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Agricultural and Environmental Chemistry
SlMYC2 Involved in MeJA-induced Tomato Fruit Chilling Tolerance Dedong Min, Fujun Li, Xinhua Zhang, Xixi Cui, Pan Shu, Lulu Dong, and Chuntao Ren J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00299 • Publication Date (Web): 12 Mar 2018 Downloaded from http://pubs.acs.org on March 12, 2018
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SlMYC2 Involved in MeJA-induced Tomato Fruit Chilling Tolerance
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Dedong Min, Fujun Li, Xinhua Zhang*, Xixi Cui, Pan Shu, Lulu Dong, Chuntao Ren
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School of Agricultural Engineering and Food Science, Shandong University of Technology, Zibo
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255049, Shandong, PR China
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* Corresponding author
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Tel: +86-533-2786398; Email:
[email protected] (Xinhua Zhang)
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ABSTRACT
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MYC2, a basic helix-loop-helix transcription factor, is a master regulator in Jasmonic acid (JA)
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signaling pathway. However, the functions of SlMYC2 in MeJA-mediated fruit chilling tolerance are
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far from being clearly understood. Thus, in present work, we constructed SlMYC2-silenced tomato
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fruit by virus-induced gene silencing (VIGS) and investigated the function of SlMYC2 in
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MeJA-induced tomato fruit chilling tolerance. The results showed that MeJA treatment markedly
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induced the SlMYC2 expression, increased proline content, lycopene content and antioxidant enzyme
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activities, including superoxide dismutase, peroxidase, catalase and ascorbate peroxidase, inhibited
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the increase of electrical conductivity and malondialdehyde content, and effectively reduced the
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chilling injury (CI) incidence and CI index. However, these effects of MeJA treatment were partially
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counteracted in SlMYC2-silenced tomato fruit, and the CI incidence and CI index in
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(SlMYC2-silenced + MeJA)-treated fruit were higher than those in MeJA-treated fruit. Our results
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indicated that SlMYC2 might be involved in MeJA-induced chilling tolerance, possibly by
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ameliorating the antioxidant enzyme system of fruit and increasing proline and lycopene levels.
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KEYWORDS: MYC2 transcription factor, methyl jasmonate, virus-induced gene silencing, chilling
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injury, tomato fruit
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INTRODUCTION
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Cold storage is one of the main methods used to prolong the storage time and maintain postharvest
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quality of horticultural crops. However, low temperature could result in chilling injury (CI) for many
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cold-sensitive fruit and vegetables.1,2 The main symptoms of CI are discoloration, surface lesion and
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abnormally ripening, which further shorten the storage time and lead to quality deterioration.3
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Therefore, it is urgent to understand the physiological mechanism of CI in fruit and optimize
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methods that alleviate the CI symptoms.
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Jasmonic acid (JA) and methyl jasmonate (MeJA), an endogenous regulator, play important roles
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in the development and defense responses of many plant,4 such as Chinese bayberry,5 bean6 and
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cotton.7 Recently, most literature suggested that MeJA could enhance the chilling tolerance in many
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fruit and vegetables, including cucumber,8 cowpea,9 loquat,10 peach,11 tomato,12 etc. In addition,
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MeJA, an important plant hormone, not only has no side effects on the health, but also could
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promote the biosynthesis of natural products with healthy characteristics in many plant, then
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improving their beneficial on human health.13 Thus, the application of MeJA is greatly promising in
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alleviating CI and maintaining fruit quality during postharvest cold storage periods. The induced
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chilling tolerance of these fruit and vegetables by MeJA might be due to reduced the H2O2 levels,
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enhanced antioxidant enzyme activity, improved proline and γ-aminobutyric acid contents, higher
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level of energy charge and cooperation with catabolism of arginine.8-12 Nevertheless, the molecular
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mechanisms of MeJA-induced fruit chilling tolerance remain poorly understood.
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MYC2, a basic helix-loop-helix transcription factor, is the master regulator in JA signaling
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pathway and induces JA-mediated responses such as wounding, water deficit stress and oxidative
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stress adaptation.14-16 Recently, zhao et al.17 pointed out that MeJA treatment could induce the 3
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expression of MaMYC2a, MaMYC2b and cold-responsive pathway genes, thus, MaMYC2 might
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participate in MeJA-induced banana chilling tolerance. However, the functions of SlMYC2 in
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MeJA-mediated fruit chilling tolerance are far from being clearly understood. Hence, it is urgent to
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find an effective method that can be used to study the function of SlMYC2 in MeJA-mediated fruit
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chilling tolerance. Virus-induced gene silencing (VIGS) is an effective, rapid and simple reverse
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genetics approach for studying plant genes function18, 19 and has been widely used in several plant
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species, such as tomato,20 spinach,21 peach,22 litchi23 and strawberry.24
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In addition, tomato fruit is a typical representative of the respiratory climacteric fruit, and is a
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model material for studying chilling injury of fruits. Thus, in present work, we firstly constructed
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SlMYC2-silenced tomato fruit by VIGS and treated with MeJA before cold storage. The indexes
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correlated with fruit chilling tolerance such as CI index, electrical conductivity, malondialdehyde
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(MDA) content and antioxidant enzyme activities were measured. Meanwhile, the expression levels
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of SlMYC2 in tomato fruit exposed to chilling were also investigated by quantitative real-time
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polymerase chain reaction (qRT-PCR). The aim of this work was to study the role of SlMYC2 in
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MeJA-induced chilling tolerance of tomato fruit.
