盐胁迫条件下杜梨叶片差异表达基因qRT-PCR内参基因筛选

doi:10.16420/j.issn.0513-353x.2021-0376

摘要/Abstract

摘要：

为了准确评价盐胁迫下杜梨（Pyrus betulaefolia Bunge.）目标基因表达量,筛选qRT-PCR适用内参基因。利用杜梨的全基因组注释信息和转录组测序数据,选择Actin2（Chr15.g01351）、EF1α-1（Chr3.g19898）、 EF1α-2（Chr4.g38173）、EF2（Chr5.g06899）、 GAPDH-1（Chr16.g30426）、 GAPDH-2（Chr13.g23532）、 TUBB（Chr5.g06472）、UBQ（Chr4.g40121）等8个候选基因,设计Actin2、EF1α-1、EF1α-2A、EF1α-2B、EF2、GAPDH-1、GAPDH-2、TUBB-A、TUBB-B、UBQE等10对qRT-PCR引物。首先对10对引物的扩增效率进行了测定,再以200 mmol · L^-1的NaCl溶液对两个杜梨家系（盐城和连云港）幼苗进行0、24、48、72 h盐胁迫处理。提取叶片的总RNA,反转录合成cDNA,使用10对引物进行qRT-PCR扩增,根据扩增产物使用delta Ct、BestKeeper、geNorm和NormFinder 等4种评价内参基因稳定性的方法,对10对引物扩增产物的稳定性进行分析,并使用RefFinder软件对4种评价方法进行综合分析,以确定最稳定内参基因及其引物。利用筛选出的2对最优内参基因引物对（TUBB-A和GAPDH-1）对1个可能参与杜梨盐胁迫响应的HKT基因（Chr16.g29024）的表达情况进行分析,8个候选内参基因的FPKM值（Fragments PerKilobase Million）都大于30,不同盐处理时间下的FPKM变异系数为0.087 ~ 0.260;10对荧光定量PCR引物的扩增效率在91.82% ~ 112.99%之间,其中引物对TUBB-A和GAPDH-1的扩增效率最接近于100%;稳定性分析显示10对引物扩增产物的稳定性排序为GΑPDH-1、TUBB-Α、EF1α-1、TUBB-B、EF1α-2Α、EF2、UBQE、GΑPDH-2、Αctin2、EF1α-2B;不同引物对（TUBB-Α、TUBB-B和EF1α-2Α、EF1α-2B）对同一基因的扩增产物的稳定性存在明显差异;利用TUBB-A和GAPDH-1分析HKT基因的表达趋势没有差别。说明基因GΑPDH和TUBB适于作为杜梨盐胁迫下进行荧光定量PCR的内参基因,对应的GAPDH-1、TUBB-A引物对的扩增效果最好。

关键词: 杜梨, 内参基因, 实时荧光定量PCR分析, 基因表达稳定性

Abstract:

Screening the appropriate reference genes for qRT-PCR analysis on different progenies of Pyrus betulaefolia Bunge. under salt stress,in order to accurately evaluate expression of target genes. In this study,eight reference genes including Actin2（Chr15.g01351）,EF1α-1（Chr3.g19898）,EF1α-2（Chr4.g38173）,EF2（Chr5.g06899）,GAPDH-1（Chr16.g30426）,GAPDH 2（Chr13.g23532）,TUBB（Chr5.g06472）,UBQ（Chr4.g40121）were selected as candidates based on the whole genome and transcriptome sequencing data of P. betulaefolia Bunge. Ten pairs of qRT-PCR primers including Actin2,EF1α-1,EF1α-2A,EF1α-2B,EF2,GAPDH-1,GAPDH-2,TUBB-A,TUBB-B,UBQE were designed or cited from previous publications,and the amplification efficiency of the primer pairs was determined. Two families（Yancheng and Lianyungang）of P. betulaefolia Bunge. were treated with 200 mmol · L^-1NaCl for 0,24,48,and 72 h respectively and leaf samples were collected. Total RNAs were extracted from 24 leaf samples and cDNAs were synthesized by reverse transcription as templates. The stability of amplification products by 10 primer pairs was analyzed by delta Ct,BestKeeper,geNorm and NormFinder respectively,and then analyzed comprehensively using RefFinder software based on four evaluation results to determine the most stable reference gene and the reference gene primers. The TUBB-A and GAPDH-1 have good stability and applicability. Finally,the expression of a HKT（Chr16.g29024）that may be involved in P. betulaefolia response to salt stress was measured by using those two selected primer pairs. The result showed that:the FPKM values（Fragments Per Kilobase Million）of the eight candidate reference genes were all greater than 30 and the FPKM variation coefficients under salt treatment at different times were 0.087-0.260;the amplification efficiency of 10 qRT-PCR primer pairs was between 91.82% and 112.99%,with primer pairs TUBB-A and GAPDH-1 being closest to 100%;The stability of 10 pairs of primer amplification products are ranked as GΑPDH-1,TUBB-Α,EF1α-1,TUBB-B,EF1α-2Α,EF2,UBQE,GAPDH-2,Actin2,EF1α-2B;Two genes（GΑPDH and TUBB）and primer pairs（GΑPDH-1 and TUBB-Α）were determined as the most suitable reference genes and the most stable primer pairs in this study. It was also found that the stability of the amplified products of different primer pairs of the same gene（such as TUBB-A and TUBB-B,EF1α-2A and EF1α-2B）is also significantly different. GAPDH-1 and TUBB-Α primer pairs are suitable for qRT-PCR analysis in P. betulaefolia under salt stress.

