无患子RT-qPCR内参基因的筛选与验证

doi:10.13560/j.cnki.biotech.bull.1985.2021-1616

摘要/Abstract

摘要：

无患子根、茎、叶、花和果皮均含有生物活性物质三萜皂苷，为了解三萜皂苷生物合成途径中相关基因的表达水平，需要筛选稳定表达的内参基因。以无患子根、茎、叶、芽、雄花、雌花和不同发育时期的果皮为材料，根据无患子转录组数据，选择Sm18S，SmACT，SmTBCC，SmEF-1α，SmRPL1，SmRPS26，SmUBC12，SmUBP等8个基因作为候选内参基因。通过实时荧光定量PCR（RT-qPCR）检测这些候选内参基因的表达量，并利用geNorm、NormFider和BestKeeper三个软件及RefFinder在线分析工具评价候选内参基因的稳定性。结果表明，8个候选内参基因的表达量在所有样本间的变化幅度存在差异；geNorm、NormFinder和BestKeeper 3个软件筛选出的最佳内参基因略有不同；综合分析结果表明SmACT、SmRPL1和SmUBP表达较为稳定，SmEF-1α稳定性最差；以SmACT、SmACT+SmRPL1组合和SmACT+SmRPL1+SmUBP组合为内参对三萜皂苷生物合成途径中的8个候选基因进行校准所得的表达量基本上保持一致，且相对表达量结果与转录组数据基本保持一致，表明SmACT、SmACT+SmRPL1组合和SmACT+SmRPL1+SmUBP组合可作为无患子三萜皂苷生物合成途径相关基因表达研究的内参基因，同时也可以为无患子及近缘植物的其他生物学过程中的基因表达研究提供参考。

关键词: 无患子, 内参基因, 实时荧光定量PCR, 三萜皂苷生物合成, 表达分析

Abstract:

The roots，stems，leaves，flowers，and pericarps of Sapindus mukorossi Gaertn. are rich in bioactive triterpenoid saponin. In order to understand the expressions of related genes in triterpenoid saponin biosynthesis pathway，it is necessary to screen the stably-expressed reference genes. In this study，8 traditional reference genes including 18S rRNA（guanine1575-N7）-methyltransferase（Sm18S），actin-related protein 8（SmACT），elongation factor 1-alpha（SmEF-1α），large subunit ribosomal protein L1（SmRPL1），small subunit ribosomal protein S26e（SmRPS26），tubulin-specific chaperone C（SmTBCC），ubiquitin-conjugating enzyme E2 M（SmUBC12），E3 ubiquitin-protein ligase BAH（SmUBP）were selected as candidate reference genes based on the RNA-seq data of roots，stems，leaves，buds，male flowers，female flowers，and developing pericarps of S. mukorossi. The expressions of these candidate reference genes were detected by RT-qPCR in the root，stem，leaf，bud，male flower，female flower，and pericarps at different developmental stages，and the stability of them were then evaluated by geNorm，NormFinder and BestKeeper software and RefFinder online analysis tool. The results showed that the expressions of 8 candidate reference genes differed in the variations among all samples. And the optimal reference genes screened by geNorm，NormFinder and BestKeeper were also slightly different. The results of comprehensive analysis showed that SmACT，SmRPL1 and SmUBP expressed quite stably，while the stability of SmEF-1α was the lowest among all candidate reference genes. The RT-qPCR results of 8 triterpenoid saponin biosynthesis related genes using SmACT，SmACT+SmRPL1 and SmACT+SmRPL1+SmUBP as the reference genes respectively were in accordance with the results of RNA-seq. Thus，it is concluded that SmACT and the reference gene combinations of SmACT+SmRPL1 or SmACT+SmRPL1+SmUBP can be used as reference genes for gene expression analysis of triterpenoid saponin biosynthesis pathway in S. mukorossi and other biological processes in S. mukorossi and related plants.

Key words: Sapindus mukorossi Gaertn., reference gene, quantitative real-time PCR, triterpenoid saponin biosynthesis, expression analysis

徐圆圆, 赵国春, 郝颖颖, 翁学煌, 陈仲, 贾黎明. 无患子RT-qPCR内参基因的筛选与验证[J]. 生物技术通报, 2022, 38(10): 80-89.

