丹参SmGRP1基因克隆及其功能研究
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摘要
丹参(Salvia miltiorrhiza Bunge)为唇形科多年生草本药用植物,其主要药理活性成分为脂溶性二萜醌类和水溶性酚酸类化合物,具有活血化淤,凉血消肿,清心除烦,消炎、抗病毒、抗肿瘤、抗溃疡等多种重要的药理活性,被广泛应用于心脑血管、月经不调以及多种炎症等疾病的治疗。近年来,随着药材需求量的增加,丹参野生资源破坏日益严重。因此,利用现有研究平台,整合基因组学、转录组学以及代谢组学等方法,筛选丹参优良种质形成相关基因,探讨优异种质形成的分子机理具有重要的意义。本研究以丹参EST数据库中一高丰度表达的新基因为研究对象,对该基因的表达及调控特征进行研究,结合体外表达蛋白的活性分析以及该基因“knock in”和“knock down”突变体的生理变化等研究结果,阐明该基因的功能,为进一步开发利用这一新的基因资源奠定基础。具体研究内容如下:
1.通过对丹参EST数据库的分析,发现一条由11个EST组成的unigene,利用blast x在NCBI的“nr”库中检索不到该基因的相似序列,推测其可能为一新基因。该基因包含一个全长456 bp的开放读码框,其编码的多肽序列中谷氨酸含量高达33.77%,因此将该基因命名为SmGRP1(Salvia miltiorrhiza Glutamic acid-Rich Protein 1)。
2.利用PCR和RT-PCR法获得SmGRP1基因DNA和cDNA全长。通过cDNA及DNA序列的比对发现,该基因由两个外显子和一个内含子组成,全长837 bp。内含子序列长度为381 bp,位于Glu~5和Val~6之间。信息学分析显示该蛋白的分子量为15.801 kDa,理论等电点为3.79,不稳定常数为103.93,为不稳定的酸蛋白。该蛋白不含Trp,Tyr和Cys等芳香族氨基酸,不具有信号肽,没有膜锚定或跨膜结构域,总体表现为亲水性蛋白。α-螺旋为其主要的二级结构单元,很难形成复杂的高级结构。
3.利用DNA Walking的方法克隆获得SmGRP1基因长为1,932 bp的5'侧翼序列,PLACE、PlantCARE等软件分析显示该区域含有大量与生物源或非生物源胁迫相关的顺式作用元件和激素应答元件,提示该基因的表达可能与激素及各种逆境胁迫相关。Real time quantitativeRT-PCR研究显示该基因在根、茎、叶中均有表达,但叶和茎中的表达丰度远高于根;外源ABA和NaCl处理以及暗培养、脱水和损伤胁迫等均可诱导SmGRP1基因的表达,其中,ABA和黑暗可使其表达丰度显著提高,低温处理则抑制该基因的表达。
4.构建SmGRP1基因的原核表达载体,并对表达的蛋白进行纯化。Ca~(2+)结合蛋白的特异性染色剂Stains-all染色结果显示SmGRP1蛋白可能具有Ca~(2+)结合特性,而Ca~(2+)凝胶迁移电泳实验显示,这种结合可能不会造成蛋白空间构象的改变。
5.SmGRP1基因启动子-报告基因融合载体转化丹参的结果显示,SmGRP1基因5'侧翼区具有启动子活性。启动子5'缺失实验证实报告基因的表达可以被ABA、黑暗、高温、NaCl以及低温等条件所影响,而且不同的5'缺失片段对报告基因表达影响的程度有所不同,实验结果显示,ABA响应的顺式作用元件主要分布在-1,851--760 bp区域,黑暗响应的元件主要分布在-759--252 bp的区域,高温响应元件主要分布在-251-+81 bp的区域,在-1,851--760 bp的区域存在低温响应的增强型元件,而在-759--252 bp的区域则存在低温响应的抑制性元件。NaCl响应元件则分散在-1,851--102 bp区域内。
6.通过农杆菌介导的转基因方法获得丹参SmGRP1基因“knock in”和“knock down”突变体。结果显示,在正常培养条件下,“knock in”和“knock down”各株系与对照植株相比无明显的表型差异,但“knock in”株系的气孔开度变小。利用外源ABA、NO、H_2O_2和黑暗等能够诱导气孔关闭的因素处理“knock down”株系,植株气孔维持开放,表现为不关闭的状态。此外,当暴露于空气中时,“knock down”株系脱水速率明显比对照植株快,而“knock in”株系脱水速率则慢于对照株系。
综上所述,SmGRP1基因在叶和茎中高丰度表达,而且其表达受外源ABA和NaCl,暗培养、脱水和伤害处理以及温度等因素的影响。通过对该基因“knock in”和“knock down”突变体株系进行分析,以及SmGRP1蛋白与Ca~(2+)的结合实验推测,丹参SmGRP1可能通过与Ca~(2+)的作用调节气孔运动,参与植株抵御外界不良环境的过程,具体的作用机制尚有待于进一步探讨。
Salvia miltiorrhiza Bge.is a well-known medicinal plant.Its root(called danshen or tanshen in Chinese but better known in the west as Chinese sage or red sage root) contains two major groups of identified biologically active compounds,the hydrophilic caffeic-acid-derived phenolic acids,and various lipophilic tanshinones belonging to the diterpene quinines,and has been formulated and used clinically for the treatment of various diseases such as cardiovascular and cerebrovascular disease. With continued pharmacologic studies on its secondary metabolites,more and more biological activities including antioxidant,anti-thrombosis,anti-hypertension,antivirus and antitumor have been reported.In recent years,the wild resources of S.miltiorrhiza are destroyed severely with the constantly rising demand for its pharmacologic properties.Therefore,screening genes related to the eminent characters of S.miltiorrhiza and discussing its molecular mechanism are of great significance.Here,a novel unigene with high abundance in the EST database of S.miltiorrhiza was found and its expression pattern was also studied.Furthermore,the function of this gene was elucidated via studying the physiological changes of its "knock in" and "knock down" mutants.This study will provide information for further investigating and utilizing this special gene.The main results were as follows:
