低温敏感植物白银豆中三个脂肪酸去饱和酶基因的克隆与表达分析
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摘要
植物对低温的敏感程度与其起源地密切相关,并且起源于温带的植物经过低温冷驯化后可以获得一定的抗冷能力,而起源于热带亚热带的植物却普遍缺乏这样的能力。白银豆(Phaseolus lunatus L.)则是起源于热带美洲的豆科棉豆属植物。
大量实验证明膜脂的不饱和度与植物的冷适应性关系密切,抗冷植物比冷敏感植物的膜脂肪酸不饱和度高。生物膜磷脂的脂肪酸组成及不饱和度直接取决于脂肪酸去饱和酶的种类和数量,通过酶的种类和活性的变化,调节膜脂的不饱和度进而改变其流动性,以适应外界温度的变化。本研究对白银豆进行抗冷性鉴定,并克隆了3个脂肪酸去饱和酶基因,分析了低温等逆境脂肪酸去饱和基因的表达情况,为进一步研究脂肪酸去饱和酶基因的功能以及在植物抵抗低温等非生物逆境中的作用打下基础。
1.通过表型观察以及用膜相对电解质渗漏率做Arrhenius图,得到白银豆幼苗生长临界低温为8±1℃。对白银豆幼苗进行8℃低温处理,研究不同胁迫时间下叶片中各生理生化指标的应激动态变化。结果表明在临界生长低温下,白银豆幼苗的生长受到一定的影响,但是通过增加体内氧化酶活性等能在一定程度上抵御8℃低温产生的影响。
2.利用同源序列设计简并引物,先通过RT-PCR的方法从白银豆叶片中克隆到各脂肪酸去饱和酶基因的保守区域,然后根据基因片段设计RACE引物,利用RACE技术克隆各基因末端序列,拼接得到3个脂肪酸去饱和酶基因cDNA全长序列。同时,根据全长序列设计分段引物,以基因组DNA为模板分段克隆,拼接获得各基因DNA水平全长序列。其中SAD(硬脂酰-ACP去饱和酶)cDNA全长为1777 bp,ORF为1317 bp,编码439个氨基酸,DNA全长为1939 bp,包含有一个内含子。与大豆SAD同源性为86%,但是在进化关系上,白银豆与麻疯树的SAD亲缘关系最近。FAD3 cDNA全长1510 bp,ORF为1116 bp,编码372个氨基酸,DNA全长为2663 bp,包含有7个内含子。预测为内质网FAD3,负责催化亚油酸脱氢生成油酸,与大豆中FAD3同源性达91%。白银豆FAD2 cDNA全长为1652 bp,ORF为1059 bp,编码353个氨基酸,DNA全长与cDNA一致,无内含子。预测为叶绿体FAD2,和大豆FAD2亲缘关系最近,同源性达85%。
3.组织表达分析发现：SAD与FAD2在白银豆根和茎中几乎不表达,而在叶中表达量很高；而FAD3在根、茎、叶中均有表达,以叶中表达量最高。
4.运用半定量RT-PCR法分析各基因在生长临界低温以及高温、盐和干旱处理下的表达情况。结果表明：四种胁迫均能诱导FAD2基因的表达,而高温和盐胁迫对SAD和FAD3基因的表达有抑制作用。随着低温处理时间的延长,3个基因呈现不同的表达模式。SAD基因的表达逐渐下调,到12h时表达量最低,经过随后的12h低温黑暗后其表达量逐渐回升直到最大。FAD3表达在低温处理3h时达最大值,一直保持到低温处理12h,但随后表达降低。FAD2在低温处理过程之初瞬间表达上调,低温处理1h和3h间表达量基本不变,随后逐渐下调。在低温条件下,白银豆体内生成多不饱和脂肪酸的去饱和酶FAD2、FAD3表达的增强,可以促进单不饱和及双不饱和脂肪酸进一步去饱和。
The sensitivity of plants to low temperature is closely related to their initial origins. Plants from temperate regions have the capacity of cold acclimatation, that is, to develop increased freezing tolerance after being exposed to low nonfreezing temperature, whereas species from tropical and subtropical areas nearly do not have it. Phaseolus lunatus L. belonging to legumes origins from tropical regions.
