全 文 ：第 51 卷 第 5 期
2 0 1 5 年 5 月
林 业 科 学
SCIENTIA SILVAE SINICAE
Vol. 51，No. 5
May，2 0 1 5
doi:10．11707 / j．1001-7488．20150516
Ｒeceived date:2014 － 07 － 21;Ｒevised date:2014 － 10 － 03．
Funded project:The National Natural Science Foundation of China (31071775) ;The Science and Technology Department of Zhejiang Province
(2008C24006) ;The Zhejiang Normal University Innovative Ｒesearch Team Program．
* Corresponding author:Yang Ling．
牛奶子实时定量 PCＲ分析中内参基因的评价与验证*
成龙平 胡海涛 郭卫东 杨 莉 王长春 杨 玲
(浙江师范大学化学与生命科学学院 金华 321004)
摘 要: 【目的】实时定量 PCＲ结果的精确性在很大程度上取决于选择的内参基因的稳定性。通过评估候选管
家基因的表达稳定性，筛选出用于牛奶子研究的最佳内参基因。【方法】设计简并引物从牛奶子中克隆 12 个潜在
的内参基因片段，包括 14-3-3、18S核糖体 ＲNA(18SrＲNA)、β-actin (Actin)、延长因子 1-α(EF1-α)、真核起始因子
4A (eIF4A)、3 －磷酸甘油醛脱氢酶(GAPDH)、ＲNA聚合酶 －Ⅱ (ＲPⅡ)、60S核糖体蛋白(ＲPL7)、翻译延长因子 2
(TEF2)、泛素连接酶 E2 (UBCE)、泛素(UBQ)和泛素延伸蛋白 5(UBQ5)。采集牛奶子 5 个不同器官(根、茎、叶、
花和红果)、4 个成熟期的果实(绿果、黄果、深粉果和完全成熟的红果)、2 种激素(脱落酸、赤霉素)处理 4 个时间
点的绿果、热处理 4 个时间点的离体叶片、幼苗盐胁迫 2 个时间点的根茎叶，应用实时荧光定量 PCＲ 技术检测 12
个内参基因在各样品中的表达情况，采用基于 CT值的 geNorm、Normfinder和 BestKeeper及 CT比较 4 种算法评价这
些内参基因的稳定性，最终的排名则由 ＲefFinder算法产生。【结果】器官组稳定性好的前 2 位内参基因为 UBCE
和 ＲPL7，果实成熟期组排名前 2 的为 eIF4A和 UBCE，激素处理组排名前 2 的为 eIF4A和 UBCE，非生物胁迫组排名
前 2 的则为 Actin和 EF1-α，综合分析所有样品排名前 3 位的是 eIF4A、ＲPL7 和 UBCE。分别用筛选出的稳定的
eIF4A、ＲPL7、UBCE和不稳定的 ＲPⅡ作为内参基因评价目的基因八氢番茄红素合成酶基因 EutPsy 在果实成熟过
程中的表达，结果显示分别以 3 个稳定的内参基因为单内参基因与以 eIF4A 同 UBCE 组合做内参基因所得到的结
论一致，而 ＲPⅡ给出的则不同。【结论】在应用荧光实时定量 PCＲ技术研究牛奶子基因转录表达时，通常情况下
eIF4A、ＲPL7 和 UBCE相对于其他 9 个候选内参基因更为可靠。研究结果为牛奶子及胡颓子属其他物种的基因表
达分析奠定基础。
关键词: 牛奶子;基因表达;实时定量 PCＲ;标准化;内参基因
中图分类号:S718. 46 文献标识码:A 文章编号:1001 － 7488(2015)05 － 0135 － 10
Evaluation and Validation of Potential Ｒeference Genes for
Quantitative Ｒeal-Time PCＲ Analysis in Elaeagnus umbellata
Cheng Longping Hu Haitao Guo Weidong Yang Li Wang Changchun Yang Ling
(College of Chemistry and Life Sciences，Zhejiang Normal University Jinhua 321004)
Abstract: 【Objective】The accuracy of quantitative real-time polymerase chain reaction (qＲT-PCＲ)analysis strongly
depends on transcript normalization using stably expressed reference genes． The present study was conducted to evaluate
the stability of candidate housekeeping genes and identify the most reliable gene or a set of genes to be used as reference
genes in qPCＲ analysis of Elaeagnus umbellata．【Method】Twelve potential reference genes were selected among the most
common reference genes reported in literature and their fragments were cloned by degenerate primers from E． umbellata，
including 14-3-3，18S ribosomal ＲNA gene (18SrＲNA) ，β-actin (Actin) ，elongation factor 1-α (EF1-α) ，eukaryotic
initiation factor 4A (eIF4A) ，glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ，ＲNA polymerase-Ⅱ (ＲPⅡ) ，60S
ribosomal protein L7 (ＲPL7) ，translation elongation factor 2 (TEF2) ，ubiquitin-conjugating enzyme E2 (UBCE) ，
ubiquitin (UBQ)and ubiquitin extension protein 5 (UBQ5)． Samples were collected from five types of organs (root，
stem，leaf，flower and red fruit) ，fruits at four different ripening stages (green，yellow，dark yellow and fully matured red
fruit) ，green fruits at four time points after hormone ABA or GA3 treatments，detached leaves at four time points under
40 ℃，and three types of organs (root，stem and leaf)of the seedlings treated with salt stress at three time points． The
expression stability of these 12 genes was evaluated based on the CT values using four statistical algorithms including
林 业 科 学 51 卷
geNorm，Normfinder，BestKeeper，and the comparative ΔCT ． Overall ranking of four sets aforementioned was generated
using ＲefFinder software．【Ｒesult】UBCE and ＲPL7 were ranked as the two best reference genes for organ set，eIF4A and
UBCE for fruit-ripening samples，eIF4A and UBCE for hormone treatment set，and Actin and EF1-α for abiotic stress set．
When including the data obtained from all the 29 samples into the analysis，eIF4A，ＲPL7，and UBCE were identified as
