Cloning and Primary Functional Analysis of PeGRF1 in Phyllostachys edulis

To explore the role of Growth regulating-factor (GRF) in the growth of bamboo shoots, one GRF gene was isolated from moso bamboo ( Phyllostachys edulis ) by RT-PCR. Bioinformatics methods were used for gene sequence analysis, and the expression pattern of the GRF gene in bamboo shoots at different development stages was analyzed by qRT-PCR. Simultaneously, ectopic expression in  Arabidopsis  was conducted to validate the gene function. The result showed that the isolated gene from moso bamboo was 1 164 bp, encoding 387 amino acids, which was named as  PeGRF1 . Protein sequence analysis showed that PeGRF1 had the complete typical domains (WRC and QLQ) of GRF family. The phylogenetic analysis demonstrated that PeGRF1 were clustered closer to the GRFs of monocotyledonous plants such as  Oryza sativa , indicating they had close relationship.  PeGRF1  expressed predominantly in young bamboo shoots. Meanwhile, the expression level of  PeGRF1  was significantly higher during rapid growth stage than that during slow growth stage of bamboo shoots, and the expression level of  PeGRF1  in the upper and middle of bamboo shoots was significantly higher than that in the base. Overexpression of  PeGRF1  could increase the plant height of transgenic  Arabidopsis . Taken together, our results demonstrated that  PeGRF1  was involved in development of bamboo shoots, which provided references for elucidating the biological functions of GRF genes in bamboo.

The Growth regulating factor (GRF) is a plant-specific transcription factor, and that playing an important role in regulating plant growth and development (Omidbakhshfard et al., 2015;Yuan et al., 2017;He et al., 2018). The OsGRF1 of Oryza sativa is the first member of GRF gene family was identified, and it perform a regulatory role in rice stem growth by GA ( Van der Knaap et al., 2000). At present, more and more GRF gene families have been identified. For example, the Oryza sativa genome harbours at least 12 GRF genes and the Arabidopsis thaliana genome harbours at least 9 GRF genes (Kim et al., 2003;Choi et al., 2004). In recent years, with the continuous publication of plant genome, many non-model plants GRF gene families have also been identified and analyzed, such as Brassica rapa , Citrus sinensis (Liu et al., 2016), Camellia sinensis (Wu et al., 2017) and Brassica juncea (Zhang et al., 2020). GRF proteins consist of two conserved domains, the QLQ and WRC domains, in the N-terminal regions. The QLQ domain acts as a transcriptional co-activator, while the WRC domain comprises a functional nuclear localization signal and a zinc-finger motif that functions in DNA binding, which play an important role in the implementation of biological functions of GRF genes (Cao et al., 2016;He et al., 2018).
Previous studies have shown that the GRF genes are strongly expressed in actively growing and developing tissues, and thus participate in the regulation of the growth and development of plant, such as stem elongation (Van der Knaap et al., 2000), leaf development (Kim et al., 2003), flower development (Lee et al., 2014) and root growth (Bao et al., 2014). Overexpression of AtGRF1, AtGRF2 or AtGRF5 results in larger leaves in Arabidopsis (Kim et al., 2003;Horiguchi et al., 2005); meanwhile, overexpression of OsGRF1 lead to plant height in Arabidopsis ( Van der Knaap et al., 2000). In addition, overexpression of ZmGRF10 results in smaller leaves, decreased cell number and increased cell size (Wu et al., 2014). These results indicated that the function of GRF gene was diverse.
Stem is the main structural of the plant, supporting the above-ground tissues or organs of the plant, and its growth largely effect the size of the plant. In grasses, the stem is composed of nodes and internodes. Bamboo belongs to the subfamily Bambusoideae of the Poaceae family, as an important group of forest, many bamboo species can complete their height growth in the short term. For example, the moso bamboo can grow more than 10 m in 2 months (Yuan et al., 2015), the Dendrocalamus hamiltonii height-growth lasts for about 3 months (Li et al., 2010). In order to explore the mechanism of the rapid growth of bamboo, there are many studies about moso bamboo from various aspects, such as morphological (Cui et al., 2012), physiology and biochemistry (Sun et al., 2018), molecular regulation (Bu et al., 2020). The moso bamboo genome harbours at least 18 GRF genes, and among which at least one GRF1 homologous gene exists (He et al., 2018). Therefore, Based on previous studies, a GRF gene, PeGRF1, was cloned from moso bamboo, and its expression pattern in the rapid growth stage of bamboo shoots was preliminarily analyzed in this study. Meanwhile, ectopic expression in Arabidopsis provided reference for the functional analysis of moso bamboo GRF gene in the future.

