Cloning and Expression Patterns of <italic style="font-style: italic">LmC</italic>3<italic style="font-style: italic">H</italic>1 Gene in <italic style="font-style: italic">Lonicera macranthoides</italic> and Its Correlation with Chlorogenic Acid Content

Ya CHEN; An-qi WANG; Cong LIU; Yu-qing LONG; Xia LIU; Juan ZENG; Can LI; Xiang-dan LIU; Ri-bao ZHOU

doi:10.13422/j.cnki.syfjx.20200913

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Cloning and Expression Patterns of LmC3H1 Gene in Lonicera macranthoides and Its Correlation with Chlorogenic Acid Content

Pharmacy Fundamentals | 更新时间：2021-02-09

- Cloning and Expression Patterns of LmC3H1 Gene in Lonicera macranthoides and Its Correlation with Chlorogenic Acid Content
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 26, Issue 9, Pages: 167-175(2020)
- 作者机构：
  
  1.湖南中医药大学　药学院，长沙　 410208
  2.湖南省中药饮片标准化及功能工程技术研究中心，长沙　 410208
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20200913
  CLC： R22; R289; R2-031; R284.2
- Received：08 October 2019，
  
  Published Online：16 January 2020，
  
  Published：05 May 2020
- 稿件说明：
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Ya CHEN, An-qi WANG, Cong LIU, et al. Cloning and Expression Patterns of LmC3H1 Gene in Lonicera macranthoides and Its Correlation with Chlorogenic Acid Content[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(9): 167-175.
DOI：

Ya CHEN, An-qi WANG, Cong LIU, et al. Cloning and Expression Patterns of LmC3H1 Gene in Lonicera macranthoides and Its Correlation with Chlorogenic Acid Content[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(9): 167-175. DOI： 10.13422/j.cnki.syfjx.20200913.

摘要

目的：

以灰毡毛忍冬为材料，克隆对-香豆酸3-羟化酶(

LmC

1)基因，进行生物信息学和表达模式分析，结合绿原酸含量，研究推测灰毡毛忍冬

LmC

1基因的功能。

方法：

通过逆转录聚合酶链式反应(RT-PCR)和RACE技术克隆

LmC

1基因的全长cDNA序列，对该序列进行生物信息学分析，并利用实时荧光定量PCR(Real-time PCR)和HPLC分别测定灰毡毛忍冬茎、叶及不同花期花中

LmC

1的相对表达量及绿原酸含量。

结果：

克隆得

LmC

1(GenBank：MN177695)基因，开放阅读框(ORF)长度为1 533 bp，编码510个氨基酸，推测其分子式为C

2618

4134

718

727

，相对分子质量为58 005.32，等电点8.92，为亲水性蛋白，定位于叶绿体中，具有跨膜区域LLLIPAVLFLISLVYPLI，含有细胞色素P450的保守结构域CYTOCHROME_P450(422-433 aa)；Real-time PCR结果显示，

LmC

1在灰毡毛忍冬茎、叶及不同花期花有不同程度的表达，其中在花发育阶段，白色花蕾期相对表达量最高，花蕾初期及白色开花期次之；白色花蕾期花与茎、叶比，花的相对表达量最高，叶的最低；HPLC结果显示，从绿白色花蕾期到金黄色开花期绿原酸含量呈上升趋势，金黄色开花期含量最高，不同器官中，花中绿原酸最高，茎最低。

结论：

克隆得到灰毡毛忍冬

LmC

1基因，推测

LmC

1可能参与灰毡毛忍冬花绿原酸的生物合成。该研究为进一步研究该基因的功能及探究灰毡毛忍冬绿原酸生物合成和调节机制提供了依据，同时为遗传改良灰毡毛忍冬品质奠定了基础。

Abstract

Objective:

To clone

-coumaroyl quinate/shikimate 3' -hydroxylase gene from

Lonicera macranthoides

and analyze its bioinformatics and expression patterns with chlorogenic acid content

in order to speculate the functions of

LmC

1 gene from

macranthoides

Method:

The full-length cDNA sequence of

LmC

1 gene was cloned by reverse trascription polymerase chain reaction(RT-PCR) and RACE techniques. The bioinformatics analysis of the gene sequence was carried out by using relevant software.Real-time fluorescence quantification PCR(Real-time PCR) and HPLC were used to determine relative expression of

LmC

1 and content of chlorogenic acid in stems

leaves and flowers of different flowering stages.