XU Yuan-yuan, ZHAO Guo-chun, HAO Ying-ying, WENG Xue-huang, CHEN Zhong, JIA Li-ming. Reference Genes Selection and Validation for RT-qPCR in Sapindus mukorossi[J]. Biotechnology Bulletin, 2022, 38(10): 80-89.

图/表 8

图1 无患子采样材料 R：根；ST：茎；L：叶；B：芽；MF：雄花；FF：雌花；S1：花后15 d的果皮；S2：花后45 d的果皮；S3：花后75 d的果皮；S4：花后90 d的果皮；S5：花后105 d的果皮；S6：花后120 d的果皮；S7：花后135 d的果皮；S8：花后150 d的果皮

Fig. 1 Sample materials of S. mukorossi R：root；ST：stem；L：leaf；B：bud；MF：male flower；FF：female flower；S1：pericarp at 15 d after flower pollination；S2：pericarp at 45 d after flower pollination；S3：pericarp at 75 d after flower pollination；S4：pericarp at 90 d after flower pollination；S5：pericarp at 105 d after flower pollination；S6：pericarp at 120 d after flower pollination；S7：pericarp at 135 d after flower pollination；S8：pericarp at 150 d after flower pollination

表1 候选内参基因的引物

Table 1 Primers of candidate reference genes

内参基因 Reference genes	基因ID Gene ID	产物长度 Length of product /bp	正向引物序列 Forward primer sequences（5'- 3'）	反向引物序列 Reverse primer sequences（5'- 3'）
Sm18S	Samuk09G0136600	221	AGATTGAAGGCGTTCTTTGGAT	TCAGTGTTTATGGACGGTGGAC
SmACT	Samuk13G0061200	129	AGAAAGTTGGCCTCGCTGAA	CAGGAACCAGACCACCTGTC
SmEF-1α	Samuk14G0055700	101	TCCAAGGCCAGGTACGATGA	AAACCAGAGATGGGGACGAAG
SmRPL1	Samuk07G0011500	206	CTGACCACCCCACCACTCTC	TCCTCCTCATCTGCCACCTC
SmRPS26	Samuk08G0043900	230	GCGAAGAAATGGAGGAAGGA	AGTGGATGGCACACGAAACA
SmTBCC	Samuk01G0035500	130	AGCCTGACACCCTTTCTCTACC	CCAACCTCTTCTGCAGCCTT
SmUBC12	Samuk11G0037500	102	TTGTCTGGACCAACCAAAGGA	TGCTTCTTGCCAGGTGTTTTC
SmUBP	Samuk09G0089700	180	TGCACAGTTGTTGCTCATGC	CTGCTTCATTTTCCCCGTGC

表2 目的基因的引物

Table 2 Primers of objective genes

内参基因 Reference gene	基因ID Gene ID	产物长度 Length of product /bp	正向引物序列 Forward primer sequences（5'- 3'）	反向引物序列 Reverse primer sequence（5'- 3'）
SmAACT4	Samuk13G0079600	132	CCTGTTTTGAGGGCATTGATTG	ACCAGCATAGAACGCAGCCA
SmDXS4	Samuk14G0066700	194	AACAATGCTCAGAAGCCGAGA	GCGATCCATCCAGTAATCCAC
SmFPS	Samuk04G0156300	131	GAGGCAGAAGTAGGAACTCACCA	CCATTGTAGCAGTCAAGTGGTAAG
SmbAS1	Samuk02G0323400	113	GAGTGGGATACTGGTTTCGCTAT	TCCTTGACCTGAGATGCTTTGA
SmCYP716A-5	Samuk08G0024600	146	GCTGTTTTCTGTGGCCCTTC	CGAGTCTTGGCAATTTTCCC
SmUGT73C-14	Samuk12G0007500	120	ACCTACAAGTGCCCAAACAGAT	ATTCCCAGTTCACCACATTCC
SmbHLH8	Samuk10G0068800	107	CAACCTTTGACTCCGACTCCA	CCTTCCCTTACCCTAACCTCC
SmERF25	Samuk11G0050000	129	CAACAGCCACACCAACAGCA	TCTTCTCGGGTCACGGATCTC

