In order to reveal which role the callose played in R. rugosa pollination incompatibility, the full-length cDNA sequence of β-1,3-glucanase gene was cloned for the first time from the stylus of Rosa rugosa “Tanghong” with RT-PCR and RACE methods and named as RrGlu. The full-length cDNA is 1380 bp with an open reading frame of 1041 bp, encoding 346 amino acids. The derived protein has a molecular weight of 37.85 kD, a calculated pI of 9.12, a pfam00332 conserved domain at position 36 - 345, and belongs to glycosyl hydrolase family 17. The derived protein is a hydrophilic protein secreted into the vacuole. There is a signal peptide cleavage site at position 34 - 35, a transmembrane domain at position 13 - 32, six Ser phosphorylation sites, three Thr phosphorylation sites, three Tyr phosphorylation sites, one N-glycosylation site, and five O-glycosylation sites. There are 31.50% α-helixes, 30.92% random coil, 25.14% extended peptide chain, and 12.43% β-corner structure. This protein and the Glu protein from eight other species, including Prunus persica, share a sequence homology of greater than 72%; all of the proteins contain a pfam00332 conserved domain and a β-1,3-glucanase active center sequence (LIVM)-X-(LIVMFYW)3-(STAG)-E-(ST)-G-W-P-(ST)-X-G. Furthermore, their phylogenetic relationships are consistent with their traditional classifications. These results were meaningful to reveal the molecular mechanism of R. rugosa pollination incompatibility and improve the theory and techniques of breeding ornamental R. rugosa.
The Rosa rugosa is a famous traditional Chinese flower. It is fragrant as well as resistant to cold, drought, pest, disease, salt, and alkali [
The hydrolysis and synthesis of callose are catalyzed by β-1,3-glucanase (Glu) and β-1,3-glucan synthase (Cals) [
The plant material, Chinese representative R. rugosa “Tanghong”, was from the rose germplasm resources garden at Shandong Agricultural College. R. rugosa “Tanghong” is the most representative traditional rose in China.
Between May 2015 and June 2015, the robust “Tanghong” buds were parchment isolated at 5:00-6:00 pm the day before blooming. At 6:00-7:00 am the next morning, self-pollination was performed, and the buds were parchment isolated when the anthers started to release pollen. Twelve hours after pollination, the styles were collected and flash frozen with liquid nitrogen and then stored in a −80˚C freezer.
An EASYspin plant RNA Rapid Extraction Kit from Adlai Biotechnology Co., Ltd. was used to extract the total RNA from the R. rugosa style tissue. Agarose gel electrophoresis and spectrophotometer were used to determine the quality and concentration of the RNA. An EasyScript First-Strand cDNA Synthesis SuperMix Kit from Beijing TransGen Biotech Co., Ltd. was used to synthesize the first-strand cDNA.
According to the reported Glu sequences of Prunus persica, Prunus mume, Malus domestica, Malus hupehensis, and Pyrus bretschneideri, the degenerate primers F1 (5’-TACATYGCBGTWGGAAAYGAA-3’) and R1 (5’- GGCCAACCRSTYTCSGATA-3’) were designed with Primer Premier 5.0. PCR amplification was conducted using the synthesized cDNA in Section 2.2.2 as a template and F1 and R1 as the primers. The reaction system included 1 µL cDNA, 1 µL F1 primer (10 µmol/L), 1 µL R1 primer (10 µmol/L), and 12.5 µL PCR MIX, with ddH2O added to a total volume of 25 µL. The reaction conditions were: 94˚C for 3 min; 94˚C for 30 s, 55˚C for 30 s, and 72˚C for 30 s for a total of 36 cycles; and then extension at 72˚C for 10 min. Next, 1% agarose gel electrophoresis was used to detect the PCR products. The target PCR fragment was recovered with the MiniBEST Agarose Gel DNA Extraction Kit Ver. 3.0 (TaKaRa). The recovered fragment was ligated to the pMD18-T vector and then transformed into E. coli DH5a. The positive clones were selected and sent to BGI for sequencing.
The 3’ RACE specific primers MG1 (5’-GCGCTGCTCGATCCCATTATACGCT-3’) and MG2 (5’- CGATGCCATGTTGGACGCTGTGTAT-3’) and the 5’ RACE specific primers GSP1 (5’- CAGACTTGAAGGAACC-3’), GSP2 (5’-GTGTCGATGGCTGTGGAAAC-3’), and GSP3 (5’- CAGCATTGGAAATTGCGGTT-3’) were all designed with Primer Premier 5.0. Nested PCR was conducted using MG-1, MG-2, and the SMARTer™ RACE cDNA Amplification Kit (Clontech) in order to obtain the 3’- terminal sequence of the target gene. Nested PCR was also conducted using GSP1, GSP2, GSP3, and the 5’ RACE System for Rapid Amplification of cDNA Ends (Version 2.0, Invitrogen) in order to obtain the 5’-ter- minal sequence of the target gene.
DNAstar software was used to splice the middle fragment, the 5’-terminal sequence, and the 3’-terminal sequence in order to obtain the full-length cDNA sequence of the gene. The 5’- and 3’-primers for the spliced sequence were designed with Primer Premier 5 as follows: F2 (5’-GCTCTAGAATGTCTAAATGCAATTCTTCAG-3’) and R2 (5’-CGGGATCCATTGAAATTGATAGGGTATTTTGG-3’). The spliced sequence was amplified using the reverse transcription product of cDNA as a template, and then, it was further validated and verified.