the top three reference candidates． The expression levels of phytoene synthase (EutPsy)were further assessed during fruit
ripening by using the top three reference genes in comparison to the worst one ＲPⅡ ． When the three stable reference
genes eIF4A，UBCE and ＲPL7 or the combination of top two eIF4A and UBCE were used for normalization，the trend of
the relative transcript abundance of EutPsy were consistent． However，when the worst reference gene ＲPⅡwas employed
for normalization，the expression profile was different． 【Conclusion】eIF4A followed by ＲPL7 and UBCE were found to be
more reliable than other nine genes for normalization purposes in measuring gene expression of E． umbellata． This was the
first systematic analysis for the selection of superior reference genes for qＲT-PCＲ in E． umbellata under different
conditions，which benefits future studies on gene expression in E． umbellata and other species of the genus Elaeagnus．
Key words: Elaeagnus umbellata;gene expression;qＲT-PCＲ;normalization;reference gene
Elaeagnus umbellata is a rapid-growing large
shrub that has been widely planted to prevent erosion
and provide screening along highways due to its ability
to fix nitrogen，drought and disease resistance，and
tolerance of poor soil (Fordham et al．，2003)． Most of
the extracts from the flowers，leaves and berries display
broad-spectrum antibacterial activity (Sabir et al．，
2007)． In addition，its red fruit is rich in minerals，
vitamins， essential fatty acids， flavonoids and
carotenoids，especially lycopene，which is about 5 －
20 times higher than that of ordinary tomato (Fordham
et al．，2003;Ahmad et al．，2006;Guo et al．，2009)．
The sweet-tart fruit can be eaten fresh or processed for
preserves，condiments，fruit-rolls and juice，or used as
a substitute for tomato products (Fordham et al．，2003;
Ahmad et al．， 2006)． Because of these unique
features， E． umbellata has attracted considerable
scientific interest， not only among nutritionist and
agricultural scientists，but also ecologist and molecular
biologists (Fordham et al．，2003;Ahmad et al．，2006;
Guo et al．，2009)． We have analyzed the expression
changes of seven genes encoding enzymes of the main
steps of carotenoid biosynthetic pathway during fruit
ripening． The results revealed that the accumulation of
lycopene in the fruit was concomitant with the up-
regulation of upstream gene phytoene synthase
(EutPsy)and the silence of downstream gene lycopene
ε-cyclase (Guo et al．， 2009 )． Therefore， the
understanding of expression patterns of some key genes
will help elucidate the mechanism involved in the
specific physiological and biochemical process．
Quantitative real-time polymerase chain reaction
(qＲT-PCＲ) has rapidly become one of the most
important tools in measuring gene expression levels due
to its sensitivity，accuracy and specificity (Chao et al．，
2012)． However，to avoid experimental deviations or
errors that inevitably occur during sample preparation
and data analysis，all of which make quantitation of
gene transcripts unreliable，normalization of the qＲT-
PCＲ data is essential． Among several normalization
strategies，the use of one or more internal reference
genes is currently the preferred way (Hong et al．，
2008)． An ideal reference gene should be stably
expressed among various samples including those from
developmental stages，organ types and external stimuli
(Han et al．，2012)． Housekeeping genes are routinely
employed for this purpose，but these genes are not
always stably expressed when tested in other species or
under various experimental conditions． Ｒecent studies
have shown that no single reference gene is universal
for all experiments，and using different reference genes
can result in appreciable errors even up to 20-fold (Lu
et al．，2013)． Thus，it is crucial to systematically
evaluate the expression stability of potential reference
