偏穗鹅观草RT-PCR内参基因筛选及验证

doi:10.13304/j.nykjdb.2021.0960

中国农业科技导报 ›› 2023, Vol. 25 ›› Issue (6): 32-39.DOI: 10.13304/j.nykjdb.2021.0960

偏穗鹅观草RT-PCR内参基因筛选及验证

张宝蓉¹^,²(), 解继红³, 金丹青¹^,², 汪军成²^,⁴, 姚立蓉²^,⁴, 司二静²^,⁴, 尚勋武²^,⁴, 王化俊²^,⁴, 李葆春¹^,²()

^1.甘肃农业大学生命科学技术学院，兰州 730070
^2.甘肃农业大学甘肃省干旱生境作物学国家重点实验室，兰州 730070
^3.中国农业科学院草原研究所，呼和浩特 010010
^4.甘肃农业大学农学院，兰州 730070

收稿日期:2021-11-09 接受日期:2022-03-27 出版日期:2023-06-01 发布日期:2023-07-28
通讯作者: 李葆春
作者简介:张宝蓉 E-mail： serendipityfjzbr@163.com；
基金资助:
国家自然科学基金项目(31860377);甘肃省自然科学基金重点项目(21JR7RA801);财政部和农业农村部国家现代农业产业技术体系建设项目(CARS-05-04B-2)

Selection and Validation of Reference Genes for RT-PCR Analysis in Roegneria komarovii

Baorong ZHANG¹^,²(), Jihong XIE³, Danqing JIN¹^,², Juncheng WANG²^,⁴, Lirong YAO²^,⁴, Erjing SI²^,⁴, Xunwu SHANG²^,⁴, Huajun WANG²^,⁴, Baochun LI¹^,²()

^1.College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China
^2.Gansu Provincial Key Lab of Aridland Crop Science, Gansu Agricultural University, Lanzhou 730070, China
^3.Institute of Grassland Research of Chinese Academy of Agricultural Sciences, Hohhot 010010, China
^4.Agronomy College, Gansu Agricultural University, Lanzhou 730070, China

Received:2021-11-09 Accepted:2022-03-27 Online:2023-06-01 Published:2023-07-28
Contact: Baochun LI

摘要/Abstract

摘要：

偏穗鹅观草（Roegneria komarovii Nevski）是小麦族鹅观草属的植物，具有抗旱、抗病等特点，是多年生优良牧草，也是小麦三级资源库。为分析偏穗鹅观草耐盐机制及抗病基因的表达，筛选出偏穗鹅观草在不同非生物胁迫条件下稳定表达的内参基因，以偏穗鹅观草根、茎、叶对照组及盐胁迫处理组的偏穗鹅观草为材料，利用实时荧光定量PCR （qRT-PCR）技术，对肌动蛋白（Actin1、Actin2、Actin3）、微管蛋白（BUTB）、亲环素（Cyclophilin）、甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate，GAPDH）、组蛋白（histone 3， H3）、延伸因子（EF1a1）8个常用小麦内参基因进行筛选；并借助geNorm、BestKeeper 软件对内参基因表达的稳定性进行评价，进一步利用SnRK2基因对获得的内参基因的稳定性和可靠性进行验证。结果表明，2个软件筛选出的内参基因基本一致，8个候选内参基因在偏穗鹅观草不同胁迫处理下的表达丰度及稳定性存在差异，盐处理及干旱处理下Cyclophilin、GAPDH稳定性表现最好，高温处理下EF1a1、Cyclophilin稳定性最好。综合分析表明，Cyclophilin、GAPDH及EF1a1在偏穗鹅观草中表达丰度相对较高，并且在器官中表达较为稳定。通过对盐、高温及干旱处理组的稳定性评价，确定Cyclophilin为偏穗鹅观草最适内参基因。研究结果为后续探讨偏穗鹅观草抗逆性分子机制奠定了基础。

关键词: 偏穗鹅观草, 内参基因, 实时荧光定量PCR

Abstract:

Roegneria komarovii is a plant of Roegneria spp in Triticeae with the characteristics of drought and disease resistance， and it also is an excellent perennial forage. In order to analyze the mechanism of salt tolerance and the expression of disease resistance genes by real-time quantitative PCR （qRT-PCR）， the reference genes that expressed stably under different abiotic stress were screened. The expression of 8 wheat internal reference genes were assessed including Actin protein （Actin1， Actin2， Actin3）， beta-tubulin （BTUB）， Cyclophilin， glyceraldehyde-3-phosphate （GAPDH）， histone 3 （H3） and elongation factor-1-alpha-subunit （EF1a1） in Roegneria komarovii samples under the stresses of salt， drought and high temperature， and using geNorm and BestKeeper softwares to evaluate the stability of them. SnRK2 gene was used to verify the stability and reliability of the selected internal reference genes. Based on the statistical analysis of qRT-PCR results，the expression abundance and stability of 8 candidate reference genes were different under different stress treatments. The stability of Cyclophilin and GAPDH were the best under salt and drought treatments. EF1a1 and Cyclophilin had the best stability under high temperature treatment. The results showed that Cyclophilin， GAPDH and EF1a1 were highly expressed and stable in the various organs. Cyclophilin was selected as the most suitable reference gene through the stability evaluation of abiotic stress. The results provided foundation for investigating the molecular mechanism of salt stress tolerance in Roegneria komarovii.

Key words: Roegneria komarovii, reference gene, qRT-PCR

中图分类号:

张宝蓉, 解继红, 金丹青, 汪军成, 姚立蓉, 司二静, 尚勋武, 王化俊, 李葆春. 偏穗鹅观草RT-PCR内参基因筛选及验证[J]. 中国农业科技导报, 2023, 25(6): 32-39.

Baorong ZHANG, Jihong XIE, Danqing JIN, Juncheng WANG, Lirong YAO, Erjing SI, Xunwu SHANG, Huajun WANG, Baochun LI. Selection and Validation of Reference Genes for RT-PCR Analysis in Roegneria komarovii[J]. Journal of Agricultural Science and Technology, 2023, 25(6): 32-39.

图/表 8

表1 偏穗鹅观草候选内参基因引物序列

Table 1 Sequence of the primers for candidate reference genes in Roegneria komarovii

基因名称 Gene symbol	GenBank 登录号 GenBank ID	引物序列 Primer sequence（3’-5’）	产物长度 Product length/bp
Actin1	AB181991	F：GTTCCAATCTATAAGGGATACACGC R：GAACCTCCACTGAGAACAACATTACC	422
BTUB	U7689	F：GGACCGTACGGGCAGATCT R：CACCAGACTGCCCAAACACA	210
Cyclophilin	AF262984	F：CAAGCCGCTGCACTACAAGG R：AGGGGACGGTGCAGATGAA	227
Actin2	AK458277	F：GTTCTACAACGAGCTCCGTGTC R：GACATACATTGCTGGGCAAC	116
Actin3	MF405765.1	F：GTCGGGATCTCACGGACTC R：CATAATCAAGGGCAACATAC	106
GAPDH	FN429985	F：CTGCATCATACGATGACATC R：TGTCACCGACAAAGTCAGTG	216
Histone H3	XM_03760136	F：GTCACCATCATGCCCAAG R：CAACACATTCCACTTCCG	150
EF1α1	M90077	F：AGAAGACTCACATCAACATC R：CGTACTTGAAAGACCTCTTG	163

图1 RNA监测及候选基因扩增A：总RNA；B：8个候选基因在偏穗鹅观草中扩增条带；C：8个候选基因在中国春中扩增条带； 1—Actin1； 2—BTUB； 3—Cyclophilin； 4—Actin2； 5—Actin3； 6—GAPDH； 7—HistoneH3； 8—EF1a1

Fig. 1 RNA detection and candidate gene amplificationA： Total RNA； B： Amplified bands of 8 candidate genes in Roegneriakomarovii； C： Amplified bands of 8 candidate genes in Chinese Spring； 1—Actin1； 2—BTUB； 3—Cyclophilin； 4—Actin2； 5—Actin3； 6—GAPDH； 7—HistoneH3； 8—EF1a1

图2 偏穗鹅观草部分候选内参基因序列对比

Fig. 2 Comparison of the sequence of some candidate internal reference genes

表2 8个候选内参基因在偏穗鹅观草不同器官中的Ct值范围

Table 2 Range of Ct values of 8 candidate internal reference genes in different organs of Roegneria komarovii

基因Gene	Ct值 Ct value	基因Gene	Ct值Ct value
Actin1	31.80~32.70	Actin3	30.90~31.40
BTUB	30.40~31.90	GAPDH	23.70~24.15
Cyclophilin	20.87~21.34	Histone H3	26.82~28.11
Actin2	27.85~29.31	EF1a1	21.52~21.83

