甜瓜实时荧光定量PCR分析中内参基因的筛选

章丽珍; 韩晓云; 吴菁华; GefuWANG-PRUSKI; 张志忠

doi:10.19303/j.issn.1008-0384.2020.11.002

甜瓜实时荧光定量PCR分析中内参基因的筛选

doi: 10.19303/j.issn.1008-0384.2020.11.002

1.
福建农林大学园艺学院/福建农林大学-戴尔豪西大学联合实验室，福建　福州　350002
2.
加拿大戴尔豪西大学农学院，新斯科舍省　特鲁罗　B2N5E3

基金项目: 福建省闽江学者科研基金（116-114120019）；福建农林大学创新专项基金（CXZX2016108、CXZX2017168）

详细信息

作者简介:
章丽珍（1994−），女，硕士研究生，研究方向：农艺与种业（E-mail：651260775@qq.com）

通讯作者:
张志忠（1976−），男，博士，副教授，研究方向：园艺植物逆境生理和生物技术（E-mail：zeada2001@163.com）

中图分类号: S 652
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出版历程
- 收稿日期: 2020-07-29
- 修回日期: 2020-10-04
- 网络出版日期: 2020-11-13
- 刊出日期: 2020-11-30

Reference Gene Selection for RT-qPCR Analysis on Cucumis melo

1.
Joint FAFU-Dalhousie Lab, College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
2.
Faculty of Agriculture, Dalhousie University, Truro, NS, Canada　B2N5E3

摘要

摘要: 目的筛选可分别在甜瓜不同组织器官和不同胁迫处理下稳定表达的内参基因，用于对靶基因表达量的实时荧光定量分析，保证相关试验的准确性及可靠性。方法以甜瓜品种新银辉为试验材料，通过实时荧光定量PCR技术分析18s rRNA、TUA、EF1a、Actin1、Actin2、Actin3、Actin4、CYC和UBI-ep共9个候选内参基因在甜瓜不同组织器官及不同胁迫处理下的表达稳定性，包括甜瓜根、叶、种子和果实4种不同组织材料，以及水分胁迫、肉桂酸胁迫、盐碱胁迫和ABA胁迫4种处理。同时使用Best-Keeper、Norm Finder和ge Norm软件对9个候选内参基因进行稳定性分析。结果对不同组织器官而言，Best-Keeper评估排名前5的内参基因依次为CYC＞18s rRNA＞UBI-ep＞EF1a＞TUA；Norm Finder计算排名前5的内参基因依次为EF1a＞UBI-ep＞Actin4＞CYC＞Actin3；ge Norm分析排名前5的基因依次为Actin4=Actin3＞Actin1＞EF1a＞UBI-ep。不同胁迫处理中，Best-Keeper计算排名前5的基因依次为18s rRNA＞Actin3＞Actin4＞EF1a＞UBI-ep；Norm Finder分析排名前5的基因依次为EF1a＞UBI-ep＞Actin4＞CYC＞18s rRNA；ge Norm分析排名前5的基因依次为EF1a=UBI-ep＞Actin4＞CYC＞Actin1。总体而言EF1a在不同组织器官和不同胁迫处理中的综合排名均较为稳定；Actin4、Actin3、Actin1和EF1a是不同组织器官中较为稳定的内参基因组合；EF1a和UBI-ep是4种胁迫条件下较稳定的内参基因组合。结论 EF1a在甜瓜不同组织器官及不同胁迫条件下均可稳定表达，是较为合适的内参基因；同时可通过设置双内参基因进一步降低试验误差。
- 甜瓜 /
- 内参基因 /
- 实时荧光定量PCR /
- 组织器官 /
- 胁迫
Abstract: Objective In search for internal reference genes of Cucumis melo L. that could stably express in different tissues and under stress conditions to warrant accuracy and reliability of the RT-qPCR analysis on target gene expression. Method Expression stabilities of 9 candidate genes, 18srRNA, TUA, EF1a, Actin1, Actin2, Actin3, Actin4, CYC, and UBI-ep, from the roots, leaves, seeds, and fruits of Xinyinhui melon being treated by water, cinnamic acid, saline alkali or ABA were determined by RT-qPCR and analyzed using the BestKeeper, NormFinder, and geNorm software. Result In different tissues, the top 5 choice genes identified by BestKeeper ranked as CYC＞18s rRNA＞UBI-ep＞EF1a＞TUA, those by NormFinder EF1a＞UBIep＞Actin4＞CYC＞Actin3, and those by geNorm Actin4=Actin3＞Actin1＞EF1a＞UBI-ep. Under various stresses, they were 18s rRNA＞Actin3＞Actin4＞EF1a＞UBI-ep as ranked by BestKeeper, EF1a＞UBI-ep＞Actin4＞CYC＞18s rRNA by NormFinder, and EF1a=UBI-ep＞Actin4＞CYC＞Actin by geNorm. Overall, EF1a appeared to be most stable among the 9 genes. Insofar as variety of tissues is concerned, Actin4, Actin3, Actin1, and EF1a were more stable than the others; and, under stress, EF1a and UBI-ep tended to be superior. Conclusion Stably expressed in the tissues under the stresses as tested, EF1a was selected as the reference gene for studies on C. melo to reduce experimental errors. To further ensure accuracy, application of dual reference genes in RT-qPCR analysis might be considered.
- Cucumis melo L. /
- reference gene /
- RT-qPCR /
- Tissuestissues and organs /
- stress

