灰毡毛忍冬LmPAL1基因的克隆及表达分析
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摘要
目的克隆灰毡毛忍冬苯丙氨酸解氨酶(LmPAL1)基因全长序列,并进行生物信息学和表达模式分析。方法提取灰毡毛忍冬花总RNA,通过实时荧光定量PCR(qRT-PCR)和RACE技术克隆LmPAL1基因的全长cDNA序列;运用相关软件对该基因序列进行生物信息学分析;利用qRT-PCR测定灰毡毛忍冬茎、叶和不同花期花中该基因的相对表达量。结果克隆的LmPAL1基因开放阅读框(ORF)长度为2 145 bp,编码714个氨基酸,生物信息学分析预测为亲水性蛋白,定位于叶绿体中,包含PAL shielding结构域(527～641 aa),并且含有PAL/HAL活性中心序列GTITASGDLVPLSYIAG(196～212aa),与其他LmPAL1具有较高的相似度;LmPAL1基因q RT-PCR结果显示,7个花期材料以金黄色开花期相对表达量较高;与茎、叶比,白色花蕾期花的相对表达量最高,叶的最低。结论成功克隆了LmPAL1基因,为进一步研究该基因的功能以及遗传改良灰毡毛忍冬品质奠定基础,同时为探究灰毡毛忍冬绿原酸生物合成和调节机制提供了依据。
Objective To clone the full length of LmPAL1 gene and analyze bioinformatics and expression patterns from Lonicera macranthoides. Methods The total RNA of L. macranthoides was extracted. The full-length cDNA sequence of LmPAL1 gene was cloned by RT-PCR and RACE technique; The genome sequence in bioinformatics was analyzed by using the relevant software; The relative expression of the gene in stem, leaf, and different flower period was determined by using real-time PCR. Results The cloned LmPAL1 gene open reading frame(ORF) was 2 145 bp, encoding 714 amino acids. It was predicted by bioinformatics analysis as hydrophilic protein, being located in the chloroplasts, containing PAL shielding structure domain(527-641 aa). This gene contained PAL/HAL active center sequence GTITASGDLVPLSYIAG(196-212 aa), which was highly similar to other phenylalanine ammonia-lyase. Real-time PCR results showed that the relative expression level of golden yellow flowering flower was higher in seven florescence periods. When comparing the stem, leaf, and white flower bud period, the relative expression of flower was the highest and the leaf was the lowest. Conclusion In this study, PAL1 gene of L. macranthoides was cloned successfully, laying a foundation for further study of the function of this gene and genetic improvement of L. macranthoides quality and providing the research basis for exploring the biosynthesis and regulation of chlorogenic acid in L. macranthoides.
Objective To clone the full length of LmPAL1 gene and analyze bioinformatics and expression patterns from Lonicera macranthoides. Methods The total RNA of L. macranthoides was extracted. The full-length cDNA sequence of LmPAL1 gene was cloned by RT-PCR and RACE technique; The genome sequence in bioinformatics was analyzed by using the relevant software; The relative expression of the gene in stem, leaf, and different flower period was determined by using real-time PCR. Results The cloned LmPAL1 gene open reading frame(ORF) was 2 145 bp, encoding 714 amino acids. It was predicted by bioinformatics analysis as hydrophilic protein, being located in the chloroplasts, containing PAL shielding structure domain(527-641 aa). This gene contained PAL/HAL active center sequence GTITASGDLVPLSYIAG(196-212 aa), which was highly similar to other phenylalanine ammonia-lyase. Real-time PCR results showed that the relative expression level of golden yellow flowering flower was higher in seven florescence periods. When comparing the stem, leaf, and white flower bud period, the relative expression of flower was the highest and the leaf was the lowest. Conclusion In this study, PAL1 gene of L. macranthoides was cloned successfully, laying a foundation for further study of the function of this gene and genetic improvement of L. macranthoides quality and providing the research basis for exploring the biosynthesis and regulation of chlorogenic acid in L. macranthoides.
引文
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[13]刘湘丹,徐玉琴,王珊,等.适用于基因全长克隆的灰毡毛忍冬不同器官总RNA提取方法筛选[J].湖南中医药大学学报,2015,35(12):60-63.
[14]蔡嘉洛,朱贻霖,谢舒平,等.灰毡毛忍冬内参基因筛选和Mads-box家族基因AGL15的时空表达分析[J].中草药,2016,47(15):2727-2733.
[15]彭美晨,徐玉琴,王珊,等.灰毡毛忍冬Lm-XL-AP1基因克隆、生物信息学和时空表达分析[J].中草药,2018,8(7):1652-1660.
[16]杨银菊,陈爱国,刘光亮,等.烟草绿原酸生物合成途径关键酶基因的研究进展[J].现代农业科技,2018(13):5-8.
[17]李隆云,张应,马鹏,等.山银花(灰毡毛忍冬)适宜采收期研究[J].中国中药杂志,2014,39(16):3060-3064.
[18]吴飞燕,卿志星,曾建国.HPLC测定灰毡毛忍冬不同部位中绿原酸和木犀草苷的含量[J].中国现代中药,2014,16(8):614-617.