Molecular characterization of PeGRF1 in moso bamboo
A cDNA fragment of 1 200 bp was obtained from moso bamboo (Ph. edulis) leaves using the primers, which was consistent with the expected size ( Figure 1). The sequencing results showed that the isolated gene from moso bamboo was 1 164 bp, which was 99.57% consistent with that of PH01087379G0010 in the BambooGDB database. The gene encoded 387 amino acids with a calculated molecular mass of 42.3 kDa and a theoretical isoelectric point of 7.66, named PeGRF1.
The secondary structure of PeGRF1 protein was predicted by SOMPA software, and the results showed that the random coil was about 67.44%, the extended strand was about 11.89%, and the α helix and β turn were about 18.09% and 2.58%, respectively. There are similar in the N and C terminal regions between PeGRF1and OsGRF1 proteins, but it is different in their middle region.

Alignment and phylogenetic analysis of PeGRF1
The deduced amino acid sequences of PeGRF1 were further examined for homology analysis with other known GRF1 sequences using BLASTP program in NCBI. The result showed that PeGRF1 was more than 75% homologous with those of other monocots, such as Oryza sativa, Brachypodium distachyum and Hordeum vulgare. And about 55% homologous with those of other dicots, such as Arabidopsis thaliana, Brassica rapa and Populus trichocarpa. Similar to other GRF, PeGRF1 had the complete typical domains (WRC and QLQ) of GRF family ( Figure 2). To investigate the phylogenetic relationship of GRF1 among different species, a phylogenetic tree was constructed based on the amino acid sequence of PeGRF1 and GRF from other plants. In the phylogenetic tree, GRF from monocots and dicots were clustered into two clades (Figure 3), Furthermore, the PeGRF1 and OsGRF1 were in the same clades, PeGRF1 homology with the OsGRF1 was the highest (84%), indicating that they are closely relationship.

Expression analysis of PeGRF1
The expression patterns of PeGRF1 in different tissues of moso bamboo were analyzed according to the RPKM value of PH01087379G0010 in moso bamboo transcriptome data. It is found that RPKM value of PeGRF1 in leaves, early panicles, advanced panicles, roots, rhizomes, 20-cm and 50-cm bamboo shoots were 2.42, 1.83, 1.52, 4.12, 5.92, 55.33 and 72.14, respectively. This showed that PeGRF1 are expressed in all tissues, however, the expression levels of PeGRF1 are difference in different tissues. PeGRF1 expressed predominantly in young bamboo shoots, such as 20 cm and 50 cm bamboo shoots.
The expression patterns of PeGRF1 in different development stage of bamboo shoots were analyzed using qRT-PCR. The results demonstrated that the expression levels of PeGRF1 increased significantly along with the shoot growth, and reached the highest expression level in 3.0 m bamboo shoots. The expression levels of PeGRF1 in 6.7 m bamboo shoots decreased slightly compared with that of 3.0m bamboo shoots, but it was still significantly higher than that of 0.2 m bamboo shoots ( Figure 4A). Further analysis showed that PeGRF1 had the highest expression in the upper internodes and nodes of 3.0 m and 6.7m bamboo shoots, and it was significantly higher than that of the middle and the basal (Figure 4 B; Figure 4C).

Generation of PeGRF1 transgenic Arabidopsis plants and phenotypic analysis
To investigate whether heterologous expression of PeGRF1 is functional, overexpression vector of PeGRF1 was constructed and transformed into Arabidopsis (Col-0). After hygromycin resistance screening and verified by PCR, two transgenic lines (L4 and L8) were obtained ( Figure 5A). In addition, the transcript levels of PeGRF1 in L1 and L8 transgenic lines were determined using semi quantitative RT-PCR. The results showed that PeGRF1 was successfully introduced into the Arabidopsis genome and expressed successfully ( Figure 5B). It was found that the plant height of the PeGRF1 transgenic plants increased significantly compared with the Arabidopsis (Col-0) ( Figure  6), indicating that the overexpression of PeGRF1 affect the growth of transgenic plants.

