霍山石斛的分子分类、NO生理调节作用研究及FPS基因的克隆
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摘要
石斛属(Dendrobium)植物是兰科多年生附生植物、兼性CAM植物,为名贵中药材。其中近40种可以入药,但不同种类石斛药用成分及含量差异较大,药理活性也不尽相同。传统的系统分类和进化研究,主要依据形态学和地理分布,得到的结论不一致,易引起争论。目前,已有多种分子标记方法应用于石斛属植物的遗传多样性及亲缘关系的研究,但序列相关扩增多态性(SRAP)分子标记技术在该属植物中的应用鲜见报道。
霍山石斛(D.huoshanense)是安徽省霍山县的特有石斛,属药用石斛中的特级珍品,是国家重点保护的药用植物和名贵中药材。由于其生境独特,繁殖困难,加之过度采收,野生资源已濒临绝迹。为增加其药源,前人在霍山石斛人工栽培、组织培养快繁方面做了大量研究工作,但对其生理代谢的调控、关键酶基因的克隆以及利用基因工程的方法调节功能基因表达的研究较少。一氧化氮(nitric oxide,NO)作为植物体一种新的信号分子,参与植物许多生理代谢的调节和次生代谢产物合成调控,而且可以作为胁迫响应的关键信使。但NO对霍山石斛这种兼性CAM植物生理代谢调节的研究未见报道。
本课题利用两种分子标记技术分析药用石斛的遗传多样性及亲缘关系,确立属下分类系统,为更好地开发利用药用石斛资源奠定基础。同时,本课题以霍山石斛为材料,研究在逆境与非逆境条件下NO对霍山石斛的生理调节作用,为了解NO对兼性CAM植物生理代谢的调节提供理论依据。此外,本研究还通过基于同源性的RT-PCR方法,克隆霍山石斛倍半萜类物质合成途径中的关键酶法呢基焦磷酸合酶基因(FPS),为进一步利用基因工程调控霍山石斛生物碱合成提供基础。主要研究结果如下:
1.利用SRAP和RAPD两种分子标记技术,研究霍山石斛与其它药用石斛主要产区代表性石斛的亲缘关系。试验以用36个RAPD引物和40个SRAP引物组合分别对9个供试石斛的基因组DNA进行扩增。RAPD引物共产生281条带,多态性带占87.09%,遗传相似性系数在0.4942～0.7731之间;SRAP引物组合共产生1977条带,多态性带占90.2%,遗传相似性系数在0.3302～0.7892之间。基于两种标记的聚类结果存在一定的差异,SRAP聚类结果能较好地体现供试品种的亲缘关系、地域特性和树型特点,与传统的基于形态特征建立的分类结果比较一致。相对而言,SRAP比RAPD可检测到更高的遗传变异、更大的标记效率和较高的多样性检测能力。
2.以不同浓度的SNP处理霍山石斛野生苗和试管苗,研究外源NO对霍山石斛膜脂过氧化和抗氧化系统的影响,筛选了0.1mmol·L~(-1)和0.5mmol·L~(-1)SNP作为霍山石斛野生苗的处理浓度,而0～75μmol·L~(-1)SNP为霍山石斛试管苗的适宜处理浓度。进一步研究表明,(1)强光胁迫下,0.1mmol·L~(-1)SNP处理增强了霍山石斛野生苗的SOD、POD和CAT三种酶活,提高了抗氧化系统清除活性氧能力,膜脂过氧化程度减轻,缓解了强光的胁迫作用,而0.5mmol·L~(-1)SNP处理后,霍山石斛的SOD、POD和CAT的活性降低,丙二醛含量升高,SNP处理加剧了强光胁迫造成的氧化损伤。(2)以SNP处理霍山石斛试管苗发现,在0～50μmol·L~(-1)SNP处理浓度间,随着SNP浓度的提高,霍山石斛的叶绿素含量、可溶性蛋白、苯丙氨酸解氨酶活和生物碱含量均呈现出逐步的递增,NO可诱导霍山石斛次生代谢产物的合成积累。作为诱导子,50μmol·L~(-1)SNP是处理霍山石斛试管苗的适宜浓度。
3.总结了强光胁迫下霍山石斛光抑制的生理变化,研究了外源NO在光抑制及暗恢复中的作用。试验采用“lake model”和“puddle model”两种模式,对800μmol·m~(-2)·s~(-1)光强下的霍山石斛叶绿素荧光参数进行了测定。800μmol·m~(-2)·s~(-1)光强下,霍山石斛的Fv/Fm、Φ_(psⅡ)、qP、qL、NPQ、Φ_(NPQ)、ETR显著下降,Φ_(NO)显著上升,PSⅡ反应中心已不能通过有效调控对过剩光能进行耗散,霍山石斛发生严重的光抑制;暗恢复24h后,霍山石斛的Fv/Fm、Φ_(psⅡ)、qP、qL、NPQ、Φ_(NPQ)、ETR上升,PSⅡ反应中心逐渐恢复。通过两种模式下叶绿素荧光参数的比较发现,“lake model”更能简单有效的反映霍山石斛的光能转换系统的运转状态。
