葱鳞葡萄孢菌诱导下韭菜RT-qPCR内参基因的筛选和验证

doi:10.13560/j.cnki.biotech.bull.1985.2022-0851

生物技术通报 ›› 2023, Vol. 39 ›› Issue (3): 101-115.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0851

葱鳞葡萄孢菌诱导下韭菜RT-qPCR内参基因的筛选和验证

宋海娜¹(), 吴心桐¹, 杨鲁豫¹, 耿喜宁¹, 张华敏²(), 宋小龙³

1.平顶山学院化学与环境工程学院河南省生态经济型木本植物种质创新与利用重点实验室，平顶山 467000
2.河南城建学院生命科学与工程学院，平顶山 467036
3.河南科技大学化工与制药学院，洛阳 471000

收稿日期:2022-07-10 出版日期:2023-03-26 发布日期:2023-04-10
通讯作者: 张华敏，男，博士，讲师，研究方向：植物遗传与分子育种；E-mail: hmzhang111@126.com
作者简介:宋海娜，女，博士，讲师，研究方向：植物分子育种；E-mail: shn1126@126.com
基金资助:
国家自然科学基金青年项目(31401463);河南省重点研发与推广专项（科技攻关）项目(222102110071);平顶山学院博士科研启动项目(PXY-BSQD-202109);河南城建学院博士科研启动项目(K-Q2021010)

Selection and Validation of Reference Genes for RT-qPCR in Allium tuberosum Infected by Botrytis squamosa

SONG Hai-na¹(), WU Xin-tong¹, YANG Lu-yu¹, GENG Xi-ning¹, ZHANG Hua-min²(), SONG Xiao-long³

1. School of Chemical and Environmental Engineering, Pingdingshan University, Henan Key Laboratory of Germplasm Innovation and Utilization of Eco-economic Woody Plant, Pingdingshan 467000
2. College of Life Sciences and Engineering, Henan University of Urban Construction, Pingdingshan 467036
3. School of Chemical Engineer & Pharmacy, Henan University of Science and Technology, Luoyang 471000

Received:2022-07-10 Published:2023-03-26 Online:2023-04-10

摘要/Abstract

摘要：

葱鳞葡萄胞菌引起的韭菜灰霉病是影响韭菜产量和品质的主要因素之一。为了筛选出感染灰霉病后韭菜叶片中稳定表达的内参基因用于基因定量表达分析，以模拟接种和接种葱鳞葡萄孢菌24、48、72 h的韭菜叶片为材料，基于前期的转录组测序结果选取 UBC1、UBC2、UBQ1、UBQ2、GAPDH3、GAPDH4、TUB、EF-1α、40S RP、DDX、eIF-1A、PABP和DnaJ共13个基因为候选内参基因，利用实时荧光定量PCR（RT-qPCR）技术检测13个基因的表达情况，采用geNorm、NormFinder、BestKeeper软件和Reffinder在线程序对候选内参基因的表达稳定性进行评估。结果表明，13个候选内参基因中UBQ1的Ct值变化范围最小，表达水平最稳定。GeNorm、NormFinder和BestKeeper 软件筛选出的最佳内参基因不同，RefFinder综合评估显示，UBC2和UBQ1是韭菜叶片接种葱鳞葡萄孢菌后表达稳定性较好的基因，DDX是稳定性较差的基因。为了验证所筛选内参基因的可靠性，选择6个稳定性不同的候选内参基因分别作为定量分析的内部参照，对接种葱鳞葡萄孢菌后不同时间韭菜叶片中GST676和PRP902基因的表达水平进行归一化处理。结果显示，以稳定性较高的UBC2或UBQ1为内参基因时，校正的GST676和PRP902基因的表达趋势与转录组测序结果一致，而以稳定性相对较差的40S RP、eIF-1A、GAPDH4或DDX为内参基因时，校正的目的基因的表达趋势与转录组测序结果稍有差异。软件分析和实验验证结果表明，UBC2和UBQ1基因可作为葱鳞葡萄孢菌侵染韭菜叶片后相关基因定量表达分析的最合适内参基因。研究结果为后续开展韭菜抗灰霉病关键基因表达分析和功能研究奠定了基础。

关键词: 韭菜, 葱鳞葡萄孢菌, 内参基因, 实时荧光定量PCR, 表达稳定性, 灰霉病

Abstract:

