全 文 ：浙江农业学报 Acta Agriculturae Zhejiangensis，2016，28(5):782 － 789 http:/ /www． zjnyxb． cn
金微微，陈功楷，朱建军，等． 瓯柑 NIN 和 Sus 基因克隆、序列分析与表达(英)［J］． 浙江农业学报，2016，28(5):782
－ 789．
DOI:10. 3969 / j． issn． 1004-1524. 2016. 05. 12
Ｒeceived date:2015-12-14
Foundation item:Wenzhou Technology Bureau Project(N20130013)
Biography:JIN Wei-wei(1982—)， female，Cangnan，Zhejiang Province，PhD，lecturer，majored in fruit postharvest biology． E-mail:
wwjin2003@ 163． com
* Corresponding author:CHEN Gong-kai，E-mail:32328665@ qq． com
Gene clone，sequence analysis and gene expression of NIN
and Sus from Ougan fruit
JIN Wei-wei，CHEN Gong-kai* ，ZHU Jian-jun，GAO Ai-ling
(Institute of Pomology，Wenzhou Academy of Agricultural Sciences，Wenzhou 325006，China)
Abstract:The purpose of this work was to separate NIN and Sus gene from Ougan fruit and analyze expression pat-
terns of both two genes in different tissues at different development stages of fruits，which will provide some basic in-
formation for sugar metabolism of Ougan fruit and enrich NIN and Sus study on citrus fruit． Two full length sequences
encoding NIN and Sus genes were isolated． They were 1 932 and 2 418 bp in length，respectively，encoding putative
proteins of 643 and 805 amino acid． Sequence alignment results showed that both NIN and Sus were highly con-
served，and shared more than 70% sequence homology with other plants from NCBI and almost 100% homology with
citrus fruit． GenBank accession number for NIN and Sus from Ougan fruit was KF694988 and KF694989，respective-
ly． Ｒelative molecular mass of the putative NIN and Sus protein was 72. 18 and 92. 19 ku，respectively，and the iso-
electric point (pI)was 6. 882 and 6. 051，respectively． Phylogenetic analysis showed that putative NIN protein be-
longed to Group α，which might localize in plastid，and putative Sus protein belongs to Sus Ⅰ． Ｒesults of real-time
quantitative PCＲ showed that the mＲNA level of both two genes accumulated highly in stem，then leaf，peel and
flesh in descending order． The transcription level of NIN in peel was higher than that in flesh，while the transcription
level of Sus in peel was relative to that in flesh． At different development stages of fruit，both genes expressed the
highest at 120 after full bloom and then maintained at a low level until fruit maturation． The study showed that both
NIN and Sus genes expressed with tissue-specificity，and both expressed at a higher level at the immature stage and
decreased towards the ripening stage．
Key words:Ougan fruit;neutral invertase;sucrose synthase;gene clone;gene expression
CLC number:S666. 1 Document:A Article ID:1004-1524(2016)05-0782-08
瓯柑 NIN和 Sus基因克隆、序列分析与表达:金微微，陈功楷* ，朱建军，郜爱玲(温州市农业科学研究院 果树
研究所，浙江 温州 325006)
摘 要:以瓯柑果肉为材料，分离瓯柑中性转化酶(NIN)和蔗糖合成酶基因(Sus)，了解其序列特征，并对瓯
柑 NIN，Sus在不同组织的表达进行分析。结果显示:该研究首次从瓯柑果实中克隆获得 NIN和 Sus基因 CDS
全长序列，分别为 1 932 bp和 2 418 bp，预测分别编码 643 和 805 个氨基酸。序列比对结果显示:3 个基因均
高度保守，与 NCBI 网站上其他物种的序列同源性均高于 70%，与柑橘类物种的同源性接近 100%。NIN 和
Sus蛋白的相对分子量分别为 72. 18 和 92. 19 ku，理论等电点(pI)分别为 6. 882 和 6. 051。系统进化树结果
显示:瓯柑 NIN属于 α组，推测可能定位于质体;瓯柑 Sus属于 Sus Ⅰ组。实时荧光定量 PCＲ结果显示:瓯柑
NIN，Sus基因在茎中表达量均最高，叶其次，果肉中表达量最低;NIN在果皮中的表达量比果肉高，Sus则处于
相同水平。在果实不同发育阶段，NIN，Sus基因均在花后 120 d表达最强，随后下降，接近成熟表达维持在较
低的水平。结果表明:2 个基因在瓯柑不同组织间的表达具有不同程度的差异性，在果实发育前期表达水平
略高，随着成熟表达水平降低。
关键词:瓯柑;中性转化酶;蔗糖合成酶;基因克隆;基因表达
中图分类号:S666. 1 文献标志码:A 文章编号:1004-1524(2016)05-0000-00
Sugar metabolism plays a very important role in
final fruit quality［1］，which is always the focus point
of pomology research． Sucrose is the main transpor-
ting product of photosynthesis in higher plants，so it
is the most important soluble sugar in fruit，and re-
search on sucrose metabolism is a hot topic． Trans-
portation of sucrose from source to sink tissues in
plants mainly depends on invertase (Inv)and su-