1.A unigene composed by 11 ESTs were found by screening the EST library of S.miltiorrhiza. It showed no significant similarities with the sequences in NCBI "nr" database by blast x analysis, which implied that this putative gene may be a novel gene.Very interestingly,this novel gene encoded a peptide,glutamic acid residue content of which was as high as 33.77%.Based on this,the new gene was designated as SmGRP1(Salvia miltiorrhiza Glutamic acid-Rich Protein 1).
2.The entire DNA and cDNA sequence of SmGRP1 were cloned using PCR and RT-PCR methods.Comparation of these two sequences suggested that SmGRP1 consisted of two extrons and one intron of 381 bp whose position was between Glu~5 and Val~6.Bioinformatics analysis showed that the molecular weight of SmGRP1 was 15.801 kDa,the theoretical pI was 3.79 and the instability index is 103.93,which implied that SmGRP1 was an unstable acidic protein.Interestingly,there were no aromatic amino acids such as Trp,Tyr and Cys in the protein.Also no signal peptide and transmembrane domain existed in SmGRP1.Prediction by ProtScale showed that SmGRP1 was a hydrophilic protein.Besides,alpha helix was the main secondary structure unit of the protein.
3.Totally,1,932 bp-long 5' flanking region of SmGRP1 gene was obtained by DNA walking. Plant growth regulators responsive elements and biotic or abiotic responsive elements were found in this region using PLACE and PlantCARE softwares.Real time quantitative RT-PCR technique was performed to characterize the expression patterns of the gene.It showed that SmGRP1 expressed in all examined tissues of S.miltiorrhiza but most highly in leaves and stems.The expression level of SmGRP1 was prominently enhanced under ABA and dark treatments.Also the expression level can be induced by high temperature,NaCl,and dehydration treatments,while low temperature suppressed its expression.
4.A recombinant vector pQE30-SmGRP1 was constructed and transferred into E.coli M15 for protein expression and purification.Stains-all staining implied that the protein may be a Ca~(2+) binding protein,but this binding activity could not arouse the conformation change of SmGRP1 as revealed by Ca~(2+)-dependent mobility shift assay.
5.According to cis-acting elements' localization predicted by bioinformatical tools,five 5' deleted promoter segments were obtained by PCR and cloned into pCAMBIA-1391z vector.All of the "Promoter-reporter" constructs were transformed into S.miltiorrhiza and a large number of positive transformants of each construct were obtained.Consistent with the result of real-time quantitative RT-PCR analysis,the reporter gene's expression could be induced by ABA,dark,high temperature,NaCl and low temperature treatments by GUS staining analysis.Moreover,the transformants of different 5'-deletion construct responsed differently under diverse treatments.This result implied that the ABA responsive elements were mainly located in -1,851- -760 bp,dark responsive elements mainly in -759- -252 bp region,high temperature responsive elements maily in -251- +81 bp region and NaCl responsive elements mainly in -1,851- -102 bp region.
6.The "knock in" and "knock down" mutants of SmGRP1 were achieved by Agrobacterium tumefaciens-mediated gene transformation method.Results indicated that all the mutants showed no obvious phenotypic differences compared with the control plantlets,except that the stoma aperture of the "knock in" mutants were always smaller.While the epidermal strips of both mutants and control plantlets were treated by ABA,dark,NO and H_2O_2,the stoma of the "knock down" lines remained open.Besides,the water loss rate of "knock in" mutants was slower than control lines when exposed to air,while that of"knock down" mutants was much quicker.