Result:

The

LmC

1 (GenBank: MN177695) gene was cloned

and the open reading frame (ORF) of it was 1 533 bp in length and encoded 510 amino acids. The molecular formula was C

2618

4134

718

727

the relative molecular mass was 58 005.32

and the isoelectric point was 8.92.It was a hydrophilic protein located in the chloroplast with a transmembrane region LLLIPAVLFLISLVYPLI

and contained a conserved domain CYTOCHROME_P450(433-422 aa) in cytochrome P450.The results of Real-time PCR showed that

LmC

1 was expressed in different degrees in stems

leaves and different flowering stages of

macranthoides

. In the flower development stage

the relative expression of white bud stage was the highest

followed by flower buds and white flowering stage. The ratio of flower to stem and leaf was the highest

and the relative expression of flower was the highest. The HPLC results showed that the content of chlorogenic acid increased from greenish white to golden yellow in flowering stage and golden yellow flowering stage. Among the different organs

the flower had the highest chlorogenic acid

and the stem showed the lowest.

Conclusion:

The

LmC

1 gene of

macranthoides

is cloned

suggesting that

LmC

1 might be involved in the biosynthesis of

macranthoides

chlorogenic acid. This study provides a basis for further studying the functions of the gene and exploring the biosynthesis and regulation mechanism of

macranthoides

chlorogenic acid

while laying the foundation for the genetic improvement of

macranthoides

关键词

Keywords

references

国家药典委员会．中华人民共和国药典：一部 [M]．北京：中国医药科技出版社， 2015 ： 30 - 31 .

赵留存，叶世芸，杨春，等． UPLC-PDA-ELSD测定贵州山银花中绿原酸、灰毡毛忍冬皂苷乙和川续断皂苷乙的含量 [J]．中国实验方剂学杂志， 2015 ， 21 ( 7 )： 64 - 67 .

李莉，李燕君，王森弘，等．灰毡毛忍冬花蕾抑菌及抗炎作用研究 [J]．食品工业科技， 2013 ， 34 ( 23 )： 65 - 69 .

白仲杰，任群利，俸婷婷，等．基于炎症细胞模型的灰毡毛忍冬抗炎活性研究 [J]．现代中药研究与实践， 2015 ， 29 ( 5 )： 35 - 39 .

史秀玲，高银辉．绿原酸对小鼠急性肝损伤的保护作用 [J]．中国实验方剂学杂志， 2011 ， 13 ( 19 )： 199 - 202 .

L WU , Z J ZHANG , Z S ZHANG . Characterization of antioxidant activity of extracts from Flos Lonicerae [J]． Drug Dev Ind Pharm ， 2007 ， 33 ( 8 )： 841 - 847 .

J WANG , X Z ZHAO , Q QI , et al . Macranthoside B，a hederagenin saponin extracted from Lonicera macranthoides ，and its anti-tumor activities in vitro ，and in vivo [J]． Food Chem Toxicol ， 2009 ， 47 ( 7 )： 1716 - 1721 .

张小娜．灰毡毛忍冬与忍冬化学成分及药理作用的比较研究 [D]．重庆：西南大学， 2014 .

张小娜，唐清，侯敏，等．灰毡毛忍冬与忍冬的药理作用比较研究 [J]．中国药理学通报， 2012 ， 28 ( 11 )： 1582 - 1585 .

刘文娟，陈雨，马鑫，等．灰毡毛忍冬化学成分研究进展 [J]．中国野生植物资源， 2013 ， 32 ( 1 )： 6 - 10 .

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 .

那袭雪，张文涛，谈远锋，等．绿原酸及其异构体药理作用及不良反应研究进展 [J]．辽宁中医药大学学报， 2018 ， 20 ( 3 )： 140 - 144 .

