青花菜MYB28、MYB29基因克隆与功能分析

发布时间：2018-08-14 09:07

【摘要】：脂肪族芥子油昔(aliphatic glucosinolates, AGSL)是一种广泛存在于十字花科植物中的含氮、含硫的植物次生代谢产物,其降解产物(如萝卜硫素)不仅在植物防御、响应生物或非生物胁迫的生理过程中具有重要的作用,而且还具有极强的抗癌活性。MYB28和MYB29是调控脂肪族GSL生物合成的两类重要转录因子。在拟南芥中MYB28和MYB29直接调控脂肪族芥子油苷的合成。在十字花科蔬菜中,青花菜中的芥子油苷含量最为丰富,成分更为复杂,目前国内外对青花菜AGSL生物合成途径中的结构基因研究的较多,而对其中起调控作用的转录因子的研究则相对较为滞后。本研究以高萝卜硫素青花菜“福青1号”为材料,克隆了MYB28和MYB29两种转录因子,并对其表达模式以及功能进行初步了分析,主要研究结果如下：(1)通过同源克隆的方法,克隆出青花菜脂肪族芥子油苷合成调控转录因子MYB28的2个同源基因(BoMYB28-1, BoMYB28-2)和MYB29的2个同源基因(BoMYB29-1和BoMYB29-2)。生物信息学分析发现,这四个转录因子核苷酸序列与其他物种同类基因的同源性较高,均含有明显的MYB家族R2R3型转录因子结构,初步确定它们为青花菜BoMYB28和BoMYB29基因。上述四个转录因子提交至GenBank数据库,登录号分别为：KM262765.1、KM262766.1、 KM262767.1和KM262768.1。(2)分别构建了BoMYB28-1, BoMYB28-2 、 BoMYB29-1和BoMYB29-2基因的亚细胞定位载体,并进行瞬时表达,结果显示在洋葱表皮细胞核中均出现明显的绿色荧光,表明BoMYB28-1, BoMYB28-2 、 BoMYB29-1和BoMYB29-2基因定位于细胞核中的转录因子基因。(3)荧光定量PCR分析结果表明,四个同源基因在青花菜不同组织中基因的表达量存在显著差异。其中BoMYB28-1在花球中表达量最高,约是根中的100倍,随着茎、叶和花逐渐降低。在种子中BoMYB28-1的表达量几乎检测不到。BoMYB28-2基因在茎中表达最高,较根中的表达量略高。花球和花中的表达量略少于根,在叶中的表达量很少,种子中几乎见不到表达。BoMYB29-1基因的表达量在花球中的最高,约是根中表达量的6倍,茎和叶中的表达量相似,略低于花球,在种子中的表达量较根略高。BoMYB29-2基因的表达在花球中的表达量最高,大约是根中的3倍,在茎中的表达量较花球中略低,随着叶、根和花逐渐降低,在种子中几乎检测不到基因的表达量。用MeJA和蔗糖对青花菜进行胁迫处理分析发现,四个同源基因的表达量随着处理的时间延长逐渐增加,并分别在15 min和2 h时达到最高,随后表达量逐渐降低至初始状态。(4)构建了四个同源基因的过量表达载体,并转入野生型拟南芥中,分别获得了相应的转基因植株。结果发现,随着转录因子BoMYB28-1.BOMYB28-2. BoMYB29-1和BoMYB29.2基因表达量的增加,脂肪族芥子油苷的含量有了显著改变。其中,在BoMYB28一1和BoMYB28-2过表达的拟南芥转基因植株中,短链脂肪族芥子油苷的含量显著增加,而长链脂肪族芥子油苷的含量未有明显的改变。在BoMYB29—1和BoMYB29—2过表达的拟南芥转基因植株中,短链和长链脂肪族芥子油苷含量均有显著增加。(5)构建了BoMYB28和BoMYB29的干扰表达载体,并转入青花菜,已获得了一定数量的抗性芽,目前正处于生根阶段,但由于根系生长缓慢,尚未进行相关次生代谢产物含量的检测分析。
[Abstract]:Aliphatic glucosinolates (AGSL) are nitrogen-and sulfur-containing plant secondary metabolites widely present in cruciferous plants. Their degradation products (such as sulforaphane) play an important role not only in plant defense, but also in response to biological or abiotic stresses. MYB28 and MYB29 are two important transcription factors that regulate the biosynthesis of aliphatic GSL. In Arabidopsis, MYB28 and MYB29 directly regulate the synthesis of aliphatic glucosinolates. In cruciferous vegetables, broccoli contains the most abundant glucosinolatest glucosinolates and its components are more complex. In this study, two transcription factors, MYB28 and MYB29, were cloned from broccoli "Fuqing No. 1" with high sulfur content, and their expression patterns and functions were preliminarily analyzed. The main results were as follows: (1) Homology was used to study the transcription factors. Two homologous genes (BoMYB28-1, BoMYB28-2) and two MYB29 homologous genes (BoMYB29-1 and BoMYB29-2) were cloned from broccoli. Bioinformatics analysis showed that the nucleotide sequences of these four transcription factors had high homology with the same genes in other species. The four transcription factors mentioned above were submitted to GenBank database with the login numbers of KM262765.1, KM262766.1, KM262767.1 and KM262768.1. (2) Subcellular localization of BoMYB28-1, BoMYB28-2, BoMYB29-1 and BoMYB29-2 genes were constructed respectively. The results showed that the genes of BoMYB28-1, BoMYB28-2, BoMYB29-1 and BoMYB29-2 were located in the nucleus of onion epidermal cells. 3. The expression of four homologous genes in different tissues of broccoli was detected by fluorescence quantitative PCR. The expression of BoMYB28-1 in flower bulb was the highest, about 100 times higher than that in root, and gradually decreased with stem, leaf and flower. The expression of BoMYB28-1 in seed was almost undetectable. The expression level of BoMYB29-1 gene in flower bulb was the highest, about 6 times that in root, similar in stem and leaf, slightly lower than that in flower bulb, and slightly higher in seed than that in root. The expression of four homologous genes in broccoli increased gradually with the prolongation of treatment time, and reached the highest level at 15 min and 2 h, respectively, and then gradually decreased to the beginning. (4) Overexpression vectors of four homologous genes were constructed and transformed into wild-type Arabidopsis, and corresponding transgenic plants were obtained. The results showed that the content of aliphatic glucosinolates changed significantly with the increase of the expression of the transcription factors BoMYB28-1.BOMYB28-2.BoMYB29-1 and BoMYB29.2. Short-chain and long-chain aliphatic glucosinolates increased significantly in transgenic Arabidopsis plants overexpressed by BoMYB28-2 and BoMYB29-1, while long-chain aliphatic glucosinolates did not change significantly. In transgenic Arabidopsis plants overexpressed by BoMYB29-1 and BoMYB29-2, both short-chain and long-chain aliphatic glucosinolates increased significantly. The interference expression vectors of BoMYB28 and BoMYB29 were constructed and transformed into broccoli, and a certain number of resistant buds were obtained. At present, they are in the rooting stage, but the content of related secondary metabolites has not been detected and analyzed because of the slow growth of roots.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S635.3

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