强光胁迫下,0.1mmol·L~(-1)SNP处理提高了石斛PSⅡ的光能转换效率和潜在活性,增加了过剩光能的非光化学耗散,缓解了光抑制的发生,有效保护了光合机构免受强光胁迫的伤害,PSⅡ反应中心得以较快恢复。而经0.5mmol·L~(-1)SNP处理后,霍山石斛的光能转换系统未能通过有效的光能转换和非光化学反应耗散过剩的光能,加剧了PSⅡ反应中心光抑制的发生。
4.以霍山石斛为材料,根据在GenBank已登录的其它植物法呢基焦磷酸合酶基因的氨基酸序列,设计简并引物,利用RT-PCR技术,克隆出了石斛FPS基因片段(GenBank登录号:EU681273),sh-fps的cDNA序列长为522bp,编码174个氨基酸。sh-fps推断的氨基酸序列与多种植物FPS基因的氨基酸序列具有较高的同源性,同源性为79%-88%,且该基因片段在推测的氨基酸序列中含有FPS基因家族中高度保守的、与其活性有关的2个特异性的富含天冬氨酸的区段。
Dendrobium Sw.,perennial herbages in orchidaceae family,normally grow in shadow environment as facultative CAM plants.Many wild species of Dendrobium are rare traditional Chinese medicine.But there is great difference in medicinal components and pharmacological activities between different species of Dendrobium Sw.The original taxonomy and evolution research are based on morphology and geographical distribution, which make the results different.At present,there are several molecular marker methods has been used in the Dendrobium Sw.to study the genetic diversity and relationship. Sequence-related amplified polymorphism(SRAP) is a molecular marker technology, newly developed by Li and Quiros in 2001,however this new technology has not been applied in research of Dendrobium.
Dendrobium.huoshanense,a precious wild medicinal plant in China,distributes in Huoshan County,Anhui Province.Because the request for environment comparatively stringent,outgrowth propagate frequency is highly low,adding to lasting collection,the wildness plant is close to annihilation.More attention was paid to its tissue culture and artificial cultivation in order to increase its production.However,there is little research about how to increase its production and the contents of medicinal components by regulating physiological metabolism and molecular regulation.Nitric oxide(NO) is a freely diffusible,gaseous compound and an important signal molecule in living organisms. In plants,NO influences aspects of growth and development,as well as the ability of plant responses to stress.Increasing evidences indicated that NO is a key signal messenger in disease resistance in plants.
