CLONING AND EXPRESSION ANALYSIS OF TRANSCRIPTION FACTOR RrTTG 1 RELATED TO PRICKLE DEVELOPMENT IN ROSE ( Rosa rugosa )

A prickle is an acuminate protuberance formed by the deformation of plant trichomes together with a few cortical cells. It is a type of multicellular eglandular trichome with special morphology, which originates from the phloem but is not connected to the xylem. Rosa rugosa is an important ornamental/commercial plant and an important raw material in the food and perfume industries. However, the firm prickles on its stems are inconvenient to field management, the harvesting of flowers and garden management. The TTG1 transcription factor related to the development of prickle was isolated from R. rugosa in the present study. Its expression patterns in different tissues and varieties were analyzed. Results showed the expression level of the RrTTG1 gene was highest in the leaves, followed by the stems, but was lower in the pericarps and petals. Moreover, the higher expression level of the RrTTG1 gene in all tissues of the ‘Ciguo rose’, as compared with that of the ‘Weihai wild rose’, follows the results of field morphological observation. Therefore, the RrTTG1 transcription factor is likely to regulate the development of rose prickles. This study allows for further discussion on the molecular mechanisms of prickle formation and development in R. rugosa and provides a molecular basis for the cultivation of roses with fewer or no prickles via genetic engineering.


INTRODUCTION
Prickles are widely distributed in many plants (Du, 1999).Each prickle is an acuminate protuberance formed by the deformation of plant trichomes with a few cortical cells.This type of multicellular eglandular trichome has a special morphology, which originates from the phloem and is not connected to the xylem.Most terrestrial plants have trichomes, which are specialized structures on the plant epidermis that develop from epithelial cells.The major function of trichomes is to increase the thickness of the epidermis, reduce heat and water dissipation, and protect plants against the invasion of insects and pathogens or mechanical injury (Szymanski et al., 2000).Common plants with prickles are found in the Rosaceae, Araliaceae, Rutaceae, and Leguminosae families, including the species Rosa rugosa, Rosa chinensis, Rosa multiflora, Zanthoxylum bungeanum, and Aralia chinensis.Prickles are distributed on the stems and branches, as well as the leaves or fruits.Trichomes, especially prickles, in plant parts such as fruits or stems are critical for defending against predation by natural enemies.However, these structures cause inconveniences during cultivation, management, harvesting, transportation and processing.The prickles of plants have become the new focus of research with the development of the horticulture industry.The cultivation of new plant varieties with fewer or no trichomes or prickles can promote the production, processing and utilization of such plants, as well as increase the economic benefits and enrich plant germplasm resources.
Great progress has been made in the study of the genetic regulatory mechanisms of plant trichome development, especially in Arabidopsis thaliana.TTG1 (TRANSPARENT TESTA GLABRA1, WD40) is an important transcription factor that controls the de-velopment of trichomes in A. thaliana (Walker et al., 1999).Its mutation will cause almost complete loss of trichomes during the early developmental stages.Studies of the cloned TTG1 gene likewise prove its importance.Guan (2008) designed the primers from the TTG1 gene sequences of Gossypium hirsutum and A. thaliana to perform homologous cloning.The CsTTG1 gene that is related to the formation of prickles in cucumbers was isloated.RT-PCR analysis indicated that this gene was expressed in important organs and meristematic tissues.Functional complementation assays verified the complementation between the CsTTG1 gene and the AtTTG1 mutant, thereby suggesting their functional homology.Dressel and Hemleben (2009) cloned the TTG1 gene using wild G. hirsutum and its white-flowered trichomefree mutant.Their results found a point mutation on the TTG1 nucleotide sequence of the mutant, which caused the substitution of tryptophan to arginine, thereby directly influencing the function of the WD gene.This mutation is believed to cause the loss of function in the regulatory factor, which interferes with the formation of trichomes and biosynthesis of anthocyanin as well as the development of pigments and seed coats.Wang (2009) designed primers based on the AtTTG1 gene that regulates the development of trichomes of A. thaliana and cloned the homologous gene NtTTG1 in tobacco trichomes.Yeast twohybrid assays indicated the interaction between the NtTTG1 gene product and the elicitor ParA1 secreted by oomycetes.Additionally, this group cloned a novel NtTTG1-like gene of tobacco via RT-PCR technology conjugated with RACE technology, and obtained plants with the silenced NtTTG1-like gene.Their work has laid the foundation for further study of the development and functions of tobacco trichomes.
The mechanism of trichome development of A. thaliana is similar with that of other plants, but some differences still exist.Therefore, these plants need to be further studied with regard to their genetic regulation of trichome development, especially the important economic crops.Several researchers have recently increased their attention on the morphological structure, histochemical features, origin and genetic pattern of prickles in Rosaceae species.(Asano et al., 2008;Kellogg et al., 2011;Li et al., 2012;Rajapakse et al., 2001).However, the study of rosaceous plants is insufficient with respect to their prickle development.
R. rugosa is a deciduous shrub of the genus Rosa in the Rosaceae family.It is one of the important ornamental flowers in the world because of its graceful flower shape, bright color and redolent odor.It is also an important raw material in the food and perfume industries.However, the firm prickles on the stems of these plants causes inconveniences in the field management, harvesting of flowers and garden management.A R. rugosa variety with fewer or no prickles would greatly improve the efficiency of R. rugosa field-management and harvesting.In the present study, the TTG1 transcription factor related to the development of prickles was isolated from R. rugosa 'Fenzizhi' (named the 'Fenzizhi rose'), which is a widely cultivated variety with pink flowers and beautiful branches in China, but it does not bear fruit.The expression patterns of the TTG1gene in different tissues and varieties were also analyzed.This work paves the way for further discussion on the molecular mechanisms of the formation and development of prickles in R. rugosa and provides a molecular basis for the cultivation of thornless roses via genetic engineering.

