卷叶贝母环阿屯醇合成酶基因的克隆及表达分析
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摘要
目的对卷叶贝母Fritillaria cirrhosa中参与生物碱合成的关键酶环阿屯醇合成酶(cycloartenol synthase,CAS)基因进行克隆,并对其进行生物信息学和表达分析。方法基于转录组测序结果,通过PCR技术克隆卷叶贝母CAS基因(FcCAS)开放阅读框(open reading frame,ORF)序列,并基于在线工具对cDNA序列进行生物信息学分析。通过荧光定量(q RT-PCR)手段检测FcCAS在野生鳞茎与愈伤组织(通过激素组合刺激获得的组织培养物)中的表达情况并测定总生物碱的含量。结果 FcCAS编码区ORF长为2 271 bp,编码756个氨基酸,并与NCBI上公布的天门冬、芭蕉、油棕等植物CAS蛋白的相似性达80%以上;qRT-PCR与总生物碱含量测定实验表明FcCAS的表达水平与总生物碱含量的变化趋势一致,都是愈伤组织高于野生鳞茎。结论 FcCAS在不同组织状态下表达量差异较大,FcCAS是一个有生物学功能的蛋白质,受激素组合诱导表达,为进一步研究CAS对卷叶贝母生物碱含量的影响和表达调控奠定基础。
Objective To obtain the key enzyme gene involved in alkaloids biosynthesis pathway of Fritillaria cirrhosa, cycloartenol synthase(CAS) gene was cloned, and its bioinformatics analysis and gene expression pattern were also performed. Methods The open reading frame(ORF) of F. cirrhosa CAS(FcCAS) was amplified by PCR based on transcriptome sequencing. The bioinformatics analysis of FcCAS cDNA sequences was carried out by some online tools. Meanwhile, using real-time quantitative PCR(qRT-PCR) to analyze the gene expression patterns between wild and regeneration bulbs. Moreover, the content of total alkaloids in wild and regenerated bulbs were also investigated. Results The results showed that CAS had a length of 2 271 bpORF, which encoding 756 amino acids. The phylogenetic tree analysis showed that FcCAS were highly similar to the corresponding proteins in Asparagus officinalis, Musa acuminate, and Elaeis gunineensis from the NCBI website, and the similarities were more than 80%. The results of qRT-PCR and total alkaloids assay showed that the changing trend of the expression level of FcCAS was consistent with that of the content of total alkaloids, and higher alkaloid accumulation was in regeneration bulbs than wild bulbs. Conclusion The expression of FcCAS gene varied widely in different tissues. These findings suggested that FcCAS was a biological functional protein induced by hormone combination, which laid a theoretical foundation for the improvement of the alkaloid content by using the genetic engineering.
Objective To obtain the key enzyme gene involved in alkaloids biosynthesis pathway of Fritillaria cirrhosa, cycloartenol synthase(CAS) gene was cloned, and its bioinformatics analysis and gene expression pattern were also performed. Methods The open reading frame(ORF) of F. cirrhosa CAS(FcCAS) was amplified by PCR based on transcriptome sequencing. The bioinformatics analysis of FcCAS cDNA sequences was carried out by some online tools. Meanwhile, using real-time quantitative PCR(qRT-PCR) to analyze the gene expression patterns between wild and regeneration bulbs. Moreover, the content of total alkaloids in wild and regenerated bulbs were also investigated. Results The results showed that CAS had a length of 2 271 bpORF, which encoding 756 amino acids. The phylogenetic tree analysis showed that FcCAS were highly similar to the corresponding proteins in Asparagus officinalis, Musa acuminate, and Elaeis gunineensis from the NCBI website, and the similarities were more than 80%. The results of qRT-PCR and total alkaloids assay showed that the changing trend of the expression level of FcCAS was consistent with that of the content of total alkaloids, and higher alkaloid accumulation was in regeneration bulbs than wild bulbs. Conclusion The expression of FcCAS gene varied widely in different tissues. These findings suggested that FcCAS was a biological functional protein induced by hormone combination, which laid a theoretical foundation for the improvement of the alkaloid content by using the genetic engineering.