In conclusion,SmGRP1 expressed highly in leaves and stems of S.miltiorrhiza,and its expression can be induced by ABA,dark,dehydration,NaCl and some other stress conditions. SmGRP1 was presumed to involve in stomatal closure course as a Ca~(2+) binding protein and play important roles in plant stress resistance.
1.通过对丹参EST数据库的分析,发现一条由11个EST组成的unigene,利用blast x在NCBI的“nr”库中检索不到该基因的相似序列,推测其可能为一新基因。该基因包含一个全长456 bp的开放读码框,其编码的多肽序列中谷氨酸含量高达33.77%,因此将该基因命名为SmGRP1(Salvia miltiorrhiza Glutamic acid-Rich Protein 1)。
2.利用PCR和RT-PCR法获得SmGRP1基因DNA和cDNA全长。通过cDNA及DNA序列的比对发现,该基因由两个外显子和一个内含子组成,全长837 bp。内含子序列长度为381 bp,位于Glu~5和Val~6之间。信息学分析显示该蛋白的分子量为15.801 kDa,理论等电点为3.79,不稳定常数为103.93,为不稳定的酸蛋白。该蛋白不含Trp,Tyr和Cys等芳香族氨基酸,不具有信号肽,没有膜锚定或跨膜结构域,总体表现为亲水性蛋白。α-螺旋为其主要的二级结构单元,很难形成复杂的高级结构。
3.利用DNA Walking的方法克隆获得SmGRP1基因长为1,932 bp的5'侧翼序列,PLACE、PlantCARE等软件分析显示该区域含有大量与生物源或非生物源胁迫相关的顺式作用元件和激素应答元件,提示该基因的表达可能与激素及各种逆境胁迫相关。Real time quantitativeRT-PCR研究显示该基因在根、茎、叶中均有表达,但叶和茎中的表达丰度远高于根;外源ABA和NaCl处理以及暗培养、脱水和损伤胁迫等均可诱导SmGRP1基因的表达,其中,ABA和黑暗可使其表达丰度显著提高,低温处理则抑制该基因的表达。
4.构建SmGRP1基因的原核表达载体,并对表达的蛋白进行纯化。Ca~(2+)结合蛋白的特异性染色剂Stains-all染色结果显示SmGRP1蛋白可能具有Ca~(2+)结合特性,而Ca~(2+)凝胶迁移电泳实验显示,这种结合可能不会造成蛋白空间构象的改变。
5.SmGRP1基因启动子-报告基因融合载体转化丹参的结果显示,SmGRP1基因5'侧翼区具有启动子活性。启动子5'缺失实验证实报告基因的表达可以被ABA、黑暗、高温、NaCl以及低温等条件所影响,而且不同的5'缺失片段对报告基因表达影响的程度有所不同,实验结果显示,ABA响应的顺式作用元件主要分布在-1,851--760 bp区域,黑暗响应的元件主要分布在-759--252 bp的区域,高温响应元件主要分布在-251-+81 bp的区域,在-1,851--760 bp的区域存在低温响应的增强型元件,而在-759--252 bp的区域则存在低温响应的抑制性元件。NaCl响应元件则分散在-1,851--102 bp区域内。
6.通过农杆菌介导的转基因方法获得丹参SmGRP1基因“knock in”和“knock down”突变体。结果显示,在正常培养条件下,“knock in”和“knock down”各株系与对照植株相比无明显的表型差异,但“knock in”株系的气孔开度变小。利用外源ABA、NO、H_2O_2和黑暗等能够诱导气孔关闭的因素处理“knock down”株系,植株气孔维持开放,表现为不关闭的状态。此外,当暴露于空气中时,“knock down”株系脱水速率明显比对照植株快,而“knock in”株系脱水速率则慢于对照株系。
综上所述,SmGRP1基因在叶和茎中高丰度表达,而且其表达受外源ABA和NaCl,暗培养、脱水和伤害处理以及温度等因素的影响。通过对该基因“knock in”和“knock down”突变体株系进行分析,以及SmGRP1蛋白与Ca~(2+)的结合实验推测,丹参SmGRP1可能通过与Ca~(2+)的作用调节气孔运动,参与植株抵御外界不良环境的过程,具体的作用机制尚有待于进一步探讨。
Salvia miltiorrhiza Bge.is a well-known medicinal plant.Its root(called danshen or tanshen in Chinese but better known in the west as Chinese sage or red sage root) contains two major groups of identified biologically active compounds,the hydrophilic caffeic-acid-derived phenolic acids,and various lipophilic tanshinones belonging to the diterpene quinines,and has been formulated and used clinically for the treatment of various diseases such as cardiovascular and cerebrovascular disease. With continued pharmacologic studies on its secondary metabolites,more and more biological activities including antioxidant,anti-thrombosis,anti-hypertension,antivirus and antitumor have been reported.In recent years,the wild resources of S.miltiorrhiza are destroyed severely with the constantly rising demand for its pharmacologic properties.Therefore,screening genes related to the eminent characters of S.miltiorrhiza and discussing its molecular mechanism are of great significance.Here,a novel unigene with high abundance in the EST database of S.miltiorrhiza was found and its expression pattern was also studied.Furthermore,the function of this gene was elucidated via studying the physiological changes of its "knock in" and "knock down" mutants.This study will provide information for further investigating and utilizing this special gene.The main results were as follows:
1.A unigene composed by 11 ESTs were found by screening the EST library of S.miltiorrhiza. It showed no significant similarities with the sequences in NCBI "nr" database by blast x analysis, which implied that this putative gene may be a novel gene.Very interestingly,this novel gene encoded a peptide,glutamic acid residue content of which was as high as 33.77%.Based on this,the new gene was designated as SmGRP1(Salvia miltiorrhiza Glutamic acid-Rich Protein 1).