表3 候选内参基因扩增效率和标准曲线参数

Table 3 Amplification efficiency and standard curve para-meters of candidate reference genes

内参基因 Reference gene	扩增效率 Amplification efficiency /%	相关系数R² Correlation coefficient R²	斜率K Slope K
Sm18S	97.872	0.998	-3.374
SmACT	93.727	0.999	-3.482
SmEF-1α	99.789	0.999	-3.327
SmRPL1	100.206	0.999	-3.317
SmRPS26	94.469	0.999	-3.462
SmTBCC	97.196	0.997	-3.391
SmUBC12	96.881	0.999	-3.399
SmUBP	98.032	0.999	-3.370

图2 候选内参基因的Ct值箱须的上、下边分别表示Ct值中的最大值和最小值，箱体的上、下边分别表示Ct值中的上四分位数和下四分位数，箱体中的横线表示Ct值的中位数，实心菱形表示异常值，空心正方形表示平均值

Fig. 2 Ct values of candidate reference genes The upper and lower parts of the box whiskers indicate the maximum and minimum values of the Ct value；the upper and lower parts of the box body indicate the upper and lower quartiles of the Ct value；lines across the boxes depict the medians；solid diamonds represent outliers and hollow squares represent averages

表4 GeNorm，NormFinder和BestKeeper分析结果和排名

Table 4 GeNorm，NormFinder and BestKeeper analysis results and rankings

内参基因 Reference gene	geNorm		NormFinder		BestKeeper						Delta Ct		RefFinder综合排序 Comprehensive ranking
内参基因 Reference gene	M值 M value	排序 Rank	SV值 SV value	排序 Rank	SD值 SD value	排序 Rank	CV 值 CV value/%	排序 Rank	r	排序 Rank	平均标准偏差 STDEV	排序 Rank	基因稳定性Gene stability	排序 Rank
Sm18S	0.710	6	0.722	7	1.41	1	5.71	4	0.950	5	0.900	7	4.300	5
SmACT	0.373	1	0.347	1	1.51	4	6.04	5	0.985	1	0.670	1	1.410	1
SmEF-1α	0.799	7	0.943	8	1.54	6	7.19	7	0.922	6	1.070	8	7.740	8
SmRPL1	0.373	1	0.373	3	1.53	5	5.65	3	0.985	1	0.680	2	2.450	2
SmRPS26	0.656	5	0.666	6	1.68	7	7.65	8	0.982	2	0.860	6	6.450	7
SmTBCC	0.615	4	0.501	5	1.45	3	5.18	1	0.973	4	0.770	5	4.400	6
SmUBC12	0.508	2	0.480	4	1.53	5	6.31	6	0.979	3	0.740	4	3.940	4
SmUBP	0.547	3	0.362	2	1.43	2	5.59	2	0.985	1	0.700	3	2.630	3

图3 geNorm软件分析内参基因最适数目

Fig. 3 Analysis of optimal number of reference genes for normalization by geNorm

图4 无患子三萜皂苷生物合成途径中相关基因的RT-qPCR结果 R，根；ST，茎；L，叶；B，芽；MF，雄花；FF，雌花；S1，花后15 d的果皮；S2，花后45 d的果皮；S3，花后75 d的果皮；S4，花后90 d的果皮；S5，花后105 d的果皮；S6，花后120 d的果皮；S7，花后135 d的果皮；S8，花后150 d的果皮

Fig. 4 RT-qPCR expression pattern of genes related to the triterpenoid saponin biosynthesis of S. mukorossi R，root；ST，stem；L，leaf；B，bud；MF，male flower；FF，female flower；S1，pericarp at 15 d after flower pollination；S2，pericarp at 45 d after flower pollination；S3，pericarp at 75 d after flower pollination；S4，pericarp at 90 d after flower pollination；S5，pericarp at 105 d after flower pollination；S6，pericarp at 120 d after flower pollination；S7，pericarp at 135 d after flower pollination；S8，pericarp at 150 d after flower pollination

