大豆MYB转录因子GmMYBJ6的功能初探
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摘要
植物在生长过程中,为了适应内部和外部环境的刺激,逐渐形成了一些适应环境的生理生态功能,从而对逆境和发育信号作出反应,产生各种类型的次生代谢产物。其中,通过转录因子在转录水平上对相关功能基因进行复杂而精细的表达调控,是植物对其生长发育及生理代谢调控的一种重要方式。近年来,众多国内外研究者报道相继从高等植物中分离出一系列调控干旱、高盐、低温、抗病反应及发育等相关基因表达的转录因子。因此,植物转录因子的结构与功能已成为植物分子生物学领域的研究热点。
类黄酮化合物是一类多酚化合物,是植物中一类重要的次生代谢物,是植物在长期的生态适应过程中为抵御恶劣生态条件、动物、微生物等胁迫而形成的一大类次生代谢产物。有研究表明在植物组织器官中,黄酮类化合物的积累是植物应答逆境胁迫的一个重要特性,如保护植物免受辐射伤害、结合植物毒素、增强植物耐寒性、抗虫性、清除自由基活性、抗血管收缩活性、抗溶血因子等。大豆是重要的油料作物,同时也是蛋白和异黄酮的重要来源。大豆异黄酮作为黄酮类化合物中的一类重要化合物,是天然存在的植物性雌激素,具有很强的生理和药理活性。MYB转录因子作为植物中最大的转录因子家族之一,广泛参与植物次生代谢调控、激素和环境因子的应答,并对细胞周期调控、形态建成起着重要作用。近年来,MYB转录因子被证实广泛参与类黄酮代谢途径的调控,植物中MYB家族基因的表达受到多种逆境条件的诱导,在植物异黄酮代谢途径中发挥重要的调控作用。因此利用MYB转录因子调控植物体内类黄酮的含量,增强植物对环境胁迫耐受性,已成为近年来植物基因工程的又一新热点。
本文以实验室前期从大豆品种吉林3号中分离克隆的MYB转录因子新基因GmMYBJ6(DQ902863)为研究材料,在前期研究基础上,分别对其核酸序列和所编码蛋白的结构特征及其表达特进行了进一步的分析鉴定;并将目的基因在烟草中进行了过表达,进一步研究鉴定GmMYBJ6在模式植物烟草中,对类黄酮生物合成及转基因烟草抗逆胁迫方面的调节作用。以期能为后续全面解析目的基因在植物类黄酮次生代谢调控网络中的功能及其调控机理奠定理论基础。本研究论文的主要研究内容概述如下:
(1)对GmMYBJ6编码蛋白进行生物信息学分析,发现其含有两个保守的MYB结构域,即N-端含有一个保守的DNA结合域(R2R3-type),C-端具有典型的转录因子转录激活域;对GmMYBJ6编码蛋白进行功能预测和鉴定,发现其在转录调控等方面起着主要作用,说明其为典型的R2R2-MYB转录因子;并通过酵母单杂交实验进一步验证了GmMYBJ6具有转录激活活性;
(2)分析了GmMYBJ6在不同胁迫处理下的表达特性。结果显示,失水条件下GmMYBJ6表达量先下降随后升高,6-BA处理24h后GmMYBJ6表达量有所增加,其他处理下GmMYBJ6表达量无明显变化;
(3)对目的基因进行了原核表达和纯化,为进一步明确GmMYBJ6在植物代谢调控途径中对下游靶基因的调控机制提供基础;
(4)测定转基因烟草中类黄酮含量,结果显示与野生型相比转基因烟草类黄酮含量显著提高,说明目的基因参与植物黄酮类的代谢调控,并能显著提高其含量;
(5)将目的基因在烟草中进行过表达,RT-PCR分析了转基因烟草阳性苗中苯丙烷类化合物代谢途径各关键酶基因的表达情况。结果显示,目的基因的导入明显提高黄酮类化合物代谢途径部分关键酶基因,如PAL、CHS、CHI、FLS的表达,而木质素代谢途径各关键酶基因如COMT、4CL、CAD的表达没有明显变化;
(6)分析转基因烟草对各种非生物胁迫的耐受性。发现转基因植株对紫外、高盐和干旱胁迫的耐受性与野生型相比都有所提高,初步鉴定了GmMYBJ6在植物抗逆胁迫方面的调控作用。
In the long-term process of growth,in order to adapting external and internal stimuli, plants are capable of synthesizing a large variety of secondary metabolites which play important roles in tolerate stresses and development.Transcriptional regulation of structure gene is a crucial part of plant growth and metabolism.For the past few years,a series of plant transcription factors which involved in numerous processes,including responses to drought,salt,low temperature,pathogenic bacteria and development have cloned.Thus, the research of the structure and function of plant transcription factor have been researched in the molecular biology field.