2.The entire DNA and cDNA sequence of SmGRP1 were cloned using PCR and RT-PCR methods.Comparation of these two sequences suggested that SmGRP1 consisted of two extrons and one intron of 381 bp whose position was between Glu~5 and Val~6.Bioinformatics analysis showed that the molecular weight of SmGRP1 was 15.801 kDa,the theoretical pI was 3.79 and the instability index is 103.93,which implied that SmGRP1 was an unstable acidic protein.Interestingly,there were no aromatic amino acids such as Trp,Tyr and Cys in the protein.Also no signal peptide and transmembrane domain existed in SmGRP1.Prediction by ProtScale showed that SmGRP1 was a hydrophilic protein.Besides,alpha helix was the main secondary structure unit of the protein.
3.Totally,1,932 bp-long 5' flanking region of SmGRP1 gene was obtained by DNA walking. Plant growth regulators responsive elements and biotic or abiotic responsive elements were found in this region using PLACE and PlantCARE softwares.Real time quantitative RT-PCR technique was performed to characterize the expression patterns of the gene.It showed that SmGRP1 expressed in all examined tissues of S.miltiorrhiza but most highly in leaves and stems.The expression level of SmGRP1 was prominently enhanced under ABA and dark treatments.Also the expression level can be induced by high temperature,NaCl,and dehydration treatments,while low temperature suppressed its expression.
4.A recombinant vector pQE30-SmGRP1 was constructed and transferred into E.coli M15 for protein expression and purification.Stains-all staining implied that the protein may be a Ca~(2+) binding protein,but this binding activity could not arouse the conformation change of SmGRP1 as revealed by Ca~(2+)-dependent mobility shift assay.
5.According to cis-acting elements' localization predicted by bioinformatical tools,five 5' deleted promoter segments were obtained by PCR and cloned into pCAMBIA-1391z vector.All of the "Promoter-reporter" constructs were transformed into S.miltiorrhiza and a large number of positive transformants of each construct were obtained.Consistent with the result of real-time quantitative RT-PCR analysis,the reporter gene's expression could be induced by ABA,dark,high temperature,NaCl and low temperature treatments by GUS staining analysis.Moreover,the transformants of different 5'-deletion construct responsed differently under diverse treatments.This result implied that the ABA responsive elements were mainly located in -1,851- -760 bp,dark responsive elements mainly in -759- -252 bp region,high temperature responsive elements maily in -251- +81 bp region and NaCl responsive elements mainly in -1,851- -102 bp region.
6.The "knock in" and "knock down" mutants of SmGRP1 were achieved by Agrobacterium tumefaciens-mediated gene transformation method.Results indicated that all the mutants showed no obvious phenotypic differences compared with the control plantlets,except that the stoma aperture of the "knock in" mutants were always smaller.While the epidermal strips of both mutants and control plantlets were treated by ABA,dark,NO and H_2O_2,the stoma of the "knock down" lines remained open.Besides,the water loss rate of "knock in" mutants was slower than control lines when exposed to air,while that of"knock down" mutants was much quicker.
In conclusion,SmGRP1 expressed highly in leaves and stems of S.miltiorrhiza,and its expression can be induced by ABA,dark,dehydration,NaCl and some other stress conditions. SmGRP1 was presumed to involve in stomatal closure course as a Ca~(2+) binding protein and play important roles in plant stress resistance.
引文
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