引文
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[14]朱振洪,葛立军,王丽丽,等.浙贝母环阿屯醇合成酶基因5-RACE快速扩增及序列分析[J].浙江中医药大学学报,2012,36(5):558-562.
[15]张顺仓,夏鹏国,王幼平,等.丹参中转录因子基因Sm MYB-like 1的克隆和表达分析[J].扬州大学学报:农业与生命科学版,2017,38(3):32-38.
[16]邢朝斌,龙月红,吴鹏,等.刺五加环阿屯醇合酶基因的克隆及其表达分析[J].中草药,2012,43(7):1387-1392.
[17]朱丹妮,高山林.组织培养川贝母化学成分和药理作用的研究[J].中国药科大学学报,1992,23(2):118-121.
[18]陈敏,陈和荣.川贝母组织培养的研究[J].中国中药杂志,1995,20(8):461-462.
[19]杨杨.野生和组培川贝母的总生物碱含量测定和定位研究[D].成都:四川大学,2007.
[2]Duan B,Wang L,Dai X,et al.Identification and quantitative analysis of nucleosides and nucleobases in aqueous extracts of Fritillaria cirrhosa D.Don.using HPLC-DAD and HPLC-ESI-MS[J].Anal Lett,2011,44(15):2491-2502.
[3]王晓静.川贝母生物碱成分与品质研究[D].成都:四川大学,2004.
[4]Wang D,Zhu J,Wang S,et al.Antitussive,expectorant and anti-inflammatory alkaloids from Bulbus Fritillariae Cirrhosae[J].Fitoterapia,2011,82(8):1290-1294.
[5]王强,兰利琼,傅华龙.秋水仙素诱导川贝母(Fritillaria cirrhosa D.Don)愈伤组织多倍体的研究[J].植物科学学报,2002,20(6):449-452.
[6]孙雪.平贝母CAS基因干扰载体的构建及遗传转化体系的建立[D].长春:吉林农业大学,2014.
[7]Ghosh A,Misra S,Dutta A K,et al.Pentacyclic triterpenoids and sterols from seven species of mangrove[J].Phytochemistry,1985,24(8):1725-1727.
[8]左玉.植物甾醇研究与应用[J].粮食与油脂,2012(7):1-4.
[9]李珍,王东浩,姚伟,等.丹参环阿屯醇合酶基因克隆及表达分析[J].西北植物学报,2013,33(7):1285-1291.
[10]袁梦求,丁春邦,陶亮,等.滇重楼环阿屯醇合酶基因的克隆及序列分析[J].中草药,2012,43(11):2250-2256.
[11]涂碧梦,陈永勤,杨之帆.盾叶薯蓣环阿屯醇合酶全长基因的克隆与分析[J].西北植物学报,2010,30(1):8-13.
[12]Bubner B,Baldwin I T.Use of real-time PCR for determining copy number and zygosity in transgenic plants[J].Plant Cell Rep,2004,23(5):263-271.
[13]王跃华,徐恩琴,郭翠平,等.煎煮法提取卷叶贝母组培物总生物碱研究[J].成都大学学报:自然科学版,2015,34(2):108-110.
[14]朱振洪,葛立军,王丽丽,等.浙贝母环阿屯醇合成酶基因5-RACE快速扩增及序列分析[J].浙江中医药大学学报,2012,36(5):558-562.
[15]张顺仓,夏鹏国,王幼平,等.丹参中转录因子基因Sm MYB-like 1的克隆和表达分析[J].扬州大学学报:农业与生命科学版,2017,38(3):32-38.
[16]邢朝斌,龙月红,吴鹏,等.刺五加环阿屯醇合酶基因的克隆及其表达分析[J].中草药,2012,43(7):1387-1392.
[17]朱丹妮,高山林.组织培养川贝母化学成分和药理作用的研究[J].中国药科大学学报,1992,23(2):118-121.
[18]陈敏,陈和荣.川贝母组织培养的研究[J].中国中药杂志,1995,20(8):461-462.
[19]杨杨.野生和组培川贝母的总生物碱含量测定和定位研究[D].成都:四川大学,2007.