genes in a given set of biological samples from a
specific organism．
To date，analysis of reference gene expression in
plants has mainly focused on model and important crop
species． E． umbellata belongs to the family
Elaeagnaceae that is distant from any model plant
species． Only a traditional housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
631
第 5 期 成龙平等:牛奶子实时定量 PCＲ分析中内参基因的评价与验证
has been used as reference gene (Guo et al．，2009;Su
et al．，2013) ，but its stability has not yet been
assessed in this species． Therefore，the objective of
this study was to determine in systematic manner
reference genes which demonstrate a high degree of
expression stability under various organs，fruit ripening
stages， hormone treatments and abiotic stress
conditions in E． umbellata， to facilitate a more
accurate normalization of qＲT-PCＲ assays． The
expression profiles of 12 potential reference genes
including 14-3-3 (Barsalobres-Cavallari et al．，2009) ，
elongation factor 1-α (EF1-α) (Nicot et al．，2005;
Hong et al．，2008;Silveira et al．，2009;Han et al．，
2012;Zhu et al．，2013) ，eukaryotic initiation factor
4A (eIF4A) (Silveira et al．，2009;Lee et al．，2010;
Zhu et al．，2012) ，ＲNA polymerase-Ⅱ (ＲP Ⅱ)
(Tong et al．， 2009) ，60S ribosomal protein L7
(ＲPL7 ) (Barsalobres-Cavallari et al．， 2009 ) ，
translation elongation factor 2 (TEF2) (Tong et al．，
2009) ，ubiquitin-conjugating enzyme E2 (UBCE)
(Czechowski et al．，2005;Silveira et al．，2009) ，
ubiquitin (UBQ) (Han et al．，2012;Fan et al．，
2013) ，ubiquitin extension protein 5 (UBQ5) (Huis
et al．，2010) ，18S ribosomal ＲNA gene (18SrＲNA)
(Nicot et al．，2005) ，GAPDH (Hong et al．，2008;
Barsalobres-Cavallari et al．，2009;Tong et al．，2009;
Huis et al．，2010;Han et al．，2012) and β-actin
(Actin) (Hong et al．，2008;Han et al．，2012)which
were previously shown to have highly stable expression
levels in other plant species，were evaluated in 29
samples． Five distinct statistical algorithms have been
implemented to identify the best reference genes for
qＲT-PCＲ data normalization under different
experimental conditions． Furthermore，the expression
pattern of target gene EutPsy during fruit ripening was
assessed using the selected reference genes．
1 Materials and methods
1. 1 Plant materials and treatments Fresh roots，
stems，leaves，flowers and red fruits were harvested from
8-year-old trees grown under natural conditions at the
experimental farm at Zhejiang Normal University，Jinhua，
China． The fruits were sampled at green (approximately
19 weeks after anthesis) ，yellow (approximately 22 weeks
after anthesis) ，dark yellow (approximately 24 weeks
after anthesis) ，and full-ripe (approximately 27 weeks
after anthesis)fruit ripening stages (Guo et al．，2009)．
For ABA and GA3 treatments，green fruits were dipping in
100 μmol·L －1 solutions contained 0. 2% Tween 80，
respectively，and dried and sampled at 0，6，12 and 24
hours after hormone treatments (Tong et al．，2009)． For
heat treatment，the detached leaves with the developing
leaf blade from 8-year-old trees were incubated at 40 ℃
for 8 h． The leaves were gathered at 2，4，6 and 8 h，
respectively． For salt stress treatment， 4-week-old
seedlings about 12 cm high in pots were supplemented
with 300 mmol·L －1 NaCl． The leaves，roots and stems
were harvested at 0，6 and 12 h，respectively． All 29
samples were collected in three replicates，immediately
frozen in liquid nitrogen and stored at － 80 ℃ ．
1. 2 ＲNA extraction， quality control and cDNA
synthesis Total ＲNA was isolated according to a modified
hot borate method (Guo et al．，2009)and digested with
ＲNase-free DNaseⅠ(Takara，Toyoto，Japan)． The purity of
all ＲNA samples was assessed with a NanoDrop2000
spectrophotometer (Thermo，Wilmington，USA)． Only
ＲNA samples with an A260 / A280 ratio of 1. 8 － 2. 1 and an
A260 /A230 ratio of 2. 0 － 2. 3 were used for subsequent
analysis． The ＲNA integrity was immediately checked
