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园艺学报 ›› 2022, Vol. 49 ›› Issue (7): 1557-1570.doi: 10.16420/j.issn.0513-353x.2021-0376

• 研究报告 • 上一篇    下一篇

盐胁迫条件下杜梨叶片差异表达基因qRT-PCR内参基因筛选

张秋悦1, 刘昌来1,2,*(), 于晓晶1, 杨甲定1, 封超年1,*()   

  1. 1南京林业大学南方现代林业协同创新中心,南京林业大学生物与环境学院,南京 210037
    2南京林业大学竹类研究所,南京 210037
  • 收稿日期:2022-03-25 修回日期:2022-04-27 出版日期:2022-07-25 发布日期:2022-07-29
  • 通讯作者: 刘昌来,封超年 E-mail:clc2012@njfu.edu.cn;fcn@njfu.edu.cn

Screening of Reference Genes for Differentially Expressed Genes in Pyrus betulaefolia Plant Under Salt Stress by qRT-PCR

ZHANG Qiuyue1, LIU Changlai1,2,*(), YU Xiaojing1, YANG Jiading1, FENG Chaonian1,*()   

  1. 1-Innovation Center for Sustainable Forestry in Southern China,College of Biology and the Environment,Nanjing Forestry University,Nanjing 210037,China
    2Bamboo Research Institute,Nanjing Forestry University,Nanjing 210037,China
  • Received:2022-03-25 Revised:2022-04-27 Online:2022-07-25 Published:2022-07-29
  • Contact: LIU Changlai,FENG Chaonian E-mail:clc2012@njfu.edu.cn;fcn@njfu.edu.cn

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GΑPDHTUBB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-1 NaCl 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

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