膜荚黄芪不定根培养及苯丙氨酸解氨酶（PAL）基因的克隆与功能鉴定
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摘要
黄芪作为一种常用中药,广泛应用于复方制剂中。为了黄芪资源的可持续利用,本文利用组织培养手段研究了影响膜荚黄芪不定根生长的因素,结果发现不定根在B_5培养基中加入30 g/L白糖和2 mg/L IBA的条件下生长良好;生物反应器培养过程中,在5L气升式反应器中(3L培养基)接入鲜物重30 g,长约2 cm的不定根,注入0.10 vvm空气时,可获得大量的不定根。有效成分分析结果表明,膜荚黄芪不定根中多糖含量明显高于栽培膜荚黄芪,总皂苷和黄酮含量与三年生栽培膜荚黄芪无显著差异,但是显著高于一年生栽培膜荚黄芪。
苯丙氨酸解氨酶(phenylalanine ammoma-lyase,PAL,EC4.3.1.5)是植物苯丙烷次生代谢途径的第一个关键酶和限速酶,包括类黄酮在内的一切含苯丙烷骨架的物质都是由该代谢途径直接或间接生成。PAL作为初级代谢和苯丙烷次生代谢的纽带,不仅对于植物的生理代谢和抗逆性有很重要的作用,对医药、生物化工领域也具有重大的现实意义与应用价值。
本文利用已知的PAL基因不同植物的氨基酸序列比较分析得保守序列,设计出简并引物,应用RT-PCR和RACE技术,首次成功地从膜荚黄芪中克隆出了PAL基因,命名为AmPAL,并在Genebank登录(EF567076)。AmPAL全长为2650 bp,含有一个完整的2154 bp开放读码框。生物信息学分析表明,AmPAL是植物苯丙氨酸解氨酶家族的一个新成员,推测其编码718个氨基酸的多肽,分子量为78.05 KDa,等电点为5.96,与豆科植物已知的PAL氨基酸序列的同源并且核酸和氨基酸序列相似性都大于80%。Southern杂交分析表明,AmPAL在膜荚黄芪基因组中是一个单拷贝基因。
为了了解和鉴定膜荚黄芪PAL基因的功能及生物学活性,用基因重组技术构建了pQE30-AmPAL原核表达载体,继而转入大肠杆菌M15表达6×His融合蛋白。结果表明重组蛋白进行了大量表达,通过Ni-NTA琼脂柱纯化,分子量为78 KDa左右,每克细胞(干物重)纯化出45 mg 6×His-AmPAL。酶活性测定表明,携带6个组氨酸的重组蛋白并不影响蛋白质的功能,可保持AmPAL原有的生物活性,AmPAL比活性为6500 U/mg蛋白质,对L-本丙氨酸的米氏常数Km值为3.35×10~(-4)mM/L。
在此基础上,构建了植物表达载体pBI121-AmPAL,并通过农杆菌介导法转化到模式植物拟南芥中,经过分子鉴定证明基因已整合到基因组DNA分子中并在转录水平上进行了表达,酶活性测定结果表明转基因植株活性远远高于未转基因拟南芥植株。本研究最终为AmPAL基因转化到其它药用植物,提高转基因植物中苯丙烷代谢途径次生代谢物含量的研究奠定了基础。
Radix Astragali is one of the commonly used traditional Chinese medicines,it has been utilized in composite herbal medicines.To sustainable utilization of resources of Radix Astragali,the effect of several factors on adventitious roots of Astragalus membranaceus (Fisch.) Bge.during tissue culture was studied.The results showed that the optimal growth of adventitious roots was found in B_5 medium with 30g/L white sugar and 2mg/L IBA.During bioreactor culture,the suitable conditions were obtained by the 20g/L of inoculum size per 5 L airlift bioreactor(3 L medium),2 cm-long adventitious roots(none-cut),and 0.10 vvm of air volume.The results of analysis of effective component was showed that polysaccharides of adventitious roots was significantly higher than that of cultivated A.membranaceus,saponins and flavonoids of adventitious roots were no significant difference with those of cultivated three-year A.membranaceus,but,significantly higher than those of cultivated annual A. membranaceus.