using 1. 5% agarose gel electrophoresis． The first-strand
cDNA template was synthesized from 2 μg total ＲNA by
M-MLV reverse transcriptase (Progema，Madison，USA)
using an oligo(dT)18 as the primer according to the
manufacturer’s instructions．
1. 3 Primer design and test The specific primers were
used for the amplification of 18SrＲNA，Actin and GAPDH．
The degenerate primers were designed on the basis of
highly conserved coding sequences of other nine tested
reference genes (14-3-3，EF1-α，eIF4A，ＲPⅡ，ＲPL7，
TEF2，UBCE，UBQ and UBQ5)from the National Center
for Biotechnology Information (NCBI) database (Tab．
1)． Primer pairs were designed using Primer Primer V5. 0
software (http: ∥ www． PremierBiosoft． com /
primerdesign /)． The PCＲ conditions were 30 cycles of 15
s at 94 ℃，30 s at 52 to 60 ℃ depending on the primers
(Tab． 1) ，and 90 s at 72 ℃ ． Purified amplified products
of appropriate length were ligated to the pUCm-T vector for
sequencing． All reference genes were named based on
similarity to known protein sequences with identity ranging
from 86% to 99% ． Gene sequences were deposited in the
GenBank and accession numbers are listed in Tab． 2． The
731
林 业 科 学 51 卷831
第 5 期 成龙平等:牛奶子实时定量 PCＲ分析中内参基因的评价与验证
specific primers for qＲT-PCＲ were designed based on the
obtained sequence above of reference genes using Primer
Primer V5. 0 with melting temperature (Tm) 60 ℃，
primer lengths 20 － 22 bp and amplicon lengths 128 － 274
bp (Tab． 2)． The primers for EutPsy gene were from our
previous study (Guo et al．，2009)． The specificity of all
primer pairs was tested by reverse transcription-PCＲ and
also by qＲT-PCＲ，followed by agarose gel electrophoresis
and melting curve analysis．
1. 4 qＲT-PCＲ conditions and analyses qＲT-PCＲ
was carried out in 96-well plates with a StepOne PlusTM
Ｒeal-Time PCＲ System (Applied Biosystems，Foster，
USA)using SYBＲ Premix Ex TaqTMⅡ Kit (Takara，
Toyoto，Japan)． PCＲ reactions were prepared in 20 μL
volumes containing 2 μL of 5-fold diluted cDNA，10 μL
of 2 × Power SYBＲ Premix Ex TaqTMⅡ，0. 5 μL (10
μmol·L －1)of each primer，0. 4 μL 50 × ＲOX Ｒeference
Dye and 6. 6 μL sterile distilled water． Aliquots from the
same cDNA solutions were used with all primer sets in
each experiment． Negative PCＲ control with no templates
was performed for each primer pair． All reaction mixtures
were incubated for 30 s at 95 ℃，followed by 45 cycles of
5 s at 95 ℃ and 30 s at 60 ℃ ． The melting curve was
obtained by heating the amplicon at 95 ℃ for 15 s
followed by a constant increase in the temperature from 60
to 95 ℃，and reading at each 0. 3 ℃ ． For each primer
pair，a standard curve was generated from 5-fold serial
dilutions of the cDNA generated from green fruit． The
correlation coefficients (Ｒ2)and slope values could be
obtained from the standard curve，and the corresponding
PCＲ amplification efficiencies (E) were calculated
according to the equation E =(10 － 1 /slope － 1)× 100 (Han
et al．，2012)． The final cycle threshold (CT)values were
the mean of at least six values including two or three
biological replicates for each treatment and three technical
replicates．
1. 5 Statistical analyses of gene expression stability
Several algorithms have been developed to study the
stability of candidate reference genes． The geNorm
software derives a stability measure (M)and creates a
stability ranking via a stepwise exclusion of the least stable
gene． It also estimates the number of reference genes
necessary for an optimal normalization (Silveira et al．，
2009;Zhu et al．，2013)． NormFinder software has the
advantage of ranking the candidate reference genes both
inter-group and intra-group according to their different
expression stability (Han et al．，2012;Zhu et al．，2013)．
BestKeeper creates an index using the geometric mean of
each reference gene’s raw CT values and determines the