参考文献 39

[1]	Kou XY, Zhang L, Yang SZ, et al. Selection and validation of reference genes for quantitative RT-PCR analysis in peach fruit under different experimental conditions[J]. Sci Hortic, 2017, 225:195-203. doi: 10.1016/j.scienta.2017.07.004 URL
[2]	Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines:minimum information for publication of quantitative real-time PCR experiments[J]. Clin Chem, 2009, 55(4):611-622. doi: 10.1373/clinchem.2008.112797 URL
[3]	Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR[J]. Nat Protoc, 2006, 1(3):1559-1582. doi: 10.1038/nprot.2006.236 pmid: 17406449
[4]	Xu LF, Xu H, Cao YW, et al. Validation of reference genes for quantitative real-time PCR during bicolor tepal development in Asiatic hybrid lilies(Lilium spp. )[J]. Front Plant Sci, 2017, 8:669. doi: 10.3389/fpls.2017.00669 URL
[5]	Ferradás Y, Rey L, Martínez Ó, et al. Identification and validation of reference genes for accurate normalization of real-time quantitative PCR data in kiwifruit[J]. Plant Physiol Biochem, 2016, 102:27-36. doi: 10.1016/j.plaphy.2016.02.011 URL
[6]	Wu JY, Zhang HN, Liu LQ, et al. Validation of reference genes for RT-qPCR studies of gene expression in preharvest and postharvest longan fruits under different experimental conditions[J]. Front Plant Sci, 2016, 7:780. doi: 10.3389/fpls.2016.00780 pmid: 27375640
[7]	袁伟, 万红建, 杨悦俭. 植物实时荧光定量PCR内参基因的特点及选择[J]. 植物学报, 2012, 47(4):427-436. doi: 10.3724/SP.J.1259.2012.00427
	Yuan W, Wan HJ, Yang YJ. Characterization and selection of reference genes for real-time quantitative RT-PCR of plants[J]. Chin Bull Bot, 2012, 47(4):427-436.
[8]	王丽平, 梁瑾, 谌琴琴, 等. 千里光RT-qPCR分析中内参基因的选择[J]. 中国中药杂志, 2019, 44(3):465-471.
	Wang LP, Liang J, Shen QQ, et al. Identification of stable reference gene by RT-qPCR in Senecio scandens[J]. China J Chin Mater Med, 2019, 44(3):465-471.
[9]	徐碧霞, 郭巧生, 朱再标, 等. 老鸦瓣实时定量PCR内参基因的筛选和验证[J]. 中国中药杂志, 2021, 46(4):938-943.
	Xu BX, Guo QS, Zhu ZB, et al. Selection and validation of reference genes for quantitative Real-time PCR analysis in Amana edulis[J]. China J Chin Mater Med, 2021, 46(4):938-943.
[10]	Li DD, Hu B, Wang Q, et al. Identification and evaluation of reference genes for accurate transcription normalization in safflower under different experimental conditions[J]. PLoS One, 2015, 10(10):e0140218. doi: 10.1371/journal.pone.0140218 URL
[11]	Nicot N, Hausman JF, Hoffmann L, et al. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress[J]. J Exp Bot, 2005, 56(421):2907-2914. pmid: 16188960
[12]	贾黎明, 孙操稳. 生物柴油树种无患子研究进展[J]. 中国农业大学学报, 2012, 17(6):191-196.
	Jia LM, Sun CW. Research progress of biodiesel tree Sapindus mukorossi[J]. J China Agric Univ, 2012, 17(6):191-196.
[13]	徐圆圆, 周思维, 陈仲, 等. 无患子不同器官中的总皂苷和总黄酮含量[J]. 南京林业大学学报:自然科学版, 2021, 45(4):83-89.
	Xu YY, Zhou SW, Chen Z, et al. Contents of the total saponins and total flavonoids in different organs of Sapindus mukorossi[J]. J Nanjing For Univ Nat Sci Ed, 2021, 45(4):83-89.
