【摘要】：油桐是一種經(jīng)濟(jì)價(jià)值較高的木本油料樹種。生態(tài)環(huán)境的惡化使得油桐在生長(zhǎng)發(fā)育過(guò)程中面臨著多種逆境,造成油桐減產(chǎn)。植物在遭受逆境脅迫時(shí)會(huì)產(chǎn)生有毒的醛類物質(zhì),乙醛脫氫酶作為一種醛類物質(zhì)的消除劑,對(duì)植物抗擊逆境意義重大。本論文從油桐中分離克隆了乙醛脫氫酶家族的3個(gè)基因,并對(duì)這些基因進(jìn)行了功能表達(dá)研究,主要的研究結(jié)果如下:1.以'葡萄桐'為實(shí)驗(yàn)研究材料,基于油桐轉(zhuǎn)錄組數(shù)據(jù),設(shè)計(jì)引物,分別克隆得到油桐乙醛脫氫酶ALDH2B4、ALDH2B7、ALDH3H1基因的全長(zhǎng)CDS序列。ALDH2B4基因CDS全長(zhǎng)為1617 bp,ORF為1617 bp,編碼538個(gè)氨基酸,預(yù)測(cè)分析顯示蛋白質(zhì)分子量為58.79 kDa,等電點(diǎn)pI為7.16;ALD2B7基因CDS全長(zhǎng)為1626 bp,ORF為1626 bp,編碼541個(gè)氨基酸,預(yù)測(cè)分析顯示蛋白質(zhì)分子量為 59.01 kDa,等電點(diǎn) pI 為 6.38;ALD3H1基因 CDS 全長(zhǎng)為 1245 bp,ORF為1245bp,編碼414個(gè)氨基酸,預(yù)測(cè)分析顯示蛋白質(zhì)分子量為45.06kDa,等電點(diǎn) pI 為 9.47。2.以葡萄桐葉片為材料提取DNA,分段克隆拼接得到ALDHH2B4、ALDH2B7、ALDH3H1基因的基因組全長(zhǎng)序列。ALDH2B4基因組全長(zhǎng)3746bp,含有11段內(nèi)含子;ALDH2B7基因組全長(zhǎng)5358bp,含有11段內(nèi)含子;ALDH3H1基因組全長(zhǎng)4323bp,含有9段內(nèi)含子。3.實(shí)時(shí)定量PCR分析ALDH基因在'葡萄桐'不同組織和種子發(fā)育時(shí)期的表達(dá)量變化。結(jié)果表明,ALDH2B4基因在不同組織的表達(dá)量大小為:嫩葉老葉幼根老莖莖柱頭雄蕊子房花萼花瓣;ALDH2B7基因在不同組織的表達(dá)量大小為:花瓣老莖柱頭莖幼根嫩葉子房老葉花萼雄蕊;ALDH3H1基因在不同組織的表達(dá)量大小為:老葉嫩葉雄蕊柱頭花瓣幼根老莖莖花萼子房。4.實(shí)時(shí)定量PCR分析ALDH基因在干旱、鹽脅迫以及脫落酸條件下的表達(dá)情況。在干旱處理下,ALDH2B4、ALDH2B7和ALDH3H1基因均呈現(xiàn)上升再下降的變化趨勢(shì);在鹽脅迫條件下,ALDH2B4基因呈下降-升高的趨勢(shì),ALDH2B7表達(dá)呈現(xiàn)下降-升高-下降模式,ALDH3H1基因表達(dá)呈現(xiàn)升高-下降-升高模式。ALDH2B4、ALDH3H1基因?qū)Φ蜐舛让撀渌岬捻憫?yīng)更為顯著,ALDH2B7在低濃度脫落酸處理下呈現(xiàn)促進(jìn)作用,在高濃度情況下受到抑制。結(jié)果表明,這三個(gè)基因均受到干旱脅迫、鹽脅迫及ABA誘導(dǎo)表達(dá)。5.構(gòu)建了pCAMBI1300-335S-ALDH2B4、pCAMB1300-0535S-ALD2B7、pCAMBIA1300-35S-ALDH3H1三個(gè)植物超表達(dá)載體,通過(guò)花粉管通道法侵染擬南芥和陽(yáng)性篩選,得到轉(zhuǎn)基因擬南芥植株。
[Abstract]:Taulownia is a kind of woody oil tree with high economic value. The deterioration of ecological environment makes the growth and development of Taulownia faced with a variety of stresses, resulting in a reduction in the production of Taulownia. When plants are stressed by stress, they produce toxic aldehydes. Aldehyde dehydrogenase, as an abatement agent of aldehydes, is of great significance for plants to resist stress. In this paper, three genes of acetaldehyde dehydrogenase family were isolated and cloned from Taulownia oleifera, and the functional expression of these genes was studied. The main results are as follows: 1. The full-length CDS sequence of ALDH2B4 and ALDH2B7 ALDH3H1 gene were cloned by primer design based on Taulownia chinensis transcriptome data. The full-length CDS sequence of ALDH2B4 gene CDS was 1617 BP and 1617 BP ORF, encoding 538 amino acids, respectively, and the total length of ALDH2B4 gene was 1617 BP, encoding 538 amino acids. The predicted molecular weight of the protein was 58.79 kDa, the isoelectric point Pi was 7.16 CDS of ALD2B7 gene, the total length of the gene was 1626 BP ORF was 1626 BP, encoding 541 amino acids. The predicted protein molecular weight was 59.01 kDa.The isoelectric point Pi was 6.38 CDS of ALD3H1 gene was 1245 BP, encoding 414 amino acids. The predicted molecular weight of protein was 45.06 kDa and the isoelectric point Pi was 9.47.2. The full-length sequence of ALDH2B7 ALDH3H1 gene was obtained from the leaves of Paulownia vinifera. The full-length of ALDH2B4 genome was 3746bp. it contained 11 introns (ALDH2B7), and the total length of ALDH2B7 genome was 5358bp. the total length of ALDH2B1 gene was 4323bp, and the whole length of ALDH2B1 gene contained 9 introns (3.3bp.). Real time quantitative PCR was used to analyze the expression of ALDH gene in different tissues and seeds of 'Grape'. The results showed that the expression of ALDH2B4 gene in different tissues was as follows: the expression of ALDH2B7 gene in different tissues was as follows: the expression of ALDH2B7 gene in different tissues was as follows: young stem, young leaf, ovary and old leaf of young stem, young leaf, young stem, young leaf and young leaf. The expression of ALDH3H1 gene in different tissues was as follows: tender leaves, stamen, stigma, petal, stem, calyx ovary. 4. The expression of ALDH gene in drought, salt stress and abscisic acid was analyzed by real-time quantitative PCR. The ALDH2B7 and ALDH3H1 genes of ALDH2B4 and ALDH2B7 increased and decreased under drought treatment. Under salt stress, ALDH2B4 gene showed a down-rise trend. ALDH2B7 expression showed a down-up-down-down pattern. ALDH2B4ALDH3H1 gene response to low concentration of abscisic acid was more significant in ALDH2B7. The effect of low concentration abscisic acid on promoting effect was observed. Inhibited at high concentrations. The results showed that the three genes were all under drought stress, salt stress and ABA induced expression. Three plant superexpression vectors pCAMBI1300-335S-ALDH2B4 (pCAMB1300-0535S-ALD2B7) pCAMBIA1300-35S-ALDH3H1 were constructed.
【學(xué)位授予單位】：中南林業(yè)科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S794.3
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本文編號(hào)：2189554