most stably expressed genes based on coefficient of
correlation to the index (Tong et al．，2009;Zhang et al．，
2012;Zhu et al．，2013)． The comparative ΔCT method
evaluates the most stable reference genes by comparing
relative expression of “pairs of genes” within each
sample，which measures the stability of a gene by the
mean of standard deviation (SD) values derived from
comparison between a reference gene and other candidate
reference genes (Chao et al．，2012;Lu et al．，2013)．
Based on the rankings from each of the four computational
programs aforementioned， a user-friendly web-based
comprehensive tool ＲefFinder (http:∥ www． leonxie．
com /referencegene． php? type = reference) assigns an
appropriate weight to an individual gene and calculates the
geometric mean of their weights for the overall final
ranking (Xie et al．，2011;Chao et al．，2012)． The lower
ranking indicated genes with more stable gene expression．
2 Ｒesults
2. 1 Verification of primer specificity and PCＲ
amplification efficiency A total of 12 reference genes
were cloned (Tab． 1)． Gene name，accession number，
specific primer sequences for qＲT-PCＲ and amplicon
lengths were listed in Tab． 2． For each of the potential
reference genes， the single peak in qＲT-PCＲ melting
curves suggested that each primer pair amplified a unique
product． The primer efficiency ranged from 93. 56% for
UBCE up to 108. 22% for 14-3-3 with a correlation
coefficient (Ｒ2 ) from 0. 990 to 0. 998 (Tab． 2) ，
indicating that the primers worked successfully and gave
consistent results．
CT is defined as the number of cycles needed for
fluorescence to reach a specific threshold level of detection
and is inversely related to the amount of initial ＲNA
template present in the sample． The expression levels of
the potential reference genes were determined as CT
values． As expected，the median CT values of the genes in
the 29 samples were in the range of 16. 75 and 30. 13
cycles (Fig． 1) ，showing a high range of variation
between them． 18SrＲNA was the most abundant
transcript， while ＲP Ⅱ and UBQ5 were the least
abundant transcript with the median CT values about 30
cycles． UBQ，EF1-α，UBCE，Actin，ＲPL7，GAPDH，
931
林 业 科 学 51 卷
14-3-3，eIF4A and TEF2 displayed median CT values
ranging from 19 to 25，which was considered a moderate
level of expression． For qＲT-PCＲ normalization， a
moderately expressed reference gene is preferred because
extremely high or low expression of a reference gene
could introduce variability to data analysis．
Tab． 2 Ｒeference genes and primer pairs used for quantitative ＲT-PCＲ
Gene
name
GenBank
accession
number
Primer sequence (5—3) Amplicon
length /bp
PCＲ
efficiency
(%)
Ｒ2
14-3-3 KJ643915 F:CACGTGTCACTCGTCAAGGA 274 108. 22 0. 990
Ｒ:TCGGATGAGTGGGTGCTAGA
18SrＲNA L24090. 1 F:GAGAAACGGCTACCACATCC 250 113. 76 0. 996
Ｒ:ACAACCCAAGGTCCAACTACGA
Actin KJ643925 F:TTGCACTGGACTACGAGCAG 257 95. 60 0. 991
Ｒ:GGTCGATCCAACACTGAGCA
EF1-α KJ643917 F:AGCCTCTACGTCTCCCACTT 272 98. 96 0. 990
Ｒ:GGCTGGGTCATCCTTGGAAT
eIF4A KJ643916 F:GAAGGCAGTCACTTCGTCCA 214 99. 30 0. 995
Ｒ:CGCCTCACGAGAATCCTCAC
GAPDH KJ643924 F:GGGTGGTGCTAAGAAGGTTG 128 100. 81 0. 990
Ｒ:TGGAATAATGTTGAAGGAGGC
ＲPⅡ KJ643918 F:TGATGATGCGGGTAGCAGTG 242 105. 65 0. 991
Ｒ:GTTCTCGACAAAACCGCGAC
ＲPL7 KJ643922 F:GCTGGACAAGCAGAGAATTGC 214 95. 14 0. 996
Ｒ:ATCGCCACCCTCGACATAAT
TEF2 KJ643919 F:TGATTCACCTGGACACGTCG 215 94. 17 0. 997
Ｒ:AAGCTTCCTCACCATCGACC
UBCE KJ643923 F:GGGACCCCCTGATAGTCCTT 176 93. 56 0. 997
Ｒ:GTGCAGGACTCCATTGCTCT
UBQ KJ643920 F:ACTTGGTGCTTCGTCTTCGT 261 94. 72 0. 995
Ｒ:TCTGCATACCACCCCTCAGA
UBQ5 KJ643921 F:CCGGACCAGCAGAGGTTAAT 202 108. 06 0. 992
Ｒ:ACTGGAGGAGTGCGAGCTTA
Phytoene synthase FJ643541 F:CTGCTATTATGTTGCTGGGACTG 86 94. 78 0. 998
(EutPsy) Ｒ:CGCTTTCTGTTGTGGCTTGTG
Fig． 1 Expression levels of candidate reference genes across all samples
A line across the box is depicted as the median． The box indicates the 25th and 75th percentiles，whisker caps represent 95%