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图 1 9个候选内参基因的引物扩增特异性与扩增长度

注：M：Marker；1～9分别为内参基因Actin1、Actin2、Actin3、Actin4、CYC、UBI-ep、TUA、18s rRNA、EF1a。

Figure 1. Amplification specificities and lengths of 9 candidate reference genes

Note: M: marker; 1–9: candidate reference genes, Actin1, Actin2, Actin3, Actin4, CYC,UBI-ep, TUA, 18s rRNA, and EF1a, respectively.

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图 2 9个候选内参基因的熔解曲线

Figure 2. Melting curves for 9 candidate reference genes

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图 3 9个候选内参基因Ct值箱线图

注：箱线图箱体代表Ct值主要集中范围；上边表示上四分线；中线表示中位数；下边表示下四分线；上边缘表示本组数据最大值；下边缘表示本组数据最小值。

Figure 3. Box plot of Ct values of 9 reference genes

Note: Box: range of concentrated Ct values; top line: upper quarter line; middle line: median value; bottom line: lower quarter line; top edge: maximum value of group; bottom edge: minimum of group.

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图 4 9个候选内参基因的配对数变异指数

Figure 4. Calculated pairwise variations (V) of 9 candidate reference genes

下载: 全尺寸图片幻灯片

表 1 9个候选内参基因RT-qPCR引物序列及其扩增效率

Table 1. RT-qPCR primer sequences and amplification efficiency of 9 candidate reference genes

登录号 GenBank ID	基因 Gene	引物序列（5′-3′） Primer sequence F/R（5′-3′）	产物大小 Product size/bp	扩增效率 Efficiency（E）
AF206894.1	18s rRNA	TCTGCCCGTTGCTCTGATG	130	2.05
AF206894.1	18s rRNA	TCACCCGTCACCACCATAG	130	2.05
XM_008456199.2	TUA	ACGCTGTTGGTGGTGGTAC	106	2.06
XM_008456199.2	TUA	GAGAGGGGTAAACAGTGAATC	106	2.06
XM_008459007.2	EF1a	ACTGTGCTGTCCTCATTATTG	98	1.98
XM_008459007.2	EF1a	AGGGTGAAAGCAAGAAGAGC	98	1.98
LN713255.1	CYC	GATGGAGCTCTACGCCGATGTC	153	1.97
LN713255.1	CYC	CCTCCCTGGCACATGAAATTAG	153	1.97
NM_001282241.1	UBI-ep	CACCAAGCCCAAGAAGATC	220	2.10
NM_001282241.1	UBI-ep	TAAACCTA ATCACCACCAGC	220	2.10
XM_008442791.2	Actin1	TTCTGGTGATGGTGTGAGTC	149	2.09
XM_008442791.2	Actin1	GGCAGTGGTGGTGAACATG	149	2.09
XM_008449644.2	Actin2	GAAGGAATAACCACGCTCAG	117	2.02
XM_008449644.2	Actin2	ACACAGTTCCCATCTACGAG	117	2.02
XM_008449644.2	Actin3	GGCAGTGGTGGTGAACATG	149	1.99
XM_008449644.2	Actin3	TTCTGGTGATGGTGTGAGTC	149	1.99
LN713262.1	Actin4	TGGAAGCTGCAGGAATCCACGA	165	2.04
LN713262.1	Actin4	TGCTGGGAGCAAGGGCTGTG	165	2.04

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表 2 Best-Keeper软件分析9个候选内参基因稳定性