Chinese chive gray mold caused by Botrytis squamosa is one of the major factors affecting the production and quality of Chinese chive（Allium tuberosum）. In order to screen stable reference genes in Chinese chive leaves infected with gray mold for quantitative gene expression analysis, Chinese chive leaves mock-inoculated and inoculated with B. squamosa for 24, 48 and 72 h were taken as materials. Thirteen genes including UBC1, UBC2, UBQ1, UBQ2, GAPDH3, GAPDH4, TUB, EF-1α, 40S RP, DDX, eIF-1A, PABP and DnaJ were selected as candidate reference genes based on previous RNA-Seq data of Chinese chive. The expressions of the candidate reference genes were detected by real-time quantitative PCR（RT-qPCR）and evaluated by geNorm, NormFinder, BestKeeper and RefFinder statistical algorithms. The results showed that among the thirteen candidate reference genes, UBQ1 had the smallest variation range of Ct value and the most stable expression. The most suitable reference genes evaluated by geNorm, NormFinder and BestKeeper software were different. Comprehensive evaluation of RefFinder website showed that UBC2 and UBQ1 had the better stability in Chinese chive leaves after inoculation with B. squamosa, and DDX displayed the lower stability. In order to verify the reliability of the selected reference genes, six candidate genes with different stability were used as internal controls for quantitative analysis, and normalized the expression levels of GST676 and PRP902 in Chinese chive leaves at different time points after inoculation with B. squamosa. The expression trends of GST676 and PRP902 corrected by UBC2 or UBQ1 with fine stability were consistent with that in RNA-Seq data, while the expression trends of GST676 and PRP902 corrected by 40S RP, eIF-1A, GAPDH4 or DDX with relative poor stability had some difference with that in RNA-Seq data. These results of software analysis and experimental verification showed that UBC2 and UBQ1 were the most suitable reference genes in Chinese chive leaves infected by B. squamosa for quantitative expression analysis. The results lay the foundation for future research on the expression and function of key genes in Chinese chives leaves during infection with B. squamosa.

Key words: Allium tuberosum, Botrytis squamosa, reference gene, real-time quantitative PCR, expression stability, gray mold

宋海娜, 吴心桐, 杨鲁豫, 耿喜宁, 张华敏, 宋小龙. 葱鳞葡萄孢菌诱导下韭菜RT-qPCR内参基因的筛选和验证[J]. 生物技术通报, 2023, 39(3): 101-115.

SONG Hai-na, WU Xin-tong, YANG Lu-yu, GENG Xi-ning, ZHANG Hua-min, SONG Xiao-long. Selection and Validation of Reference Genes for RT-qPCR in Allium tuberosum Infected by Botrytis squamosa[J]. Biotechnology Bulletin, 2023, 39(3): 101-115.

图/表 12

表1 基因和引物序列信息

Table 1 Genes and primer sequences

基因名称Gene symbol	基因ID Gene ID	引物序列Primer sequence（5'-3'）	扩增长度Amplified length/bp
UBC1	F02_cb4795_c6/f1p0/677	F：CACTTCCCTCCTGACTATCCCT R：CAAATACTGCCATTGCTGTTGA	92
UBC2	F02_cb10194_c5/f1p0/683	F：AAGGTGCTCCTGTCTATCTGCT R：TGATTCATACTTGGCTCTGTCG	108
UBQ1	F02_cb4331_c39/f4p2/2064	F：GTCAACTCCGTCACCGTAAACA R：GCAACTTCGGACTTCTCCTCTA	119
UBQ2	F02_cb3159_c105/f1p2/1236	F：ACCTTGTGCTCCGTCTTCGTG R：AACATCTGGCTTACCGTCGTCT	162
GAPDH3	F02_cb2521_c25/f1p0/1405	F：GACAAGCCAGTTGCTGTATTCG R：CTTTGCACCACCCTTAATGTGA	140
GAPDH4	F02_cb2521_c44/f7p1/1415	F：AATCACTGCCACCCAGAAGACT R：ACGGAATGACATGCCAGTAAGC	163
TUB	F02_cb8195_c2075/f1p0/1651	F：GGTGGTGGTACTGGATCTGG R：TTGTATGGCTCCACAACTGC	134
EF-1α	F02_cb8884_c10/f1p3/1571	F：ACCGAGTCCATTCCTCCTGTTC R：GGGTTTAGGTGCCTCCTCTTCA	147
40S RP	F02_cb13441_c1/f3p0/558	F：TGACCTCTTGAATCCACCTGC R：AGCAACCTTGGCACTTGACA	107
PABP	F02_cb2343_c66/f1p0/2490	F：TCCTGATATGGCTGGCTTGC R：CCTCGGGCGATTCAAGAAGA	224
DnaJ	F02_cb2629_c9/f1p0/682	F：AGACTCCCTGCAATTCCAGC R：TCTTCATGAACTCCTCGGCG	223
DDX	F02_cb3613_c7/f1p2/2466	F：CCATGTTCAAGAGCAGCACG R：CATGAAGAGCACGAGACCCA	145
eIF-1A	F02_cb13117_c3/f2p1/651	F：GGCTTTGTCATATTCGGGGC R：ATCACCAGCACCATCGTCC	227
GST676	F02_cb3237_c0/f37p2/676	F：GCAAGTCGGTAAAGCTCGGA R：ACTGGCCAAAAGAGTAGCGA	160
PRP902	F02_cb14527_c723/f1p0/902	F：CGTCACAGACAAGCAAAGGC R：CTTACTGTGCCACGTGGGAT	180