In this paper,the genetic diversity and kinship of medicinal Dendrobium Sw.were analyzed using RAPD and SRAP markers,and the subordinate classification system was confirmed,which lay a basis for exploitation and utilization of medicinal Dendrobium Sw. On the other hand,the physiological regulation effects of NO on the Dendrobium.huoshanense under stress and non-stress condition were studied to provide a theoretical foundation for action mechanism of NO to facultative cam plant.In addition,a cDNA fragment of farnesyl phosphate synthase genes was amplified by RT-PCR,which provided basic information for regulation of alkaloid synthesis for Dendrobium.huoshanense by using gene engineering technology.The results were as follows:
1.This study was carried out in order to analyze genetic diversity and compare the Mark index(MI) between RAPD and SRAP markers in Dendrobium Sw.Using 36 random RAPD primers and 40 SRAP primers combinations,the genetic diversity of 9 species of Dendrobium was studied.A total of 281 loci were generated by 36 RAPD primers,of which 87.09%was polymorphic and the genetic distance ranged from 0.4942 to 0.7731.As for SRAP,1977 bands were obtained and 90.2%of them was polymorphic,and the genetic distance was from 0.3302 to 0.7892.There were some differences existing in the two cluster results revealed by RAPD and SRAP markers,they were as follows:compared to that of RAPD,the result of SRAP could more comfortably manifest the kinship、geographical origin、tree forms and so on,and also was more conformant to the traditional classification.Compared to RAPD,SRAP has higher marker efficiency and diversity detection ability.
2.Normal conditions,using the wild seedlings and tube seedlings of D.huoshanense by presoaking in different SNP concentrations,the effects of NO on membrane lipid peroxidation and antioxdant enzyme activities were studied.Accordingly,the 0.1mmol·L~(-1) and 0.5mmol·L~(-1)SNP were selected to treat wild seedlings,and 10、25、50 and 75μmol·L~(-1)SNP were determined as a appropriate concentration for tube seedlings.In addition,there were some interesting points revealed as follows.(1) Under high light intensity,the capacity of scavenging Active Oxygen for antioxidant system was increased and the level of cell membrane lipid peroxidation was decreased by presoaking with 0.1mmol/L SNP,which alleviated the photoinhibition on leaves of Dendrobium huoshanense significantly.On the contrary,the leaves presoaked in 0.5mmol/LSNP were severely inhibited by the light and the enzyme activities of SOD、POD and CAT were decreased.(2) The increase of the concentration of SNP can further improve chlorophyll content、soluble protein content、PAL activity and alkaloid content of Dendrobium. huoshanense when presoaking within 0～50μmol·L~(-1)SNP.So,NO can induce the accumulation of secondary metabolic product for Dendrobium.huoshanense.As elicitor, 50μmol·L~(-1)SNP is a suitable concentration.
3.The physiological change law of photoinhibition for Dendrobium.huoshanense at high light intensity were summarized,and the chlorophyll fluorescence parameters were determined under 800μmol·m~(-2)·s~(-1) light intensity based on lake model and puddle model. As a result,the chlorophyll fluorescence parameters Fv/Fm、Φ_(psⅡ)、qP、qL、NPQ、Φ_(NPQ)、 ETR measured under 800μmol·m~(-2)·s~(-1) light intensity decreased significantly,andΦ_(NO)rose remarkably,which showed that the Dendrobium.huoshanense suffered photoinhibition and the dissipation of excess light energy was not regulated efficiently by PSⅡreaction center. After dark recovery for 24h,the parameters Fv/Fm、Φ_(psⅡ)、qP、qL、NPQ、Φ_(NPQ)、ETR rose back,and the running state of PSⅡreaction center recovered gradually.Comparing with the original florescence parameters based on the puddle model,the new florescence parameters based on the lake model have been found more appropriate for estimating the running state of light energy conversion system of photosystemⅡ.
In the experiment,the effects of NO donor sodium nitroprusside(SNP) on the chlorophyll fluorescence under high light stress were studied too.The result showed that the efficiency of light energy conversion and regulated non-photochemical dissipation of excess light energy for PSⅡwere improved by presoaking with 0.1mmol/L SNP,which alleviated the photo inhibition on leaves of Dendrobium huoshanense significantly. Therefore the PSⅡwas protected efficiently and the recovery was faster.On the contrary, the leaves presoaked in 0.5mmol/LSNP were severely inhibited under the strong light.
4.Total RNA was extracted from young leaves of D.huoshanense.The degenerate primers were designed and synthesized according to the highly conservative sequences among the known famesyl phosphate synthase genes.A cDNA fragment(sh-fps) of 522bp was amplified by RT-PCR,which was subsequently cloned into pUCm-T Vector for sequencing.The sequence has been submitted to the GenBank data base,the accession number is EU681273.The sequence encoded 174 amino acids.By using the program of Blast on GenBank database,the amino acid sequence deduced from this fragment presented 79%-88%similarity with the FPP synthases from other plants.The amino acid sequence also contains two FPS conservative core fragments.The two areas always appear in isoprenoid transferase,which possibly represent the active center of the sort of enzymes.