Phenylalanine ammonia-lyase(PAL;EC4.3.1.5) is the first and also a rate-limiting step in phenylpropanoid pathway,which supplies precursors for a variety of secondary metabolites including flavonoids.PAL as a link of the primary metabolism and phenylpropane secondary metabolism,not only has the very vital role regarding physiological metabolism and the resistance of adverse stress in plant,but also has the great practical significance and the application value in medical application and biochemical industry.
In this study,using degenerate PCR primers that target evolutionarily conserved sequeces of PAL gene in plants,we have firsrt cloned full-length sequences of a novel PAL gene in A. membranaceus by the technique of RT-PCR and RACE,the PAL gene named as AmPAL and the genebank accsesion number is EF567076.The full-length of AmPAL cDNA was 2650bp, including a 2154 bp open reading frame(ORF).The analysis of bioinformatics showed that AmPAL was a new number of PAL family,the putative AmPAL protein consisted of 718 amino acids with prediated molecular weight of 78.05 and isoelectric point(pI) of 5.96,had the homology with PAL of known leguminous plants and shared above 80%identity of nucleotide and amino acid sequences.The analysis of Southern blot showed that the AmPAL was a singlecopy gene in A.membranaceus genome.
In order to identify the function and activity of AmPAL,we constructed pQE30- AmPAL vector and transformed it into E.coli M15 to allow expression of PAL with the N-terminal six consecutive histidine residues.The PAL proteins were purified using Ni-NTA metalaffinity chromatography with molecular weight about 78 KDa and final yield of 45mg/g dry cells,specific activity was 6 500U/mg and the Km value was 3.35×10~(-4)mM/L for L-Phe.
On the basis above,we constructed pBI-AmPAL vector and transformed it into Arabidopsis thaliana.PCR,Southern blot and Northern blot showed that the AmPAL gene was integrated into A.thalianas genome and that expressed in transcription level,enzyme activity of transformed A.thaliana was much higher than that of untransformed A.thaliana.This study provides a solid foundation and theoretical basis on improving contents of secondary metabolites of phenylpropanoid pathway by transgene(AmPAL) to other Chinise tratditional medicines.
苯丙氨酸解氨酶(phenylalanine ammoma-lyase,PAL,EC4.3.1.5)是植物苯丙烷次生代谢途径的第一个关键酶和限速酶,包括类黄酮在内的一切含苯丙烷骨架的物质都是由该代谢途径直接或间接生成。PAL作为初级代谢和苯丙烷次生代谢的纽带,不仅对于植物的生理代谢和抗逆性有很重要的作用,对医药、生物化工领域也具有重大的现实意义与应用价值。
本文利用已知的PAL基因不同植物的氨基酸序列比较分析得保守序列,设计出简并引物,应用RT-PCR和RACE技术,首次成功地从膜荚黄芪中克隆出了PAL基因,命名为AmPAL,并在Genebank登录(EF567076)。AmPAL全长为2650 bp,含有一个完整的2154 bp开放读码框。生物信息学分析表明,AmPAL是植物苯丙氨酸解氨酶家族的一个新成员,推测其编码718个氨基酸的多肽,分子量为78.05 KDa,等电点为5.96,与豆科植物已知的PAL氨基酸序列的同源并且核酸和氨基酸序列相似性都大于80%。Southern杂交分析表明,AmPAL在膜荚黄芪基因组中是一个单拷贝基因。
为了了解和鉴定膜荚黄芪PAL基因的功能及生物学活性,用基因重组技术构建了pQE30-AmPAL原核表达载体,继而转入大肠杆菌M15表达6×His融合蛋白。结果表明重组蛋白进行了大量表达,通过Ni-NTA琼脂柱纯化,分子量为78 KDa左右,每克细胞(干物重)纯化出45 mg 6×His-AmPAL。酶活性测定表明,携带6个组氨酸的重组蛋白并不影响蛋白质的功能,可保持AmPAL原有的生物活性,AmPAL比活性为6500 U/mg蛋白质,对L-本丙氨酸的米氏常数Km值为3.35×10~(-4)mM/L。
在此基础上,构建了植物表达载体pBI121-AmPAL,并通过农杆菌介导法转化到模式植物拟南芥中,经过分子鉴定证明基因已整合到基因组DNA分子中并在转录水平上进行了表达,酶活性测定结果表明转基因植株活性远远高于未转基因拟南芥植株。本研究最终为AmPAL基因转化到其它药用植物,提高转基因植物中苯丙烷代谢途径次生代谢物含量的研究奠定了基础。