Discussions
GRF gene is a plant specific transcription factor gene family, and its related studies are increasing in recent years. In previous research, our teams have identified and analyzed 18 GRF genes from moso bamboo by bioinformatics, meanwhile, which tissue expression pattern also analyzed (He et al., 2018). Based on previous studies, a GRF gene, PeGRF1, was cloned from moso bamboo, with a length of 1 164 bp, encoding 387 amino acids, and belonging to the GRF gene family. According to the results of PeGRF1 homology analysis and phylogenetic relationship, PeGRF1 had high homology and closely relationship with GRF1 from monocots, such as Oryza sativa, Hordeum vulgare, moreover, had low homology with GRF1 from dicot, such as Arabidopsis thaliana, Brassica rapa. This indicates that the evolution of the GRF1 in monocots and dicot plants may be differences.
It has reported that the GRF genes are strongly expressed in actively growing and developing tissues, but weakly expressed in mature tissues or organs (Kim et al., 2003;Liu et al., 2016;He et al., 2018), In other words the expression level of GRF gene in tissues or organs is related to the development stage of that. For example, the expression levels of CsGRF genes were higher in tender leaves and consistently downregulated during tea plant leaf development (Wu et al., 2017). Bamboo shoots are the tender stage of the stem of bamboo, and the most active parts in the growth and development of various tissues or organs of bamboo. In this study, PeGRF1 was mainly expressed Molecular Plant Breeding 2020, Vol.11, No.30, 1-9 http://genbreedpublisher.com/index.php/mpb 6 in bamboo shoots, which was consistent with the expression patterns of other plant GRF genes (Kim et al., 2003;Liu et al., 2016), meaning that PeGRF1 play the roles in the development of moso bamboo shoots.
The expression patterns of PeGRF1 in the 0.2, 1.0, 3.0 and 6.7 m bamboo shoots were analyzed, and found that PeGRF1 higher expressed in the 1.0 and 3.0 m bamboo shoots, which was similar to the expression patterns of GRF genes families. The plant height growth process of moso bamboo can be divided into four stages: the initial stage, the ascending stage, fast growth stage and maturity stage. Bamboo shoots after Unearthed, which growth from the basal to the top internode, and showed the pattern of "slow-fast-slow". The 0.2 m bamboo shoots, which just unearthed and in the initial stage of height growth process, with slow growth and less active of tissues, so the PeGRF1 low expressed. The 1.0, 3.0 and 6.7 m bamboo shoots are in the ascending and fast growth stages, the rapid growth of 1.0 and 3.0 m bamboo shoots mainly caused by the rapid elongation of the basal internode, which have the high activity, and the expression levels of PeGRF1 increased significantly. However, the basal internode of the 6.7 m bamboo shoots has basically completed the height growth, which in the "mature stage", so it activity has decreased. At the same time, PeGRF1 expression decreased. This suggests that PeGRF1 may be involved in the rapid growth of moso bamboo shoots. In addition, at fast growth stages, 3.0 m and 6.7 m bamboo shoots, the growth and development activity of the upper internode of the shoots was significantly higher than that of the middle and basal internode, and the expression level of PeGRF1 in the upper internode was higher than that of the middle and basal internode. The nodes of the bamboo shoots have intercalary meristem, which is an important reason for the elongation of bamboo shoots. Compared with the internodes, the expression level of PeGRF1 in the nodes of bamboo shoots is higher.
GRF transcription factors play an important role in regulating plant growth and development. For example, overexpression of AtGRF1 or AtGRF2 results in larger leaves and cotyledons in Arabidopsis (Kim et al., 2003), Overexpression of BnGRF2 and BrGRF8 in Arabidopsis also leads to larger leaves, and Overexpression of PagGRF15 in poplar leads to larger leaves (Liu et al., 2012;Wang et al., 2014;Zhou et al., 2019). However, overexpression of ZmGRF10 results in smaller leaves by decreased cell number and increased cell size (Wu et al. 2014). This indicating that GRF gene has both positive and negative regulatory functions. In the present study, we found that PeGRF1 affected plant height, and overexpression of PeGRF1 can increase the plant height in Arabidopsis. Combined with the previous results that PeGRF1 expressed predominantly in young bamboo shoots, meanwhile, the expression levels of PeGRF1 were significantly higher during rapid growth stage than that during slow growth stage of bamboo shoots, we deduced that PeGRF1 plays an important role in regulating bamboo shoots growth and development.
In this study, the full-length cDNA of PeGRF1 of moso bamboo was cloned, and its expression pattern in the rapid growth stage of bamboo shoots was preliminarily analyzed. Meanwhile, ectopic expression in Arabidopsis provided reference for the functional analysis of moso bamboo GRF gene in the future.