霍山石斛(D.huoshanense)是安徽省霍山县的特有石斛,属药用石斛中的特级珍品,是国家重点保护的药用植物和名贵中药材。由于其生境独特,繁殖困难,加之过度采收,野生资源已濒临绝迹。为增加其药源,前人在霍山石斛人工栽培、组织培养快繁方面做了大量研究工作,但对其生理代谢的调控、关键酶基因的克隆以及利用基因工程的方法调节功能基因表达的研究较少。一氧化氮(nitric oxide,NO)作为植物体一种新的信号分子,参与植物许多生理代谢的调节和次生代谢产物合成调控,而且可以作为胁迫响应的关键信使。但NO对霍山石斛这种兼性CAM植物生理代谢调节的研究未见报道。
本课题利用两种分子标记技术分析药用石斛的遗传多样性及亲缘关系,确立属下分类系统,为更好地开发利用药用石斛资源奠定基础。同时,本课题以霍山石斛为材料,研究在逆境与非逆境条件下NO对霍山石斛的生理调节作用,为了解NO对兼性CAM植物生理代谢的调节提供理论依据。此外,本研究还通过基于同源性的RT-PCR方法,克隆霍山石斛倍半萜类物质合成途径中的关键酶法呢基焦磷酸合酶基因(FPS),为进一步利用基因工程调控霍山石斛生物碱合成提供基础。主要研究结果如下:
1.利用SRAP和RAPD两种分子标记技术,研究霍山石斛与其它药用石斛主要产区代表性石斛的亲缘关系。试验以用36个RAPD引物和40个SRAP引物组合分别对9个供试石斛的基因组DNA进行扩增。RAPD引物共产生281条带,多态性带占87.09%,遗传相似性系数在0.4942～0.7731之间;SRAP引物组合共产生1977条带,多态性带占90.2%,遗传相似性系数在0.3302～0.7892之间。基于两种标记的聚类结果存在一定的差异,SRAP聚类结果能较好地体现供试品种的亲缘关系、地域特性和树型特点,与传统的基于形态特征建立的分类结果比较一致。相对而言,SRAP比RAPD可检测到更高的遗传变异、更大的标记效率和较高的多样性检测能力。
2.以不同浓度的SNP处理霍山石斛野生苗和试管苗,研究外源NO对霍山石斛膜脂过氧化和抗氧化系统的影响,筛选了0.1mmol·L~(-1)和0.5mmol·L~(-1)SNP作为霍山石斛野生苗的处理浓度,而0～75μmol·L~(-1)SNP为霍山石斛试管苗的适宜处理浓度。进一步研究表明,(1)强光胁迫下,0.1mmol·L~(-1)SNP处理增强了霍山石斛野生苗的SOD、POD和CAT三种酶活,提高了抗氧化系统清除活性氧能力,膜脂过氧化程度减轻,缓解了强光的胁迫作用,而0.5mmol·L~(-1)SNP处理后,霍山石斛的SOD、POD和CAT的活性降低,丙二醛含量升高,SNP处理加剧了强光胁迫造成的氧化损伤。(2)以SNP处理霍山石斛试管苗发现,在0～50μmol·L~(-1)SNP处理浓度间,随着SNP浓度的提高,霍山石斛的叶绿素含量、可溶性蛋白、苯丙氨酸解氨酶活和生物碱含量均呈现出逐步的递增,NO可诱导霍山石斛次生代谢产物的合成积累。作为诱导子,50μmol·L~(-1)SNP是处理霍山石斛试管苗的适宜浓度。
3.总结了强光胁迫下霍山石斛光抑制的生理变化,研究了外源NO在光抑制及暗恢复中的作用。试验采用“lake model”和“puddle model”两种模式,对800μmol·m~(-2)·s~(-1)光强下的霍山石斛叶绿素荧光参数进行了测定。800μmol·m~(-2)·s~(-1)光强下,霍山石斛的Fv/Fm、Φ_(psⅡ)、qP、qL、NPQ、Φ_(NPQ)、ETR显著下降,Φ_(NO)显著上升,PSⅡ反应中心已不能通过有效调控对过剩光能进行耗散,霍山石斛发生严重的光抑制;暗恢复24h后,霍山石斛的Fv/Fm、Φ_(psⅡ)、qP、qL、NPQ、Φ_(NPQ)、ETR上升,PSⅡ反应中心逐渐恢复。通过两种模式下叶绿素荧光参数的比较发现,“lake model”更能简单有效的反映霍山石斛的光能转换系统的运转状态。
强光胁迫下,0.1mmol·L~(-1)SNP处理提高了石斛PSⅡ的光能转换效率和潜在活性,增加了过剩光能的非光化学耗散,缓解了光抑制的发生,有效保护了光合机构免受强光胁迫的伤害,PSⅡ反应中心得以较快恢复。而经0.5mmol·L~(-1)SNP处理后,霍山石斛的光能转换系统未能通过有效的光能转换和非光化学反应耗散过剩的光能,加剧了PSⅡ反应中心光抑制的发生。
4.以霍山石斛为材料,根据在GenBank已登录的其它植物法呢基焦磷酸合酶基因的氨基酸序列,设计简并引物,利用RT-PCR技术,克隆出了石斛FPS基因片段(GenBank登录号:EU681273),sh-fps的cDNA序列长为522bp,编码174个氨基酸。sh-fps推断的氨基酸序列与多种植物FPS基因的氨基酸序列具有较高的同源性,同源性为79%-88%,且该基因片段在推测的氨基酸序列中含有FPS基因家族中高度保守的、与其活性有关的2个特异性的富含天冬氨酸的区段。
Dendrobium Sw.,perennial herbages in orchidaceae family,normally grow in shadow environment as facultative CAM plants.Many wild species of Dendrobium are rare traditional Chinese medicine.But there is great difference in medicinal components and pharmacological activities between different species of Dendrobium Sw.The original taxonomy and evolution research are based on morphology and geographical distribution, which make the results different.At present,there are several molecular marker methods has been used in the Dendrobium Sw.to study the genetic diversity and relationship. Sequence-related amplified polymorphism(SRAP) is a molecular marker technology, newly developed by Li and Quiros in 2001,however this new technology has not been applied in research of Dendrobium.