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MATERIALS AND METHODS
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Construction of SlMYC2-Silenced Tomato Fruit. Firstly, Total RNA was isolated from frozen
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tomato tissue (1.5 g) according to the method of Zhang et al.25 The first strand complementary DNA
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(cDNA) was obtained with 2 µg of total RNA, Oligo(dT18) and M-MLV reverse transcriptase.
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Then, a 454-bp fragment of SlMYC2 gene (GenBank Accession No. KF428776) was amplified by
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PCR from tomato cDNA sources using gene specific primes (forward: 5’-GGG GTA CCC CTG
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GTC AGG CGT TGT ATA GTT C-3’ with a KpnI restriction site and reverse: 5’-CCG CTC GAG 4
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GAG GAG GAT TCT TCT GTT GTT GC-3’ with a XhoI restriction site). The production of PCR
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was cloned into pTRV2 via a KpnI/XhoI digestion to form pTRV2-SlMYC2.
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pTRV1, pTRV2 and pTRV2-SlMYC2 were transformed into competent cells E.coli with heat
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shock method and grown on select Luria- Bertani (LB) media containing appropriate antibiotics
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overnight at 37 °C. All constructs were then purified by Fast-Plasmid Mini kit and the identity of the
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final constructs was assayed by sequencing.
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Finally, the purified plasmid of pTRV1, pTRV2 and pTRV2-SlMYC2 were transformed into
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Agrobacterium strain GV3101. A 5 mL agrobacterium culture with 50 µg mL-1 kanamycin (Kan), 50
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µg mL-1 gentamicin (Gen) and 20 µg mL-1 rifampicin (Rif) was grown overnight at 28 °C. The next
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day, the culture was transformed into 50 mL yeast extract and beef (YEB) medium that containing
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50 µg mL-1 Kan, 50 µg mL-1 Gen, 20 µg mL-1 Rif, 10 mM 2-N-morpholino ethanesulfonic acid
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(MES) and 20 µM acetosyringone (AS) and grown overnight at 28 °C. Subsequently, the
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agrobacterium cells were harvested and resuspended with infiltration media (containing 10mM
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MgCl2, 10 mM MES, 200 µM AS), adjusted to OD600≥1.0. The agrobacterium soluble containing
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pTRV1 and pTRV2 (as a control) or pTRV1 and pTRV2-SlMYC2 (as SlMYC2-silenced fruit) in a 1:1
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ratio added and cultured at room temperature for 2 h. After 2 h, the agrobacterium soluble was
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infiltrated into the carpopodium of tomato fruit about 10 d after pollination from a green house in
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Zibo, Shandong province, China.
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Fruit and Treatment. Tomato fruit (Solanum lycopersicum L. cv. Badun) containing control and
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SlMYC2-silenced fruit were hand-harvested at mature-green stage. After transported immediately to
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our laboratory, both control and SlMYC2-silenced tomato fruit were randomly divided into two
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groups. One group of control or SlMYC2-silenced fruit were treated with 0.05 mM MeJA as 5
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described in our previous work,12 the other were treated with air as control. Each treatment was
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replicated 3 times (60 fruit /replicated).
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After treatment, the container was opened and ventilated for 1 h. Then, all fruit were stored at
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2±1 °C and 80-90 % relative humidity. The mesocarp of fruit were cut at 0, 1, 3, 7, 14, 28 d, frozen
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immediately in liquid nitrogen and stored at -80 °C until further analysis of physiological indicators.
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The fifteen fruits were sampled at 14 and 28 d of storage and stored at room temperature with
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80-90 % relative humidity for 7 d to assay the CI symptom and color change index.
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CI Incidence and CI Index. CI incidence and CI index were measured that used to evaluate the
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CI degree of tomato fruit during cold storage. According to the method of Zhang et al.26 with little
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modification, the CI symptom was measured visually and CI index was calculated using following
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scale: 0=no CI symptom, 1=less than 5% of surface area, 2=from 5% to 25%; 3=from 25% to 50%,
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4=more than 50% of surface area. CI index=Σ(scale×the number of fruit within this scale)/(total
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fruit×4).
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The CI incidence (%) was calculated as the number of CI fruit divided by the total number of fruit recorded and multiplied by 100.
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Color Change Index. According to the method of Cao et al.,27 the color change index was
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assayed. Color change index=Σ(color scale value×number of the fruits within each scale)/(5×total
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number of fruits).