confidence intervals，and black dots represent outliers．
2. 2 Expression stability of potential reference genes
The stabilities of the 12 potential reference genes were
analyzed among different organs (five samples) ，fruit
ripening stages (four samples) ，hormone treatments
041
第 5 期 成龙平等:牛奶子实时定量 PCＲ分析中内参基因的评价与验证
(seven samples) ，and abiotic stresses (13 samples)． The
CT values for each potential reference gene were used for
stability comparison in the NormFinder， geNorm，
BestKeeper，and Comparative ΔCT algorithms to identify
the best reference genes for qＲT-PCＲ data normalization
(Xie et al．，2011;Han et al．，2012;Zhang et al．，2012;
Fan et al．，2013;Zhu et al．，2013)． Overall ranking of four
sets aforementioned was generated using Ｒecommended
Comprehensive Ｒanking method，i． e． ＲefFinder． Tab． 3
summarized the ranking results for 12 potential reference
genes． In the meantime，geNorm analysis was also used to
determine the optimal number of stable reference genes for
accurate normalization (Han et al．，2012)． Generally，
0. 15 was used as a cutoff value to confirm the optimal
number of reference genes (Hong et al．，2008;Han et al．，
2012)． The evaluation of the organs，fruit-ripening stages
or hormone-treated samples did not exhibit any discernible
differences in the pairwise variation with the inclusion of a
third gene． It is apparent that two stable reference genes
would be sufficient for normalizing gene expression for three
sets of E． umbellata samples． As for abiotic stress and total
samples，the use of three reference genes was recommended
although their values were higher than 0. 15 (Han et al．，
2012;Lu et al．，2013)．
In organ samples，six genes (UBCE，ＲPⅡ，ＲPL7，
18SrＲNA，UBQ and UBQ5)were classified among the top
two reference genes in each of four algorithms． The overall
ranking of the best reference genes using ＲefFinder for five
organs was UBCE and ＲPL7． In fruit-ripening samples，
five genes (eIF4A，UBCE，UBQ5，GAPDH and ＲPL7)
were identified，and the top two were eIF4A and UBCE
based on ＲefFinder． In hormone treatment set， only
eIF4A，UBCE，GAPDH，and 18SrＲNA were classified
among the top two genes，and the two best were eIF4A and
UBCE according to the ＲefFinder． Likewise，six genes
(Actin，eIF4A，EF1-α，18SrＲNA，UBQ5 and ＲPL7)
were identified as the top three reference genes for abiotic
stress set，and the three best were Actin，EF1-α，and
eIF4A based on the ＲefFinder．
When including the data obtained from all the 29
samples into the analysis，four genes (eIF4A，ＲPL7，
UBCE，and UBQ) were identified，of which eIF4A，
ＲPL7，and UBCE were ranked as the best reference
candidates (Tab． 3)．
2. 3 Ｒeference gene validation for fruit-ripening
stages To validate the selection of candidate reference
genes，the expression pattern of functional genes EutPsy
was inspected using the different reference genes at four
ripening stages of E． umbellata fruit (Fig． 2)． The top
two stable references eIF4A and UBCE in the fruit-
ripening stages selected by geNorm，comparative ΔCT and
ＲefFinder，as well as ＲPL7 selected by BestKeeper were
used as internal controls． EutPsy is a key upstream gene
for lycopene accumulation during E． umbellata fruit
ripening (Guo et al．，2009)similar to tomato(Solanum
lycopersicum) Psy-1 gene which is induced by fruit
ripening in association with elevated lycopene
accumulation (Giuliano et al．，1993)． When the three
stable reference genes eIF4A，UBCE and ＲPL7 or the
combination of top two eIF4A and UBCE were used for
normalization， the trend of the relative transcript
abundance of EutPsy were consistent． The expression
levels of EutPsy peaked in dark-yellow fruits． However，
when the worst reference gene ＲP Ⅱ was employed for
normalization，EutPsy was expressed in much higher level
in yellow fruits that resulted in distinct expression pattern
