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Cloning and Expression Analysis of RrMYB113 Gene Related to Anthocyanin Biosynthesis in Rosa rugose

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American Journal of Plant Sciences 2018年第4期9卷 701-710页

作者： Kai Zou Yang Wang Mingyuan zhao lanyong zhao Zongda Xu Forestry College of Shandong Agricultural University Tai’an China

Anthocyanin is one of water-soluble natural pigments widely existing in flowers, fruits, stems, leaves and seeds of plants, and it is the major factor conferring pink or red to the petals of Rosa rugose. MYB TFs play an important role in the anthocyanin synthesis in plants. This work aimed to clone the MYB gene related to anthocyanin synthesis in the petals of Rosa rugose, and explore the relationship between them to lay a good foundation for gene engineering improvement of R. rugose. Based on the transcriptional data, a full-length cDNA sequence of MYB Gene, RrMYB113 (GenBank accession Nos MG720012), was cloned at the first time from the petals of Rosa rugose “Zi zhi” with RT-PCR and RACE methods. The full-length cDNA is 885 bp with an open reading frame of 654 bp, encoding 216 amino acids. The derived RrMYB113 protein has a molecular weight of 25,297.64 Da, a calculated pI of 9.61, a R2R3-MYB domain and bHLH binding domain, and it also has the signature motifs ((A/S/G)NDV and KPRPR(T/S)), thus belonging to Sg6 R2R3-MYB subfamily. In the secondary structure of RrMYB113 protein, there is 37.04% α-helix, 39.81% random coil, 14.81% extended peptide chain, and 8.33% β-corner. There is no transmembrane domain and no signal peptide cleavage site, seventeen Ser phosphorylation sites, fifteen Thr phosphorylation sites, four Tyr phosphorylation sites, and no O-glycosylation sites. The expression of RrMYB113 increased with the color deepening in petals, and it expressed at a higher level in petals than in other tissues of R. rugose “Zi zhi”. These results are meaningful to reveal that RrMYB113 might be an important regulator in anthocyanin biosynthesis and coloration in the petals of R. rugose.

关键词： Rosa rugose Anthocyanin R2R3-MYB Gene Expression

Cloning and Expression of Anthocyanin Biosynthesis Related Gene RrMYB6 in Rosa rugosa

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Agricultural Sciences 2018年第3期9卷 374-383页

作者： Kai Zou Yang Wang Mingyuan zhao lanyong zhao Zongda Xu Forestry College of Shandong Agricultural University Tai’an China

R2R3-MYB transcription factor plays an important role in plant anthocyanin synthesis. Based on the transcriptional database of Rosa rugosa, one MYB transcription factor related to floral color, RrMYB6, was cloned. By using bioinformatics analysis method, cloning MYB gene and analyzing its function in anthocyanin biosynthesis regulation, we hope to lay a solid foundation for new color variety breeding of R. rugosa. Using the R. rugosa “Zi zhi” as the material, we obtained the total length of cDNA of RrMYB6 by RT-PCR and RACE. By analyzing its bioinformatics, we found that the formula of the protein was C1491H2368N452O470S17, molecular weight was 34690.97 Da, the theoretical pI was 8.74. In addition, it belonged to unstable protein with an unstable index at 50.59, and it was also a hydrophilic protein with the total average hydrophobic index at -0.847. In the secondary structure of RrMYB6 protein, the Alpha helix accounted for 32.35%, random coil was 47.39%, extended strand was 11.11%, and beta turn was 9.15%. The sequence analysis showed that RrMYB6 had a typical R2R3-MYB domain and bHLH binding domain, and it also had an N1, C1, C2 inhibitory motif, belonging to the Sg4 subfamily MYB protein. What’s more, evolutionary analysis indicated that the RrMYB6 protein was closely related with the MYB protein in Rosacea family, while it was far from those in other families. The expression analysis showed that RrMYB6 protein decreased with the color of petals deeping, and its expression was the lowest in the petals while the highest in stamens. According to the above results, it was speculated that RrMYB6 was involved in regulating the anthocyanin synthesis of R. rugosa, which belonged to negative regulatory mechanism.