Flavonoids are a important subclass of plant secondary metabolites that are derived from the phenylpropanoid pathway responding to stress,such as environmental tresses, animal and microorganism stresses et al.As one kind of flavonoids,isoflavone is a natural vegetality estrin that has many strong physiological and pharmacology activities,such as defence of radiational injury,combining of phytotoxin,resistance to tolerance and insect feeding,getting rid of free radicle activity,resistance to vasoconstriction and antihemolytic factor,et al.As one of the largest plant transcription factor families,MYB transcription factors play an important role in plant secondary metabolism,such as,responsing to hormoned and environmental factor,regulation of cell life cycle and plant morphogenesis et al.Soybean is an important oil grain,which is the principle source of isoflavone,as well. Some researches showed that the accumulation of flavonoids in plant tissues and organs plays a part in plant defense.Up to now,many MYB transcription factors were reported to be involved in the flavonoids metabolism pathway.And,using MYB transcription factors to regulate the content of isoflavone in plant and enhance the plant tolerance to environment stimulates have beening a hot spot in plant genetic engineering.
In this paper,we do some deep research of the MYB transcription factor gene GmMYBJ6(DQ902863) which was isolated from soybean cultivar Jilin 3 by our lab, resently.In this paper,we analyzed its structure and expression characteristics. Furthermore,the GmMYBJ6 was overexpressed in tobacco under the control of the CaMV 35 promoter to research its functions in the flavonoid biosynthetic pathway,as well as its roles in palnt resistance to in various defense and stress responses.The main contents of this study are as follows:
(1) Analysis the GmMYBJ6 encoding protein by bioinformatics,and the result showed that there are two conservative MYB domains in GmMYBJ6.In its C-terminal,there is a transcription activation domain which presumed that it may have a transcriptional activation.Predition of the function of GmMYBJ6 showed that it may play a crucial part of transcriptional regulation as well.All of which demonstrated that GmMYBJ6 was a typical plant R2R2-MYB transcription factor.The recombinant vectors pBridge-GmMYBJ6 was constructed and transformed into the yeast to analysiz the transcriptional activation of GmMYBJ6.
(2) The expression pattern of GmMYBJ6 in different stress treatments was studied by semi-quantitative RT-PCR.The expression of GmMYBJ6 could be decreased and later increased under drought.The Expression of GmMYBJ6 could be increased after 24 hours by 6-BA treatment.The expression of GmMYBJ6 didn't change in the others treatments.
(3) GmMYBJ6 fusion protein was expressed in E.coli and purified which will be a potential foundation for the further reseach the regulation mechanism of GmMYBJ6 in plant metabolism pathway.
(4) We analysis the content of isoflavone in transgenic tobacco.And variance analysis showed that the content of isoflavone was highly increased in transgenic tobacco.Multiple comparisons illustrate that the content of isoflavone increased remarkably,in the transformed plant.
(5) Semi-quantitative RT-PCR analysis indicated that GmMYBJ6 could improve the expressions of some flavonoid biosynthetic genes,such as PAL(Phenylalanine ammonia lyase),CHS(Chalcone Synthase),CHI(chalcone isomerase) and FLS(flavonol synthase). However,the expressions of some lignin biosynthetic genes,such as COMT (catechol-O-methyltransferase),4CL(4-coumaroyl-CoA ligase) and CAD(cinnamyl alcohol dehydrogenase) were not influenced obviously.