compared to the stable references for normalization
(Fig． 2)．
Fig． 2 Ｒelative quantification of EutPsy expression
during fruit ripening using selected reference
genes for normalization
3 Discussion
Normalization is one of the key factors affecting the
accuracy and reliability of the quantitative gene expression
analysis． This study was conducted to identify the
appropriate reference genes for gene expression analysis of
E． umbellata for studies of different organs，fruit ripening
stages，hormone treatments and abiotic stress conditions．
To our knowledge，this is the first report on evaluating the
expression stability of different potential reference genes
for qＲT-PCＲ in the family Elaeagnaceae．
eIF4A protein unwinds ＲNA secondary structure in
141
林 业 科 学 51 卷241
第 5 期 成龙平等:牛奶子实时定量 PCＲ分析中内参基因的评价与验证
the 5-UTＲ of mＲNAs，which was necessary to allow
efficient binding of the small ribosomal subunit and
subsequent scanning for the initiator codon． Here，eIF4A
was found to rank not only the first from the results of
overall ranking of the best reference genes for all samples，
but also the first for different fruit ripening stages and
hormone treatments，and the third for different abiotic
stresses among 12 genes from E． umbellata． The ＲPL7
encoding the transcription regulator and structural
constituent of the 60S subunit of the cytosolic ribosome
was the second most stable gene in both all samples and
five organ samples． The UBCE gene，which is involved in
protein degradation through ubiquitination reactions，
exhibited the third most stable expression from the results
of overall ranking for all samples，also the first one in the
different organs and the second one in both different fruit
ripening stages and hormone treatments． These genes have
been identified as reliable reference genes for transcription
normalization in other plants in previous studies． For
example，EIF4A showed highly stable expression under
most experimental conditions in papaya (Carica papaya)
(Zhu et al．，2012) ，and in the laboratory-grown samples
of perennial ryegrass (Lolium perenne) (Lee et al．，
2010)． E1F4A and UBCE were the best reference genes
for various vegetative and reproductive tissues in
Brachiaria brizantha (Silveira et al．，2009)． UBCE has
been noted as displaying superior expression stability and
lower absolute expression levels across different nutrient
stresses in Arabidopsis thaliana (Czechowski et al．，
2005)． ＲPL7 had an equivalent transcript level over five
distinct tissue types and was therefore adequate for
normalization purposes in Coffea arabica (Barsalobres-
Cavallari et al．，2009)．
To validate the suitability of the reference genes we
identified in this study，the expression profile of EutPsy
was assessed in different ripening fruits． During the fruit
ripening of E． umbellata，EutPsy is associated with the
carotenoid biosynthesis． The data showed that the use of
the most stable reference genes UBCE and eIF4A or the
combination of stable references resulted in the trend
consistency of the relative transcript abundance of EutPsy．
However， the relative abundance displayed a distinct
pattern when the most variable reference gene ＲPⅡ used
as an internal control (Fig． 2)． Thus， the use of
unsuitable references can lead to over-or under-estimation
of relative transcript abundance and misinterpretation of
data． These results reinforce the importance of validating
reference genes prior to experimental applications． The
new reference genes will enable more accurate and reliable
normalization of qＲT-PCＲ results for gene expression
studies in this important plant and other species of the
genus Elaeagnus．
参 考 文 献
Ahmad S D，Sabir S M，Zubair M． 2006． Ecotypes diversity in autumn
olive (Elaeagnus umbellata Thunb):A single plant with multiple
micronutrient genes． Chemistry and Ecology，22(6) :509 － 521．