Plant materials
Tender leaves of 'Fenzizhi rose' were randomly collected and immediately placed in liquid nitrogen for gene cloning.The samples were brought back to the laboratory and stored at -80°C.
To detect the expression levels of the TTG1 gene, three plants were randomly selected for sampling, and the petals, sepals, leaves, and stems from one branch of the 'Fenzizhi rose' were sampled (Fig. 1A).Meanwhile, the stem epidermis was quickly separated from the xylem with a sharp blade, immediately frozen in liquid nitrogen, and stored at -80°C.
The other two varieties, wild R. rugosa from Weihai city, Shandong Province (named the 'Weihai wild rose') and R. rugosa 'Ciguo' (named the 'Ciguo rose'), which are different with regard to prickle density, were selected to verify the expression patterns of the TTG1 gene.Three plants of each variety were randomly selected for sampling, and the tissues of leaves, stems and fruit peels with obvious differences in the density of prickles were sampled (Fig. 1B).The stem epidermis was quickly separated from the xylem and preserved.The pericarp was likewise rapidly separated from the fruit.Both the stem epidermis and pericarp were immediately frozen in liquid nitrogen and stored at -80°C.

RNA extraction and purification
Total RnA was isolated based on a modified CTAB method (Zhao et al., 2011).RnA samples were treated with Dnase from the Dnase I kit (TaKaRa, Japan) following the manufacturer's guidelines before reverse transcription.The cDnA product was then quantified with a spectrophotometer (Eppendorf, Germany) at 230, 260, and 280 nm.

Isolation of TTG1 transcription factor
The TTG1 transcription factor related to prickle development in R. rugosa was isolated following an experimental system established in our laboratory (Feng et al., 2014).

3ʹ RACE
Total RnA of the leaf tissue (1 mg) was used to synthesize the first strand cDnA based on the manufacturer's instructions of the 3ʹ full RACE Core Set ver.2.0 (TaKaRa, Japan).The 3ʹ ends of genes were amplified in two rounds of PCR with the gene-specific primers (Table 1).These primers were designed according to the reported TTG1 gene of other plants from GenBank.The first round of PCR was performed by initially denaturing the cDnA at 94°C for 3 min, followed by 20 cycles of amplification (94°C for 30 s, 55°C for 30 s, 72°C for 120 s) and a final extension at 72°C for 10 min.The product of the first round of PCR was used as the template of the second nested PCR amplification, and 30 cycles were run under conditions similar to the first round of PCR.

5ʹ RACE
Total RnA of leaf tissue (1 mg) was used to synthesize the first strand cDnA according to the manufacturer's instructions of the SMARTer TM RACE cDnA Amplification Kit User Manual (Clontech Laboratories, Inc., TAKARA).The gene-specific primers were designed and synthesized following the sequences of the abovementioned 3ʹ ends (Table 1).Touchdown PCR was then performed with the following program: 5 cycles of 30 s at 94°C and 90 s at 72°C, followed by 5 cycles of 30 s at 94°C, 30 s at 70°C, and 1 min at 72°C, with the final 30 cycles of 30 s at 94°C, 30 s at 68°C, and 120 s at 72°C.

