【摘要】：脂肪族芥子油昔(aliphatic glucosinolates, AGSL)是一種廣泛存在于十字花科植物中的含氮、含硫的植物次生代謝產(chǎn)物,其降解產(chǎn)物(如蘿卜硫素)不僅在植物防御、響應生物或非生物脅迫的生理過程中具有重要的作用,而且還具有極強的抗癌活性。MYB28和MYB29是調控脂肪族GSL生物合成的兩類重要轉錄因子。在擬南芥中MYB28和MYB29直接調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為豐富,成分更為復雜,目前國內外對青花菜AGSL生物合成途徑中的結構基因研究的較多,而對其中起調控作用的轉錄因子的研究則相對較為滯后。本研究以高蘿卜硫素青花菜“福青1號”為材料,克隆了MYB28和MYB29兩種轉錄因子,并對其表達模式以及功能進行初步了分析,主要研究結果如下：(1)通過同源克隆的方法,克隆出青花菜脂肪族芥子油苷合成調控轉錄因子MYB28的2個同源基因(BoMYB28-1, BoMYB28-2)和MYB29的2個同源基因(BoMYB29-1和BoMYB29-2)。生物信息學分析發(fā)現(xiàn),這四個轉錄因子核苷酸序列與其他物種同類基因的同源性較高,均含有明顯的MYB家族R2R3型轉錄因子結構,初步確定它們?yōu)榍嗷ú薆oMYB28和BoMYB29基因。上述四個轉錄因子提交至GenBank數(shù)據(jù)庫,登錄號分別為：KM262765.1、KM262766.1、 KM262767.1和KM262768.1。(2)分別構建了BoMYB28-1, BoMYB28-2 、 BoMYB29-1和BoMYB29-2基因的亞細胞定位載體,并進行瞬時表達,結果顯示在洋蔥表皮細胞核中均出現(xiàn)明顯的綠色熒光,表明BoMYB28-1, BoMYB28-2 、 BoMYB29-1和BoMYB29-2基因定位于細胞核中的轉錄因子基因。(3)熒光定量PCR分析結果表明,四個同源基因在青花菜不同組織中基因的表達量存在顯著差異。其中BoMYB28-1在花球中表達量最高,約是根中的100倍,隨著莖、葉和花逐漸降低。在種子中BoMYB28-1的表達量幾乎檢測不到。BoMYB28-2基因在莖中表達最高,較根中的表達量略高�；ㄇ蚝突ㄖ械谋磉_量略少于根,在葉中的表達量很少,種子中幾乎見不到表達。BoMYB29-1基因的表達量在花球中的最高,約是根中表達量的6倍,莖和葉中的表達量相似,略低于花球,在種子中的表達量較根略高。BoMYB29-2基因的表達在花球中的表達量最高,大約是根中的3倍,在莖中的表達量較花球中略低,隨著葉、根和花逐漸降低,在種子中幾乎檢測不到基因的表達量。用MeJA和蔗糖對青花菜進行脅迫處理分析發(fā)現(xiàn),四個同源基因的表達量隨著處理的時間延長逐漸增加,并分別在15 min和2 h時達到最高,隨后表達量逐漸降低至初始狀態(tài)。(4)構建了四個同源基因的過量表達載體,并轉入野生型擬南芥中,分別獲得了相應的轉基因植株。結果發(fā)現(xiàn),隨著轉錄因子BoMYB28-1.BOMYB28-2. BoMYB29-1和BoMYB29.2基因表達量的增加,脂肪族芥子油苷的含量有了顯著改變。其中,在BoMYB28一1和BoMYB28-2過表達的擬南芥轉基因植株中,短鏈脂肪族芥子油苷的含量顯著增加,而長鏈脂肪族芥子油苷的含量未有明顯的改變。在BoMYB29—1和BoMYB29—2過表達的擬南芥轉基因植株中,短鏈和長鏈脂肪族芥子油苷含量均有顯著增加。(5)構建了BoMYB28和BoMYB29的干擾表達載體,并轉入青花菜,已獲得了一定數(shù)量的抗性芽,目前正處于生根階段,但由于根系生長緩慢,尚未進行相關次生代謝產(chǎn)物含量的檢測分析。
[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.
【學位授予單位】：福建農林大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S635.3

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