本文選題：興安落葉松 + CesA　； 參考：《內(nèi)蒙古大學(xué)》2017年碩士論文

【摘要】：興安落葉松(Larix gmelinii)是中國(guó)東北地區(qū)荒山造林和森林更新的主要樹(shù)種,因其材質(zhì)優(yōu)良,所以也為建筑、木材加工,木纖維工業(yè)等提供原料,具有巨大的經(jīng)濟(jì)價(jià)值。纖維素合酶(Celllμlose Synthase)是纖維素生物合成途徑中的一個(gè)關(guān)鍵酶,它的表達(dá)豐度可以直接影響到纖維素的合成,進(jìn)而對(duì)木材材質(zhì)實(shí)現(xiàn)有效調(diào)控。因此研究興安落葉松CesA基因的表達(dá)調(diào)控機(jī)理和功能特性意義非凡。本研究利用兼并PCR和RACE-PCR技術(shù)分離了興安落葉松纖維素合酶基因(命名為L(zhǎng)gCesA3),并對(duì)該基因的功能和表達(dá)特性進(jìn)行了分析。LgCesA3基因的cDNA全長(zhǎng)為4220bp,其中包含3297bp的開(kāi)放閱讀框(ORF)、531bp的5,-UTR和392bp的3,-UTR,編碼1099個(gè)氨基酸。LgCesA3基因的DNA全長(zhǎng)為7255bp,包含14個(gè)外顯子和13個(gè)內(nèi)含子。亞細(xì)胞定位結(jié)果顯示LgCesA3::GFP融合蛋白信號(hào)在細(xì)胞膜周?chē)粰z測(cè)到,因此推測(cè)LgCesA3定位于細(xì)胞膜上。為了調(diào)查L(zhǎng)gCesA3的表達(dá)調(diào)控機(jī)理,利用基因組步移的技術(shù)克隆了 1180bp的LgCesA3啟動(dòng)子序列。序列分析顯示,該基因的啟動(dòng)子區(qū)域包含水楊酸、茉莉酸甲酯等植物激素應(yīng)答原件,光誘導(dǎo)元件以及干旱、機(jī)械壓力等非生物脅迫相關(guān)的應(yīng)答元件。實(shí)時(shí)熒光定量PCR檢測(cè)結(jié)果顯示,LgCesA3在興安落葉松的根、莖、葉中均穩(wěn)定表達(dá),莖中的表達(dá)量要遠(yuǎn)遠(yuǎn)高于根和葉。同時(shí)LgCesA3基因的表達(dá)受外源激素茉莉酸甲酯和水楊酸的誘導(dǎo),但在15%PEG和GA3處理?xiàng)l件下,該基因表現(xiàn)出先升高后下降的表達(dá)趨勢(shì)。為了深入研究LgCesA3的功能和異源表達(dá)特性,構(gòu)建了 pBI101-LgCesA3雙元表達(dá)載體,并通過(guò)農(nóng)瘤桿菌介導(dǎo)的花序浸染法獲得了轉(zhuǎn)基因擬南芥。RT-PCR和酶活性檢測(cè)結(jié)果證實(shí)了 LgCesA3在轉(zhuǎn)基因擬南芥中的穩(wěn)定表達(dá)。超表達(dá)LgCesA3基因擬南芥的表型分析結(jié)果顯示LgCes43基因的過(guò)量表.達(dá)促進(jìn)了轉(zhuǎn)基因擬南芥植株中纖維素的積累。以上研究結(jié)果表明,可通過(guò)對(duì)纖維素合成路徑中的關(guān)鍵酶CesA進(jìn)行遺傳修飾調(diào)控纖維素的生物合成,進(jìn)而有目的地改良材質(zhì)。該研究成果亦為深入研究興安落葉松ZgCesA3基因的表達(dá)調(diào)控機(jī)制以及利用該基因進(jìn)行木本植物的材質(zhì)改良奠定了基礎(chǔ)。
[Abstract]:Larix gmelinii (Larix gmelinii) is the main tree species for afforestation and forest regeneration in northeast China. Because of its excellent material quality, Larix gmelinii also provides raw materials for construction, wood processing and wood fiber industry, which is of great economic value. Cellulosic synthase (Celll 渭 lose Synthase) is a key enzyme in cellulose biosynthesis pathway. Therefore, it is of great significance to study the expression and regulation mechanism and functional characteristics of CesA gene in Larix gmelinii. In this study, the cellulose synthase gene of LgCesA3 (LgCesA3) gene was isolated by devolving PCR and RACE-PCR techniques. The function and expression characteristics of LgCesA3 gene were analyzed. The cDNA length of LgCesA3 gene was 4220 BP, including the open reading frame of 3297bp. The DNA encoding 1099 amino acids, LgCesA3, was 7255bp. it contained 14 exons and 13 introns. The subcellular localization results showed that the LgCesA3::GFP fusion protein signal was detected around the cell membrane, so we speculated that LgCesA3 was located on the cell membrane. In order to investigate the expression regulation mechanism of LgCesA3, the LgCesA3 promoter sequence of 1180bp was cloned by genomic step technique. Sequence analysis showed that the promoter region of the gene contained plant hormone responses such as salicylic acid methyl jasmonate photoinduced elements and abiotic stress response elements such as drought and mechanical pressure. The results of real-time fluorescence quantitative PCR showed that LgCesA3 was stably expressed in roots, stems and leaves of Larix gmelinii, and the expression of LgCesA3 in stems was much higher than that in roots and leaves. At the same time, the expression of LgCesA3 gene was induced by exogenous hormones methyl jasmonate and salicylic acid, but under the condition of 15%PEG and GA3 treatment, the expression of LgCesA3 gene increased first and then decreased. In order to study the function and heterologous expression characteristics of LgCesA3, a double expression vector of pBI101-LgCesA3 was constructed. The stable expression of LgCesA3 in transgenic Arabidopsis thaliana was confirmed by Agrobacterium tumefaciens mediated inflorescence soaking method. Phenotypic analysis of overexpression of LgCesA3 gene in Arabidopsis thaliana showed that LgCes43 gene was overrated. Da promoted the accumulation of cellulose in transgenic Arabidopsis thaliana plants. The results show that the cellulosic biosynthesis can be controlled by genetic modification of the key enzyme CesA in the cellulose synthesis pathway, and then the material can be modified purposefully. The results also laid a foundation for the further study of the regulation mechanism of ZgCesA3 gene expression and the material improvement of woody plants by using this gene.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S791.222;Q943.2

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