基于小RNA测序的风轮菜microRNA及其靶基因分析

doi:10.11913/PSJ.2095-0837.2022.20216

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摘要

本研究以风轮菜（Clinopodium chinense（Benth.） O.Kuntze）为材料，采用BGISEQ-500测序平台对风轮菜根、茎和叶的小RNA进行转¼组测序，并对其黄酮类物质合成途径中参与调控的microRNA （miRNA）及其靶基因进行了分析。结果显示，鉴定出的保守miRNA有86个，属于26个家族，新发现miRNA 8个，筛选出风轮菜黄酮类物质合成途径中调控3个关键ø的候选miRNA （novel_mir3、miR167d-5p、miR396h）。通过对靶基因编码的关键ø4-香豆酸辅øA连接ø进行序列分析和同源建模，发现其具有高度保守的底物结合区域、催化结构域及两个保守的肽基序。

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	许景垚
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	单婷玉
	赵历强
	马克龙
	吴家文

关键词 ：风轮菜, 转¼, 组测序, miRNA, 黄酮, 4-香豆素辅ø, A连接ø

Abstract：

To analyze the flavonoid biosynthesis pathway in Clinopodium chinense (Benth.) O. Kuntze and to explore the role of microRNAs (miRNAs) in target gene regulation, transcriptome sequencing of small RNAs (sRNAs) in the roots, stems, and leaves of C. chinense was carried out using the BGISEQ-500 platform. In total, 86 conserved miRNAs were identified, divided into 26 families, and eight novel miRNAs were predicted. Three candidate miRNAs (novel_mir3, miR167d-5p, and miR396h) and their target genes involved in the flavonoid biosynthesis pathway were screened out. Sequence analysis and homologous modeling of 4-coumarate-CoA ligase, a key enzyme encoded by a target gene, showed that it had two conserved peptide motifs and a highly conserved substrate binding groove and catalytic domain. This research lays a foundation for functional genomics study of C. chinense and understanding the regulation mechanism of flavonoid biosynthesis.

Key words： Clinopodium chinense RNA sequencing miRNA Flavonoids 4-Coumarate-CoA ligase

收稿日期: 2021-09-13

ZTFLH:

Q943.2

基金资助:

安徽省自然科学基金项目（2008085MH268，2108085MH315）；国家重点研发计划项目（2017YFC1701600）；安徽中医药大学国家项目培养基金项目（2020PY02）；安徽高校自然科学研究项目（KJ2019A0476）。

通讯作者: 马克龙,E-mail:makelong210@126.com;吴家文,E-mail:wujiawen@ahtcm.edu.cn E-mail: makelong210@126.com;wujiawen@ahtcm.edu.cn

作者简介: 许景垚(1996-),女,硕士研究生,研究方向为中草药分子生物学(E-mail:737126233@qq.com)。

引用本文:

许景垚, 单春苗, 单婷玉, 赵历强, 马克龙, 吴家文. 基于小RNA测序的风轮菜microRNA及其靶基因分析[J]. 植物科学学报, 2022, 40(2): 216-228. Xu Jing-Yao, Shan Chun-Miao, Shan Ting-Yu, Zhao Li-Qiang, Ma Ke-Long, Wu Jia-Wen. Analysis of microRNAs and their target genes in Clinopodium chinense (Benth.) O. Kuntze using small RNA sequencing. Plant Science Journal, 2022, 40(2): 216-228.

链接本文:

http://www.plantscience.cn/CN/10.11913/PSJ.2095-0837.2022.20216 或 http://www.plantscience.cn/CN/Y2022/V40/I2/216