Large amounts of experiments have demonstrated that the plant resistance to cold is quite related to UFAI (Unsaturated Fatty Acid Index) of cell membrane. The cell membrane of cold-resistant plant has higher UFAI than cold-sensitive ones. Fatty acid compositions and UFAI of membrane phospholipids are directly determined by the type and quantity of fatty acid desaturases (FAD). The ability to adjust membrane lipid fluidity by changing levels of unsaturated fatty acids is a feature of cold acclimating plants, which is mainly regulated by activity and categories of FADs. The growth critical temperature of cold-resistant Phaseolus lunatus L. was identified and three FAD genes were cloned from its leaf in this study to help understanding the mechanisms of cold-sensitivity. The results are summarized as follows.
1. Seedings of Phaseolus lunatus L. could resist 8±1℃according to phenotypic observation and Arrhenius plot of REC (Relative Electric Conductivity). Dynamic changes of various indexes in leaves of plant treated under 8±1℃indicated that the seedlings could truly resist low temperature of 8±1℃through increasing activities of oxidase system.
2. Primers were designed according to relative sequences on NCBI. Constant fragments of three FAD were isolated from leave of Phaseolus lunatus L. by RT-PCR. The full-lenth of their cDNAs were acquired by 5'and 3'RACE. The sequence of SAD, FAD3 and FAD2 had 1777 bp,1510 bp,1652 bp respectively with ORF of 1317 bp, 1116 bp and 1059 bp, encoding a 439,372 and 353 amino acid protein. Putative protein sequence alignments and phylogenetic analysis of three genes showed that three genes shared the highest identity of 86%,85% and 91% respectively with their counterpart species. The full-lenth DNAs of SAD and FAD3 contained 1 and 7 introns respectively, while FAD2 had no introns.
3. Tissue-specific expression analysis revealed that SAD and FAD2 expressed highly in leaves, but nearly did not express in root and sterm; while FAD3 expressed in all three tissues, especially high in leaves.
4. Expression pattern of each gene under different stresses including 8±1℃, 42℃,0.15 mmol/L NaCl and 15% PEG by RT-PCR showed that FAD2 could be induced by the stresses, while high temperature and salt somewhat inhibited the expression of SAD and FAD3. The expression patterns of three genes were different under low temperature. SAD was down-regulated in first 12 h and reached its lowest level, then returned in the following 12 h-dark till its maximum. Expression of FAD3 reached its maximum at the time point of 3 h and maintained to 12 h, then decreased. Expression of FAD2 was up-regulated from 1 h to 3 h, then decreased. To resist chilling temperature, the expression of FAD3、FAD2 in Phaseolus lunatus L. might enhance the amount of poly-unsaturated fatty acid to change the fluidity of cell membrane.
大量实验证明膜脂的不饱和度与植物的冷适应性关系密切,抗冷植物比冷敏感植物的膜脂肪酸不饱和度高。生物膜磷脂的脂肪酸组成及不饱和度直接取决于脂肪酸去饱和酶的种类和数量,通过酶的种类和活性的变化,调节膜脂的不饱和度进而改变其流动性,以适应外界温度的变化。本研究对白银豆进行抗冷性鉴定,并克隆了3个脂肪酸去饱和酶基因,分析了低温等逆境脂肪酸去饱和基因的表达情况,为进一步研究脂肪酸去饱和酶基因的功能以及在植物抵抗低温等非生物逆境中的作用打下基础。
1.通过表型观察以及用膜相对电解质渗漏率做Arrhenius图,得到白银豆幼苗生长临界低温为8±1℃。对白银豆幼苗进行8℃低温处理,研究不同胁迫时间下叶片中各生理生化指标的应激动态变化。结果表明在临界生长低温下,白银豆幼苗的生长受到一定的影响,但是通过增加体内氧化酶活性等能在一定程度上抵御8℃低温产生的影响。
2.利用同源序列设计简并引物,先通过RT-PCR的方法从白银豆叶片中克隆到各脂肪酸去饱和酶基因的保守区域,然后根据基因片段设计RACE引物,利用RACE技术克隆各基因末端序列,拼接得到3个脂肪酸去饱和酶基因cDNA全长序列。同时,根据全长序列设计分段引物,以基因组DNA为模板分段克隆,拼接获得各基因DNA水平全长序列。其中SAD(硬脂酰-ACP去饱和酶)cDNA全长为1777 bp,ORF为1317 bp,编码439个氨基酸,DNA全长为1939 bp,包含有一个内含子。与大豆SAD同源性为86%,但是在进化关系上,白银豆与麻疯树的SAD亲缘关系最近。FAD3 cDNA全长1510 bp,ORF为1116 bp,编码372个氨基酸,DNA全长为2663 bp,包含有7个内含子。预测为内质网FAD3,负责催化亚油酸脱氢生成油酸,与大豆中FAD3同源性达91%。白银豆FAD2 cDNA全长为1652 bp,ORF为1059 bp,编码353个氨基酸,DNA全长与cDNA一致,无内含子。预测为叶绿体FAD2,和大豆FAD2亲缘关系最近,同源性达85%。