Key words: Pyrus betulaefolia, reference gene, qRT-PCR, gene expression stability

中图分类号:

S661.2

张秋悦, 刘昌来, 于晓晶, 杨甲定, 封超年. 盐胁迫条件下杜梨叶片差异表达基因qRT-PCR内参基因筛选[J]. 园艺学报, 2022, 49(7): 1557-1570.

ZHANG Qiuyue, LIU Changlai, YU Xiaojing, YANG Jiading, FENG Chaonian. Screening of Reference Genes for Differentially Expressed Genes in Pyrus betulaefolia Plant Under Salt Stress by qRT-PCR[J]. Acta Horticulturae Sinica, 2022, 49(7): 1557-1570.

图/表 10

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引物名称 Primer names	基因名称 Gene name	基因编号 Gene ID	片段长度/bp Gene length	来源 Source	变异系数/% CV	引物序列 Primer sequence
EF1a-1	EF1a-1	Chr3.g19898	168	本研究设计 Designed in this study	18.0	F:CATCGAGAGGTTCGAGAAGG R:CCGGGAGCATCAATAACAGT
EF1a-2A	EF1a-2	Chr4.g38173	167	蒲小秋等,2020	26.0	F:GGTGTGAAGCAGATGATTTG R:TCACCCTCAAACCCAGATAT
EF1a-2B	EF1a-2	Chr4.g38173	201	本研究设计 Designed in this study	26.0	F:AGGTCCACCAACCTTGACTG R:TGGACCAAAAGTGACAACCA
EF2	EF2	Chr5.g06899	179	本研究设计 Designed in this study	8.7	F:CCCAAGAGATGATCCCAAGA R:ACCCAGCAACAACAGAATCC
Actin2	Actin2	Chr15.g01351	101	蒲小秋等,2020	13.8	F:CTTCAATGTGCCTGCCATGT R:TCACACCATCACCAGAGTCC
GAPDH-1	GAPDH-1	Chr16.g30426	171	本研究设计 Designed in this study	17.9	F:GTTCGTTGTTGGTGTGAACG R:GTCTTTTGGGTGGCAGTGAT
GAPDH-2	GAPDH-2	Chr13.g23532	123	张雪等,2019	11.2	F:GAGGGTCTCATGACCACAGT R:TCCAGTGCTGCTAGGAATGA
TUBB-A	TUBB	Chr5.g06472	212	张雪等,2019	20.6	F:CTGCTGTGTTCCGTGGTAAG R:CTGCTCGCTAACTCTCCTGA
TUBB-B	TUBB	Chr5.g06472	237	本研究设计 Designed in this study	20.6	F:ACCCGATAACTTCGTGTTCG R:AACATCATTCGATCCGGGTA
UBQE	UBQ	Chr4.g40121	140	本研究设计 Designed in this study	8.8	F:GGCAGAACTGCCTGCTAATC R:CGGTTTTGCTCGATAAGCTC
HKT	HKT	Chr16.g29024	197	本研究设计 Designed in this study	63.9	F:TGGGCTACTGTCATTTGCTG R:AACGGATTCACCGCTATGTC