[1] 国家药典委员会. 中华人民共和国药典:一部[S]. 北京:中国医药科技出版社, 2015:326.
[2] 张冬玲,何枢衡, 黄雁, 洪美闲, 吴建雄. 基于系统药理学分析断血流治疗出血性疾病的机制[J]. 西北药学杂志, 2020, 35(6):839-945. Zhang DL, He SH, He Y, Hong MX, Wu JX. Research on the mechanism of Duanxueliu in the treatment of bleeding diseases based on system pharmacology[J]. Northwest Pharmaceutical Journal, 2020, 35(6):839-945.
[3] 王圣男, 余世春, 许旭东, 佘守军, 范石虎. 风轮菜属三萜皂苷与黄酮研究进展及波谱特征[J]. 波谱学杂志, 2013, 30(3):447-460.
[4] 苗得足,高峰,鞠建刚. 风轮菜中黄酮苷类化合物的结构鉴定[J]. 药学与临床研究, 2014, 22(4):342-343. Miao DZ, Gao F, Ju JG. Flavonoid glycosides from Clinopodium chinense (Benth.) O. Kuntze[J]. Pharmaceutical and Clinical Research, 2014, 22(4):342-343.
[5] 祁建宏, 董芳旭. 黄酮类化合物药理作用研究进展[J]. 北京联合大学学报, 2020, 34(3):89-92. Qi JH, Dong FX. Research progress on pharmacological action of flavonoids[J]. Journal of Beijing Union University, 2020, 34(3):89-92.
[6] 田晓明, 颜立红, 向光锋, 蒋利媛. 植物4香豆酸:辅øA连接ø研究进展[J]. 生物技术通报, 2017, 33(4):19-26. Tian XM, Yan LH, Xiang GF, Jiang LY. Research progress on 4-Coumarate:coenzyme A ligase(4CL) in plants[J]. Biotechnology Bulletin, 2017, 33(4):19-26.
[7] Li SB, Liu L, Zhuang XH, Yu Y, Liu XG, et al. MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis[J]. Cell, 2013, 153(3):562-574.
[8] Qin JP, Tang ZH, Ma XX, Meng YJ. Investigating the regulatory roles of the microRNAs and the Argonaute 1-enriched small RNAs in plant metabolism[J]. Gene, 2017, 628:180-189.
[9] Zhang SX, Shi YY, Shan CM, Zhao LQ, Ma KL, et al. Analysis of the transcriptome of Polygonatum odoratum (Mill.) druce uncovers putative genes involved in isoflavonoid biosynthesis[J]. J Plant Biol, 2020, 63(10):217-228.
[10] 单春苗, 王晨凯, 施圆圆, 张声祥, 赵历强, 等. 多花黄精甾体皂苷生物合成途径分析及关键ø基因研究[J]. 中国中药杂志, 2020, 45(12):2847-2857. Shan CM, Wang CK, Shi YY, Zhang SX, Zhao LQ, et al. Identification of key enzyme genes involved in biosynthesis of steroidal saponins and analysis of biosynthesis pathway in Polygonatum cyrtonema[J]. China Journal of Chinese Materia Medica, 2020, 45(12):2847-2857.
[11] 赵历强, 单春苗, 张声祥, 施圆圆, 马克龙, 等. 基于转¼组测序的细风轮花青素合成途径及关键ø基因分析[J]. 植物研究, 2020, 40(6):886-896. Zhao LQ, Shan CM, Zhang SX, Shi YY, Ma KL, et al. Identification of key enzyme genes involved in anthocyanin synthesis pathway in Clinopodium gracile by transcriptome analysis[J]. Bulletin of Botanical Research, 2020, 40(6):886-896.
[12] Fan RY, Li YJ, Li CF, Zhang YS. Differential microRNA snalysis of glandular trichomes and young leaves in Xan-thium strumarium L. reveals their putative roles in regulating terpenoid biosynthesis[J]. PLoS One, 2015, 10(9):e0139002.
[13] Τ荣昌. 三七皂苷生物合成途径关键ø基因和miRNA的挖掘与分析[D]. 北京:北京协和医学院, 2014:96-134.
[14] Wong MM, Cannon CH, Wickneswari R. Identification of lignin genes and regulatory sequences involved in secon-dary cell wall formation in Acacia auriculiformis and Acacia mangium via de novo transcriptome sequencing[J]. BMC Genomics, 2011, 12(1):342-354.
[15] Shi YY, Zhang SX, Peng DY, Wang CK, Huang LQ. Transcriptome analysis of Clinopodium chinense (Benth.) O. Kuntze and identification of genes Involved in triterpenoid saponin biosynthesis[J]. Int J Mol Sci, 2019, 20(11):2643-2659.