crose synthase (Sus)． Inv mainly catalyzes cleavage
of sucrose irreversibly，while Sus catalyzes reversi-
ble cleavage and synthesis of sucrose［2 － 4］．
The first Inv gene was isolated from carrot ［5］．
Now this gene has been identified from a number of
plants and was found to be involved in plant devel-
opment［6 － 7］，carbohydrate partitioning［8］ and stress
response［9 － 10］． Based on the pH-optimal and isoe-
lectric point (pI)，Inv could be distinguished into 2
subfamilies:acid invertases (AIn)and neutral /al-
kaline invertases (NIN)． The two kinds apparently
performed with different subcellular locations． AIn
preferred to function in cell wall and vacuole，while
NIN preferred to target to cytosol ［3，5，8］． It was re-
ported that modern plant NIN originated in cya-
nobacterial［11］． Ｒecent research on rice showed that
NIN was also located in plant mitochondria and
plastids［12］． In contrast to AIn，NIN has not been
studied extensively，as the generally low enzyme ac-
tivity was easy to lost［8］． In recent years，more and
more researchers realized the importance of NIN，so
more and more attention was put on the research
of NIN．
The gene Sus has also been identified from a
number of plants including Arabidopsis［13］，rice［14］，
cotton［2］，citrus［4］，et al． They are also found to be
encoded by a multi-gene family． Expression patterns
and probable functions of Sus genes had also been
reported［15 － 17］． However，there was no report on
Inv and Sus genes from Ougan fruit ever．
Ougan (Citrus reticulate cv． Suavissima)is an
ancient and traditional mandarin in China［18］． Peo-
ple put more and more attention on Ougan fruit in
recent years because of its special medical effects
such as relieving a cough， lowering blood pres-
sure［19］． In recent years，the price and export vol-
ume of Ougan fruit ascended quickly． However，the
flavor of Ougan fruit needs to be improved because
children dislike it． So far research on Ougan was
mainly focused on breeding［20］ and medical
effect［18 － 19，21］． There was no report on Inv and Sus
of Ougan fruit so far． The objective of the current
study was to isolate Inv and Sus from Ougan and e-
valuate their expression patterns，which could accu-
mulate information for deep sugar metabolism study
of Ougan fruit．
1 Materials and Methods
1． 1 Materials
Fruits of Ougan mandarin (Citrus reticulate cv．
Suavissima)grown on the farm of Wenzhou Acade-
my of Agricultural Sciences (China)were used to
clone the genes for sucrose synthase and invertase．
For tissue-specific expression， samples including
young leaves，young stems，peel and flesh from ma-
ture fruit harvested at full ripening stage (210 after
full blooming，210 DAFB)were taken from 3 adult
Ougan trees． Flesh picked at 90，120，150，180
DAFB were used for gene expression test at different
·387·金微微，等． 瓯柑 NIN和 Sus基因克隆、序列分析与表达(英文)
development stages of fruit． Samples for each tissue
type were bulked and immediately frozen in liquid
nitrogen and stored at － 80 ℃ until used．
1． 2 Gene clone
Total ＲNA was extracted by microＲNA extrac-
tion kit (Qiagen miＲNeasy minikit， Cat． No．
217004)． TaKaＲa PrimerScript ＲT reagent (Code
DＲＲ047A)was used for reverse transcription． Ac-
cording to the sequence message of NIN，Sus and
Actin of sweet orange (Citrus sinensis) (or-
ange1. 1g006488m，orange1. 1g003661m，Cs1g05000. 3，
respectively)which was supplied by Citrus genome
database (http:/ /www． citrusgenomedb． org; ht-
tp:/ / citrus． hzau． edu． cn /orange / index． php) ，
primers for Ougan were designed． Primer pairs for
NIN were NIN-FP and NIN-ＲP，for Sus were Sus-
FP and Sus-ＲP，for Actin were Actin-FP and Actin-
ＲP (Table 1)． Polymerase chain reaction (PCＲ)
amplification was performed according to the method
of Zhang et al．［22］ with an annealing temperature of
