青花菜BZR1、BES1转录因子的克隆与功能分析

发布时间：2018-01-22 22:07

本文关键词： 青花菜转录因子BZR1 转录因子BES1 基因克隆功能鉴定　出处：《福建农林大学》2017年硕士论文　论文类型：学位论文

【摘要】：青花菜(Brassica oleracea var.italica)是发达国家进口量最大的蔬菜之一,含有迄今为止在蔬菜中发现的抗癌活性最强的天然活性物质—萝卜硫素(Sulforaphane)。萝卜硫素的合成前体是芥子油苷(Glucosinolate),是一种含氮和硫的次生代谢物,其广泛存在于十字花科植物中,对植物响应生物或非生物胁迫具有很重要的作用,且其部分降解产物对人体具有很强的保健功能。因此,其合成调控分子生物学近年来逐渐成为研究热点。油菜素内酯(Brassinosteroids,BRs)作为植物特有的一类类固醇激素,广泛参与植物生长发育和响应环境的过程。BZR1(brassinazole-resistant 1)和BES1(BRI1-EMS-suppressor)是油菜素内酯信号转导途径中的两个重要转录因子,已在拟南芥中证实油菜素内酯可以通过BZR1和BES1抑制植物体内芥子油苷的生物合成。萝卜硫素和芥子油苷在青花菜中含量最为丰富,BZR1和BES1转录因子是否影响其在青花菜中的合成以及如何影响?目前尚不清楚。为此,本研究从青花菜中克隆了 BZR1和BES1基因,并进一步分析了其表达模式和功能,主要研究结果如下:(1)根据青花菜同属(芸苔属)物种的BZR1和BES1转录因子核苷酸序列,设计特异性引物,通过RT-PCR技术,从高萝卜硫素青花菜"福青1号"叶片cDNA中扩增得到油菜素内酯信号转导途径中的转录因子BZR1和BES1的同源序列,其长度分别为993bp、1011bp,分别编码330和336个氨基酸,并分别命名为BoBZR1和BoBES1。生物信息学分析发现,克隆的这两个基因的核苷酸序列与NCBI上已登录的其他物种的同类基因相似性较高,含有典型的N末端结构域和C末端转录激活结构域。初步确定它们为青花菜BoBZR1和BoBES1基因。(2)分别构建了BoBZR 和BoBES1基因的亚细胞定位载体pEGAD-BoBZR1和pEGAD-BoBES1,并转化本氏烟草中进行瞬时表达。结果表明,BoBZR1和BoBES1蛋白定位在几乎整个细胞中,当外源喷施BR后,融合蛋白全部集中在核内,表明植物细胞接收到BR信号后,转录因子BoBES1和BoBZR1被激活,进入细胞核内部行使功能。(3)qRT-PCR分析表明,BoBZR1和BoBES1基因在根中表达量均最高,在花球中最少,在种子中几乎都检测不到。当对其叶片进行外源喷施500 μM的甲基茉莉酸甲酯(MeJA)、5 mM的水杨酸(SA)和3 μM的BR后,青花菜中BZR1和BES1基因的表达量分别在处理24 h、8 h、24 h后达到最大值;在处理48 h后,除了 BR处理组的基因表达量稍微降低,其他两种处理均趋于初始状态。(4)构建了以35S启动子驱动BoBZR1和BoBES1基因表达的植物超表达载体pBI-BoBZR1和pBI-BoBES1,并转化到野生型拟南芥Col-0中。对T3代转基因拟南芥纯合体的表型分析和芥子油苷含量检测发现:两种基因超表达植株均出现植株矮小以及发育迟缓的表型,而BoBES1基因超表达受到的影响更大,叶片变小且出现卷叶。与野生型拟南芥Col-0相比,BoBZR1和BoBES1基因超表达都会降低拟南芥中短链脂肪族芥子油苷(3MSOP、4MSOB、5MSOP、4MTB)和长链脂肪族芥子油苷(8MSOO)的含量,其中BoBES1基因超表达显著降低拟南芥中芥子油苷的含量。结果说明BoBZR1和BoBES1基因的表达对芥子油苷的合成具有显著的抑制作用。(5)构建了BoBZR 和BoBES1基因的干扰表达载体,并与上述超表达载体一起分别转化到青花菜中,已获得T0代抗性苗110株,其中转BZR1和BES1基因干扰表达载体的植株分别28株和26株,转超表达载体的植株均28株。
[Abstract]:Broccoli (Brassica oleracea var.italica) is one of the largest volume of imports of vegetables in developed countries, with by far the strongest anticancer activity found in the vegetables in the natural active substances - sulforaphane (Sulforaphane). The synthesis of sulforaphane is glucosinolates (Glucosinolate), is a kind of nitrogen and sulfur containing secondary metabolites and it is widely present in cruciferous plants, the plant response to biotic or abiotic stress has a very important role, and some of its degradation products on the human body has a strong health care function. Therefore, the regulation of the synthesis of molecular biology has become a research hotspot in recent years. Brassinosteroid (Brassinosteroids, BRs) is a kind of steroid the plant specific hormone, widely involved in plant development and response to environmental.BZR1 (brassinazole-resistant 1) and BES1 (BRI1-EMS-suppressor) is a brassinosteroid signal to Two important transcription factor mediated pathway of biosynthesis, has confirmed that brassinolide can inhibit plant glucosinolate by BZR1 and BES1 in Arabidopsis. Sulforaphane and glucosinolates in broccoli were the most abundant BZR1 and BES1 transcription factors in broccoli will affect its synthesis and how? It is not clear. Therefore, this study cloned BZR1 and BES1 genes from broccoli, and further analysis of the expression pattern and function, the main results are as follows: (1) according