MOLECULAR CLONING OF A DEHYDRATION-RESPONSIVE PROTEIN GENE (MRD22) FROM MULBERRY, AND DETERMINATION OF ABIOTIC STRESS PATTERNS OF MRD22 GENE EXPRESSION
© 2014 Heng Wang", Zhaoyue Liu", Feng Li", Yuhua Wang"-b, Rongjun Fang",c,
Weiguo Zhao",b- #, Long Li",b
aCollege of Biological and Chemical Engineering, Jiangsu University of Science and Technology,
Zhenjiang Jiangsu, 212018 P.R. China bSericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang Jiangsu, 212018 P.R. China cSchool of Life Science, Nanjing University, Nanjing Jiangsu, 210093 P.R. China Received July 1, 2013; in final form, June 6, 2013
A full-length cDNA sequence coding for Dehydration-responsive protein gene of mulberry tree, which we designated was MRD22 (GenBank accession number: JQ804833) was cloned based on mulberry expressed sequence tags (ESTs). MRD22 is 1503 bp long, contains a 334 bp 5'-UTR (untranslated region) and a 563 bp 3'-UTR, encodes 201 amino acids with a predicted molecular weight of 54.28 kDa and an isoelectric point of 9.35. Phylogenetic analysis based on MRD22 sequences from different species showed that mulberry has close relationship with Populus trichocarpa, Ricinus communis, Camellia sinensis, Gossypium hirsutum, Gossypium barbadense and so on. The expression level of the MRD22 gene under conditions of drought, low temperature 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, dehydration-responsive protein gene, gene cloning and expression, abiotic stress
DOI: 10.7868/S0132342314010138
INTRODUCTION
The BURP domain is a conserved amino acid motif whose name is based on four members: BNM2, USP, RD22, and PG1P [1]. BURP domain-containing proteins consist of several conserved modules: an N-terminal hydrophobic domain with a putative signal peptide; a BURP domain containing two conserved cysteine (C) residues and four repeat cysteine— histidine (CH) motifs in the C-terminal; and a variable internal region containing a short conserved segment or a short segment plus an optional segment consisting of repeated units, which is unique to each member [1]. Many BURP domain containing proteins have been reported to be ubiquitous in plant species. Examples are BNM2, an oilseed rape (Brassi-canapus L.) microspore protein [2]; RD22, an Arabi-dopsis thaliana dehydration-responsive protein [3]; PG1P, a P-subunit of polygalacturonase isoenzyme 1 of tomato (Lycopersicon esculentum L.) [4, 5]; ADR6, a soybean [Glycine max (L.) Merr.] auxin down-regulated protein [6]; and SCB1, seed coat BURP-containing protein 1 from soybean [7]. The BURP do-
1 The first two authors contributed equally. # Corresponding author (e-mail: wgzsri@126.com).
main has been found in abundant plant proteins as mentioned earlier. Even in the ancient moss Phy-scomitrella patens (Hedw.) Bruch and Schimp. in B.S.G (Grimmia funalis), BURP domain-containing proteins were identified [8]. So far, no BURP domain-containing protein has been identified in a nonplant species.
Based on phylogenetic analysis of the putative BURP domain-containing proteins in soybean and other plant species, the members of the BURP family can be classified into BNM2-like, USP-like, RD22-like, and PG1-like subfamilies [9]. The major variability among the members of the BURP family occurs in the region between the hydrophobic signal sequence and the BURP domain. This region includes a short conserved segment and an optional segment consisting of repeated units. The repeated units are absent in the BNM2-like proteins. The PG1_-like proteins can be distinguished from the members of other subfamilies by the presence of a sequence segment containing a 14 amino acid sequence, for instance, FT-NYGXXGNGGXXX (where X = any amino acid residue) in PG1p [4]. Both the USP-like and RD22-like proteins can be distinguished from other subfamilies by a sequence segment containing ~30 amino acids,
M
bp
2000 —
1000 — 750 — 500 —
250 — 100 —
Fig. 1. Electrophoretogram of amplification product of mulberry Dehydration-responsive protein gene ORF. (M): DL2000 DNA molecular marker; (1) product of RT-PCR.
but the RD22-like proteins have a segment consisting of repeated units of approximately 20 amino acids while the USP-like proteins do not contain such a segment [9].