56 ℃ ． PCＲ products were purified and then ligated
into pJET vector． Sequencing was completed by
Shanghai Biosune Biotechnology Co． Ltd (China)．
The sequences obtained were analyzed using BLAST
function on the net of National Centre of Biotechnol-
ogy Information (NCBI)，and then aligned with
Clustal W． Phylogenetic trees with neighbor-joining
(NJ)method were constructed with MEGA 5. 05．
Predicted molecular mass，isoelectric point (pI)
and secondary structure of protein were calculated
with web tool．
1． 3 Ｒeal-time quantitative PCＲ (q-PCＲ)analysis
q-PCＲ was performed in FTC-2000 real-time
PCＲ instrument (Canada)following the protocol of
Table 1 PCＲ primer sequence employed for gene clone
Gene Primer Primer sequence(5’-3’)
NIN NIN-FP
NIN-ＲP
ATGACTGCTGCTGGGGAAGCAG
TCATACTATGAAGGTCTGCTTTTTC
Sus Sus-FP
Sus-ＲP
ATGGCCGAACGTGCTTTGACC
TTACTCCACAGCCAGAGGAAC
Actin Actin-FP
Actin-ＲP
ATGGCAGATACTGAGGATATTCAG
TTAGAAGCATTTTCTGTGGACAATTG
Jin et al［23］． q-PCＲ primer pairs used for NIN，Sus
and Actin were QInv-FP and QInv-ＲP，QSus-FP
and QSus-ＲP，QActin-FP and QActin-ＲP，respec-
tively (Table 2)． Actin was used as a control gene
during expression． Gene expression levels in differ-
ent tissues were expressed as a ratio relative to that
expressed in stem，which were set as 1. 0．
Table 2 Primers used for q-PCＲ analysis
Gene Primer Primer sequence(5’-3’)
NIN QInv-FP
QInv-ＲP
CAGCATTACTCTCTGCACGT
TTCCCTGATATGGAATGACAAAG
Sus QSus-FP
QSus-ＲP
AGAGTCACACTGCTTTCACTC
GCTCATATCAGCACCAGGAG
Actin QActin-FP
QActin-ＲP
GGTCGTACAACTGGTATTGT
CAAGTCCAAACGAAGAATG
2 Ｒesults
2． 1 Sequence analysis of NIN，Sus and Actin gene
Three bands of expected sizes (around 1. 9 kb，
2. 4 kb and 1. 1 kb)were got by PCＲ from flesh tis-
sue of ripening Ougan fruit with primer pairs for
NIN，Sus and Actin． The bands were purified and
cloned and 10 recombinant plasmids for each gene
were sequenced． Three individual sequences with
1 932，2 418 and 1 134 bp in size were obtained
and were highly homologous to plant NIN，Sus and
Actin gene，respectively． With NCBI blast server，it
turned out that the nucleotide sequence of NIN had
83%，82% and 73% identity with that from litchi
(JQ773414. 1)，cassava (JQ339933. 1)and peach
(JQ412750. 1) ，respectively． The nucleotide se-
quence of Sus had 99%，85%，79% identity with that
from citrus (AB022092. 1)，papaya (AF420224. 1)
and Arabidopsis (NM _ 122090. 3) ，respectively．
Actin from Ougan shared both 92% homology with
that from litchi (HQ588865. 1) and bayberry
(AB650589. 1)． The results of blast above sugges-
ted that the 3 sequences obtained did encode Inv，
Sus and Actin，respectively． And the predicted pro-
tein of Inv and Sus was 643 and 805 amino acid in
length，respectively． GenBank accession number for
·487· 浙江农业学报 第 28 卷 第 5 期
NIN and Sus from Ougan fruit was KF694988 and
KF694989，respectively． As the two genes were
cloned according to the message from Citrus (sweet
orange)genome database，the two sequences of Ou-
gan and sweet orange were aligned with Clustal W，
and it was found that they share almost 100% ho-
mology for each gene (as both genes were so long，
results were not shown)． With the calculation based
on web tool，predicted molecular mass of the puta-
tive Inv and Sus protein were 72. 18 and 92. 19 ku，
respectively;and the pI were 6. 882 and 6. 051，re-
spectively． With the use of web tool SOPMA，it was
found that α-helix was constituted of 37. 17% amino
acid residues，while random coil 34. 53%，extend-
ed strand 20. 37% and turn 7. 93%，respectively，
in predicted NIN protein (Fig． 2)． In predicted
Sus protein (Fig． 3)，it was found that α-helix was
constituted of 50. 43% amino acid residues，while
random coil 24. 35%，extended strand 16. 15% and
turn 9. 07%，respectively．
2． 2 Phylogenetic analysis of Ougan NIN and
Sus gene
