MULBERRY (MORUS L.) METHIONINE SULFOXIDE RREDUCTASE GENE CLONING, SEQUENCE ANALYSIS, AND EXPRESSION IN PLANT DEVELOPMENT AND STRESS RESPONSE
© 2013 Wei Tong", Yinghua Zhang", Heng Wang", Feng Li", Zhaoyue Liu", Yuhua Wang", b,
Rongjun Fang", c, Weiguo Zhao", b, #, Long Li", b, #
a College of Biological and Chemical Engineering, Jiangsu University of Science and Technology,
Zhenjiang, Jiangsu, 212018P.R. China b Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu, 212018 P.R. China c School of Life Science, Nanjing University, Nanjing, Jiangsu, 210093 P.R. China Received February 27, 2013; in final form, April 23, 2013
Methionine sulfoxide reductase plays a regulatory role in plant growth and development, especially in scavenging reactive oxygen species by restoration of the oxidation of methionine in protein. A full-length cDNA sequence encoding methionine sulfoxide reductase (MSR) from mulberry, which we designated MMSR, was cloned based on mulberry expressed sequence tags (ESTs). Sequence analysis showed that the MMSR is 810 bp long, encoding 194 amino acids with a predicted molecular weight of 21.6 kDa and an isoelectric point of 6.78. The expression level of the MMSR gene under conditions of drought and salt stresses was quantified by qRT-PCR. The results show that the expression level changed significantly under the stress conditions compared to the normal growth environment. It helps us to get a better understanding of the molecular basis for signal transduction mechanisms underlying the stress response in mulberry.
Keywords: mulberry, methionine sulfoxide reductase, gene cloning, induced expression
DOI: 10.7868/S0132342313050151
INTRODUCTION
Methionine sulfoxide reductase plays a regulatory role not only in scavenging reactive oxygen species by restoration of the oxidation of methionine in protein, but also in plant growth and development [1]. It can repair some intracellular oxidative proteins and restore their biological functions [2]. In addition, it is also involved in many intracellular signal transduction pathways [3] directly or indirectly, through the maintenance of heat shock protein activity or the conformation of calmodulin. In 1992, MSRA gene was first cloned from E. coli [4], and later, from other bacteria [5, 6], yeast [7], plants [8, 9], animals [10, 11], human [12, 13], and other 20 kinds of organisms. It indicates that MSR gene exists in most biological organisms. Except for methionine sulfoxide reductase in Neisseria gonorrhoeae with a molecular weight of about 58 kDa, the others are mostly between 25—35 kDa. However, few studies of MSR have been carried out in plants. The first MSR gene was isolated from Brassica napus [8] and now, MSR gene has been cloned from Arabidopsis [14], cotton [15], and rice [16]. The differ-
#Corresponding author (e-mail: wgzsri@126.com; seri68@hot-mail.com). 1 The first two authors contributed equally.
ences between MSR in plants and other organisms are that plants usually contain multiple copies of MSR gene. Thus, arabidopsis is reported to contain five MSR genes [17]. MSR gene plays an important role in plant antioxidative mechanisms; it can reduce yield losses of some crops under stresses.
Mulberry (Morus L.), a perennial tree or shrub, is an economically important plant used not only for sericulture as the sole food plant for the domesticated silkworm (Bombyx mori), but also for a variety of other purposes such as the production of edible fruits or useful timber. The growth and productivity of mulberry is adversely affected by abiotic and biotic stresses. Higher plants always adapt to the environmental stresses by the activation of cascades of molecular networks involved in stress perception, signal transduction, and expression of specific stress-related genes, but no study has investigated the role of MSR in mulberry. In this study, we cloned the MMSR gene based on the expressed sequence tags (ESTs) from mulberry cDNA library constructed previously [18, 19], and got the full-length sequence by RT-PCR [20]. Furthermore, changes in the transcription level of MMSR under drought and salt stresses were detected by qRT-PCR. The results will lay a good foundation for understanding of the signal transduction mechanism underlying
stress response and design new breeding strategies for improving mulberry production through the resistant transgenes in the future.
MATERIALS AND METHODS
Plant Materials and Reagents
To analyze the gene expression under different conditions, mulberry variety Yu711 (Morus multicau-lis) was grown under standard conditions in the National Mulberry Gene Bank of the Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu Province, China. The mulberry shoots were grafted, and the grafted plants were transferred into an incubator and maintained at 25°C for a 12-h photoperiod to induce tender buds until the winter buds grew to about 30 cm in length (50 days).
For the study and analysis of the expression level of MMSR at different developmental stages and in different tissues, samples were collected from a 10-year old mulberry tree and of mulberry grown under the same conditions as the plants mentioned above.
RNAiso Plus, Reverse Transcriptase M-MLV (RNaseH-), RNase Inhibitor, dNTP, rTaq poly-merase,T4 DNA ligase, Agarose Gel DNA Purification Kit, and pMD18-T vectors were purchased from Takara (TAKARA Bio Co, Ltd). All PCR primers were synthesized by Shanghai Sangong Biological Engineering Technology and Services Co. Ltd, and all chemicals used were analytical grade reagents.