关键词： Rosa rugosa Anthocyanin R2R3-MYB Gene Expression

Cloning and Bioinformatics Analysis of Rosa rugosaTFL1 Gene (RrTFL1)

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Advances in Bioscience and Biotechnology 2017年第6期8卷 185-194页

作者： Dandan zhao Xiao Wei Leilei Wang lanyong zhao Xiaoyan Yu Forestry College of Shandong Agricultural University Tai’an China Shandong Provincial Research Center of Landscape Engineering Technology for Urban and Rural Tai’an China Agricultural Company Shengda Group Dongying China Logistic Department of Harbin Engineering University Harbin China

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.

关键词： Rosa rugosa TFL1 Gene Clone Biological Analysis

Cloning and Expression Analysis of RrG-Beta1 Gene Related to Anthocyanin Biosynthesis in Rosa rugose

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American Journal of Plant Sciences 2018年第11期9卷 2244-2255页

作者： Yenan Wang Mingyuan zhao Xu Han lanyong zhao Zongda Xu College of Forestry Shandong Agricultural University Taian China

As an important signal transduction protein, G protein beta subunit gene encoded by oligonucleotides plays an important role in many physiological, biochemical and environmental stresses in plants. In order to understand the action mode of G protein beta subunit gene, this paper cloned a Wd40 gene related to G protein beta subunit gene, named RrG-beta1, based on the R. rugose—transcriptome data, using Rosa rugose “Zi zhi” as experimental materials. The full length of cDNA of the gene was obtained by RT-PCR and RACE methods. The total length of this gene is 981 bp, and it encodes 326 amino acids. After bioinformatics analysis, the molecular formula C1601H2520N450O486S11 was predicted;the relative molecular weight was 36,201.00 Da;the theoretical isoelectric point PI value was 6.71;and its instability index was 30.44. The total average hydrophobic index was -0.847. In the secondary structure of RrG-beta1 protein, there are 17 α-helix, 131 Random coil, and 141 extended peptide chain. Gene Bank Blast results showed that the amino acid sequence encoded by RrG-beta1 was more than 90% homologous with the beta-like protein of Rosa chinensis, Fragaria, Malus, Pyrus, Prunus, Arabidopsis and tobacco, so it can be inferred that the RrG-beta1 Gene is guanine nucleotide-binding protein subunit beta-like protein. Fluorescence quantitative expression analysis of RrG-beta1 protein decreased with the development of flower color, and it was speculated that it could exert negative regulation effect on flower color. The leaf expression was highest in the tissue part, so it was inferred that the signal was transmitted through the stoma on the leaf.

关键词： Rosa rugose Gβ Signal Transduction Drought Stress Gene Expression

Cloning and Expression Analysis of TTG1 Gene Related to Rosa rugosaTrichomes Formation

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American Journal of Plant Sciences 2019年第2期10卷 265-275页

作者： Yu Wang Mingyuan zhao Zongda Xu lanyong zhao Xu Han College of Forestry Shandong Agricultural University Tai’an China