图3 geNorm分析候选内参基因表达稳定性A：CK；B：盐处理；C：干旱组；D：高温组

Fig. 3 Analysis of the expression stability of candidate reference genes by geNormA： CK； B： Salt stress； C. Drought stress； D： High temperature stress

图4 geNorm分析确定候选内参基因数目A：CK；B：盐处理；C：干旱组；D：高温组

Fig. 4 Determined the optimal number of candidate reference genes by geNormA： CK； B： Salt stress； C. Drought stress； D： High temperature stress

表3 BestKeeper分析候选内参基因表达稳定性排名

Table 3 Expression stability values of candidate reference genes determined through BestKeeper

基因名称Gene name	CK	盐处理Salt stress	干旱处理Drought stress	高温处理 High temperature stress
EF1a1	0.81±0.17	0.88±0.20	1.79±0.36	1.00±0.20
Histone H3	2.86±0.78	1.20±0.33	2.08±0.70	0.99±0.35
GAPDH	0.35±0.09	1.19±0.31	0.84±0.22	2.06±0.56
Actin3	0.88±0.28	1.11±0.35	1.15±0.36	0.97±0.28
Actin2	2.07±0.59	2.43±0.64	1.51±0.42	2.81±0.80
Cyclophilin	1.93±0.41	1.12±0.24	0.83±0.18	1.11±0.24
BTUB	3.15±0.98	2.60±0.79	2.41±0.64	3.04±0.78
Actin1	2.02±0.65	1.92±0.64	1.31±0.44	2.42±0.81

图5 Cyclophilin分析偏穗鹅观草不同非生物胁迫处理中SnRK2表达水平

Fig. 5 Expression level of SnRK2 in Roegneriakomarovii under different abiotic stress treatments using Cyclophilin