RNA Isolation and Cloning of the Full-Length cDNA
Total RNA was isolated from tender buds (net weight about 90 mg) of grafted mulberry seedlings using RNAiso Plus reagent following the manufacturer's protocol, resuspended in DEPC-treated water, and stored at —80°C. The quality of total RNA was determined by ultraviolet spectrophotometer combined with electrophoresis. RNA sample of1 |L was diluted with 99 |L DEPC water, and the absorbance (A) was measured by ultraviolet spectrophotometer (UVS). The ratio of A260/A280 was used to express the RNA purity, and the mass concentration was calculated according to A value. In all samples, the ratio of A260/A280 was 1.8-1.9, so all RNAs could be used in the experiments.
The cDNA was synthesized from 9 |L total RNA by Reverse Transcriptase M-MLV (RNaseH-) at 42°C for 60 min with 4 |L oligo-dT-adaptor primer in 20 |L system following the manufacturer's protocol. The cDNA was used as a template for PCR in gene cloning. Forward and reverse primers were designed according to the EST with the inference function from mulberry cDNA library.
Forward primer (20 |M): M-MSR-F:
M
3-
2000 bp
1000 bp 750 bp 500 bp
250 bp 100 bp
Fig. 1. RT-PCR of mulberry gene MMSR. (M) DL2000 DNA molecular marker; (1) the first PCR amplification products; (2) the second PCR amplification products.
Reverse primer (20 |M): M-MSR-R: 5'-GCA-CATAACACGGATTACACAACT-3'
The RT-PCR reactions were performed in a total volume of 50 |L, including 1 |L first-strand cDNA, 41 |L ddH2O, 1 |L each of the gene-specific primers, 0.5 |L dNTP (10mM), 5|L buffer, and 0.5 |L rTaq DNA polymerase (5U/|L). The RT-PCR amplifications were performed using the following parameters: DNA was denatured at 94°C for 5 min followed by 30 amplification cycles (94°C for 30 s, 55°C for 1min, 72°C for 1min), with a final extension step of 7 min at 72°C. The RT-PCR products were analyzed in 1% agarose gels and purified using Takara Agarose Gel DNA Purification Kit following the manufacturer's protocol. The purified fragment, which was confirmed to have the predicted length, was then cloned into pMD18-T vector and sequenced to confirm the presence of an open reading frame (ORF) related to the tentative consensus sequence.
Analysis of the Full-Length cDNA Sequence of MMSR
Sequence encoding MMSR was determined by homology searches in the NCBI (http://www.ncbi. nlm.gov/) databases using the BLAST program, and the homologous sequences were downloaded from the database. Alignment of the MMSR protein with other structurally related MSR proteins was performed using the Clustal X program. Some other basic properties were accomplished by tools at the ExPASy website (http://www.expasy.org) and those of the DNAStar
TATCGGCCATTACGGCCGGGGCTTTTTCTCTCTAACACAAAGAA 45
ATGGCGACTTCTTCGGACAAGAACGGGACCAACCCGGCTCTTGACCCGGACTTGGACTCC CCGGGCGACCCGGCT 1
A 120
CACGAGTTGGCTCAGTTCGGAGCTGGGTGCTTCTGGGGCGTAGAGCTCACGTTCCAGCGA GTGGTCGGCGTCGTC
26
V V 195
AAGACCGAGGTTGGCTACTCGCAGGGCCACGCGCCGGACCCGAATTACAAACTGGTTTGC
TCTGGATCCACCAAC
51
N
270
CACGTCGAGGTGATTCGGGTCGAGTTCGACCCGAAAGTTTGTCCTTACACTCATCTCCTCTC TGTTTTCTGGGGT
76 II V l:. V I R V К I- П lJ К V Г P Y 'I И I. I. S V I W
G
345
CGCCATGATCCCACGACTCTCAATCGCCAGGGTGGTGATGTTGGGGCTCAATATAGATCAGG AATATACTACTAC
101 R [| П [> T Г I. N R g (., <.i I) V G Л O Y R S (.i [ Y Y
V
420
AATGAAACTCAAGCTCGTTTAGCTCAGGAATCAAAGGAAGCTAAGCAGTTGGAGTTGAAG GATAAGAAGATAGTG
126 \ i: T g л R [. л g i: s к с л к g i к i. к n К К I
V
495
ACAGAAATTCTTCCAGCAAAGAGATTCTACAGGGCTGAGGAGTACCATCAGCAGTATCTTG AGAAGGGTGGCGGC
151 I" I-. I 1. 1' Л К R J- Y R Л J-. К Y II Q Q Y 1. К К (i (i
G
570
CAAGGCCGGAAACAATCCGCCGAGAAGGGCTGCACTGATCCCATCAGATGCTATGGCTAG
176 g t¡ R к g s л i-, 1ч Ci Г Т l.) Г 1 R t" Y G -
630
GGCTAAGG CTAACCTAAATA AAAATA AAAC AAATAAAAAAG ATTGCA AGTTTGG AAC ATAA
ATTCCAACAGTTGT
705
GTAATCCGTGTTATGTGCACTAATTATGTATGCATAATTTGGAGTCTATCCTTGTTTTAAGGAC TCTTTTGCGTA
780 TAACTTTCTTGTTCATAAAAAAAAAAAAAAA
БИООРГАНИЧЕСКАЯ ХИМИЯ том 39 № 5 2013
Fig. 2. The full-length cDNA sequence and deduced amino acids of mulberry gene MMSR. ATG indicates the start codon; TAG indicates the stop codon.
software, such as ORF searching and translation of nucleotide sequence, as well as prediction of isoelectric point and molecular weight of MMSR. To determine the relationship between MMSR and other M
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