中间锦鸡儿油酸脱氢酶（FAD2）基因克隆及酵母表达调控的研究
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摘要
中间锦鸡儿(Caragana intermedia)广泛分布于我国西部、西北部干旱地区,具有改良土壤和保持水土等重要生态价值。为了在兼顾生态价值的同时创造更大的经济价值,结合中间锦鸡儿种子油脂含量较高的特点,对其脂肪酸生物合成进行研究,探讨内在的调控机理,对培育抗寒性强、油脂品质高的锦鸡儿优良新品种具有重要的意义和价值。本研究采用RT-PCR和RACE方法从中间锦鸡儿未成熟种子中克隆了多不饱和脂肪酸合成的关键酶——油酸脱氢酶(FAD2)基因;采用DNAMAN、DNAStar软件及http://www.us.expasy.Org、http://www.ch.embnet.org等网络资源分析了4个FAD2蛋白的二级结构特征、蛋白质修饰等生物学特性;通过定量PCR的方法研究了FAD2基因在组织中的表达谱,并将3个FAD2基因转化酵母,从mRNA、蛋白、产物三个水平对其进行表达、调控分析,初步探讨了其内在的调控机理。主要研究结果如下:
1.从中间锦鸡儿未成熟种子中克隆了3个Δ12脂肪酸脱氢酶基因,分别命名为FAD2-2A、FAD2-1A和FAD2-1B(GenBank No. EF503621、EU433557、EU433558 )。对这三个基因以及本实验室前期研究过的中间锦鸡儿CaFAD2基因(GenBank No. AY957394,本文中命名为FAD2-2B)进行同源性比较分析可知,FAD2-1A与FAD2-1B同源性很高, FAD2-1A编码的前283个氨基酸与FAD2-1B蛋白的同源性高达98.9%,前者比后者多编码97个氨基酸,全长基因编码氨基酸的同源性为70.71%。FAD2-2A与FAD2-2B的氨基酸序列同源性也较高,为81.6%。FAD2-2B与FAD2-1A的氨基酸序列同源性最低,为64.7%。
2.生物信息学分析表明,4个蛋白中只有FAD2-2B有信号肽,其余3个都没有信号肽剪切位点。二级结构FAD2-2A与FAD2-1A相似,以无规卷曲为主,含少量直链,间或有α螺旋;FAD2-2B与FAD2-1B相似,主要以无规卷曲和直链为主。4个蛋白是典型的膜结合蛋白,都有5~6个跨膜疏水区。在蛋白的稳定性方面,FAD2-2A蛋白半衰期是220h,其余的半衰期仅30h或31h;在蛋白翻译相对效率方面,FAD2-2B为1,其余3个均为5。FAD2-1A、FAD2-1B、FAD2-2A和FAD2-2B四个蛋白的棕榈酰化位点分别是5、4、8和9个。
3、Southern检测结果表明,在中间锦鸡儿基因组中FAD2基因至少有4个拷贝。FAD2-2A、FAD2-1A、FAD2-1B三个基因的荧光定量PCR结果显示,FAD2-2A基因在根和发育中期、后期种子中低水平表达,在幼嫩的茎、叶以及发育早期的种子中高水平表达。FAD2-1A基因在根中的表达量最低,在种子发育早期、中期大量表达,在幼嫩叶子中表达水平也较高。FAD2-1B基因仅在种子发育中期大量表达,别的组织、发育阶段表达量都保持在本底水平。在不同组织以及种子的不同发育时期,FAD2-1A表达量的变化幅度最大,达到了近100倍,FAD2-1B次之,FAD2-2A的变化幅度最小。结合序列比对分析我们推断:FAD2-2A基因可能主要负责合成膜脂中亚油酸,FAD2-1A主要负责种子及叶子贮脂中亚油酸的合成,FAD2-1B负责种子贮脂中油酸去饱和作用。
4、通过双酶切将FAD2-2A、FAD2-1A和FAD2-1B三个基因构建到酵母表达载体pYES2中转化酵母。20℃诱导表达菌株样品的定量PCR、SDS-PAGE以及脂肪酸含量三个层次的分析可见,FAD2-2A基因在诱导表达的前期转录水平迅速增加,后期表达量保持平稳,蛋白质、产物与转录水平变化趋势一致;FAD2-1A基因表达的3个层次上的变化趋势也一样,都是表达量逐渐升高,且升高速度在整个过程中比较均衡; FAD2-1B的三个表达层次都表现出初期少量表达,中期表达量迅速增加的趋势。
5、进行15℃低温处理后,FAD2-2A、FAD2-1A表达量在三个层次都有明显升高,FAD2-1A的升高幅度比FAD2-2A小;FAD2-1B转录水平略有升高,但蛋白和产物水平都没有明显变化。在受到高温胁迫时,FAD2-2A、FAD2-1A基因表达在三个层次都表现出先升高后降低的趋势,FAD2-1B转录水平和产物水平都是先升高后降低,但蛋白水平无明显变化。
6、三个处理温度下基因表达三个层次的分析表明,FAD2-2A、FAD2-1A在各处理条件下的表达调控可能主要通过转录调控机制实现。FAD2-1B基因在20℃条件下主要是转录水平调控,在低温条件下可能是通过转录和翻译调控来共同实现的,而高温条件下可能是通过转录、翻译以及翻译后三个层次进行调控的。
7、在温度条件变化时,FAD2-2A基因感应外界条件变化的速度最快,最先出现表达量的变化,FAD2-1A次之,FAD2-1B最慢。