Dendrobium.huoshanense,a precious wild medicinal plant in China,distributes in Huoshan County,Anhui Province.Because the request for environment comparatively stringent,outgrowth propagate frequency is highly low,adding to lasting collection,the wildness plant is close to annihilation.More attention was paid to its tissue culture and artificial cultivation in order to increase its production.However,there is little research about how to increase its production and the contents of medicinal components by regulating physiological metabolism and molecular regulation.Nitric oxide(NO) is a freely diffusible,gaseous compound and an important signal molecule in living organisms. In plants,NO influences aspects of growth and development,as well as the ability of plant responses to stress.Increasing evidences indicated that NO is a key signal messenger in disease resistance in plants.
In this paper,the genetic diversity and kinship of medicinal Dendrobium Sw.were analyzed using RAPD and SRAP markers,and the subordinate classification system was confirmed,which lay a basis for exploitation and utilization of medicinal Dendrobium Sw. On the other hand,the physiological regulation effects of NO on the Dendrobium.huoshanense under stress and non-stress condition were studied to provide a theoretical foundation for action mechanism of NO to facultative cam plant.In addition,a cDNA fragment of farnesyl phosphate synthase genes was amplified by RT-PCR,which provided basic information for regulation of alkaloid synthesis for Dendrobium.huoshanense by using gene engineering technology.The results were as follows:
1.This study was carried out in order to analyze genetic diversity and compare the Mark index(MI) between RAPD and SRAP markers in Dendrobium Sw.Using 36 random RAPD primers and 40 SRAP primers combinations,the genetic diversity of 9 species of Dendrobium was studied.A total of 281 loci were generated by 36 RAPD primers,of which 87.09%was polymorphic and the genetic distance ranged from 0.4942 to 0.7731.As for SRAP,1977 bands were obtained and 90.2%of them was polymorphic,and the genetic distance was from 0.3302 to 0.7892.There were some differences existing in the two cluster results revealed by RAPD and SRAP markers,they were as follows:compared to that of RAPD,the result of SRAP could more comfortably manifest the kinship、geographical origin、tree forms and so on,and also was more conformant to the traditional classification.Compared to RAPD,SRAP has higher marker efficiency and diversity detection ability.