Table 2. Stabilities of 9 reference genes analyzed by BestKeeper software

样品分组　　 Sample group	基因 Gene	几何平均值 Geometric mean value	最大值 Max	最小值 Mix	标准差 Stardand deviaton（SD）	变异差 Coefficient ofvariation（CV）	稳定性排名 Ranking of stable value
不同组织器官 Different tissues	CYC	20.88	21.55	19.72	0.57	2.72	1
	18s rRNA	10.21	11.46	8.90	0.77	7.55	2
	UBI-ep	20.12	22.10	18.69	0.94	4.67	3
	EF1a	20.42	22.03	19.11	0.95	4.65	4
	TUA	22.69	24.80	21.10	0.98	4.31	5
	Actin1	21.92	23.98	19.23	1.37	6.21	6
	Actin4	20.63	23.14	18.99	1.38	6.65	7
	Actin3	21.61	24.03	19.39	1.50	6.92	8
	Actin2	31.38	33.95	29.40	1.86	5.92	9
不同胁迫处理 Different stress conditions	18s rRNA	11.82	12.31	11.16	0.22	1.86	1
	Actin3	22.85	23.30	21.54	0.37	1.62	2
	Actin4	21.85	22.77	21.23	0.43	1.97	3
	EF1a	21.24	22.20	20.36	0.46	2.18	4
	UBI-ep	21.38	22.29	20.49	0.46	2.14	5
	Actin1	23.20	24.22	22.28	0.54	2.35	6
	CYC	18.87	19.89	17.95	0.59	3.44	7
	TUA	22.05	23.35	21.27	0.60	2.71	8
	Actin2	36.68	35.63	28.84	1.86	5.67	9

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表 3 Norm Finder和ge Norm软件分析候9个选内参基因稳定性

Table 3. Stabilities of 9 reference genes analyzed by NormFinder and geNorm software

样品分组　　 Sample group	Norm Finder			ge Norm
样品分组　　 Sample group	基因 Gene	稳定值 Stability value	稳定性排名 Ranking of stable value	基因 Gene	稳定值 Stability value	稳定性排名 Ranking of stable value
不同组织器官 Different tissues	EF1a	0.107	1	Actin4	0.526	1
	UBI-ep	0.153	2	Actin3	0.526	2
	Actin4	0.228	3	Actin1	0.640	3
	CYC	0.243	4	EF1a	0.794	4
	Actin3	0.291	5	UBI-ep	0.894	5
	18s rRNA	0.315	6	18s rRNA	0.961	6
	Actin1	0.319	7	CYC	1.101	7
	TUA	0.345	8	TUA	1.318	8
	Actin2	0.423	9	Actin2	1.462	9
不同胁迫处理 Different stress conditions	EF1a	0.023	1	EF1a	0.138	1
	UBI-ep	0.053	2	UBI-ep	0.138	2
	Actin4	0.059	3	Actin4	0.145	3
	CYC	0.078	4	CYC	0.207	4
	18s rRNA	0.128	5	TUA	0.242	5
	TUA	0.128	6	18s rRNA	0.321	6
	Actin1	0.214	7	Actin1	0.373	7
	Actin3	0.236	8	Actin3	0.445	8
	Actin2	0.264	9	Actin2	0.781	9

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表 4 9个选内参基因稳定性综合排名

Table 4. Overall ranking on expression stabilities of 9 reference genes

样品分组 Sample group	基因 Gene	Best-Keeper	Norm-Finder	ge Norm	几何平均值 Geometric mean value	综合排名 Comprehensive rankings
不同组织器官 Different tissues	EF1a	4	1	4	2.520	1
	Actin4	7	3	1	2.759	2
	CYC	1	4	7	3.037	3
	UBI-ep	3	2	5	3.107	4
	18s rRNA	2	6	6	4.160	5
	Actin3	8	5	2	4.309	6
	Actin1	6	7	3	5.013	7
	TUA	5	8	8	6.840	8
	Actin2	9	9	9	9.000	9
不同胁迫处理 Different stress conditions	EF1a	4	1	1	1.587	1
	UBI-ep	5	2	2	2.714	2
	Actin4	3	3	3	3.000	3
	18s rRNA	1	5	6	3.107	4
	CYC	7	4	4	4.820	5
	Actin3	2	8	8	5.040	6
	TUA	8	6	5	6.214	7
	Actin1	6	7	7	6.649	8
	Actin2	9	9	9	9.000	9

下载: 导出CSV

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