油桐乙醛脱氢酶ALDH基因家族的克隆与功能表达研究
发布时间：2018-08-18 13:05
【摘要】：油桐是一种经济价值较高的木本油料树种。生态环境的恶化使得油桐在生长发育过程中面临着多种逆境,造成油桐减产。植物在遭受逆境胁迫时会产生有毒的醛类物质,乙醛脱氢酶作为一种醛类物质的消除剂,对植物抗击逆境意义重大。本论文从油桐中分离克隆了乙醛脱氢酶家族的3个基因,并对这些基因进行了功能表达研究,主要的研究结果如下:1.以'葡萄桐'为实验研究材料,基于油桐转录组数据,设计引物,分别克隆得到油桐乙醛脱氢酶ALDH2B4、ALDH2B7、ALDH3H1基因的全长CDS序列。ALDH2B4基因CDS全长为1617 bp,ORF为1617 bp,编码538个氨基酸,预测分析显示蛋白质分子量为58.79 kDa,等电点pI为7.16;ALD2B7基因CDS全长为1626 bp,ORF为1626 bp,编码541个氨基酸,预测分析显示蛋白质分子量为 59.01 kDa,等电点 pI 为 6.38;ALD3H1基因 CDS 全长为 1245 bp,ORF为1245bp,编码414个氨基酸,预测分析显示蛋白质分子量为45.06kDa,等电点 pI 为 9.47。2.以葡萄桐叶片为材料提取DNA,分段克隆拼接得到ALDHH2B4、ALDH2B7、ALDH3H1基因的基因组全长序列。ALDH2B4基因组全长3746bp,含有11段内含子;ALDH2B7基因组全长5358bp,含有11段内含子;ALDH3H1基因组全长4323bp,含有9段内含子。3.实时定量PCR分析ALDH基因在'葡萄桐'不同组织和种子发育时期的表达量变化。结果表明,ALDH2B4基因在不同组织的表达量大小为:嫩叶老叶幼根老茎茎柱头雄蕊子房花萼花瓣;ALDH2B7基因在不同组织的表达量大小为:花瓣老茎柱头茎幼根嫩叶子房老叶花萼雄蕊;ALDH3H1基因在不同组织的表达量大小为:老叶嫩叶雄蕊柱头花瓣幼根老茎茎花萼子房。4.实时定量PCR分析ALDH基因在干旱、盐胁迫以及脱落酸条件下的表达情况。在干旱处理下,ALDH2B4、ALDH2B7和ALDH3H1基因均呈现上升再下降的变化趋势;在盐胁迫条件下,ALDH2B4基因呈下降-升高的趋势,ALDH2B7表达呈现下降-升高-下降模式,ALDH3H1基因表达呈现升高-下降-升高模式。ALDH2B4、ALDH3H1基因对低浓度脱落酸的响应更为显著,ALDH2B7在低浓度脱落酸处理下呈现促进作用,在高浓度情况下受到抑制。结果表明,这三个基因均受到干旱胁迫、盐胁迫及ABA诱导表达。5.构建了pCAMBI1300-335S-ALDH2B4、pCAMB1300-0535S-ALD2B7、pCAMBIA1300-35S-ALDH3H1三个植物超表达载体,通过花粉管通道法侵染拟南芥和阳性筛选,得到转基因拟南芥植株。
[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.
【学位授予单位】：中南林业科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S794.3
，
本文编号：2189554
[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.
【学位授予单位】：中南林业科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S794.3
，
本文编号：2189554
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