Even though the BURP family can be easily classified based on the sequence features, the functions of most members in this family are largely unknown. but previous reports have attributed two possible functions. First, they are important in plant development and metabolism. PG1P, the non-catalytic P-subunit of the polygalacturonase isozyme I, can regulate pectin metabolism by limiting the extent of pectin solubi-lization and depolymerization during tomato ripening [4, 5]. SCB1, a seed coat-specific protein in soybean (Glycine max), is involved in the formation of the seed coat by regulating the differentiation of parenchyma cells [7]. OsRAFTINi, an anther-specific protein in rice (Oryza sativa), plays an important role in transition of sporopollenin from the tapetum to developing microspores via Ubisch bodies [10]. It has been reported that the pollen grains collapse and the mature anthers become nondehiscent and shortened when the OsRAFTINl gene is inhibited. Another anther-specific protein, RA8, is also involved in the dehiscence of anther and microspore development [11]. AtUSPLi may play a role in seed development and has been identified in distinct cellular compartments [12]. Second, many BURP domain-containing proteins are responsive to stress treatments. RD22 can be induced by drought, ABA and salt stress [13—15]. BnBDCl, a shoot-specific gene in oilseed rape, is up regulated by NaCl, ABA and mannitol, and down regulated by salicylic acid and UV irradiation. BnBDC1 expression is also involved in plant response to abiotic and biotic stresses [16]. The expressions of SALI3—2 and SALI5— 4a are induced by aluminum stress [17] and the BURP
domain of SALI3—2 is crucial for its tolerance to salt [18]. ADR6, which is expressed in G. max, is down regulated by auxin [6]. Recently, seven BURP genes from Zea mays (ZmBURPs) were shown to be up and down-regulated by ABA and cold, respectively; moreover, two of these genes were up- and down regulated by NaCl.
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 morii) but also for a variety of other purposes such as the production of edible fruits or useful timber [19]. The growth and productivity of mulberry is adversely affected by abiotic and biotic stresses [20], but only a few studies have investigated the role of MRD22 in other plants, and the exact function of MRD22 genes and encoded proteins in the stress response is still not fully understood in mulberry. Therefore, efforts to investigate the molecular adaptation mechanisms to stresses and to strengthen stress tolerance in this plant are of fundamental importance to mulberry production.
In this study, we cloned the MRD22 gene based on the expressed sequence tags (ESTs) from mulberry cDNA library constructed previously [21], and got the full-length sequence by RT-PCR [22] and RACE method [23] and analyzed the expression of the gene in different tissues and under different kinds of stresses in mulberry. The purpose of this study is to lay a good foundation for understanding the complicated signal transduction mechanism underlying the stress response and present new breeding strategies for improving mulberry production.
RESULTS AND DISCUSSION
Results
Amplification of Full-Length cDNAs and Sequence Analysis of the MRD22 Gene
To clone the full-length cDNA of the mulberry MRD22 gene (GenBank accession number: JQ804833), a cloning strategy combining bioinformatics analysis and the mulberry cDNA library screening technique was used. RT-PCR products were detected by 1% agarose gel, and yielded the expected size (Fig. 1). Sequencing showed that it was basically the same sequence as the relevant EST. Nested amplification yielded a specific bright band of about 550 bp, which was also in consensus with homology-based length prediction (Fig. 2). Sequence analysis showed that the isolated cDNA designated MRD22 is 1503 bp long and contains a 334 bp 5' untranslated region (5'-UTR) and a 563 bp 3'-UTR. Its open reading frame (ORF) is 606 bp long, encoding 201 amino acids with a predicted molecular weight of 54.28 kD and an isoelectric point of9.35. Online software Batch CD-search (http://www.ncbi.nlm.nih.gov/Struc-ture/cdd/wrpsb.cgi) analysis showed that the gene structure containing a conserved domain. Conserved domain
1
database (CDD) is a protein annotation resource that consists of a collection of well-annotated multiple sequence alignment models for ancient domains and full-length proteins. These are available as position-specific score matrices (PSSMs) for fast identification of conserved domains in protein sequences via RPS-BLAST. CDD content includes NCBI-curated domains, which use 3D-structure information to explicitly define domain boundaries and provide insights into sequence, structure and function relationships, as well as domain models imported from a number of external source database.
Homologous Alignment and Phylogenetic Analysis of the MRD22 Gene
According to the encoded amino acid sequence of the target gene, selected from the NCBI website homologous amino acid sequence homology analysis to establish the evolutionary tree using MEGA 4.0 Software (Fig. 3). The results show that: the participation of more than 26 homologous sequences on the length and structure are very conservative, shows that the
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