The TTG1 transcription factor plays an important role in the formation of plant trichomes. Based on the R. rugosa transcriptome data, this study cloned a R. rugosa TTG1 gene, named RrTTG1, and carried out bioinformatics analysis and fluorescence quantitative analysis to explore the relationship between TTG1 gene and R. rugosa trichomes formation, in order to lay a good foundation to cultivate a thornless plant in the family Rosaceae. In this experiment, six hybrid cultivars of R. rugosa “Zizhi”, R. rugosa “Xizi”, R. rugosa “Tang fen”, R. rugosa “Hun chun”, R. rugosa “Zi long wo chi” and R. rugosa “Tian e huang” were used as experimental materials, and the cDNA full length of this gene was obtained by RT-PCR and RACE, and the full length of the cDNA was 1348 bp. After bioinformatics analysis, it is predicted that its molecular formula is C1723H2661N465O529S12, the molecular weight is 38.71 KB, and the isoelectric point is 5.00. Its instability index is 54.30, which belongs to unstable protein;and its hydrophilic amino acid distribution is relatively uniform, and the amount is larger than hydrophobic amino acid, which belongs to hydrophilic protein. Phylogenetic tree was constructed for the TTG1 gene. Evolutionary analysis indicated that RrTTG1 is closely related to the TTG1 protein of Rosaceae family, and has a close relationship with other families. The expression analysis showed that the expression of RrTTG1 protein was negatively correlated with the trichome content of R. rugosa stems and leaves. The expression levels of the three spiny varieties of R. rugosa “Hun chun”, R. rugosa “Xizi” and R. rugosa “Zi long wo chi” were lower, and the expressions of the three less thorn varieties of R. rugosa “Zizhi”, R. rugosa “Tian e huang” and R. rugosa “Tang fen” were higher. According to the above results, it was speculated that RrTTG1 is involved in the synthesis of R. rugosa trichomes and belongs to the negative regulation mechanism.

关键词： R. rugosa Trichome RrTTG1 Gene Expression

Cloning and Expression Analysis of RrRUP2 Gene Related to Photomorphogenesis Biosynthesis in Rosa rugosa

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American Journal of Plant Sciences 2018年第13期9卷 2533-2544页

作者： Yenan Wang Mingyuan zhao Xu Han lanyong zhao Zongda Xu College of Forestry Shandong Agricultural University Taian China

Plants have evolved and perfected a series of light receptors to feel the light at different bands and regulate the expression, modification and interaction of related genes in plants through signal transduction. So far, many photoreceptors have been identified in plants, UVR8 has recently been identified as a receptor for UV-B light. This paper cloned a WD40 gene related to UVR8 protein subunit, named RrRUP2, based on the Rosa rugose transcriptome data, using Rosa rugose “Zi zhi” as experimental materials. The full length of cDNA of the gene was obtained by RT-PCR and RACE methods. The total length of this gene is 1173 bp, and it encodes 390 amino acids. After bioinformatics analysis, the molecular formula C3415H5659N1173O1434S313 was predicted;the relative molecular weight was 96129.27 Da;the theoretical isoelectric point PI value was 5.00;and its instability index was 47.06. The total average hydrophobic index was 0.750. In the secondary structure of RrRUP2 protein, there are 10 α-helix, 45 β-helix, 181 Random coil, and 154 Extended strand. Gene Bank Blast results showed that the amino acid sequence encoded by RrRUP2 was more than 90% homologous with the RUP2 protein of Rosa chinensis, Fragaria, Malus, Pyrus, Prunus, Juglans, Arabidopsis and Tobacco, so it can be inferred that the RrRUP2 gene is a WD repeat-containing protein. Regarding to fluorescence quantitative expression analysis of RrRUP2, we find its experssion pattern is corresponded with the accumulation of anthocyanins.

关键词： Rosa rugose UV-B UVR8 RUP2 Photomorphogenesis Anthocyanin Gene Expression

Establishment of Virus-Induced Gene Silencing (VIGS) System in Perennial RosaPlants under Field Conditions

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Natural Science 2018年第9期10卷 319-328页

作者： Xiaoming Sui Shikuan Yan Xu Han Mingyuan zhao lanyong zhao Zongda Xu College of Forestry Shandong Agricultural University Tai’an China Shandong Jing Liang Industry and Trade Co. Ltd. Jinan China

