本文選題：白樺 + BpBEE2�。� 參考：《東北林業(yè)大學(xué)》2016年碩士論文

【摘要】：Brassinolide Enhanced Expression(BEE)基因編碼植物所特有的bHLH轉(zhuǎn)錄因子,是調(diào)控油菜素內(nèi)酯信號(hào)轉(zhuǎn)導(dǎo)的重要元件。本實(shí)驗(yàn)通過構(gòu)建白樺BpBEE2基因的植物過表達(dá)載體和抑制載體,進(jìn)行白樺的遺傳轉(zhuǎn)化,對(duì)獲得的轉(zhuǎn)基因株系進(jìn)行生長(zhǎng)量測(cè)定和鹽、旱脅迫,驗(yàn)證白樺BpBEE2基因所發(fā)揮的作用。主要研究結(jié)果如下：1.根據(jù)白樺轉(zhuǎn)錄組數(shù)據(jù),獲得了3條白樺BpBEE基因,對(duì)3條白樺BpBEE基因進(jìn)行生物信息學(xué)分析,結(jié)果表明,3條白樺BpBEE基因均具有典型的bHLH結(jié)構(gòu)域,基因序列特性非常相似,且均有5個(gè)內(nèi)含子與6個(gè)外顯子。2.通過實(shí)時(shí)熒光定量PCR的方法,對(duì)3條白樺BpBEE基因的表達(dá)模式進(jìn)行分析,結(jié)果表明,這3條基因在葉片,雌花,雄花,莖和頂芽中均有表達(dá),葉中BpBEE基因表達(dá)量明顯高于其他部位。在生長(zhǎng)旺盛時(shí)期,葉和頂芽中BpBEE基因均呈現(xiàn)上調(diào)表達(dá),表明BpBEE基因可能參與到白樺的生長(zhǎng)過程。BR激素處理之后,BpBEE1在早期的芽和木質(zhì)部中呈顯著上調(diào)；BpBEE2在早期的木質(zhì)部和韌皮部顯著上調(diào)外,其他處理時(shí)間也呈現(xiàn)不同程度的上調(diào)。表明3個(gè)白樺BpBEE基因參與BR激素應(yīng)答,且BpBEE1和BpBEE2呈現(xiàn)早期應(yīng)答響應(yīng)。3.應(yīng)用Gateway技術(shù)構(gòu)建白樺BpBEE2基因的植物過表達(dá)載體和酶切連接方法構(gòu)建的白樺BpBEE2基因的植物抑制載體,采用根瘤農(nóng)桿菌介導(dǎo)的合子胚法進(jìn)行白樺的遺傳轉(zhuǎn)化,通過PCR鑒定轉(zhuǎn)基因植株,結(jié)果表明,白樺BpBEE2基因已經(jīng)成功整合到了植物基因組上,獲得了1個(gè)超表達(dá)株系和5個(gè)抑制表達(dá)株系。4.對(duì)獲得的轉(zhuǎn)基因植株進(jìn)行生長(zhǎng)量測(cè)定。結(jié)果表明：超表達(dá)株系的生長(zhǎng)量顯著高于野生型,而抑制表達(dá)株系則明顯低于野生型。表明超表達(dá)BpBEE2基因能提高植株的生長(zhǎng)量。5.抗鹽和抗旱的逆境脅迫分析,分別選取0.3%和0.6% NaCl;5%和10%PEG的脅迫處理,進(jìn)行鮮重和生根率統(tǒng)計(jì)。結(jié)果表明：轉(zhuǎn)基因株系并沒有顯著的提高抗鹽旱性。6.克隆BpBEE1基因的啟動(dòng)子,通過原件預(yù)測(cè),發(fā)現(xiàn)：該啟動(dòng)子含有很多與激素相關(guān)的元件；轉(zhuǎn)基因擬南芥株系進(jìn)行不同生長(zhǎng)時(shí)期的組織特異性染色和激素處理后的GUS活性檢測(cè)和染色,結(jié)果表明：轉(zhuǎn)基因株系中GUS染色在葉脈處染色更重,MeJA處理后的GUS酶活性較未處理的明顯提高,IAA和ABA處理后明顯降低,與GUS染色結(jié)果相符。
[Abstract]:Brassinolide Enhanced expression bee (Bee) gene is a unique bHLH transcription factor in plants, which is an important element in the regulation of rapesinolide signal transduction. In this experiment, the plant overexpression vector and inhibition vector of Bai Hua BpBEE2 gene were constructed, and the genetic transformation of Bai Hua was carried out, and the growth of the transgenic lines was measured, salt and drought stress were used to verify the role of Bai Hua BpBEE2 gene. The main results are as follows: 1. Three Bai Hua BpBEE genes were obtained from Bai Hua transcriptome data, and three Bai Hua BpBEE genes were analyzed by bioinformatics. The results showed that all three Bai Hua BpBEE genes had typical bHLH domains, and the gene sequence characteristics were very similar. There were 5 introns and 6 exons. The expression patterns of three Bai Hua BpBEE genes were analyzed by real-time fluorescence quantitative PCR. The results showed that the three genes were expressed in leaves, female flowers, male flowers, stems and terminal buds. The expression of BpBEE gene in leaves was significantly higher than that in other parts. During the period of vigorous growth, the expression of BpBEE gene was up-regulated in both leaves and terminal buds. It was suggested that BpBEE gene might be involved in the growth process of Bai Hua. Br hormone treatment showed that BpBEE1 was upregulated significantly in early bud and xylem, while BpBEE2 was upregulated in early xylem and phloem, and other treatment time was also up-regulated in different degree. The results indicated that three Bai Hua BpBEE genes were involved in Br hormone response, and BpBEE1 and BpBEE2 showed early response. 3. The plant overexpression vector of Bai Hua BpBEE2 gene and the plant inhibition vector of Bai Hua BpBEE2 gene constructed by restriction endonuclease ligation were constructed by Gateway technique. The Bai Hua genetic transformation was carried out by zygotic embryo mediated by Agrobacterium tumefaciens (Agrobacterium tumefaciens). The results of identification of transgenic plants by PCR showed that Bai Hua BpBEE2 gene had been successfully integrated into the plant genome, and one super-expression strain and five suppressive expression lines .4were obtained. The growth of transgenic plants was measured. The results showed that the growth of overexpression lines was significantly higher than that of wild type, while the growth of suppressive expression lines was significantly lower than that of wild type. The results showed that overexpression of BpBEE2 gene could increase plant growth. The stress treatments of 0.3% and 0.6% NaClO 5% and 10%PEG were used to calculate the fresh weight and rooting rate respectively. The results showed that the transgenic lines did not significantly improve salt drought tolerance. 6. The promoter of BpBEE1 gene was cloned. By original prediction, it was found that the promoter contained a lot of hormone related elements, and the transgenic Arabidopsis thaliana lines were tested for tissue specific staining and GUS activity after hormone treatment at different growth stages. The results showed that the GUS enzyme activity of the transgenic lines treated with GUS at the leaf vein was significantly higher than that of the untreated ones, which was consistent with the results of GUS staining.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S792.153

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