参考文献 24

1	蔡联柄.鹅观草属的地理分布[J].西北植物学报, 2002, 22(4): 189-199.
	CAI L B. Geographical distribution of Roegneria [J]. Acta Bot. Bor-Occid. Sin., 2002, 22(4):189-199.
2	罗粤川,邓雪雪,吴丹丹,等.鹅观草不同居群赤霉病抗性评价及抗病种质鉴定[J].植物遗传资源学报,2021,22(3):725-733.
	LUO Y C, DENG X X, WU D D, et al.. Evaluation of resistance to head blight and identification of resistant germplasm in different populations of Gooseguancao [J]. J. Plant Genetic Resour.,2021,22(3):725-733.
3	周永红,万永芳.鹅观草、大麦及其属间杂种赤霉病抗性研究初报[J].四川农业大学学报, 1994, 12(1): 34-36.
	ZHOU Y H, WAN Y F. Preliminary study on scab resistance of Gossypium, barley and their intergeneric hybrids [J]. J. Sichuan Agric. Univ.,1994,12(1):34-36.
4	陈景芋, 解继红, 徐柱,等. NaCl胁迫对3种鹅观草幼苗耐盐性研究[J].中国农学通报, 2011, 27(1):383-388.
	CHEN J Y, XIE J H, XU Z, et al.. Effects of NaCl stress on salt tolerance of three species of gossamer seedlings [J]. Chin. Agric. Sci. Bull.,2011,27(1):383-388.
5	万永芳,刘法圈.小麦近缘野生植物的赤霉病抗性研究[J].植物病理学报, 1997, 27(2):107-111.
	WAN Y F, LIU F Q. Study on scab resistance of wild plants related to wheat [J]. Acta Phytopathol. Sin.,1997,27(2): 107-111.
6	赵富强,张海琴,孙宗华,等.鹅观草不同居群条锈病和白粉病抗性评价[J].草业学报, 2016, 25(4):149-158.
	ZHAO F Q, ZHANG H Q, SUN Z H, et al.. Resistance of Roegenria kamoji (Poaceae: Triticeae) populations to stripe rust and powdery mildew [J]. Acta Pratac. Sin., 2016, 25(4):149-158.
7	马玉宝, 闫伟红, 姜超,等.鹅观草属野生牧草资源的搜集与评价[J].草业与畜牧, 2016(5):25-32.
	MA Y B, YAN W H, JIANG C, et al.. The collection and evaluation of Roegneria wild forage resources [J]. Pratac. Anim. Husbandry, 2016(5):25-32.
8	黄雪玲,冯浩,康振生.小麦条锈菌实时荧光定量PCR 分析中内参基因的选择[J].农业生物技术学报,2012,20(2):181-187.
	HUANG X L, FENG H, KANG Z S. Selection of internal reference genes in real-time fluorescence quantitative PCR analysis of wheat stripe rust [J]. J. Agric. Biotechnol.,2012,20(2):181-187.
9	胡瑞波,范成明,傅永福.植物实时荧光定量PCR内参基因的选择[J].中国农业科技导报, 2009,11(6):30-36.
	HU R B, FAN C M, FU Y F. Reference gene selection in plant realtime quantitative reverse transcription PCR (qRT-PCR) [J]. J. Agric. Sci. Technol., 2009,11(6):30-36.
10	李晖,牛宏伟,刘娜,等.小麦与叶锈菌互作过程中实时定量PCR内参基因的选择及TaSGR基因的表达分析[J].植物生理学报,2015,51(5):642-648.
	LI H, NIU H W, LIU N, et al.. The selection of internal reference genes in real-time quantitative PCR and the expression analysis of TaSGR gene in the process of interaction between wheat and leaf rust fungus [J]. Plant Physiol. J., 2015,51(5):642-648.
11	ROGERS S O, BENDICH A J. Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues [J]. Plant Mol. Biol.,1985,5(2): 69-76.
12	VANDESOMPELE J, PRETER K D, PATTYN F, et al.. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes [J/OL]. Genome Biol., 2002, 3(7):00341 [2021-10-18]. .
13	PFAFFL M W, 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.
14	朱健康,倪建平.植物非生物胁迫信号转导及应答[J].中国稻米, 2016,22(6):52-60.
	ZHU J K, NI J P. Abiotic stress signaling and responses in plants [J]. China Rice, 2016,22(6):52-60.
15	ZHANG X, SUN X B, LIU X Q, et al.. Selection of reliable reference genes for gene expression srudies on Rhododendron mille G. Don [J/OL]. Front Plant Sci., 2016, 7:1547 [2021-10-18]. .
16	HOSSAIN M S, AHMED R, HAQUE M S, et al.. Identification and validation of reference genes for real-time quantitative RT-PCR analysis in jute [J/OL]. BMC Mol. Biol., 2019,20:13 [2021-10-18]. .
17	POSTOLLEC F, FALENTIN H, PAVAN S, et al.. Recent advances in quantitative PCR (qPCR) applications in food microbiology [J]. Food Microbiol., 2011, 9(8):848-861.
18	齐香玉,陈双双,冯景,等.茉莉花实时荧光定量PCR内参基因的筛选与验证[J].华北农学报,2020,35(6):22-30.
	QI X Y, CHEN S S, FENG J, et al.. Selection and validation of candidate reference genes for quantitative real-time PCR in Jasminum sambac Aiton [J]. Acta Agric. Boreali-Sin., 2020, 35(6):22-30.
19	RADONI A, THULKE S, MACKAY I M, et al.. Guideline to reference gene selection for quantitative real-time PCR [J]. Biochem. Biophys. Res. Commun., 2004, 313(4):856-862.
20	SAGUN C M, GRANDMOTTET F, SUACHAOWNA N, et al.. Validation of suitable reference genes for normalization of quantitative reverse transcriptase-polymerase chain reaction in rice infected by Xanthomonas oryzae pv. Oryzae [J/OL]. Plant Gene, 2019, 21:100217 [2021-10-18]. .
21	TANG X, ZHANG N, SI H, et al.. Selection and validation of reference genes for RT-qPCR analysis in potato under abiotic stress [J]. Plant Methods, 2017, 13(1):85-93.
22	NICOT N, HAUSMAN J F, HOFFMANN L, et al.. House-keeping gene selection for real-time RT-PCR normalization in Glycine max during biotic and abiotic stress [J]. J. Exp. Bot., 2005, 56(421):7-14.
23	吴建阳,何冰,杜玉洁,等.利用geNorm、NormFinder和BestKeeper软件进行内参基因稳定性分析的方法[J].现代农业科技, 2017(5): 278-281.
	WU J Y, HE B, DU Y J, et al.. Analysis method of systematically evaluating stability of reference genes using geNorm, NormFinder and BestKeeper [J]. Modern Agric. Sci. Technol., 2017(5): 278-281.
24	MAFRA V, KUBO K S, ALVES-FERREIRA M, et al.. Reference genes for accurate transcript normalization in Citrus genotypes under different experimental conditions [J/OL]. PLoS One, 2012, 7(2): e31263 [2021-10-18]. .

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偏穗鹅观草RT-PCR内参基因筛选及验证

Selection and Validation of Reference Genes for RT-PCR Analysis in Roegneria komarovii

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