Barsalobres-Cavallari C F，Severino F E，Maluf M P，et al． 2009．
Identification of suitable internal control genes for expression studies
in Coffea arabica under different experimental conditions． BMC
Molecular Biology，10:1．
Chao W S，Dogˇramaci M，Foley M E，et al． 2012． Selection and
validation of endogenous reference genes for qＲT-PCＲ analysis in
leafy spurge (Euphorbia esula)． PLoS One，7(8) :e42839．
Czechowski T， Stitt M， Altmann T， et al． 2005． Genome-wide
identification and testing of superior reference genes for transcript
normalization in Arabidopsis． Plant Physiology，139(1) :5 － 17．
Fan C，Ma J，Guo Q，et al． 2013． Selection of reference genes for
quantitative real-time PCＲ in bamboo (Phyllostachys edulis)． PLoS
One，8(2) :e56573．
Fordham I M，Zimmerman Ｒ H，Black B L，et al． 2003． Autumn olive:
A potential alternative crop． Acta Horticulturae，626:437 － 439．
Giuliano G，Bartley G E，Scolnik P A． 1993． Ｒegulation of carotenoid
biosynthesis during tomato development． Plant Cell， 5 (4) :
379 － 387．
Guo X L，Yang L，Hu H T，et al． 2009． Cloning and expression
analysis of carotenogenic genes during ripening of autumn olive fruit
(Elaeagnus umbellata )． Journal of Agricultural and Food
Chemistry，57(12) :5334 － 5339．
Han X，Lu M，Chen Y，et al． 2012． Selection of reliable reference
genes for gene expression studies using real-time PCＲ in tung tree
during seed development． PLoS One，7(8) :e43084．
Hong S Y，Seo P J，Yang M S，et al． 2008． Exploring valid reference
genes for gene expression studies in Brachypodium distachyon by
real-time PCＲ． BMC Plant Biology，8:112．
Huis Ｒ，Hawkins S，Neutelings G． 2010． Selection of reference genes
for quantitative gene expression normalization in flax (Linum
usitatissimum L．)． BMC Plant Biology，10:71．
Lee J M，Ｒoche J Ｒ，Donaghy D J，et al． 2010． Validation of reference
genes for quantitative ＲT-PCＲ studies of gene expression in
perennial ryegrass (Lolium perenne L．)． BMC Molecular Biology，
11:8．
Lu Y，Yuan M，Gao X，et al． 2013． Identification and validation of
reference genes for gene expression analysis using quantitative PCＲ
in Spodoptera litura (Lepidoptera:Noctuidae)． PLoS One，8(7) :
e68059．
Nicot N，Hausman J F，Hoffmann L，et al． 2005． Housekeeping gene
selection for real-time ＲT-PCＲ normalization in potato during biotic
341
林 业 科 学 51 卷
and abiotic stress． Journal of Experimental Botany，56(421) :
2907 － 2914．
Sabir M S，Ahmad D S，Hussain I M，et al． 2007． Antibacterial activity
of Elaeagnus umbellata (Thunb．)a medicinal plant from Pakistan．
Saudi Medical Journal，28(2) :259 － 263．
Silveira E D，Alves-Ferreira M，Guimares L A，et al． 2009． Selection
of reference genes for quantitative real-time PCＲ expression studies
in the apomictic and sexual grass Brachiaria brizantha． BMC Plant
Biology，9:84．
Su Ｒ L，Hu H T，Wu M，et al． 2013． Isolation and characterisation of a
UDP-glucose pyrophosphorylase gene associated with fruit ripening in
autumn olive (Elaeagnus umbellata Thunb．)． Journal of
Horticultural Science ＆ Biotechnology，88(5) :617 － 623．
Tong Z，Gao Z，Wang F，et al． 2009． Selection of reliable reference
genes for gene expression studies in peach using real-time PCＲ．
BMC Molecular Biology，10:71．
Xie F，Sun G，Stiller J W，et al． 2011． Genome-wide functional analysis
of the cotton transcriptome by creating an integrated EST database．
PLoS One，6(11) :e26980．
Zhang Y，Chen D，Smith M A，et al． 2012． Selection of reliable
reference genes in Caenorhabditis elegans for analysis of
nanotoxicity． PLoS One，7(3) :e31849．
Zhu J，Zhang L，Li W，et al． 2013． Ｒeference gene selection for
quantitative real-time PCＲ normalization in Caragana intermedia
under different abiotic stress conditions． PLoS One，8(1) :e53196．
Zhu X，Li X，Chen W，et al． 2012． Evaluation of new reference genes
in papaya for accurate transcript normalization under different
experimental conditions． PLoS One，7(8) :e44405．
(责任编辑 徐 红)
441