(6) The analysis of anti-stress experiments showed that overexpression of GmMYBJ6 could improve transgenic tobaccos' resistance to UV-C radiation,salt and drought obviously.It confirmed that GmMYBJ6 may involved in the regulaotion of plant defense which domenstrants the primarily function of GmMYBJ6 as well.
类黄酮化合物是一类多酚化合物,是植物中一类重要的次生代谢物,是植物在长期的生态适应过程中为抵御恶劣生态条件、动物、微生物等胁迫而形成的一大类次生代谢产物。有研究表明在植物组织器官中,黄酮类化合物的积累是植物应答逆境胁迫的一个重要特性,如保护植物免受辐射伤害、结合植物毒素、增强植物耐寒性、抗虫性、清除自由基活性、抗血管收缩活性、抗溶血因子等。大豆是重要的油料作物,同时也是蛋白和异黄酮的重要来源。大豆异黄酮作为黄酮类化合物中的一类重要化合物,是天然存在的植物性雌激素,具有很强的生理和药理活性。MYB转录因子作为植物中最大的转录因子家族之一,广泛参与植物次生代谢调控、激素和环境因子的应答,并对细胞周期调控、形态建成起着重要作用。近年来,MYB转录因子被证实广泛参与类黄酮代谢途径的调控,植物中MYB家族基因的表达受到多种逆境条件的诱导,在植物异黄酮代谢途径中发挥重要的调控作用。因此利用MYB转录因子调控植物体内类黄酮的含量,增强植物对环境胁迫耐受性,已成为近年来植物基因工程的又一新热点。
本文以实验室前期从大豆品种吉林3号中分离克隆的MYB转录因子新基因GmMYBJ6(DQ902863)为研究材料,在前期研究基础上,分别对其核酸序列和所编码蛋白的结构特征及其表达特进行了进一步的分析鉴定;并将目的基因在烟草中进行了过表达,进一步研究鉴定GmMYBJ6在模式植物烟草中,对类黄酮生物合成及转基因烟草抗逆胁迫方面的调节作用。以期能为后续全面解析目的基因在植物类黄酮次生代谢调控网络中的功能及其调控机理奠定理论基础。本研究论文的主要研究内容概述如下:
(1)对GmMYBJ6编码蛋白进行生物信息学分析,发现其含有两个保守的MYB结构域,即N-端含有一个保守的DNA结合域(R2R3-type),C-端具有典型的转录因子转录激活域;对GmMYBJ6编码蛋白进行功能预测和鉴定,发现其在转录调控等方面起着主要作用,说明其为典型的R2R2-MYB转录因子;并通过酵母单杂交实验进一步验证了GmMYBJ6具有转录激活活性;
(2)分析了GmMYBJ6在不同胁迫处理下的表达特性。结果显示,失水条件下GmMYBJ6表达量先下降随后升高,6-BA处理24h后GmMYBJ6表达量有所增加,其他处理下GmMYBJ6表达量无明显变化;
(3)对目的基因进行了原核表达和纯化,为进一步明确GmMYBJ6在植物代谢调控途径中对下游靶基因的调控机制提供基础;
(4)测定转基因烟草中类黄酮含量,结果显示与野生型相比转基因烟草类黄酮含量显著提高,说明目的基因参与植物黄酮类的代谢调控,并能显著提高其含量;
(5)将目的基因在烟草中进行过表达,RT-PCR分析了转基因烟草阳性苗中苯丙烷类化合物代谢途径各关键酶基因的表达情况。结果显示,目的基因的导入明显提高黄酮类化合物代谢途径部分关键酶基因,如PAL、CHS、CHI、FLS的表达,而木质素代谢途径各关键酶基因如COMT、4CL、CAD的表达没有明显变化;
(6)分析转基因烟草对各种非生物胁迫的耐受性。发现转基因植株对紫外、高盐和干旱胁迫的耐受性与野生型相比都有所提高,初步鉴定了GmMYBJ6在植物抗逆胁迫方面的调控作用。
In the long-term process of growth,in order to adapting external and internal stimuli, plants are capable of synthesizing a large variety of secondary metabolites which play important roles in tolerate stresses and development.Transcriptional regulation of structure gene is a crucial part of plant growth and metabolism.For the past few years,a series of plant transcription factors which involved in numerous processes,including responses to drought,salt,low temperature,pathogenic bacteria and development have cloned.Thus, the research of the structure and function of plant transcription factor have been researched in the molecular biology field.