Plant materials
To examine the expression levels of PeGRF1 in moso bamboo shoots at different development stages, the basal parts of bamboo shoots with different heights (0.2, 1.0, 3.0, and 6.7 m) were collected from the Nanchang Botanical Garden of Jiangxi Academy of Forestry in April to May 2018. In addition, the nodes and internodes of the basal, middle and upper parts with the 3.0 m and 6.7 m moso bamboo shoots were collected, respectively. Each sample was three Biological replicates, frozen immediately in liquid nitrogen and stored at -80℃.

Total RNA Isolation and cDNA synthesis
The total RNA was extracted using the plant total RNA extraction kit (TaKaRa MiniBEST Plant RNA Extraction Kit, TaKaRa, Dalian, China) according to the manufacturer's instructions. High quality total RNA was selected and used for the synthesis of cDNA by a reverse transcription system (PrimeScript™ 1st Strand cDNA Synthesis Kit, TaKaRa, Dalian, China).

Gene isolation and bioinformatics analysis
To identify the putative GRF1 homologue sequence in moso bamboo, the BLAST search was performed using the known sequences of OsGRF1 (AF201895) as queries in the database of BambooGDB. Primers were designed according to the GRF1 homologue sequences (PH01087379G0010) in moso bamboo. GRF1-001: 5'-ATGATGATGATGAGCGGTCGC-3' and GRF1-501: 5'-TCACTCATCGTTGTGGTAGCGG -3'. PCR amplification reactions were performed in a volume of 25μL, which contained 3.0 μL of cDNA, 2.5 μL of 10 × LA Buffer, 2.5 μL of dNTPs Mixture (2.5 μmol/L each), 1.5 μL of forward and reverse primer (10 μmol/L), 0.3 μL of LA Taq DNA polymerase (5 U/μL) and 13.7 μL of ddH2O. PCRs were performed with an initial denaturation at 94°C for 3 min; followed by 35 cycles of denaturation at 94°C for 30 s, annealing at the optimal temperature of 65°C for 30 s, and extension at 72°C for 90 s; followed by a final extension at 72°C for 10 min. The PCR products were inserted into the pMD19-T vector, and subsequently confirmed by sequencing.
Sequence homology was searched using BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple alignments of PeGRF1 and GRF1s from other plants were conducted using DNAMAN. Phylogenetic tree was constructed using MEGA 7.0 software. The open reading frame (ORF), molecular weight (MW), and isoelectric point (pI) parameters of PeGRF1 were calculated by the online program ExPasy (http://www.expasy.org/tools/).
In addition, to investigate the expression of PeGRF1 genes in the different tissues and developmental stages of shoots, RNA-seq data generated from leaves, early panicles, advanced panicles, roots, rhizomes, 20 cm shoots and 50-cm shoots were downloaded from the NCBI Short Read Archive (SRA) (Peng et al., 2013) and used for gene expression analysis.

Construction of plant expression vectors and transformation
The ORF sequences of PeGRF1 was amplified using the primer pairs of GRF1-003 and GRF-503, which harbored BamH I and Xba I restriction sites in forward and reverse primers respectively. The fragments were subcloned into the corresponding sites of modified pCAMBIA1301 vector, and construction of plant expression vector pCAMBIA1301-PeGRF1. After the validation of integrity by sequencing, the vector pCAMBIA1301-PeGRF1 was introduced into Agrobacterium tumefaciens EHA105 by electroporation, and the positive clones were selected for transform Arabidopsis plants.
Transformation Arabidopsis plants by the floral dip method. T0 generation transgenic Arabidopsis plants were selected using 1/2 MS medium with 30 mg/L hygromycin. T1 generation resistant lines were obtained, and then which DNA extracted for PCR validation. Finally, T2 transgenic lines were selected and used in subsequent research.