Virus-induced gene silencing (VIGS) technique, which is developed in recent years, is a rapid identification of plant gene function from reverse genetics. It is a manifestation of post-transcriptional gene silencing mechanism. Compared with the traditional transgenic technology, VIGS is a transient expression system, which can achieve good results in a short time. At present, it is widely used to study the function of plant genes, but most of them are model plants, and the experiments are carried out always in the indoor environment with controlled light and temperature conditions. In this study, we creatively provided a method to establish VIGS system using perennial Rosa plants as experimental materials under field conditions. The recombinant virus vector was constructed with RrGT1 gene as reporter gene and modified TRV-GFP virus as vector, and the perennial R. rugosa “Zizhi” and R. davurica were used as experimental verification materials. According to the growth conditions of Rosa plants, the natural environment in the field and the optimal conditions for the occurrence of VIGS, the technical problems such as the confirmation of the inoculation period, the preparation of the infective fluid, the inoculation technology of the virus vector and the light and temperature conditions of plant materials cultured after inoculation were solved one by one. When the RrGT1 gene was silenced, the Rosa plants showed a pale petal color phenotype. By detection, it was found that the expression of endogenous RrGT1 gene was significantly down-regulated, and the content of all anthocyanins also decreased significantly. Therefore, we believed that the attempt to establish VIGS system in perennial Rosa plants under field conditions was very successful.

关键词： VIGS Rosa RrGT1 Gene Field Conditions Anthocyanin

Protection and Development Strategies of Urban Ancient Cultural Remains and Its Surroundings——A Case Study of Mountain Haoli in Tai'an City

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Journal of Landscape Research 2015年第2期7卷 67-70页

作者： FAN Yong MIAO Botao LV Guiju zhao lanyong Shandong Agricultural University Highway Bureau of Weifang City Shandong University of Art and Design

Urban historical and cultural heritage has been gradually formed in the long history, it is a witness of the city development history and a realistic carrier of the local civilization. However, protection of urban cultural heritage has been long neglected, with the rapid economic development and urbanization process, a lot of urban cultural heritage that bears important historical and cultural value had gone forever. Ancient cultural remains(ACRs) are an important type of urban cultural heritages, the renaissance of ACRs means the revival of the whole city, how to make ACRs in gradual decline state to be protected with proper planning and renewed vigor has great signifi cance to the urban development. Taking protection and development planning and design of ACRs in Mountain Haoli and its surroundings for example, this study put forward the objectives and principles of ACRs protection and development, extending and deepening the protection methods and renewal strategies, so as to achieve overall cultural, economic and spatial revival of urban ACRs, and sustainable development of ACRs.

关键词： Ancient cultural remains(ACRs) Protection and development Strategy

Cloning and Expression of One Anthocyanin-Related R2R3-MYB Gene in Rosa rugosa

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American Journal of Plant Sciences 2018年第10期9卷 2020-2032页

作者： Yang Wang Xiaoming Sui Mingyuan zhao Xu Han lanyong zhao Zongda Xu Forestry College of Shandong Agricultural University Taian China

Based on the transcriptome of Rosa rugosa, one anthocyanin-promoting R2R3-MYB gene, RrMYB10.1 (Accession Nos:MH717244), was cloned from the petals of Rosa rugosa ‘Zizhi’. Sequence analysis results showed that RrMYB10.1 had a full length opening reading frame of 747bp, encoding 249 amino acids. Sequence analysis revealed that RrMYB10.1 contained the conserved R2R3-MYB domain, two atypical anthocyanin-promoting motifs and a conserved amino acid signature for the interaction with bHLH protein. The results of phylogenic tree revealed that RrMYB10.1 showed high homology with other anthocyanin-promoting proteins in Rosacea, and sharing the highest identity (98.39%) with RhMYB10. RT-PCR results showed that RrMYB10.1 was mainly expressed in petals among various tissues and expressed significantly higher in petals in bud stage than in opening period. To sum up, these results showed that RrMYN10.1 may play a key role in regulating anthocyanin concentration, thus providing a certain foundation on regulating flower color formation in Rosa rugosa.

关键词： Rosa rugosa Anthocyanin R2R3-MYB Gene Expression