Cloning and Bioinformatics Analysis of Rosa rugosa β-1,3-Glucanase Gene (RrGlu)

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.

Cloning and Bioinformatics Analysis of <i>Rosa rugose S</i>Locus F-Box Gene (<i>RrSLF</i>)

In order to reveal the phenomenon of R. rugose pollination incompatibility, the full-length cDNA sequence of S Locus F-box Gene was cloned for the first time from the pollen of Rosa rugose “Zilong wochi” with RT-PCR and RACE methods and named as RrSLF. The full-length cDNA is 1236 bp with an open reading frame of 1122 bp, encoding 343 amino acids. The derived protein has a molecular weight of 43.7 kD, a calculated pI of 6.24, an F-box conserved domain at position 343 - 741, and belongs to F-box family. The derived protein is a Hydrophobicity protein secreted into the cytoplasm. There is no transmembrane domain and no signal peptide cleavage site, twenty-one Ser phosphorylation sites, seven Thr phosphorylation sites, seven Tyr phosphorylation sites, two N-glycosylation sites, and no O-glycosylation sites. There are 22.25% α-helixes, 31.37% random coil, 32.17% extended peptide chain, and 14.21% β-corner structure. This protein and the SFB/SLF protein from Rosaceae Prunus fruit, including Prunus speciose, share a sequence homology of 59% - 61%;all of the proteins contain an F-box conserved domain, two hypervariable regions HVa, HVb, and two variable regions V1, V2. Furthermore, their phylogenetic relationships are consistent with their traditional classifications. These results were meaningful to reveal the molecular mechanism of Rosa rugose pollination incompatibility and improve the theory and techniques of breeding ornamental Rosa rugose.

Cloning and Bioinformatics Analysis of <i>Rosa rugosa</i>TFL1 Gene (<i>RrTFL1</i>)

In order to determine if the TFL1 is related with the continuous flowering phenotype of wild Rosa rugosa from Muping, the full-length cDNA sequence of TFL1 Gene was cloned for the first time from the flower buds of wild Rosa rugosafrom Muping with RT-PCR and RACE methods and named as RrTFL1. The full-length cDNA is 973 bp with an open reading frame of 519 bp, encoding 172 amino acids. The derived protein has a molecular weight of 19.48 kD, a calculated pI of 9.13, a c100227 conserved domain at position 1-172, and belongs to PEBP family. The derived protein is a Hydrophilic protein secreted into the cytoplasmic. There is no transmembrane domain and no signal peptide cleavage site, five Ser phosphorylation sites, seven Thr phosphorylation sites, three Tyr phosphorylation sites, one O-glycosylation site, and no N-glycosylation sites. There are 24.42% α-helixes, 36.63% random coil, 27.91% extended peptide chain, and 11.05% β-corner structure. This protein and the TFL1 protein from Rosaceae plants, including Rosa chinensis, share a sequence homology of 87% - 96%. All of the proteins contain a c100227 conserved domain, two highly conserved modules D-P-D-x-P, G-x-H-R, and two functional sites His, Asp. Furthermore, their phylogenetic relationships are consistent with their traditional classifications. These results not only laid a foundation for further researching the expression and function of RrTFL1, but also cultivating new varieties of R. rugosawhich can flower continuously by gene engineering.

A Study on the Aseptic Germination Method for <i>Rosa rugose</i>Seeds

In order to find a method to break dormancy of Rosa rugose seeds and interspecific hybrid seeds between Rosa rugose and Rosa hybrid quickly, and accelerate the breeding process of interspecific hybridization between Rosa rugose and Rosa hybrid, the influence of concentrated acid, seed maturity, GA3 (gibberellin) and low temperature (4°C) on seed germination of Rosa rugose from Muping was researched under aseptic condition. The results showed that aseptic germination can significantly shorten the germination time of Rosa rugose seeds and raise its germination ratio. Before inoculation, concentrated acid treatment greatly increased the germination rate and reduced the contamination rate of the seeds. The higher the degree of maturity of seeds is, the lower the germination rate would be, and the best time for seed to aseptic germination is 60 d after pollination. The addition of GA3 in 1/2MS medium could promote seeds germination better, and when the concentration of GA3 was 0.15 mg/L, the seed germination ratio was the highest;the germination time decreased and the seed germination ratio increased gradually as the treatment time at 4°C lasted longer.