Eight members of NIN gene family were isola-
ted from rice and nine from Arabidopsis［24］．
OsNIN1-3 and OsNIN6-8 were full-length cDNA se-
quences among 8 members． Phylogenetic analysis of
1-4:NIN gene fragments (1. 9 kb);5-8:Sus gene fragments
(2. 4 kb) ;9-12:Actin gene fragments (1. 1 kb)． M1:1 kb lad-
der，the bands from top to bottom was 10 kb /8 kb /6 kb /5 kb /4
kb /3 kb /2 kb (the most bright)/1. 5 kb / 1. 0 kp /500. 0 bp，re-
spectively;M2:Maker I，the bands from top to bottom was 600
bp /500 bp /400 bp /300 bp /200 bp /100 bp，respectively．
Fig． 1 Electrophoretogram of NIN， Sus and Actin
gene fragments
The letters h，e，c and t represent the α-helix，extend strand，
random coil and turn，respectively．
Fig． 2 Secondary structure prediction of predicted protein
encoded by Ougan NIN
The letters h，e，c and t represent the α-helix，extend strand，
random coil and turn，respectively．
Fig． 3 Secondary structure prediction of predicted protein
encoded by Ougan Sus
rice and Arabidopsis NIN members suggested that
NIN could be clearly categorized into 2 major
groups:α and β． Group α could be further divided
into 2 subgroups，whose members may localize in
plastids and mitochondria［3，12］． In the current stud-
y，6 full-length sequences from rice and nine from
Arabidopsis took part in amino acid sequence align-
ment with that from Ougan mandarin． The results
showed that Ougan NIN clustered with group α NIN
such as At5g22510，OsNIN3 (Fig． 4-A)． Seven
members of Sus gene family were isolated from rice
·587·金微微，等． 瓯柑 NIN和 Sus基因克隆、序列分析与表达(英文)
and 6 from Arabidopsis，three from satsuma orange．
It was reported that Sus proteins could be divided
into 3 groups (SusⅠ，SusⅡ and Sus Ⅲ)with dis-
tinct function［14］． The results showed that the Sus
seperated belong to SusⅠ which also contained Cit-
Sus1，AtSus1 and AtSus4 (Fig． 4-B)．
2． 3 Expression analysis of Ougan NIN and Sus
gene in different tissues
A:The phylogenetic tree was based on 9 Arabidopsis，6 rice and
some kinds of fruit (peach，apple，grape and litchi)full-length
NIN deduced amino acids from NCBI． Ougan NIN is highlighted
with rectangle． B:The phylogenetic tree was based on 6 Arabi-
dopsis，7 rice，2 satsuma orange and 1 red radish full-length Sus
deduced amino acids from NCBI． Ougan Sus is highlighted with
rectangle．
Fig． 4 The phylogenetic tree of NIN and Sus deduced a-
mino acids
In this study，qPCＲ was performed to determine
the levels of Ougan NIN(Fig． 5-A)and Sus(Fig． 5-
B)gene expression in young leaf，young stem，peel
and flesh from mature fruit (210 DAFB)． The results
showed that both two genes were expressed across all
4 kinds of tissue，but with tissue-specific difference．
Both NIN and Sus genes accumulated the highest a-
bundance in stem，and both expressed much stronger
in leaf than those in peel and flesh． The mＲNA tran-
script level of Sus in peel was almost equivalent to
that in flesh，while NIN expression level in peel was
almost 4. 26-fold to that in flesh．
2． 4 Expression analysis of Ougan NIN and Sus
gene in different stage of fruit development
Fruit were picked at 90，120，150，180 DAFB
for expression test． Ｒesults showed that expression
level of NIN gene increased the highest at 120
DAFB，and then decreased quickly． The gene ex-
pressed lower while fruit become more ripened (Fig．
6-A)． The gene abundance of NIN at 180 DAFB was
only 27% of that at 90 DAFB． Expression level of Sus
gene increased just a little at 120 DAFB，and then de-
creased quickly at the later stage of fruit development．
The abundance of Sus declined 55% at 180 DAFB，
while compared with that of 90 DAFB (Fig． 6-B)．
3 Discussion and Conclusions
In recent years，more and more NIN had been
separated from fruits like peach［25］，litchi［26］，and
grape［24］． However，there was no report about NIN