表2 候选内参基因在RNA-Seq中的FPKM值

Table 2 FPKM values of candidate reference genes in the samples by RNA-Seq

基因名称 Gene symbol	模拟接菌不同时间的样品 Mock-inoculated samples			接种葱鳞葡萄孢菌不同时间的样品 Samples inoculated with B. squamosa
基因名称 Gene symbol	24 h	48 h	72 h	24 h	48 h	72 h
UBC1	14.63	14.47	15.48	21.21	14.66	21.60
UBC2	45.80	53.35	59.64	72.14	51.73	83.00
UBQ1	8.75	7.90	10.06	12.67	8.00	10.81
UBQ2	187.15	189.18	197.98	223.79	139.12	245.41
GAPDH3	17.86	28.28	23.98	25.81	27.96	15.07
GAPDH4	111.50	168.16	154.12	214.92	132.19	155.86
TUB	49.37	58.77	50.06	58.57	52.65	47.37
EF-1α	28.34	18.20	26.84	48.63	13.83	22.83
40S RP	183.95	252.63	198.60	206.80	235.64	163.86
PABP	87.77	97.44	88.98	97.07	97.91	72.27
DnaJ	516.66	480.78	478.24	429.37	332.07	522.24
DDX	48.00	43.42	35.19	51.43	38.59	33.68
eIF-1A	31.35	44.60	34.58	38.97	33.39	33.45

图1 韭菜叶片提取的总RNA 1-3分别是模拟接种24、48和72 h的样品；4-6分别是接种24、48和72 h的样品

Fig. 1 Extracted total RNA in the leaves of Chinese chive A. tvberosum 1-3 are the mock-inoculated samples at 24, 48 and 72 h post inoculation respectively. 4-6 are the samples inoculated with B. squamosa at 24, 48 and 72 h post inoculation respectively

图2 韭菜候选内参基因PCR 扩增产物的琼脂糖凝胶电泳检测

Fig. 2 Agarose electrophoresis analysis of PCR amplified products of candidate reference genes in Chinese chive 1: UBC1; 2: UBC2; 3: UBQ1; 4: UBQ2; 5: GAPDH3; 6: GAPDH4; 7: TUB; 8: EF-1a; 9: 40S RP; 10: DDX; 11: eIF-1A; 12: PABP; 13: DnaJ

图3 韭菜候选内参基因实时荧光定量PCR扩增的熔解曲线 -dF/dT是荧光对温度的一阶负导数

Fig. 3 Melting curves of candidate reference genes in Chinese chive for RT-qPCR amplification -dF/dT is the first negative derivative of fluorescence（F）with respect to temperature（T）

表3 候选内参基因扩增效率分析

Table 3 Amplification efficiency of candidate reference genes

基因名称 Gene symbol	线性相关系数Linear correlation coefficient（R²）	扩增效率Amplification efficiency/%
UBC1	0.993 9	104.83
UBC2	0.963 0	101.12
UBQ1	0.994 1	110.70
UBQ2	0.990 7	106.64
GAPDH3	0.995 5	109.88
GAPDH4	0.996 6	110.65
EF-1α	0.991 2	110.36
TUB	0.991 7	107.54
40S RP	0.995 3	100.80
DDX	0.990 3	99.19
eIF-1A	0.997 5	106.30
PABP	0.990 9	96.06
DnaJ	0.998 9	106.97