Flavonoids are a important subclass of plant secondary metabolites that are derived from the phenylpropanoid pathway responding to stress,such as environmental tresses, animal and microorganism stresses et al.As one kind of flavonoids,isoflavone is a natural vegetality estrin that has many strong physiological and pharmacology activities,such as defence of radiational injury,combining of phytotoxin,resistance to tolerance and insect feeding,getting rid of free radicle activity,resistance to vasoconstriction and antihemolytic factor,et al.As one of the largest plant transcription factor families,MYB transcription factors play an important role in plant secondary metabolism,such as,responsing to hormoned and environmental factor,regulation of cell life cycle and plant morphogenesis et al.Soybean is an important oil grain,which is the principle source of isoflavone,as well. Some researches showed that the accumulation of flavonoids in plant tissues and organs plays a part in plant defense.Up to now,many MYB transcription factors were reported to be involved in the flavonoids metabolism pathway.And,using MYB transcription factors to regulate the content of isoflavone in plant and enhance the plant tolerance to environment stimulates have beening a hot spot in plant genetic engineering.
In this paper,we do some deep research of the MYB transcription factor gene GmMYBJ6(DQ902863) which was isolated from soybean cultivar Jilin 3 by our lab, resently.In this paper,we analyzed its structure and expression characteristics. Furthermore,the GmMYBJ6 was overexpressed in tobacco under the control of the CaMV 35 promoter to research its functions in the flavonoid biosynthetic pathway,as well as its roles in palnt resistance to in various defense and stress responses.The main contents of this study are as follows:
(1) Analysis the GmMYBJ6 encoding protein by bioinformatics,and the result showed that there are two conservative MYB domains in GmMYBJ6.In its C-terminal,there is a transcription activation domain which presumed that it may have a transcriptional activation.Predition of the function of GmMYBJ6 showed that it may play a crucial part of transcriptional regulation as well.All of which demonstrated that GmMYBJ6 was a typical plant R2R2-MYB transcription factor.The recombinant vectors pBridge-GmMYBJ6 was constructed and transformed into the yeast to analysiz the transcriptional activation of GmMYBJ6.
(2) The expression pattern of GmMYBJ6 in different stress treatments was studied by semi-quantitative RT-PCR.The expression of GmMYBJ6 could be decreased and later increased under drought.The Expression of GmMYBJ6 could be increased after 24 hours by 6-BA treatment.The expression of GmMYBJ6 didn't change in the others treatments.
(3) GmMYBJ6 fusion protein was expressed in E.coli and purified which will be a potential foundation for the further reseach the regulation mechanism of GmMYBJ6 in plant metabolism pathway.
(4) We analysis the content of isoflavone in transgenic tobacco.And variance analysis showed that the content of isoflavone was highly increased in transgenic tobacco.Multiple comparisons illustrate that the content of isoflavone increased remarkably,in the transformed plant.
(5) Semi-quantitative RT-PCR analysis indicated that GmMYBJ6 could improve the expressions of some flavonoid biosynthetic genes,such as PAL(Phenylalanine ammonia lyase),CHS(Chalcone Synthase),CHI(chalcone isomerase) and FLS(flavonol synthase). However,the expressions of some lignin biosynthetic genes,such as COMT (catechol-O-methyltransferase),4CL(4-coumaroyl-CoA ligase) and CAD(cinnamyl alcohol dehydrogenase) were not influenced obviously.
(6) The analysis of anti-stress experiments showed that overexpression of GmMYBJ6 could improve transgenic tobaccos' resistance to UV-C radiation,salt and drought obviously.It confirmed that GmMYBJ6 may involved in the regulaotion of plant defense which domenstrants the primarily function of GmMYBJ6 as well.
引文
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