on citrus fruit but trifoliate orange (Poncirus trifolia-
ta)． PtrNINV，which was separated from trifoliate
orange，might locate in mitochondria， and was
found to be related with low temperature stress re-
sponse［27］． In this paper a NIN gene was got from
Ougan fruit，and it belonged to Group α． According
to Murayama and Handa［12］，the NIN belonging to
Group α localized in plastids． So the NIN seperated
from Ougan fruit might localize in plastids． The results
will enrich the NIN research on citrus fruit and will lay
foundation for flavor improvement of Ougan fruit．
·687· 浙江农业学报 第 28 卷 第 5 期
Transcript levels are expressed as fold changes relative to a calibrator tissue，in this case the stem． Bars represent standard deviation of three
technical replicates． The same as below．
Fig． 5 Expression patterns of Ougan NIN(A)and Sus(B)gene in young stem，young leaf，peel and flesh from
mature fruit
Fig． 6 Expression analysis of NIN(A)and Sus(B)in Ougan fruit at different development stages
Ｒesults showed that Ougan NIN exhibited a tis-
sue-specific expression． However，expression analy-
ses of OsNIN1-4 and OsNIN8 showed little differ-
ence among different tissues，while OsNIN5-7 and
PtNIN1-16 showed tissue-specific characteris-
tic［3，28］． PtNIN2-4 expressed stronger in mature leaf
than those in node and internodes，but PtNIN8-9
and PtNIN12 showed an opposite result． In this pa-
per，Ougan NIN accumulated higher abundance in
stem than that in leaf，which was similar to Pt-
NIN13．
VvNI4 and VvNI5 were clustered with OsNIN3
and At5g22510 as Ougan NIN． Ｒesults showed that
VvNI5 maintained at low level from 70 DAFB to
fruit maturation while VvNI4 expressed with ascend-
ing tendency in general． PpNI1 from peach fruit was
also clustered with Group α，as Ougan NIN［25］．
The accumulated mＲNA level of PpNI1 was rela-
tively low during fruit maturation，no matter in the
early ripening cultivar‘Springcrest or in the middle
ripening cultivar ‘Ｒedhaven’． Ougan NIN ex-
pressed highest at 120 DAFB and then maintained at
a low level until fruit maturation，which was differ-
ent from that of VvNI5 and VvNI4，but more similar
as that of PpNI1． Functions of Ougan NIN during
fruit maturation remain to be determined and we
hope more NINs could be separated in the further
research．
Plant sucrose synthase had been widely and
deeply understood． However，research about Sus on
citrus fruit developed slowly． So far there were 3 Sus
family members separated from satsuma orange (Cit-
rus unshiu)［29］． CitSus2 (which had not get full
length sequence)and CitSus1 belong to SusⅠ fam-
ily，like Ougan Sus． CitSus1 expressed higher in
leaf than that in mature fruit and showed little differ-
ence between peel and flesh． Transcript of CitSus1
was found to be maximal at the immature stage (89
DAFB)，and then decreased throughout develop-
ment of edible tissue． Expression of Ougan Sus a-
·787·金微微，等． 瓯柑 NIN和 Sus基因克隆、序列分析与表达(英文)
mong different tissues and stages were similar to Cit-
Sus1． More details remain to be deeply exploded．
In summary， the genes NIN and Sus were
cloned from Ougan fruit in current study． They were
both highly conserved． Phylogenetic analysis showed
that putative NIN protein belongs to Group α，which
might localize in plastid，and putative Sus protein
belongs to Sus I． Expression analysis turned out that
both genes showed expression difference among tis-
sues，and both expressed at a higher level at the im-
mature stage and decreased towards the ripening
stage． The work will provide some basic information
for sugar metabolism of Ougan fruit，which will fi-
nally serve for improvement research on flavor and
breeding of Ougan fruit in future．
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(责任编辑 张 韵)
·987·金微微，等． 瓯柑 NIN和 Sus基因克隆、序列分析与表达(英文)