Purification, cloning, and sequencing
The isolated PCR products were recovered from 1% agarose gels, purified using the TaKaRa Agarose Gel DnA Purification Kit ver.2.0 (Japan), cloned into the PMD18-T vector (TaKaRa, Japan), and used to transformed competent Escherichia coli DH5a cells (Trans, China).The recombinant plasmids were identified with the restriction enzymes BamHI and HindIII (Ta-KaRa, Japan).The positive clones were then sequenced by the Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.(Shanghai, China).

Sequence analysis
The open reading frame (ORF) fragments amplified by PCR were spliced using DnAMAn software.The nucleotide sequences and the derived amino acid sequences were analyzed for sequence identity with BLASTn and BLASTp in the nCBI database.Prot-Param was used to analyze the molecular weight, isoelectric point and amino acid composition of the encoded proteins.ProtScale was used to predict the hydrophilic or hydrophobic nature of these proteins.The TMHMM 2.0 Server was used to predict the membrane-spanning domain of the protein.The DSC program was used to predict the secondary structure of the encoded protein.DnAMAn software was used to construct the phylogenetic tree.

Gene expression analysis
The relative mRnA expression of RrTTG1 was analyzed by real-time quantitative RT-PCR with the BIO-RAD CFX96 TM Real-Time System (C1000 TM Thermal Cycler; Bio-Rad, USA).The cDnA was synthesized from 1 μg RnA using the PrimeScript® RT reagent Kit with gDnA Eraser (TaKaRa, Japan).The Rosa hybrid α-tubulin subunit actin gene (GenBank accession no.AF394915.1.Forward primer (5ʹ-3ʹ): TGAGGCCATT-TACGACAT; Reverse primer (5ʹ-3ʹ): AGATCACAG-GAGCATAGGAG) was used as a constitutively expressed internal control during the real-time qRT-PCR analysis.PCR was performed with the gene-specific forward primer (5ʹ-3ʹ): ACATCCTCGCCTCTTCCG and reverse primer (5ʹ-3ʹ): GGGCTCGATCTCGTTC-CA. Quantitative real-time PCR experiments were performed according to the manufacturer's instructions for the SYBR® Premix Ex Taq TM kit (Perfect Real Time; TaKaRa, Japan).Each PCR reaction contained 12.5 μl of 2× SYBR Premix Ex Taq TM , 0.5 μl of 50× ROX Reference Dye II, 2 μl of the cDnA solution as a template, 1 μl mixed solution of the target gene primers, and 9 μl ddH 2 O in a total volume of 25 μl.The amplification had initial incubation steps at 50°C for 2 min and 95°C for 5 min, followed by 40 cycles of 15 s at 95°C, 15 s at 53°C and 40 s at 72°C.The expression level of each gene was calculated with the 2 -ΔΔCt comparative threshold cycle (Ct) method (Schmittgen and Livak, 2008).The Ct values were computed by Bio-Rad CFX Manager v1.6.541.1028software.All samples were analyzed thrice, and all data were presented as the average value of three replicates with the corresponding standard deviation (SD).

Isolation and sequence analysis of RrTTG1 transcription factor from R. rugosa
The RnA extracted from the leaves of 'Fenzizhi rose' was used as the template to amplify the full-length cDnA sequence of the RrTTG1 transcription factor using the RACE method (GenBank accession num-ber: JX878895 ProtParam analysis of the amino acids encoded by the RrTTG1 gene predicted that its molecular weight was 38.71 kDa and its isoelectric point was 5.00.The protein product was composed of 13.01%acidic, 11.27% basic, 44.80% hydrophobic, 21.38% charged and 30.92% polar amino acids.ProtScale analysis indicated that the hydrophilic amino acids were uniformly distributed among the amino acids encoded by the RrTTG1 gene, and their number was greater than that of hydrophobic amino acids.Therefore, the RrTTG1-encoded protein was inferred to be a hydrophilic protein.The prediction by TMHMM 2.0 Server indicated that the RrTTG1-encoded protein did not have a membrane-spanning domain.The DSC prediction of the secondary structure of the RrTTG1 protein indicated that 41.04% of the protein was composed of extended strands, whereas 58.96% were random coils.

Phylogenetic analysis of RrTTG1 transcription factor and homology analysis of its encoded proteins
The phylogenetic relationship of RrTTG1 gene to the TTG1 genes of 12 other plants is illustrated in Fig. 2. The phylogenetic relationship of the 13 species essentially conformed to the original taxonomic groups.The RrTTG1 gene is most similar to the strawberry (Fragaria × ananassa).
The coding sequences of TTG1 genes from different species were aligned using the DnAMAn software.The results indicated that the amino acid sequences encoded by the TTG1 gene of 13 species had a consistent length.Multiple sequence alignment revealed the high conservation of the TTG1 genes.The protein sequences encoded by the different TTG1 genes all contained four WD40 repeats.InterProScan was employed to further search for the structural domain of the WD40 repeat in the RrTTG1 gene.Its results similarly identified four WD40 repeats in RrTTG1.