BLASTX (NCBI) was used to study the homology of the nucleotide sequence and the deduced amino acid sequence. The ORF finder (NCBI) was used to search for an open reading frame, and the Conserved Domains database (NCBI) was used to analyze the conserved domains. The ProtParam Tool was used to analyze protein physical and chemical properties. Post Prediction, WOLF PSORT, and SubLocv were used to predict protein sub-cellular localization. Furthermore, ProtScale was used to predict hydrophilic or hydrophobic protein properties. The SignalP 4.0 Server was used to predict the protein signal peptide. The TMHMM Server v2.0 was used to predict the protein transmembrane domain. The NetPhos 2.0 Server was used to predict potential protein phosphorylation sites, and the NetNGlyc 1.0 Server and NetOGlyc 4.0 Server were used to predict potential protein glycosylation sites. ExPaSy-SOPMA was used to predict protein secondary structure. DNAMAN5. 2.2 was used to conduct multiple sequence alignment. The Neighbor-Joining method from Mega5 was used to create the phylogenetic tree.
The cloned middle fragment is 560 bp (
The RrGlu gene has a full length of 1380 bp, an open reading frame of 1041 bp, a 5’ UTR of 131 bp, and a 3’ UTR of 208 bp, encoding 346 amino acids. The derived protein (the RrGlu protein) has a molecular weight of 37.85 kD, an isoelectric point of 9.12, a pfam00332 conserved domain at position 36 - 345, and a conserved β-1,3-glucanase motif LEIVVSDSGWPTAG in the activity center at position 264 - 277. Thus RrGlu protein
belongs to the glycosyl hydrolase family 17. Furthermore, the subcellular localization prediction result indicated that the protein is a secreted protein and is probably located at the vacuole. The hydrophilicity analysis further showed that the overall average hydrophobic index is −0.191, thus indicating a hydrophilic protein. The signal peptide prediction result demonstrated that a signal peptide cleavage site (ADA-QI) exists at position 34 - 35. The transmembrane domain analysis showed that a transmembrane domain exists at position 13 - 32. The phosphorylation site prediction results demonstrated that there are six Ser phosphorylation sites, three Thr phosphorylation sites, and three Tyr phosphorylation sites, thereby providing a reference for the future study of the regulation of gene expression and protein modification. The glycosylation site prediction results showed that there is one N-glycosylation site and five O-glycosylation sites. The secondary structure prediction result demonstrated that there is 31.50% α-helix, 30.92% random coil, 25.14% extended peptide chain, and 12.43% β-corner. The BLAST results showed that the protein shares 72% - 82% homology with the Glu amino acid sequences of Prunus persica (AAL30426.1), Prunus mume (XP 008240769.1), Malus hupehensis (ADR71671.1), Malus domestica (XP 008351633.1), Pyrus bretschneideri (XP 009363234.1), Morus notabilis (XP 010090235.1), Eucalyptus grandis (XP 010056683.1), and Citrus sinensis (CAA03908.1). The multiple sequence alignment result demonstrated that the RrGlu protein and the above plant Glu amino acid sequences all have a pfam00332 conserved domain and a conserved β-1,3-glucanase enzyme active site sequence (LIVM)-X-(LIVMFYW)3-(STAG)- E-(ST)-GWP-(ST)-XG (
Nearly all higher plants contain β-1,3-glucanase genes, and they belong to a large gene family. At present, the Glu genes of dozens of plants have been cloned, and different species have different Glu genes [
β-1,3-glucanase gene is an important class of pathogenesis-related proteins that can be induced in pathological or related conditions. Previous studies showed that the plants of β-1,3-glucanase gene expression level is very low in normal circumstances. The gene expression increased, activity was significantly enhanced when it is induced by abiotic factors such as ethylene, cytokinins, mechanical damage and metal ions as well as subjected to biological factors, for example, pathogens, insect feeding and so on [
fruit ripening, and seed germination. For example, Arabidopsis anther specifically expressed β-1,3-glucanase gene expressed in advance can cause pre-dissolution of the microsporocyte callose wall at anther development process, which affected the microspores further to develop into mature pollen grains, result in male sterility [
In addition, previous studies on the Glu phylogenetic relationships of bananas, cotton, tobacco, grapes, and other plants have demonstrated highly conserved Glu sequences in evolution and consistency between the Glu evolution and the kinship of the plant sources. This study also found that RrGlu shares 72% sequence homology with nine other species, including Prunus persica. Furthermore, RrGlu is closely related to Glu from the same family Malus domestica, Malus hupehensis, and Pyrus bretschneideri, whereas it is relatively distant from Eucalyptus grandis and Citrus sinensis, which are from different families. This indicates that the results of this study are consistent with previous studies.
In this study, the full-length cDNA sequence of β-1,3-glucanase gene was cloned successfully from R. rugosa and named as RrGlu. The bioinformatics characteristics of the RrGlu gene were analyzed, which provided the basis for further research on the function of RrGlu gene.
This work was funded by the National Science Foundation of China (NSFC) (31200524).
YananFu,JuanjuanSun,YanMa,ShutangXing,LanyongZhao,ZongdaXu,XiaoyanYu, (2016) Cloning and Bioinformatics Analysis of Rosa rugosa β-1,3-Glucanase Gene (RrGlu). American Journal of Plant Sciences,07,461-468. doi: 10.4236/ajps.2016.73040