGs(NtF3Ha,Nt DFR和NtANS)的表达量明显高于野生型、35Spro:TT8、35Spro:TTG1及35Spro:TT8TTG1植株。显示BoMYB2转录因子是调控花青素合成的关键因子。3.凡表达BoMYB2基因的转基因植株的花器官中表现出强烈的色素积累。尽管35Spro:TT8和35Spro:TT8TTG1转基因烟草植株在营养器官无花青素积累,但在花器官中可见明显的花色素积累。针对花青素合成途径中的结构基因表达分析发现,所有基因型的转基因植株EBGs的表达没有差异,而LBGs(NtDFR和NtANS)的表达量得到上调。表明BoTT8转录因子能单独调控花器官中花青素的积累。4.在种子发芽以及苗期阶段,转录因子BoTT8和BoTTG1不影响花青素的合成和积累。BoMYB2单独表达促进花青素在种子发芽后的子叶以及幼嫩的真叶中积累。但BoMYB2和BoTTG1共表达后抑制花青素在发芽的子叶中积累而促进其在幼嫩的真叶中积累。BoMYB2和BoTT8共表达,BoMYB2、BoTT8和BoTTG1共表达均导致花青素在萌发后的子叶、胚轴、根尖、幼苗真叶中大量积累。5.比较转基因植株的T1种子萌发后的根系结果显示,BoTTG1基因型T1代植株的根系长于野生型,而BoTT8和BoTT8TTG1基因型T1代植株的根系短于BoTTG1基因型和野生型,BoMYB2、BoMYB2TTG1的T1代根系长度也明显短于野生型。而共表达BoMYB2和BoTT8以及共表达BoMYB2,BoTT8和BoTTG1转基因的T1代植株根系的长度最短。以上结果表明异源表达BoTTG1转录因子对根系的生长具有促进作用,而BoTT8和BoMYB2对根系的生长具有抑制作用。
[Abstract]:Anthocyanins are a class of flavonoids, have positive effect on plant growth and animal health. The plant itself, anthocyanins give the nutritive organs, reproductive organs and other bright colors, avoid plant cells by disease, light, oxidative damage, prolong the shelf life of fruit plants and increase the ability to adapt to stress conditions. Anthocyanin as a natural pigment, safe, non-toxic, rich in resources, but also has certain nutritional and pharmacological effects in food, cosmetic, has great potential applications in medicine. In recent years, the molecular mechanism underlying plant anthocyanin synthesis pathway is more and more clear. The MYB, bHLH and WD40 of these three kinds of transcription factors the composition of MBW (MYB-bHLH-WD40) transcription complex is widely shown involved in anthocyanin synthesis regulation. In view of the fact that from different species of heterologous transcription factor after show with the original The regulation of anthocyanin different patterns of plants, the synthesis mechanism and how these three transcription factors in heterologous plants co regulation of anthocyanin remains unclear. Therefore, the separation of three transcription factor BoMYB2 from purple broccoli, BoTT8 and BoTTG1 under the control of 35S promoter in tobacco alone or co expression, comparison the morphological characteristics, expression of anthocyanin accumulation pattern and anthocyanin synthesis gene structure, revealing the function and role of three genes in anthocyanin accumulation in tobacco. The main results obtained in this study are as follows: 1. the extraction of vegetable varieties Graffiti curd total RNA by reverse transcription cDNA, according to Genbank released GU219986, GU219990 and GU21999 sequence information, design the corresponding specific primers. CDNA were isolated from 744bp BoMYB2 1566bp gene, BoTT8 gene, 1014bp BoTTG1 gene. The nucleotide sequence and amino acid sequence A, phylogenetic analysis, display constructed using Bar gene as a selection marker of the plant expression vector 35Spro:MYB2,35Spro:TT8 and 35Spro:MYB2TT8 and Hpt gene as selection marker 35Spro:TTG1 plant expression vector obtained gene into three important transcription factor.2. regulates anthocyanin synthesis. Separate transformed by Agrobacterium mediated leaf disc transformation method or the total the import of tobacco, respectively 35Spro:MYB2,35Spro:TT8 and 35Spro:TTG1 three single plant transcription factor expression; 35Spro:MYB2TT8,35Spro:MYB2TTG1 and 35Spro:TT8TTG1 three double co expression of transcription factor.35Spro:TT8,35Spro:TTG1 and 35Spro:TT8TTG1 transgenic plants; plants in callus co expression of a 35Spro:MYB2TT8TTG1 transcription factor three, the accumulation of root and leaves had no anthocyanin. All expression of transcription factor BoMYB2 gene tobacco, callus, root The leaves are visible in anthocyanin accumulation and 35Spro:MYB2TT8 and 35Spro:MYB2TT8TTG1, callus, root, stem and leaf were purple anthocyanin accumulation. Higher expression of structural genes in the anthocyanin synthesis pathway analysis showed that transgenic tobacco leaves of all genotypes in the EBGs expression has no significant difference between the wild type and quantity that transgenic tobacco leaves 35Spro:MYB2,35Spro:MYB2TTG1,35Spro:MYB2TT8 genotype and anthocyanin accumulation of LBGs (NtF3Ha, Nt, DFR and NtANS) expression was significantly higher than that of the wild type, 35Spro:TT8,35Spro:TTG1 and 35Spro:TT8TTG1 display plants. BoMYB2 transcription factor shows a strong accumulation of pigment.3. key factor in regulating the anthocyanin synthesis and expression of BoMYB2 gene in the transgenic plants in floral organs although 35Spro:TT8 and 35Spro:TT8TTG1 transgenic tobacco plants in vegetative organs but no anthocyanin accumulation. The accumulation of anthocyanin was observed in floral organs. According to the structural genes involved in anthocyanin synthesis and expression analysis found that there is no difference between the expression of EBGs in transgenic plants of all genotypes, and LBGs (NtDFR and NtANS) expression were gained. The results indicated that BoTT8 transcription factor can control single flower anthocyanin accumulation in.4. seed germination and seedling stage, transcription factor BoTT8 and BoTTG1 do not affect the synthesis and accumulation of anthocyanin.BoMYB2 alone increased expression of anthocyanin accumulation in the cotyledons after seed germination and young leaf. But the BoMYB2 and BoTTG1 co expression inhibited anthocyanin accumulation in germinating cotyledons and promote it in young leaf and the accumulation of.BoMYB2 Co expression of BoTT8, BoMYB2, BoTT8 and BoTTG1 co expression resulted in anthocyanin after germination of cotyledon, hypocotyl and root, the accumulation of.5. in leaves of transgenic seedlings T1 after the seed germination root showed that the BoTTG1 genotype of T1 generation plants roots longer than wild type, while BoTT8 and BoTT8TTG1 genotypes of T1 generations were root shorter than BoTTG1 genotype and the wild type, BoMYB2, length T1 generation root BoMYB2TTG1 was significantly shorter than that of wild type. The co expression of BoMYB2 and BoTT8 and co the expression of BoMYB2, BoTT8 and BoTTG1 transgenic T1 generation plant root length is the shortest. The results above showed that heterologous expression can promote the growth of BoTTG1 transcription factor in root, while BoTT8 and BoMYB2 can inhibit the growth of roots.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q946;Q943.2

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