RrTTG1 gene expression pattern in different tissues of the 'Fenzizhi rose'
The 'Fenzizhi rose' was selected to explore the expression characteristics of the RrTTG1 gene.The expression levels of the RrTTG1 gene in different plant tissues were detected with real-time PCR.The results indicated that the RrTTG1 gene is clearly differently expressed in various plant tissues; the expression levels were highest in the leaves, followed by the sepals and then the petals (Fig. 3).

Expression analysis of the RrTTG1 gene in different varieties of R. rugosa
To investigate the expression characteristics of the RrTTG1 gene in different varieties of R. rugosa, two thorny varieties, 'Weihai wild rose' and 'Ciguo rose' , were sampled for gene analysis.The results from the two varieties revealed the consistent variation of RrTTG1 gene expression in different tissues.That is, the expression was highest in the leaves, followed by the stems and then the pericarps.The expression level of RrTTG1 was higher in the 'Ciguo rose' compared with the 'Weihai wild rose' (Fig. 4).

DISCUSSION
The TTG1 gene is a member of the WD40-repeat protein family; this gene was isolated from A. thaliana mutants by map-based cloning.Among the developmental and biosynthetic pathways of plants, the TTG1 gene mainly regulates the formation of leaf trichomes, stems and roots, the development of the seed coat and its pigments, and the biosynthesis of flavonoids and anthocyanidins (Ramsay and Glover, 2005).The stem, leaf epidermis and subepidermal layer of the TTG1 mutant do not contain anthocyanidins (Larkin et al., 1994).Its seed coat cannot synthesize anthocyanidins, which makes the testa transparent.Moreover, the TTG1 mutant has fewer or no trichomes because of gene malfunction.These physiological changes in the TTG1 mutant suggested the extensive involvement of the TTG1 gene in several regulatory pathways (Broun, 2005).The TTG1 gene was isolated from R. rugosa by RACE technology in this paper.The analysis of its structural characteristics revealed four WD40 repeats in the protein sequence encoded by this gene.This structural domain contains a small peptide of approximately 40 amino acids, which usually begins with Gly-His and ends with Trp-Asp (WD) (Simon et al., 1991).The WD40-repeat proteins constitute a large protein family with diversified cellular, biological and biochemical functions.Specifically, these genes participate in light signal reception and transduction, cell division and cytoplasm flow, flower development, blooming, cell migration and programmed cell death (Smith et al., 1999).A search for homologous species in the nCBI databases revealed the amino acid homology of the RrTTG1 gene with the TTG1 genes of cucumber (Guan, 2008), Arabidopsis (Walker et al., 1999) and tobacco (Wang, 2009), which reached up to 81%, 79%, and 79%, respectively.The TTG1 genes of the three species all participate in the regulation of trichome development, which suggests that the RrTTG1 gene may have similar functions.
Morphological observation showed that the trichomes grow on the leaves and sepals of R. rugosa in their original morphology, and some trichomes on stems and fruit peels are specialized into prickles.Although they are very short, the trichomes densely cover the leaves of the 'Fenzizhi rose' , 'Weihai wild rose' and 'Ciguo rose' , especially on the reverse side of the leaves.They are also denser than the stem prickles of 'Weihai wild rose' and 'Ciguo rose' .The fruit peels of the 'Ciguo rose' have more prickles, whereas those of the 'Weihai wild rose' only have a few prickles (Fig. 1B).Real-time PCR analysis showed that the RrTTG1 gene was expressed in different varieties of R. rugosa  and in different plant organs.The expression level was highest in the leaves, followed by the stems, but was lower in the pericarps and petals.The expression level of the RrTTG1 gene in all tissues of the 'Ciguo rose' was higher than in the 'Weihai wild rose' (Fig. 4).These results were consistent with the results of morphological observation.Therefore, the RrTTG1 gene is likely to influence and regulate the formation of rose prickles.However, its role as a critical gene and the existence of other genes that regulate the development of rose prickles need to be verified by additional gene function analyses.

Fig. 4 .
Fig. 4. Expression level of RrTTG1 gene in different tissues of 'Weihai wild rose' and 'Ciguo rose' by real-time PCR.

Fig. 3 .
Fig. 3. Expression level of RrTTG1 gene in different tissues of 'Fenzizhi rose' by real-time PCR.

Table 1 .
Primers used for RrTTG1 transcription factor isolation from R. rugosa