2.Normal conditions,using the wild seedlings and tube seedlings of D.huoshanense by presoaking in different SNP concentrations,the effects of NO on membrane lipid peroxidation and antioxdant enzyme activities were studied.Accordingly,the 0.1mmol·L~(-1) and 0.5mmol·L~(-1)SNP were selected to treat wild seedlings,and 10、25、50 and 75μmol·L~(-1)SNP were determined as a appropriate concentration for tube seedlings.In addition,there were some interesting points revealed as follows.(1) Under high light intensity,the capacity of scavenging Active Oxygen for antioxidant system was increased and the level of cell membrane lipid peroxidation was decreased by presoaking with 0.1mmol/L SNP,which alleviated the photoinhibition on leaves of Dendrobium huoshanense significantly.On the contrary,the leaves presoaked in 0.5mmol/LSNP were severely inhibited by the light and the enzyme activities of SOD、POD and CAT were decreased.(2) The increase of the concentration of SNP can further improve chlorophyll content、soluble protein content、PAL activity and alkaloid content of Dendrobium. huoshanense when presoaking within 0～50μmol·L~(-1)SNP.So,NO can induce the accumulation of secondary metabolic product for Dendrobium.huoshanense.As elicitor, 50μmol·L~(-1)SNP is a suitable concentration.
3.The physiological change law of photoinhibition for Dendrobium.huoshanense at high light intensity were summarized,and the chlorophyll fluorescence parameters were determined under 800μmol·m~(-2)·s~(-1) light intensity based on lake model and puddle model. As a result,the chlorophyll fluorescence parameters Fv/Fm、Φ_(psⅡ)、qP、qL、NPQ、Φ_(NPQ)、 ETR measured under 800μmol·m~(-2)·s~(-1) light intensity decreased significantly,andΦ_(NO)rose remarkably,which showed that the Dendrobium.huoshanense suffered photoinhibition and the dissipation of excess light energy was not regulated efficiently by PSⅡreaction center. After dark recovery for 24h,the parameters Fv/Fm、Φ_(psⅡ)、qP、qL、NPQ、Φ_(NPQ)、ETR rose back,and the running state of PSⅡreaction center recovered gradually.Comparing with the original florescence parameters based on the puddle model,the new florescence parameters based on the lake model have been found more appropriate for estimating the running state of light energy conversion system of photosystemⅡ.
In the experiment,the effects of NO donor sodium nitroprusside(SNP) on the chlorophyll fluorescence under high light stress were studied too.The result showed that the efficiency of light energy conversion and regulated non-photochemical dissipation of excess light energy for PSⅡwere improved by presoaking with 0.1mmol/L SNP,which alleviated the photo inhibition on leaves of Dendrobium huoshanense significantly. Therefore the PSⅡwas protected efficiently and the recovery was faster.On the contrary, the leaves presoaked in 0.5mmol/LSNP were severely inhibited under the strong light.
4.Total RNA was extracted from young leaves of D.huoshanense.The degenerate primers were designed and synthesized according to the highly conservative sequences among the known famesyl phosphate synthase genes.A cDNA fragment(sh-fps) of 522bp was amplified by RT-PCR,which was subsequently cloned into pUCm-T Vector for sequencing.The sequence has been submitted to the GenBank data base,the accession number is EU681273.The sequence encoded 174 amino acids.By using the program of Blast on GenBank database,the amino acid sequence deduced from this fragment presented 79%-88%similarity with the FPP synthases from other plants.The amino acid sequence also contains two FPS conservative core fragments.The two areas always appear in isoprenoid transferase,which possibly represent the active center of the sort of enzymes.
引文
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