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Electrical Conductivity and Malondialdehyde Content. The electrical conductivity was
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measured by DDS-307A conductivity meter (Leici Inc., Shanghai, China) as described in our
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previous work.28 The MDA content was assayed with UV-2102 PCS spectrophotometer (Unico Inc.,
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Shanghai, China) according to the method of Jin et al.29 The amount of MDA expressed as nanomole 6
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MDA per gram of fresh weight (FW).
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Extraction and Measurement of Enzyme Activities. Frozen tomato tissue (1.0 g) was
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homogenized with 5 mL of sodium phosphate buffer (SPB, pH=7.0) containing 1% (w/v)
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polyvinylpyrrolidone and centrifuged at 10000g for 15 min at 4 °C. Then, the supernatant was
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collected and used for measurement of enzymes activities including superoxide dismutase (SOD),
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peroxidase (POD) and catalase (CAT) and ascorbate peroxidase (APX).
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SOD activity was assayed according to the method of Zeng et al.30 One unit of SOD activity was
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defined as the amount of enzyme causing 50% inhibition of photochemical reduction of nitroblue
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tetrazolium.
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POD activity was measured as described by Luo et al.31 One unit of POD activity was defined as the absorbance increase of 0.01 units per minute under assay condition.
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CAT and APX activity was determined according to the method of Jiang et al.32 One unit was
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defined as the amount of enzyme causing an absorbance change of 0.1 per minute under assay
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conditions. One unit of APX activity was defined as the absorbance change of 0.001 units per
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minute.
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Protein content was measured according to the method of Bradford33 with bovine serum albumin
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as a standard. All the activities of antioxidant enzymes were expressed as unit per milligram protein
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(U mg−1 protein).
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Proline and Lycopene content. Proline content was determined according to the method of Li et
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al.34 and Liu et al.35 Fruit tissue (1.5 g) was homogenized with 5 mL 3% (w/v) sulfosalicylic acid and
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incubated at 100 °C for 10 min. After incubation, the supernatant (0.25mL) mixed with glacial acetic
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acid (0.25 mL) and ninhydrin (0.5 mL), then boiled at 100 °C for 40 min. After cooled to room 7
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temperature, the reaction mixture was added 0.5 mL of toluene and the absorbance of organic phase
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was measured at 520 nm. The resulting values were compared to a standard curve constructed with
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known amounts of proline. The proline content was expressed as µg g-1 FW.
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Lycopene content was determined according to the method of Kuti and Konuru.36 Tomato fruit
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tissue (1.5 g) was homogenized in 10 mL of hexane/ methanol/ acetone (2:2:1) and centrifuged at
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10000g for 10 min. The upper hexane layer was diluted 10 times and measured at 505 nm using an
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UV-2102 PCS spectrophotometer against a hexane black. The concentration of total lycopene was
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calculated from the data using a specific extinction coefficient of 3400. The results were expressed
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as g kg−1 FW.
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Semi-quantitative Reverse Transcription PCR. Semi-quantitative reverse transcription PCR
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(SqRT-PCR) was performed as described of Chi et al.37 The specific primer was designed outside the
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region targeted for silencing, the SlUbi3 gene was used as reference gene and the results were as
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follows:
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SlMYC2-forward: 5’-CAG TTT TGC CTT CTT CGG GC-3’
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SlMYC2-reverse: 5’-TTC GCT GGC TTT CTA CCT CG-3’
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SlUbi3-forward: 5’-TCCATCTCGTGCTCCGTCT-3’
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SlUbi3-reverse: 5’-CTGAACCTTTCCAGTGTCATCAA-3’
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The SqRT-PCR program was processed with denaturation step at 95 °C for 4 min, followed by 18,
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21, 24, 27, 30 and 35 cycles of 30 s at 94 °C, 30 s at 60 °C and 45 s at 72 °C, and extension at 72 °C
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for 10 min. Finally, the PCR production was run on a 1 % agarose gel with 0.5 µg ml-1 ethidium
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bromide.
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qRT-PCR assay. qRT-PCR was performed according to our previous method.38 The relative 8
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expression of SlMYC2 was measured by qRT-PCR using the SYBR Green I Master Mix
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(Toyobo, Osaka, Japan) on a LineGene 9600 detection system (Bioer, HangZhou, China). The
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specific primers of qRT-PCR were same with primes that used in SqRT-PCR.
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And, the qRT-PCR program was processed with preliminary step of 2 min at 95 °C, followed by
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40 cycles 15 s at 95 °C and 20 s at 60 °C, and 45 s at 72 °C. The melting curves were measured from
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55 °C to 95 °C at 0.5 °C increments. The gene expression level of SlMYC2 was calculated using the
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method of 2-△△Ct.
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Data Analysis. The experiments were performed with absolutely randomized design. All the data
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were analyzed with SPSS 19.0 (SPSS Inc., chicago, LC, USA). One-way analysis of variance
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(ANOVA) was used for assay of date and the significant differences were conducted by Ducan’s
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multiple comparisons. Differences at P