[14]	刘济铭, 陈仲, 孙操稳, 等. 无患子属种质资源种实性状变异及综合评价[J]. 林业科学, 2019, 55(6):44-54.
	Liu JM, Chen Z, Sun CW, et al. Variation in fruit and seed properties and comprehensive assessment of germplasm resources of the genus Sapindus[J]. Sci Silvae Sin, 2019, 55(6):44-54.
[15]	徐圆圆, 贾黎明, 陈仲, 等. 无患子三萜皂苷研究进展[J]. 化学通报, 2018, 81(12):1078-1088.
	Xu YY, Jia LM, Chen Z, et al. Advances on triterpenoid saponin of Sapindus mukorossi[J]. Chemistry, 2018, 81(12):1078-1088.
[16]	Sun CW, Wang LC, Liu JM, et al. Genetic structure and biogeographic divergence among Sapindus species:an inter-simple sequence repeat-based study of germplasms in China[J]. Ind Crops Prod, 2018, 118:1-10. doi: 10.1016/j.indcrop.2018.03.029 URL
[17]	Zhao GC, Gao YH, Gao SL, et al. The phenological growth stages of Sapindus mukorossi according to BBCH scale[J]. Forests, 2019, 10(6):462. doi: 10.3390/f10060462 URL
[18]	Gao Y, Gao SL, Jia LM, et al. Canopy characteristics and light distribution in Sapindus mukorossi Gaertn. are influenced by crown architecture manipulation in the hilly terrain of Southeast China[J]. Sci Hortic, 2018, 240:11-22. doi: 10.1016/j.scienta.2018.05.034 URL
[19]	Hu QW, Chen YY, Jiao QY, et al. Triterpenoid saponins from the pulp of Sapindus mukorossi and their antifungal activities[J]. Phytochemistry, 2018, 147:1-8. doi: 10.1016/j.phytochem.2017.12.004 URL
[20]	陈莎莎, 陈丽萍, 林继辉. 无患子果皮皂苷提取工艺研究[J]. 云南民族大学学报:自然科学版, 2019, 28(6):558-562.
	Chen SS, Chen LP, Lin JH. The extraction technology of saponins from the soapberry pericarp[J]. J Yunnan Minzu Univ Nat Sci Ed, 2019, 28(6):558-562.
[21]	Xu YY, Gao Y, Chen Z, et al. Metabolomics analysis of the soapberry(Sapindus mukorossi Gaertn.)pericarp during fruit development and ripening based on UHPLC-HRMS[J]. Sci Rep, 2021, 11(1):11657. doi: 10.1038/s41598-021-91143-0 URL
[22]	Vandesompele J, de Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biol, 2002, 3(7):RESEARCH0034.
[23]	Andersen CL, Jensen JL, Ørntoft TF. Normalization of real-time quantitative reverse transcription-PCR data:a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[J]. Cancer Res, 2004, 64(15):5245-5250. doi: 10.1158/0008-5472.CAN-04-0496 URL
[24]	Pfaffl MW, Tichopad A, Prgomet C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity:BestKeeper—Excel-based tool using pair-wise correlations[J]. Biotechnol Lett, 2004, 26(6):509-515. doi: 10.1023/B:BILE.0000019559.84305.47 URL
[25]	Xie F, Xiao P, Chen D, et al. miRDeepFinder:a miRNA analysis tool for deep sequencing of plant small RNAs[J]. Plant Mol Biol, 2012. DOI: 10.1007/s11103-012-9885-2. doi: 10.1007/s11103-012-9885-2
[26]	蒋婷婷, 高燕会, 童再康. 石蒜属植物实时荧光定量PCR内参基因的选择[J]. 园艺学报, 2015, 42(6):1129-1138.
	Jiang TT, Gao YH, Tong ZK. Selection of reference genes for quantitative real-time PCR in Lycoris[J]. Acta Hortic Sin, 2015, 42(6):1129-1138.
[27]	Dudziak K, Sozoniuk M, Szczerba H, et al. Identification of stable reference genes for qPCR studies in common wheat(Triticum aestivum L.)seedlings under short-term drought stress[J]. Plant Methods, 2020, 16:58. doi: 10.1186/s13007-020-00601-9 URL