[19]Howles P A,Dixon R A.Overexpression of L-phenylalanine ammonia-lyase in transgenic tobacco plants reveals control points for flux into phenylpropanoid biosynthesis[J].Plant Physiol,1996,112(4):1617-1624.
[20]Chang J L,Luo J,He G Y.Regulation of polyphenols accumulation by combined overexpression/silencing key enzymes of phyenylpropanoid pathway[J].Acta Biochim Biophys Sin,2009,41(2):123-130.
[21]周日宝,童巧珍.灰毡毛忍冬与正品金银花的绿原酸含量比较[J].中药材,2003,26(6):399-400.
[22]张猛,姚本玉,汪先锋,等.灰毡毛忍冬产业现状及发展对策[J].现代农业科技,2014(16):97-99.
[23]魏颖,李朋收,时晓娟,等.金银花与山银花鉴别方法研究进展[J].中医药导报,2015,21(16):91-95.
[2]张浩超,郝宝燕,孙皓熠,等.绿原酸研究进展[J].食品与药品,2017,19(3):222-227.
[3]王玲娜,姚佳欢,马超美.绿原酸的研究进展[J].食品与生物技术学报,2017,36(11):1121-1130.
[4]Yuan Y,Wang Z,Jiang C,et al.Exploiting genes and functional diversity of chlorogenic acid and luteolin biosyntheses in Lonicera japonica and their substitutes[J].Gene,2014,534(2):408-416.
[5]李铁柱,杜红岩,刘慧敏.杜仲PAL基因c DNA全长序列特征分析[J].经济林研究,2014,32(1):40-44.
[6]乔枫,耿贵工,张丽,等.枸杞苯丙氨酸解氨酶基因的克隆与表达分析[J].中国农业大学学报,2017,22(12):64-73.
[7]高杰,谷巍,周娟娟,等.黑三棱苯丙氨酸解氨酶基因克隆与序列分析[J].中草药,2014,45(3):403-409.
[8]林懋怡,郑柳,刘晋杰,等.华细辛苯丙氨酸解氨酶基因的克隆与生物信息学分析[J].中国实验方剂学杂志,2018,24(1):38-43.
[9]刘建福,钟书淳,王明元,等.姜黄苯丙氨酸解氨酶基因的克隆与序列分析[J].中草药,2014,45(21):3141-3148.
[10]雒军,王引权,温随超,等.当归苯丙氨酸解氨酶基因片段克隆和组织特异性表达分析[J].草业学报,2014,23(4):130-137.
[11]郝向阳,孙雪丽,王天池,等.植物PAL基因及其编码蛋白的特征与功能研究进展[J].热带作物学报,2018,39(7):1452-1461.
[12]汪周勇,黄璐琦,袁媛,等.金银花苯丙氨酸解氨酶(LJPAL1)抗体制备及酶联免疫吸附分析[J].药学学报,2013,48(9):1498-1502.
[13]刘湘丹,徐玉琴,王珊,等.适用于基因全长克隆的灰毡毛忍冬不同器官总RNA提取方法筛选[J].湖南中医药大学学报,2015,35(12):60-63.
[14]蔡嘉洛,朱贻霖,谢舒平,等.灰毡毛忍冬内参基因筛选和Mads-box家族基因AGL15的时空表达分析[J].中草药,2016,47(15):2727-2733.
[15]彭美晨,徐玉琴,王珊,等.灰毡毛忍冬Lm-XL-AP1基因克隆、生物信息学和时空表达分析[J].中草药,2018,8(7):1652-1660.
[16]杨银菊,陈爱国,刘光亮,等.烟草绿原酸生物合成途径关键酶基因的研究进展[J].现代农业科技,2018(13):5-8.
[17]李隆云,张应,马鹏,等.山银花(灰毡毛忍冬)适宜采收期研究[J].中国中药杂志,2014,39(16):3060-3064.
[18]吴飞燕,卿志星,曾建国.HPLC测定灰毡毛忍冬不同部位中绿原酸和木犀草苷的含量[J].中国现代中药,2014,16(8):614-617.
[19]Howles P A,Dixon R A.Overexpression of L-phenylalanine ammonia-lyase in transgenic tobacco plants reveals control points for flux into phenylpropanoid biosynthesis[J].Plant Physiol,1996,112(4):1617-1624.
[20]Chang J L,Luo J,He G Y.Regulation of polyphenols accumulation by combined overexpression/silencing key enzymes of phyenylpropanoid pathway[J].Acta Biochim Biophys Sin,2009,41(2):123-130.
[21]周日宝,童巧珍.灰毡毛忍冬与正品金银花的绿原酸含量比较[J].中药材,2003,26(6):399-400.
[22]张猛,姚本玉,汪先锋,等.灰毡毛忍冬产业现状及发展对策[J].现代农业科技,2014(16):97-99.
[23]魏颖,李朋收,时晓娟,等.金银花与山银花鉴别方法研究进展[J].中医药导报,2015,21(16):91-95.