本研究全面、完整的阐述了FAD2-2A、FAD2-1A和FAD2-1B 3个基因的表达规律,揭示了这三个基因内在的调控机理,为进一步开展中间锦鸡儿种子油脂品质的定向遗传改良,培育适合生物柴油等各种化工生产原料的新品种创造条件和提供理论基础。同时,也能为培育抗寒性强的锦鸡儿新品种奠定基础。
Caragana intermedia is widespread and is very important for ecological environment which can improve the soil and hold water at arid region of China northwest. In order to make more production,oleic acid desaturase (FAD2) gene which code the key enzyme of polyunsaturated fatty acid synthesis were cloned from young seed of C. intermedia by RACE. And they were expressed and analyzed in yeast. These studies found the rationale for C. intermedia genetics improvement and breeding the new fuchsins with good quality oil or cold resistance. The main results are as follows:
1. Three full-lengthΔ12 fatty acid desaturases genes were cloned from pedo-seeds of C. intermedia., which named FAD2-2A(GenBank No. EF503621), FAD2-1A(GenBank No. EU433557) and FAD2-1B(GenBank No. EU433558 ), respectively. The sequence analysis of these three genes and fad2-2B gene(CaFAD2, GeneBank accession AY957394) which was cloned from caragana intermedia leaves by Wang Y.D. have done. The deduced first 283 amino acid sequences of Fad2-1A and fad2-1B showed high identity which was 98.9%, and the FAD2-1A protein is 97aa longer than FAD2-1B protein. The identity of FAD2-2A and FAD2-2B deduced amino acid sequences was 81.6%, and that of FAD2-2B and FAD2-1A was lowest 64.7%.
2. The four FAD2 proteins structure and features were analyzed by bioinformatics methods, such as DNAMAN, DNAStar software, and http://www.us.expasy.Org, http://www.ch.embnet.org internet means and so on. The results indicated that FAD2-2B had signal peptide and the others had not signal peptide cleaved sites. The secondary structure of FAD2-2A and FAD2-1A were similar and coil was main with little strand and helix. FAD2-2B and FAD2-1B secondary structure are coil and strand mainly. The four proteins were membrane-bound proteins and have five or six transmembrane domains, respectively. The half life of FAD2-2A protein was 220 h, and that of the others were 30 h or 31 h. FAD2-2B relative translation effectiveness was 1 and that of the other proteins were 5. The palmitoylation sites of FAD2-1A, FAD2-1B, FAD2-2A and FAD2-2B were 5, 4, 8, 9, respectively.