图4 候选内参基因在所有样品中的Ct值分布方框内的横线表示Ct值的平均值

Fig. 4 Distribution of Ct values of candidate reference genes across all samples The horizontal line in the box indicates the average Ct values. 1: EF-1α; 2: UBQ1; 3: TUB; 4: UBQ2; 5: 40S RP; 6: eIF-1A; 7: DnaJ; 8: PABP; 9: DDX; 10: GAPDH4; 11: UBC1; 12: UBC2; 13: GAPDH3

图5 geNorm软件分析候选内参基因的表达稳定性 A：候选内参基因表达稳定性分析；B：归一化内参基因数目的确定

Fig. 5 Expression stabilities of candidate reference genes assayed by geNorm software A is the average expression stability M value of candidate reference genes. The least stable genes are on the left and the most stable genes on the right. B is the normalized number of reference genes. In figure A, 1: eIF-1A; 2: UBQ2; 3: UBC2; 4: 40S RP; 5: PABP; 6: UBC1; 7: UBQ1; 8: DnaJ; 9: GAPDH4; 10: TUB; 11: GAPDH3; 12: EF-1a; 13: DDX

表4 NormFinder软件分析候选内参基因的表达稳定性

Table 4 Expression stabilities of candidate reference genes assayed by NormFinder software

基因名称Gene symbol	稳定值 Stability	排名Rank
UBQ1	0.195	1
UBC2	0.261	2
40S RP	0.28	3
UBC1	0.374	4
UBQ2	0.375	5
eIF-1A	0.443	6
GAPDH4	0.456	7
TUB	0.478	8
PABP	0.48	9
GAPDH3	0.51	10
DnaJ	0.581	11
EF-1α	0.642	12
DDX	0.654	13

表5 BestKeeper软件分析候选内参基因的表达稳定性

Table 5 Expression stabilities of candidate reference genes assayed by BestKeeper software

基因名称Gene symbol	标准偏差Standard deviation（SD）	变异系数Coefficient of variation（CV）	调节系数标准差Std dev[± x-fold]	排名Rank
TUB	0.327	1.156	1.254	1
UBC2	0.390	1.560	1.311	2
40S RP	0.396	1.647	1.316	3
eIF-1A	0.411	1.615	1.330	4
UBQ1	0.416	1.320	1.334	5
PABP	0.435	1.856	1.352	6
UBQ2	0.436	1.726	1.353	7
UBC1	0.472	1.666	1.387	8
GAPDH3	0.477	1.768	1.391	9
DnaJ	0.494	1.811	1.408	10
DDX	0.560	2.225	1.474	11
GAPDH4	0.596	2.359	1.512	12
EF-1α	0.727	2.567	1.655	13

图 6 RefFinder分析候选内参基因的表达稳定性

Fig. 6 Expression stabilities of candidate reference genes assayed by RefFinder 1: UBC2; 2: UBQ1; 3: 40S RP; 4: UBQ2; 5: eIF-1A; 6: TUB; 7: UBC1; 8: PABP;9: GAPDH4; 10: GAPDH3; 11: DnaJ; 12: EF-1α; 13: DDX

图7 GST676和PRP902基因分别以UBC2、UBQ1、40S RP、eIF-1A、GAPDH4和DDX为内参基因时在模拟接种和接种不同时间韭菜叶片中的相对表达量及其在转录组测序中的FPKM值 Y轴左侧是基因的相对表达量，右侧是基因的FPKM值

Fig. 7 Relative expressions of GST676 and PRP902 in Chinese chive leaves suffered by mock-inoculated and inoculated with B. squamosa when UBC2, UBQ1, 40S RP, eIF-1A, GAPDH4 or DDX used as the internal reference gene, and the FPKM values of GST676 and PRP902 in the samples by RNA-Seq The left side of Y axis is the relative expression, the right side of Y axis is the FPKM values of genes

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葱鳞葡萄孢菌诱导下韭菜RT-qPCR内参基因的筛选和验证

Selection and Validation of Reference Genes for RT-qPCR in Allium tuberosum Infected by Botrytis squamosa

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