引物名称 Primer names	基因名称 Gene name	基因编号 Gene ID	片段长度/bp Gene length	来源 Source	变异系数/% CV	引物序列 Primer sequence
EF1a-1	EF1a-1	Chr3.g19898	168	本研究设计 Designed in this study	18.0	F:CATCGAGAGGTTCGAGAAGG R:CCGGGAGCATCAATAACAGT
EF1a-2A	EF1a-2	Chr4.g38173	167	蒲小秋等,2020	26.0	F:GGTGTGAAGCAGATGATTTG R:TCACCCTCAAACCCAGATAT
EF1a-2B	EF1a-2	Chr4.g38173	201	本研究设计 Designed in this study	26.0	F:AGGTCCACCAACCTTGACTG R:TGGACCAAAAGTGACAACCA
EF2	EF2	Chr5.g06899	179	本研究设计 Designed in this study	8.7	F:CCCAAGAGATGATCCCAAGA R:ACCCAGCAACAACAGAATCC
Actin2	Actin2	Chr15.g01351	101	蒲小秋等,2020	13.8	F:CTTCAATGTGCCTGCCATGT R:TCACACCATCACCAGAGTCC
GAPDH-1	GAPDH-1	Chr16.g30426	171	本研究设计 Designed in this study	17.9	F:GTTCGTTGTTGGTGTGAACG R:GTCTTTTGGGTGGCAGTGAT
GAPDH-2	GAPDH-2	Chr13.g23532	123	张雪等,2019	11.2	F:GAGGGTCTCATGACCACAGT R:TCCAGTGCTGCTAGGAATGA
TUBB-A	TUBB	Chr5.g06472	212	张雪等,2019	20.6	F:CTGCTGTGTTCCGTGGTAAG R:CTGCTCGCTAACTCTCCTGA
TUBB-B	TUBB	Chr5.g06472	237	本研究设计 Designed in this study	20.6	F:ACCCGATAACTTCGTGTTCG R:AACATCATTCGATCCGGGTA
UBQE	UBQ	Chr4.g40121	140	本研究设计 Designed in this study	8.8	F:GGCAGAACTGCCTGCTAATC R:CGGTTTTGCTCGATAAGCTC
HKT	HKT	Chr16.g29024	197	本研究设计 Designed in this study	63.9	F:TGGGCTACTGTCATTTGCTG R:AACGGATTCACCGCTATGTC

引物名称 Primer name	斜率（K） Slope	决定系数 R²	扩增效率/% Amplification efficiency
EF1a-1	-3.43	0.998	95.66
EF1a-2A	-3.15	0.999	107.96
EF1a-2B	-3.54	0.999	91.82
EF2	-3.43	0.998	95.66
Actin2	-3.43	0.998	95.66
GAPDH-1	-3.35	0.999	98.77
GAPDH-2	-3.21	0.999	104.95
TUBB-A	-3.32	0.996	100.16
TUBB-B	-3.05	0.989	112.99
UBQE	-3.52	0.999	92.49

引物名称 Primer name	斜率（K） Slope	决定系数 R²	扩增效率/% Amplification efficiency
EF1a-1	-3.43	0.998	95.66
EF1a-2A	-3.15	0.999	107.96
EF1a-2B	-3.54	0.999	91.82
EF2	-3.43	0.998	95.66
Actin2	-3.43	0.998	95.66
GAPDH-1	-3.35	0.999	98.77
GAPDH-2	-3.21	0.999	104.95
TUBB-A	-3.32	0.996	100.16
TUBB-B	-3.05	0.989	112.99
UBQE	-3.52	0.999	92.49

引物 Primer	几何平均值 Geo mean	算术平均值 AM	最小值 Min	最大值 Max	相关系数 r	标准偏差 SD	变异系数/% CV
GAPDH-1	24.24	24.26	21.71	25.79	0.870	0.75	3.09
EF2	26.83	26.85	24.46	29.27	0.806	0.89	3.32
UBQE	28.81	28.84	26.52	32.87	0.697	0.86	2.98
EF1α-1	25.39	25.40	24.15	28.52	0.688	0.56	2.21
EF1α-2A	25.18	25.19	23.44	26.18	0.640	0.57	2.27
TUBB-B	29.56	29.58	27.32	32.08	0.817	1.02	3.45
TUBB-A	27.79	27.82	24.68	31.04	0.890	1.04	3.72
GAPDH-2	22.47	22.53	19.99	29.04	0.912	1.06	4.69
Actin2	25.01	25.05	22.93	28.41	0.792	1.27	5.05
EF1α-2B	23.71	23.83	20.70	32.10	0.648	1.75	7.34