3. Southern-blotting results indicated there are four copies of FAD2 gene in C. intermedia. genome at least, and these are coincident with the four FAD2 genes were cloned. The real-time PCR was performed to detect the relative expression levels of FAD2-2A, FAD2-1A and FAD2-1B mRNA. FAD2-2A transcript showed a low level in the root, middle-stage seeds and pre-mature seeds, and FAD2-2A was expressed more in the first-stage seeds and tender leaves than in the other tissues. FAD2-1A transcripts showed the lowest level in the root and the highest level in the first-stage and middle-stage seeds, and it was expressed abundantly in the tender leaves too. FAD2-1B was expressed abundantly in the middle-stage seeds and its transcript was very low in the other tissues. In all tissues the FAD2-1A transcript level changed most, and that of FAD2-2A changed lest. According to all of these, we deduced that FAD2-2A takes responsibility of desaturating oleic acid in the membrane lipid mainly; FAD2-1A is in charge of desaturating oleic acid in the store lipid of seeds and leaves; and FAD2-1B is in charge of desaturating oleic acid in the store lipid of seeds, too.
4. The Expression vectors pYES2-FAD2-2A, pYES2-FAD2-1A and pYES2-FAD2-1B were constructed by double enzymes digesting and transformed yeast. The yeasts with pYES2-FAD2-2A, pYES2-FAD2-1A and pYES2-FAD2-1B were inducted to express the exogenous genes at 20℃and were treated by cold(15℃) and high temperature(30℃). FAD2-2A transcription increased quickly at first stage and then keep the high level with induction medium at 20℃. The change tendencies of FAD2-2A protein and fatty acid productions are similar with that of transcription. The transcription, protein and fatty acid productions of FAD2-1A increased gradually at the whole process. And that of FAD2-1B kept low level at the first stage and increased at the middle stage.
5. When the yeasts were treated by low temperature, the expression of FAD2-2A and FAD2-1A increased obviously, and the FAD2-1B transcription increased a little.But the FAD2-1B protein and fatty acid production did not change. If the yeasts were treated by high temperature, the expression of FAD2-2A and FAD2-1A increased firstly and then decreased, but FAD2-1B protein did not change.
6. The analysis results of real-time PCR, SDS-PAGE and fatty acid content suggested that FAD2-2A and FAD2-1A expression were regulated in transcript level with all three kinds of treating methods; FAD2-1B were regulated in transcript level with 20℃, were regulated at transcript and translation stages with cold, and at transcript, translation and after translation stages with high temperature treatment.
7. FAD2-2A change was fastest to response to temperature and FAD2-1B was slowest. In this study, the FAD2 genes were researched from transcripts, translation and productions, the inherent regulating mechanism of the three genes were provided. All of these give the conditions and the theory guidance for Caragana intermedia gene engineering breeding to improve the seed oil quality.