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 .

何柳，陈士林．植物中绿原酸合成途径研究进展 [J]．药物生物技术， 2013 ， 20 ( 5 )： 463 - 466 .

刘颖，郭明晔，白根本．绿原酸的研究进展 [J]．中药材， 2012 ， 35 ( 7 )： 1180 - 1185 .

王雪霞，薛永常，赵文超．木质素生物合成中C3H/HCT的研究进展 [J]．生命的化学， 2008 ， 28 ( 5 )： 650 - 653 .

亓希武，徐道华，于盱，等．金银花香豆酸-3-羟化酶基因 LjC 3 H 2的克隆及表达分析 [J]．分子植物育种， 2018 ， 16 ( 4 )： 1092 - 1099 .

G SCHOCH , S GOEPFERT , M MORANT , et al . CYP98A3 from Arabidopsis thaliana is a 3′-hydroxylase of phenolic esters，a missing link in the phenylpropanoid pathway [J]． J Biol Chem ， 2018 ， 276 ( 39 )： 36566 - 36574 .

M MORANT , G A SCHOCH , P ULLMANN , et al . Catalytic activity，duplication and evolution of the CYP98 cytochrome P450 family in wheat [J]． Plant Mol Biol ， 2007 ， 63 ( 1 )： 1 - 19 .

袁有美，郭清泉 . 苎麻香豆酸-3-羟化酶基因的原核表达 [J]．湖南农业大学学报：自然科学版， 2018 ， 44 ( 2 )： 157 - 161 .

A M ANTEROLA , J H JEON , L B DAVIN , et al . Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism [J]． J Biol Chem ， 2002 ， 277 ( 21 )： 18272 - 18280 .

X LIU , Z DENG , S GAO , et al . A new gene coding forp-coumarate 3-hydroxylase from Ginkgo biloba [J]． Russian J Plant Physiol ， 2008 ， 55 ( 1 )： 82 - 92 .

樊二勤，刘彩霞，付鹏跃，等 . 84K杨 PagC 3 H 3基因表达模式分析 [J]．植物研究， 2019 ， 39 ( 4 )： 521 - 528 .

李洋，唐雪冰，李晓峰，等． NtC 3 H 基因对烟草类黄酮及绿原酸合成的影响 [J]．中国烟草科学， 2016 ， 37 ( 1 )： 8 - 13 .

周美娟，胡尚连，曹颖，等．慈竹 C 3 H 基因克隆及其生物信息学分析 [J]．植物研究， 2012 ， 32 ( 1 )： 38 - 46 .

蒋向辉．金银花绿原酸合成途径关键酶基因克隆与功能分析 [D]．长沙：湖南大学， 2013 .

陈勋，刘畅宇，陈娅，等．灰毡毛忍冬LmPAL1基因的克隆及表达分析 [J]．中草药， 2019 ， 50 ( 1 )： 178 - 187 .

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 .

Z CHEN , G LIU , Y LIU , et al . Overexpression of the LmHQT 1 gene increases chlorogenic acid production in Lonicera macranthoides Hand-Mazz [J]． Acta Physiol Plant ， 2017 ， 39 ( 1 )： 27 .

J RAES , A ROHDE , J H CHRISTENSEN , et al . Genome-wide characterization of the lignification toolbox in Arabidopsis [J]． Plant Physiol ， 2003 ， 133 ( 3 )： 1051 - 1071 .

G PU , P WANG , B ZHOU , et al . Cloning and characterization of Lonicera japonica p -coumaroyl ester 3-hydroxylase which is involved in the biosynthesis of chlorogenic acid [J]． Biosci Biotechnol Biochem ， 2013 ， 77 ( 7 )： 1403 - 1409 .

范丽．桑树木质素合成基因的生物信息和功能分析 [D]．重庆：西南大学， 2013 .

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Cloning and Expression Patterns of LmC3H1 Gene in Lonicera macranthoides and Its Correlation with Chlorogenic Acid Content

DOI：10.13422/j.cnki.syfjx.20200913

摘要

Abstract

关键词

Keywords

references