[28]	曹映辉, 郑燕, 张燕萍, 等. 建兰花香物质合成相关基因RT-qPCR内参基因筛选[J]. 分子植物育种, 2021, http://kns.cnki.net/kcms/detail/46.1068.S.20210510.1555.008.html.
	Cao YH, Zheng Y, Zhang YP, et al. Selection of suitable RT-qPCR reference genes for floral scent biosynthesis in Cymbidium ensifolium[J]. Mol Plant Breed, 2021, http://kns.cnki.net/kcms/detail/46.1068.S.20210510.1555.008.html.
[29]	章颖佳, 程少禹, 王卓为, 等. 紫玉兰‘红元宝’花芽分化阶段基因定量分析的内参基因筛选[J]. 广西植物, 2022, 42(1):113-121.
	Zhang YJ, Cheng SY, Wang ZW, et al. Selection of reference genes in Magnolia liliflora ‘Hongyuanbao’ during flower bud differentiation[J]. Guihaia, 2022, 42(1):113-121.
[30]	de Spiegelaere W, Dern-Wieloch J, Weigel R, et al. Reference gene validation for RT-qPCR, a note on different available software packages[J]. PLoS One, 2015, 10(3):e0122515. doi: 10.1371/journal.pone.0122515 URL
[31]	张玉芳, 赵丽娟, 曾幼玲. 基因表达研究中内参基因的选择与应用[J]. 植物生理学报, 2014, 50(8):1119-1125.
	Zhang YF, Zhao LJ, Zeng YL. Selection and application of reference genes for gene expression studies[J]. Plant Physiol J, 2014, 50(8):1119-1125.
[32]	Hwang HS, Lee H, Choi YE. Transcriptomic analysis of Siberian ginseng(Eleutherococcus senticosus)to discover genes involved in saponin biosynthesis[J]. BMC Genomics, 2015, 16(1):180. doi: 10.1186/s12864-015-1357-z URL
[33]	Han JY, Chun JH, Oh SA, et al. Transcriptomic analysis of Kalopanax septemlobus and characterization of KsBAS, CYP716A94 and CYP72A397 genes involved in hederagenin saponin biosynthesis[J]. Plant Cell Physiol, 2018, 59(2):319-330. doi: 10.1093/pcp/pcx188 URL
[34]	Shan CM, Wang CK, Zhang SX, et al. Transcriptome analysis of Clinopodium gracile(Benth.)Matsum and identification of genes related to Triterpenoid Saponin biosynthesis[J]. BMC Genomics, 2020, 21(1):49. doi: 10.1186/s12864-020-6454-y URL
[35]	Xiao Z, Sun XB, Liu XQ, et al. Selection of reliable reference genes for gene expression studies on Rhododendron molle G. don[J]. Front Plant Sci, 2016, 7:1547.
	徐圆圆, 陈仲, 贾黎明, 等. 植物三萜皂苷生物合成途径及调控机制研究进展[J]. 中国科学:生命科学, 2021, 51(5):525-555.
	Xu YY, Chen Z, Jia LM, et al. Advances in understanding of the biosynthetic pathway and regulatory mechanism of triterpenoid saponins in plants[J]. Sci Sin Vitae, 2021, 51(5):525-555. doi: 10.1360/SSV-2020-0230 URL
[37]	Zhao YJ, Li C. Biosynthesis of plant triterpenoid saponins in microbial cell factories[J]. J Agric Food Chem, 2018, 66(46):12155-12165. doi: 10.1021/acs.jafc.8b04657 URL
[38]	Augustin JM, Kuzina V, Andersen SB, et al. Molecular activities, biosynthesis and evolution of triterpenoid saponins[J]. Phytochemistry, 2011, 72(6):435-457. doi: 10.1016/j.phytochem.2011.01.015 pmid: 21333312
[39]	Xu YY, Zhao GC, Ji XQ, et al. Metabolome and transcriptome analysis reveals the transcriptional regulatory mechanism of triterpenoid saponin biosynthesis in soapberry(Sapindus mukorossi Gaertn.)[J]. J Agric Food Chem, 2022, DOI: 10.1021/acs.jafc.2c01672. doi: 10.1021/acs.jafc.2c01672