[16] Zhao SR, Ye Z, Stanton R. Misuse of RPKM or TPM normalization when comparing across samples and sequencing protocols[J]. RNA, 2020, 26(8):903-909.
[17] Tang C, Xie YM, Guo M. AASRA:An anchor alignment-based small RNA annotation pipelin[J]. Biol Reprod, 2021, 105(1):267-277.
[18] Evers M, Huttner M, Dueck A, Meister G, Engelmann JC. miRA:adaptable novel miRNA identification in plants using small RNA sequencing data[J]. BMC Bioinformatics, 2015, 16(1):370-379.
[19] Friedl?nder MR, Mackowiak SD, Li N, Chen W, Rajewsky N. miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades[J]. Nucleic Acids Res, 2012, 40(1):37-52.
[20] Midway S, Robertson M, Flinn S, Kaller M. Comparing multiple comparisons:practical guidance for choosing the best multiple comparisons test[J]. PeerJ, 2020, 8:e10387.
[21] Benjamini Y, Yekutieli D. The control of the false discovery rate in multiple testing under dependency[J]. Ann Stat, 2001, 29(4):1165-1188.
[22] Murtagh F, Legendre P. Ward's hierarchical agglomerative clustering method:which algorithms implement ward's criterion[J]. J Classif, 2014, 31:274-295.
[23] Wu HJ, Ma YK, Chen T, Wang M, Wang XJ. PsRobot:a web-based plant small RNA meta-analysis toolbox[J]. Nucleic Acids Res, 2012(W1):22-28.
[24] Fahlgren N. miRNA target prediction in plants[M]. Berlin:Springer-Verlag, 2010:51-57.
[25] Liu J, Liu XN, Zhang SJ, Liang SS, Luan WJ, Ma X. TarDB:an online database for plant miRNA targets and miRNA-triggered phased siRNAs[J]. BMC Genomics, 2021, 22(1):348-359.
[26] Rice P, Longden I, Bleasby A. EMBOSS:the European molecular biology open software suite[J]. Trends Genet, 2000, 16(6):276-277.
[27] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, et al. MEGA5:molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Mol Biol Evol, 2011, 28(10):2731-2739.
[28] Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG.The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools[J]. Nucleic Acids Res, 1997, 25(24):4876-4882.
[29] Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server[J]. Nucleic Acids Res, 2014, 42:W320-W324.
[30] Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, et al. SWISS-MODEL:homology modelling of protein structures and complexes[J]. Nucleic Acids Res, 2018, 46(W1):W296-W303.
[31] Bertoni M, Kiefer F, Biasini M, Bordoli L, Schwede T. Modeling protein quaternary structure of homo-and he-tero-oligomers beyond binary interactions by homology[J]. Sci Rep, 2017, 7(1):10480.
[32] Cabrera áC, Gil-Redondo R, Perona A, Gago F, Morreale A. VSDMIP 1.5:an automated structure-and ligand-based virtual screening platform with a PyMOL graphical user interface[J]. J Comput Aided Mol Des, 2011, 25(9):813-824.
[33] Oliveira SH, Ferraz FA, Honorato RV, Xavier-Neto J, Sobreira TJ, de Oliveira PS. KVFinder:steered identification of protein cavities as a PyMOL plugin[J]. BMC Bioinformatics, 2014, 15(1):1-8.
[34] 于小凤. 经霜对桑叶黄酮类成分积累影响的分子机制研究[D]. 镇江:江苏大学, 2018:10-12.
[35] Lavhale SG, Kalunke RM, Giri AP. Structural, functional and evolutionary diversity of 4-coumarate-CoA ligase in plants[J]. Planta, 2018, 248(5):1063-1078.
[36] 昝丽霞, 孙文基. 断血流的化学成分及药理作用研究进展[J]. 西北药学杂志, 2008, 23(2):126-128.
