红梨实时荧光定量PCR内参基因的筛选

doi:10.3969/j.issn.1674-7968.2019.02.019

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摘要红梨(Pyrus pyrifolia)果皮花青苷的合成积累是影响红梨果实着色的关键因素,其生物合成由各步酶促反应基因调控,筛选出稳定可靠的内参基因对于研究红梨果实着色关键基因及其调控机制具有重要意义。本研究旨在筛选红梨的不同品种(早白蜜, 中熟32, 云红梨1号),在不同生长发育时期(幼果期, 缓慢生长期, 快速生长期, 成熟期)和不同遮光处理(自然光照, 部分遮光及完全遮光)条件下均稳定表达的内参基因,以用于qRT-PCR分析。选取红梨果皮为实验材料,以梨树(Pyrus communis)中常见的6个管家基因—β-肌动蛋白基因(β-actin, ACT)、18S核糖体RNA基因(18S ribosomal RNA, 18S rRNA)、甘油醛三磷酸脱氢酶基因(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)、组蛋白基因(histone, His)、转录延伸因子基因(elongation factor 1α, EF-1α)及α微管蛋白基因(α-tubulin, TUB)作为候选内参基因,分别进行qRT-PCR扩增效率、内参基因稳定性参数及不同样品中相对表达量分析。以BIO-RAD CFX Manager v2.0软件对稳定性参数指标—循环阈值(cycle threshold, Ct)、平均表达系数(M)和变异系数(variation coefficient, CV)进行分析和综合对比,结果显示,不同候选内参基因的表达稳定性存在差异,其中EF-1α和His在红梨不同品种、不同生长发育时期及不同遮光处理条件下均稳定表达;对于不同样本中内参基因相对表达量的分析显示,EF-1α和His在不同样本中的表达量相对稳定。上述结果提示,EF-1α和His可作为红梨果皮组织qRT-PCR分析的内参基因,为后续开展红梨果实着色相关基因的表达分析提供了校正和标准化基因选择。

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	张雪
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	瞿飞
	杨胜俊

关键词 ：红梨, qRT-PCR, 内参基因, 筛选

Abstract：Synthesis and accumulation of cyanine nucleoside in Red pear (Pyrus pyrifolia) peel are key factors influencing red pear fruit coloring and biosynthesis regulated by enzymatic reaction genes, screening of stable and reliable reference genes has important significance to research the key genes and their regulatory mechanism in red pear fruit coloring. In order to select reference genes for qRT-PCR analysis in different growth period (young fruit period,slow growth period, fast growth period, mature period) and different shading conditions (natural light, partial shading, complete shading) in different varieties (Zaobaimi, Zhongshu32, Yunhongli No.1) of red pear fruit, 6 common house-keeping genes were chosen as candidate for internal gene which included β-actin (ACT), 18S ribosomal RNA (18S rRNA), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), histone (His), elongation factor 1α (EF-1α), and α-tubulin (TUB). The qRT-PCR amplification efficiency, the stability parameters and the relative expression in different samples of internal reference genes were analyzed. The BIO-RAD CFX Manager v2.0 software was used to analyze and compare the stability parameter index such as cycle threshold (Ct), average expression coefficient (M) and variation coefficient (CV). The results showed different expression stability in different candidate genes, and EF-1α and His showed stable expression trend in different samples from different varieties, growth stages, and processing conditions. Analysis of relative expression of reference genes in different samples showed that the expression of EF-1α and His in different samples were relatively stable. As a result, EF-1α and His could be used as reference gene for qRT-PCR analysis in red pear peel tissues which might standardize genetic selection for relative quantitation about coloring related gene expression analysis in red pear.

Key words： Red pear qRT-PCR Reference gene Screening

收稿日期: 2018-07-17

ZTFLH:

S661.2

基金资助:贵州省科学技术基金(黔科合LH字[2014]7686号)

通讯作者: *, zhangxuenl@126.com。

引用本文:

张雪, 王荔, 瞿飞, 杨胜俊. 红梨实时荧光定量PCR内参基因的筛选[J]. 农业生物技术学报, 2019, 27(2): 361-370.
ZHANG Xue, WANG Li, QU Fei, YANG Sheng-Jun. Reference Gene Screening for Real-time Quantitative PCR in Red Pear (Pyrus pyrifolia). 农业生物技术学报, 2019, 27(2): 361-370.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.02.019 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I2/361

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