本文關(guān)鍵詞： 青花菜 轉(zhuǎn)錄因子BZR1 轉(zhuǎn)錄因子BES1 基因克隆 功能鑒定　出處：《福建農(nóng)林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：青花菜(Brassica oleracea var.italica)是發(fā)達(dá)國家進(jìn)口量最大的蔬菜之一,含有迄今為止在蔬菜中發(fā)現(xiàn)的抗癌活性最強(qiáng)的天然活性物質(zhì)—蘿卜硫素(Sulforaphane)。蘿卜硫素的合成前體是芥子油苷(Glucosinolate),是一種含氮和硫的次生代謝物,其廣泛存在于十字花科植物中,對植物響應(yīng)生物或非生物脅迫具有很重要的作用,且其部分降解產(chǎn)物對人體具有很強(qiáng)的保健功能。因此,其合成調(diào)控分子生物學(xué)近年來逐漸成為研究熱點。油菜素內(nèi)酯(Brassinosteroids,BRs)作為植物特有的一類類固醇激素,廣泛參與植物生長發(fā)育和響應(yīng)環(huán)境的過程。BZR1(brassinazole-resistant 1)和BES1(BRI1-EMS-suppressor)是油菜素內(nèi)酯信號轉(zhuǎn)導(dǎo)途徑中的兩個重要轉(zhuǎn)錄因子,已在擬南芥中證實油菜素內(nèi)酯可以通過BZR1和BES1抑制植物體內(nèi)芥子油苷的生物合成。蘿卜硫素和芥子油苷在青花菜中含量最為豐富,BZR1和BES1轉(zhuǎn)錄因子是否影響其在青花菜中的合成以及如何影響?目前尚不清楚。為此,本研究從青花菜中克隆了 BZR1和BES1基因,并進(jìn)一步分析了其表達(dá)模式和功能,主要研究結(jié)果如下:(1)根據(jù)青花菜同屬(蕓苔屬)物種的BZR1和BES1轉(zhuǎn)錄因子核苷酸序列,設(shè)計特異性引物,通過RT-PCR技術(shù),從高蘿卜硫素青花菜"福青1號"葉片cDNA中擴(kuò)增得到油菜素內(nèi)酯信號轉(zhuǎn)導(dǎo)途徑中的轉(zhuǎn)錄因子BZR1和BES1的同源序列,其長度分別為993bp、1011bp,分別編碼330和336個氨基酸,并分別命名為BoBZR1和BoBES1。生物信息學(xué)分析發(fā)現(xiàn),克隆的這兩個基因的核苷酸序列與NCBI上已登錄的其他物種的同類基因相似性較高,含有典型的N末端結(jié)構(gòu)域和C末端轉(zhuǎn)錄激活結(jié)構(gòu)域。初步確定它們?yōu)榍嗷ú薆oBZR1和BoBES1基因。(2)分別構(gòu)建了BoBZR 和BoBES1基因的亞細(xì)胞定位載體pEGAD-BoBZR1和pEGAD-BoBES1,并轉(zhuǎn)化本氏煙草中進(jìn)行瞬時表達(dá)。結(jié)果表明,BoBZR1和BoBES1蛋白定位在幾乎整個細(xì)胞中,當(dāng)外源噴施BR后,融合蛋白全部集中在核內(nèi),表明植物細(xì)胞接收到BR信號后,轉(zhuǎn)錄因子BoBES1和BoBZR1被激活,進(jìn)入細(xì)胞核內(nèi)部行使功能。(3)qRT-PCR分析表明,BoBZR1和BoBES1基因在根中表達(dá)量均最高,在花球中最少,在種子中幾乎都檢測不到。當(dāng)對其葉片進(jìn)行外源噴施500 μM的甲基茉莉酸甲酯(MeJA)、5 mM的水楊酸(SA)和3 μM的BR后,青花菜中BZR1和BES1基因的表達(dá)量分別在處理24 h、8 h、24 h后達(dá)到最大值;在處理48 h后,除了 BR處理組的基因表達(dá)量稍微降低,其他兩種處理均趨于初始狀態(tài)。(4)構(gòu)建了以35S啟動子驅(qū)動BoBZR1和BoBES1基因表達(dá)的植物超表達(dá)載體pBI-BoBZR1和pBI-BoBES1,并轉(zhuǎn)化到野生型擬南芥Col-0中。對T3代轉(zhuǎn)基因擬南芥純合體的表型分析和芥子油苷含量檢測發(fā)現(xiàn):兩種基因超表達(dá)植株均出現(xiàn)植株矮小以及發(fā)育遲緩的表型,而BoBES1基因超表達(dá)受到的影響更大,葉片變小且出現(xiàn)卷葉。與野生型擬南芥Col-0相比,BoBZR1和BoBES1基因超表達(dá)都會降低擬南芥中短鏈脂肪族芥子油苷(3MSOP、4MSOB、5MSOP、4MTB)和長鏈脂肪族芥子油苷(8MSOO)的含量,其中BoBES1基因超表達(dá)顯著降低擬南芥中芥子油苷的含量。結(jié)果說明BoBZR1和BoBES1基因的表達(dá)對芥子油苷的合成具有顯著的抑制作用。(5)構(gòu)建了BoBZR 和BoBES1基因的干擾表達(dá)載體,并與上述超表達(dá)載體一起分別轉(zhuǎn)化到青花菜中,已獲得T0代抗性苗110株,其中轉(zhuǎn)BZR1和BES1基因干擾表達(dá)載體的植株分別28株和26株,轉(zhuǎn)超表達(dá)載體的植株均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.

【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2;S635.3

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