1.从中间锦鸡儿未成熟种子中克隆了3个Δ12脂肪酸脱氢酶基因,分别命名为FAD2-2A、FAD2-1A和FAD2-1B(GenBank No. EF503621、EU433557、EU433558 )。对这三个基因以及本实验室前期研究过的中间锦鸡儿CaFAD2基因(GenBank No. AY957394,本文中命名为FAD2-2B)进行同源性比较分析可知,FAD2-1A与FAD2-1B同源性很高, FAD2-1A编码的前283个氨基酸与FAD2-1B蛋白的同源性高达98.9%,前者比后者多编码97个氨基酸,全长基因编码氨基酸的同源性为70.71%。FAD2-2A与FAD2-2B的氨基酸序列同源性也较高,为81.6%。FAD2-2B与FAD2-1A的氨基酸序列同源性最低,为64.7%。
2.生物信息学分析表明,4个蛋白中只有FAD2-2B有信号肽,其余3个都没有信号肽剪切位点。二级结构FAD2-2A与FAD2-1A相似,以无规卷曲为主,含少量直链,间或有α螺旋;FAD2-2B与FAD2-1B相似,主要以无规卷曲和直链为主。4个蛋白是典型的膜结合蛋白,都有5~6个跨膜疏水区。在蛋白的稳定性方面,FAD2-2A蛋白半衰期是220h,其余的半衰期仅30h或31h;在蛋白翻译相对效率方面,FAD2-2B为1,其余3个均为5。FAD2-1A、FAD2-1B、FAD2-2A和FAD2-2B四个蛋白的棕榈酰化位点分别是5、4、8和9个。
3、Southern检测结果表明,在中间锦鸡儿基因组中FAD2基因至少有4个拷贝。FAD2-2A、FAD2-1A、FAD2-1B三个基因的荧光定量PCR结果显示,FAD2-2A基因在根和发育中期、后期种子中低水平表达,在幼嫩的茎、叶以及发育早期的种子中高水平表达。FAD2-1A基因在根中的表达量最低,在种子发育早期、中期大量表达,在幼嫩叶子中表达水平也较高。FAD2-1B基因仅在种子发育中期大量表达,别的组织、发育阶段表达量都保持在本底水平。在不同组织以及种子的不同发育时期,FAD2-1A表达量的变化幅度最大,达到了近100倍,FAD2-1B次之,FAD2-2A的变化幅度最小。结合序列比对分析我们推断:FAD2-2A基因可能主要负责合成膜脂中亚油酸,FAD2-1A主要负责种子及叶子贮脂中亚油酸的合成,FAD2-1B负责种子贮脂中油酸去饱和作用。
4、通过双酶切将FAD2-2A、FAD2-1A和FAD2-1B三个基因构建到酵母表达载体pYES2中转化酵母。20℃诱导表达菌株样品的定量PCR、SDS-PAGE以及脂肪酸含量三个层次的分析可见,FAD2-2A基因在诱导表达的前期转录水平迅速增加,后期表达量保持平稳,蛋白质、产物与转录水平变化趋势一致;FAD2-1A基因表达的3个层次上的变化趋势也一样,都是表达量逐渐升高,且升高速度在整个过程中比较均衡; FAD2-1B的三个表达层次都表现出初期少量表达,中期表达量迅速增加的趋势。
5、进行15℃低温处理后,FAD2-2A、FAD2-1A表达量在三个层次都有明显升高,FAD2-1A的升高幅度比FAD2-2A小;FAD2-1B转录水平略有升高,但蛋白和产物水平都没有明显变化。在受到高温胁迫时,FAD2-2A、FAD2-1A基因表达在三个层次都表现出先升高后降低的趋势,FAD2-1B转录水平和产物水平都是先升高后降低,但蛋白水平无明显变化。
6、三个处理温度下基因表达三个层次的分析表明,FAD2-2A、FAD2-1A在各处理条件下的表达调控可能主要通过转录调控机制实现。FAD2-1B基因在20℃条件下主要是转录水平调控,在低温条件下可能是通过转录和翻译调控来共同实现的,而高温条件下可能是通过转录、翻译以及翻译后三个层次进行调控的。
7、在温度条件变化时,FAD2-2A基因感应外界条件变化的速度最快,最先出现表达量的变化,FAD2-1A次之,FAD2-1B最慢。
本研究全面、完整的阐述了FAD2-2A、FAD2-1A和FAD2-1B 3个基因的表达规律,揭示了这三个基因内在的调控机理,为进一步开展中间锦鸡儿种子油脂品质的定向遗传改良,培育适合生物柴油等各种化工生产原料的新品种创造条件和提供理论基础。同时,也能为培育抗寒性强的锦鸡儿新品种奠定基础。
Caragana intermedia is widespread and is very important for ecological environment which can improve the soil and hold water at arid region of China northwest. In order to make more production,oleic acid desaturase (FAD2) gene which code the key enzyme of polyunsaturated fatty acid synthesis were cloned from young seed of C. intermedia by RACE. And they were expressed and analyzed in yeast. These studies found the rationale for C. intermedia genetics improvement and breeding the new fuchsins with good quality oil or cold resistance. The main results are as follows:
1. Three full-lengthΔ12 fatty acid desaturases genes were cloned from pedo-seeds of C. intermedia., which named FAD2-2A(GenBank No. EF503621), FAD2-1A(GenBank No. EU433557) and FAD2-1B(GenBank No. EU433558 ), respectively. The sequence analysis of these three genes and fad2-2B gene(CaFAD2, GeneBank accession AY957394) which was cloned from caragana intermedia leaves by Wang Y.D. have done. The deduced first 283 amino acid sequences of Fad2-1A and fad2-1B showed high identity which was 98.9%, and the FAD2-1A protein is 97aa longer than FAD2-1B protein. The identity of FAD2-2A and FAD2-2B deduced amino acid sequences was 81.6%, and that of FAD2-2B and FAD2-1A was lowest 64.7%.