[37] 陈艳琳. 长白山人参microRNA鉴定及靶基因分析研究[D]. 广州:广东药科大学. 2017:6-18.
[38] 王琪月, 孟淑君, 张柯, 张战辉, 汤继华, 等. 玉米雌穗发育杂种优势相关miRNA的研究[J]. 作物学报, 2018, 44(6):796-813. Wang QY, Meng SJ, Zhang K, Zhang ZH, Tang JH, et al. Investigation of maize miRNA involved in developing-ear heterosis[J]. Acta Agronomica Sinica, 2018, 44(6):796-813.
[39] 孙平. 茶叶儿茶素合成相关miRNA及靶基因的验证和表达分析[D]. 福州:福建农林大学, 2017:40-52.
[40] Zhang SF, Yan SS, Zhao JL, Xiong HH, An PQ, et al. Identification of miRNAs and their target genes in Larix olgensis and verified of differential expression miRNAs[J]. BMC Plant Biol, 2019, 19(1):247-266.
[41] ë家旺, 杨艳华, 陈克平, 谭小力. 植物激素与microRNA调控种子大小和发育的分子机制研究进展[J]. 植物生理学报, 2021, 57(2):274-292. Mao JW, Yang YH, Chen KP, Tan XL. Research progress in molecular mechanisms of plant hormone and microRNA regulating seed size and development[J]. Plant Physiology Journal, 2021, 57(2):274-292.
[42] Yin YC, Zhang XD, Gao ZQ, Hu T, Liu Y. The research progress of chalcone isomerase (CHI) in plants[J]. Mol Biotechnol, 2019, 61(1):32-52.
[43] Goetz M, Smith AV, Johnson SD, Koltunow AM. AUXIN RESPONSE FACTOR8 is a negative regulator of fruit initiation in Arabidopsis[J]. Plant Cell, 2006, 18(8):1873-1886.
[44] 陈媞颖, 刘娟, 袁媛, 周骏辉, 黄璐琦. 黄芩ARF基因家族生物信息学及表达分析[J]. 药学学报, 2017, 52(11):1770-1776. Chen TY, Liu J, Yuan Y, Zhou JH, Huang LQ. Bioinformatics analysis and expressed level of Auxin response factor genes in Scutellaria baicalensis[J]. Acta Pharmaceutica Sinica, 2017, 52(11):1770-1776.
[45] 鲍ï林. 拟南芥MIR396家族对靶基因的调控及对根发育的影响[D]. 杭州:浙江大学, 2011:4-6.
[46] 叶超楠, 沈栎阳, 方春, 曹跃芬, 於金生. 热胁迫下水稻miR396家族及靶基因OsGRFs的表达研究[J]. 农业生物技术学报, 2018, 26(3):393-400. Ye CN, Shen LY, Fang C, Cao YF, Yu JS. Expression analysis of rice (Oryza sativa)miR396 family and target gene OsGRFs under heat stress[J]. Journal of Agricultural Biotechnology, 2018, 26(3):393-400.
[47] 沈朝斌, 蔡红, 郁兰. 植物药miRNA396家族的应用[P]. 中国专利:105903035. 2015-07-09.
[48] Gupta OP, Dahuja A, Sachdev A, Kumari S, Jain PK, et al. Conserved miRNAs modulate the expression of potential transcription factors of isoflavonoid biosynthetic pathway in soybean seeds[J]. Mol Biol Rep, 2019, 46(4):3713-3730.
[49] Liu H, Jia SH, Shen DF, Liu J, Li J, et al. Four AUXIN RESPONSE FACTOR genes downregulated bymicroRNA167 are associated with growth and development in Oryza sativa[J]. Funct Plant Biol, 2012:39(9):736-744.
[50] Chandran V, Wang H, Gao F, Cao XL, Chen YP, et al. miR396-OsGRFs module balances growth and rice blast disease-resistance[J]. Front Plant Sci, 2019, 1999(9):1-16.
[51] 黄满芬. 桑树C4H、4CL及CHS基因克¡及其在不同桑种质间的表达差异[D]. 镇江:江苏科技大学, 2014:41-62.
[52] 杨晓云, 杨智敏, 罗小娇, 孔德媛, 袁金娥, 等.青稞4-香豆酸辅øA连接ø基因4CL的克¡及表达分析[J]. 麦类作物学报, 2014, 34(12):1603-1610. Yang XY, Yang ZM, Luo XJ, Kong DY, Yuan JE, et al. Cloning and expression analysis of 4-Ceoumarate:CoA ligase gene 4CL in hulless barley[J]. Journal of Triticeae Crops, 2014, 34(12):1603-1610.
[53] Sun HY, Li Y, Feng SQ, Zou WH, Guo K, et al. Analysis of five rice 4-coumarate:coenzyme A ligase enzyme acti-vity and stress response for potential roles in lignin and flavonoid biosynthesis in rice[J]. Biochem Biophys Res Commun, 2013, 430(3):1151-1156.