to broccoli (Brassica species) belong to the BZR1 and BES1 transcription factor nucleotide sequence, specific primers were designed by RT-PCR technology, from high sulforaphane in broccoli "homologous sequences of brassinolide in signal transduction pathway of the transcription factor BZR1 and BES1 were amplified by Fu Qing No. 1 leaf cDNA, the length were 993bp and 1011bp respectively, encoding 3 30 and 336 amino acids, and were named as BoBZR1 and BoBES1. analysis found that the biological information of similar genes in other species and nucleotide sequences of NCBI gene cloning of the two logged the high similarity, containing terminal N terminal domain and typical C transcription activation domain. Initially identified them as BoBZR1 and broccoli (2). BoBES1 gene and BoBES1 gene were constructed BoBZR subcellular localization vector pEGAD-BoBZR1 and pEGAD-BoBES1, and the expression of transforming of the instantaneous's tobacco. The results showed that BoBZR1 and BoBES1 protein in almost the entire cell, when exogenous BR fusion protein, all concentrated in the nucleus of that plant cells when receives the BR signal, the transcription factors BoBES1 and BoBZR1 are activated into the nucleus internal function. (3) qRT-PCR analysis showed that BoBZR1 and BoBES1 gene expression level in root was the highest in flowers The ball at least, in seeds almost undetectable. When Methyl Jasmonate on the leaf spraying 500 M (MeJA), 5 mM salicylic acid (SA) and 3 M after BR, expression of BZR1 and BES1 genes in broccoli, respectively at 24 h, 8 h after 24 h, reached a maximum value; after 48 h BR treatment group, in addition to the expression of genes was slightly reduced, the other two treatments tended to be the initial state. (4) constructed an over expression vector of pBI-BoBZR1 and pBI-BoBES1 in 35S promoter and the expression of BoBZR1 and BoBES1 genes, and transformed into wild type quasi Arabidopsis Col-0. It was found that mustard oil glycoside content phenotype analysis and detection of T3 generation of transgenic Arabidopsis plants were homozygous: dwarf and growth retardation phenotype expression of two genes, and the overexpression of BoBES1 is more affected, smaller leaves and leaf and wild type Arabidopsis. Mustard compared to Col-0, overexpression of BoBZR1 and BoBES1 genes in Arabidopsis thaliana will reduce the short chain aliphatic glucosinolates (3MSOP, 4MSOB, 5MSOP, 4MTB) and long chain aliphatic glucosinolate (8MSOO) content, the overexpression of BoBES1 significantly decreased the content of glucosinolates in Arabidopsis thaliana. Results showed that the expression of BoBZR1 and BoBES1 gene synthesis of glucosinolates was significantly inhibited. (5) constructed the interference of BoBZR and BoBES1 gene expression vector, and the expression vector were transformed into broccoli together, has obtained 110 strains of resistant seedlings of T0 generation, the BZR1 and BES1 gene interference expression vector of plants were 28 and 26 strains, transgenic overexpression vector of plants were 28 strains.

【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S635.3

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