2. The four FAD2 proteins structure and features were analyzed by bioinformatics methods, such as DNAMAN, DNAStar software, and http://www.us.expasy.Org, http://www.ch.embnet.org internet means and so on. The results indicated that FAD2-2B had signal peptide and the others had not signal peptide cleaved sites. The secondary structure of FAD2-2A and FAD2-1A were similar and coil was main with little strand and helix. FAD2-2B and FAD2-1B secondary structure are coil and strand mainly. The four proteins were membrane-bound proteins and have five or six transmembrane domains, respectively. The half life of FAD2-2A protein was 220 h, and that of the others were 30 h or 31 h. FAD2-2B relative translation effectiveness was 1 and that of the other proteins were 5. The palmitoylation sites of FAD2-1A, FAD2-1B, FAD2-2A and FAD2-2B were 5, 4, 8, 9, respectively.
3. Southern-blotting results indicated there are four copies of FAD2 gene in C. intermedia. genome at least, and these are coincident with the four FAD2 genes were cloned. The real-time PCR was performed to detect the relative expression levels of FAD2-2A, FAD2-1A and FAD2-1B mRNA. FAD2-2A transcript showed a low level in the root, middle-stage seeds and pre-mature seeds, and FAD2-2A was expressed more in the first-stage seeds and tender leaves than in the other tissues. FAD2-1A transcripts showed the lowest level in the root and the highest level in the first-stage and middle-stage seeds, and it was expressed abundantly in the tender leaves too. FAD2-1B was expressed abundantly in the middle-stage seeds and its transcript was very low in the other tissues. In all tissues the FAD2-1A transcript level changed most, and that of FAD2-2A changed lest. According to all of these, we deduced that FAD2-2A takes responsibility of desaturating oleic acid in the membrane lipid mainly; FAD2-1A is in charge of desaturating oleic acid in the store lipid of seeds and leaves; and FAD2-1B is in charge of desaturating oleic acid in the store lipid of seeds, too.
4. The Expression vectors pYES2-FAD2-2A, pYES2-FAD2-1A and pYES2-FAD2-1B were constructed by double enzymes digesting and transformed yeast. The yeasts with pYES2-FAD2-2A, pYES2-FAD2-1A and pYES2-FAD2-1B were inducted to express the exogenous genes at 20℃and were treated by cold(15℃) and high temperature(30℃). FAD2-2A transcription increased quickly at first stage and then keep the high level with induction medium at 20℃. The change tendencies of FAD2-2A protein and fatty acid productions are similar with that of transcription. The transcription, protein and fatty acid productions of FAD2-1A increased gradually at the whole process. And that of FAD2-1B kept low level at the first stage and increased at the middle stage.
5. When the yeasts were treated by low temperature, the expression of FAD2-2A and FAD2-1A increased obviously, and the FAD2-1B transcription increased a little.But the FAD2-1B protein and fatty acid production did not change. If the yeasts were treated by high temperature, the expression of FAD2-2A and FAD2-1A increased firstly and then decreased, but FAD2-1B protein did not change.
6. The analysis results of real-time PCR, SDS-PAGE and fatty acid content suggested that FAD2-2A and FAD2-1A expression were regulated in transcript level with all three kinds of treating methods; FAD2-1B were regulated in transcript level with 20℃, were regulated at transcript and translation stages with cold, and at transcript, translation and after translation stages with high temperature treatment.
7. FAD2-2A change was fastest to response to temperature and FAD2-1B was slowest. In this study, the FAD2 genes were researched from transcripts, translation and productions, the inherent regulating mechanism of the three genes were provided. All of these give the conditions and the theory guidance for Caragana intermedia gene engineering breeding to improve the seed oil quality.
引文
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