3.组织表达分析发现：SAD与FAD2在白银豆根和茎中几乎不表达,而在叶中表达量很高；而FAD3在根、茎、叶中均有表达,以叶中表达量最高。
4.运用半定量RT-PCR法分析各基因在生长临界低温以及高温、盐和干旱处理下的表达情况。结果表明：四种胁迫均能诱导FAD2基因的表达,而高温和盐胁迫对SAD和FAD3基因的表达有抑制作用。随着低温处理时间的延长,3个基因呈现不同的表达模式。SAD基因的表达逐渐下调,到12h时表达量最低,经过随后的12h低温黑暗后其表达量逐渐回升直到最大。FAD3表达在低温处理3h时达最大值,一直保持到低温处理12h,但随后表达降低。FAD2在低温处理过程之初瞬间表达上调,低温处理1h和3h间表达量基本不变,随后逐渐下调。在低温条件下,白银豆体内生成多不饱和脂肪酸的去饱和酶FAD2、FAD3表达的增强,可以促进单不饱和及双不饱和脂肪酸进一步去饱和。
The sensitivity of plants to low temperature is closely related to their initial origins. Plants from temperate regions have the capacity of cold acclimatation, that is, to develop increased freezing tolerance after being exposed to low nonfreezing temperature, whereas species from tropical and subtropical areas nearly do not have it. Phaseolus lunatus L. belonging to legumes origins from tropical regions.
Large amounts of experiments have demonstrated that the plant resistance to cold is quite related to UFAI (Unsaturated Fatty Acid Index) of cell membrane. The cell membrane of cold-resistant plant has higher UFAI than cold-sensitive ones. Fatty acid compositions and UFAI of membrane phospholipids are directly determined by the type and quantity of fatty acid desaturases (FAD). The ability to adjust membrane lipid fluidity by changing levels of unsaturated fatty acids is a feature of cold acclimating plants, which is mainly regulated by activity and categories of FADs. The growth critical temperature of cold-resistant Phaseolus lunatus L. was identified and three FAD genes were cloned from its leaf in this study to help understanding the mechanisms of cold-sensitivity. The results are summarized as follows.
1. Seedings of Phaseolus lunatus L. could resist 8±1℃according to phenotypic observation and Arrhenius plot of REC (Relative Electric Conductivity). Dynamic changes of various indexes in leaves of plant treated under 8±1℃indicated that the seedlings could truly resist low temperature of 8±1℃through increasing activities of oxidase system.
2. Primers were designed according to relative sequences on NCBI. Constant fragments of three FAD were isolated from leave of Phaseolus lunatus L. by RT-PCR. The full-lenth of their cDNAs were acquired by 5'and 3'RACE. The sequence of SAD, FAD3 and FAD2 had 1777 bp,1510 bp,1652 bp respectively with ORF of 1317 bp, 1116 bp and 1059 bp, encoding a 439,372 and 353 amino acid protein. Putative protein sequence alignments and phylogenetic analysis of three genes showed that three genes shared the highest identity of 86%,85% and 91% respectively with their counterpart species. The full-lenth DNAs of SAD and FAD3 contained 1 and 7 introns respectively, while FAD2 had no introns.
3. Tissue-specific expression analysis revealed that SAD and FAD2 expressed highly in leaves, but nearly did not express in root and sterm; while FAD3 expressed in all three tissues, especially high in leaves.
4. Expression pattern of each gene under different stresses including 8±1℃, 42℃,0.15 mmol/L NaCl and 15% PEG by RT-PCR showed that FAD2 could be induced by the stresses, while high temperature and salt somewhat inhibited the expression of SAD and FAD3. The expression patterns of three genes were different under low temperature. SAD was down-regulated in first 12 h and reached its lowest level, then returned in the following 12 h-dark till its maximum. Expression of FAD3 reached its maximum at the time point of 3 h and maintained to 12 h, then decreased. Expression of FAD2 was up-regulated from 1 h to 3 h, then decreased. To resist chilling temperature, the expression of FAD3、FAD2 in Phaseolus lunatus L. might enhance the amount of poly-unsaturated fatty acid to change the fluidity of cell membrane.
引文
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