Radix Astragali is one of the commonly used traditional Chinese medicines,it has been utilized in composite herbal medicines.To sustainable utilization of resources of Radix Astragali,the effect of several factors on adventitious roots of Astragalus membranaceus (Fisch.) Bge.during tissue culture was studied.The results showed that the optimal growth of adventitious roots was found in B_5 medium with 30g/L white sugar and 2mg/L IBA.During bioreactor culture,the suitable conditions were obtained by the 20g/L of inoculum size per 5 L airlift bioreactor(3 L medium),2 cm-long adventitious roots(none-cut),and 0.10 vvm of air volume.The results of analysis of effective component was showed that polysaccharides of adventitious roots was significantly higher than that of cultivated A.membranaceus,saponins and flavonoids of adventitious roots were no significant difference with those of cultivated three-year A.membranaceus,but,significantly higher than those of cultivated annual A. membranaceus.
Phenylalanine ammonia-lyase(PAL;EC4.3.1.5) is the first and also a rate-limiting step in phenylpropanoid pathway,which supplies precursors for a variety of secondary metabolites including flavonoids.PAL as a link of the primary metabolism and phenylpropane secondary metabolism,not only has the very vital role regarding physiological metabolism and the resistance of adverse stress in plant,but also has the great practical significance and the application value in medical application and biochemical industry.
In this study,using degenerate PCR primers that target evolutionarily conserved sequeces of PAL gene in plants,we have firsrt cloned full-length sequences of a novel PAL gene in A. membranaceus by the technique of RT-PCR and RACE,the PAL gene named as AmPAL and the genebank accsesion number is EF567076.The full-length of AmPAL cDNA was 2650bp, including a 2154 bp open reading frame(ORF).The analysis of bioinformatics showed that AmPAL was a new number of PAL family,the putative AmPAL protein consisted of 718 amino acids with prediated molecular weight of 78.05 and isoelectric point(pI) of 5.96,had the homology with PAL of known leguminous plants and shared above 80%identity of nucleotide and amino acid sequences.The analysis of Southern blot showed that the AmPAL was a singlecopy gene in A.membranaceus genome.
In order to identify the function and activity of AmPAL,we constructed pQE30- AmPAL vector and transformed it into E.coli M15 to allow expression of PAL with the N-terminal six consecutive histidine residues.The PAL proteins were purified using Ni-NTA metalaffinity chromatography with molecular weight about 78 KDa and final yield of 45mg/g dry cells,specific activity was 6 500U/mg and the Km value was 3.35×10~(-4)mM/L for L-Phe.
On the basis above,we constructed pBI-AmPAL vector and transformed it into Arabidopsis thaliana.PCR,Southern blot and Northern blot showed that the AmPAL gene was integrated into A.thalianas genome and that expressed in transcription level,enzyme activity of transformed A.thaliana was much higher than that of untransformed A.thaliana.This study provides a solid foundation and theoretical basis on improving contents of secondary metabolites of phenylpropanoid pathway by transgene(AmPAL) to other Chinise tratditional medicines.
引文
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