Molecular Cloning and Expression Analysis of C Function Gene CjPLE in Double Flower Varieties of Camellia japonica

The C function gene in the classic ABC model is the important factor that determines the stamen and pistil organs in the floral axis, and is also the regulatory gene of floral meristem termination differentiation. A 901 bp cDNA sequence of C function gene was cloned from flower bud of Camellia japonica , named CjPLE (MF278983), which contained an opening reading frame of 780 bp. The “Genome Walking” experiment showed that it contained two introns. Through phylogenetic analysis, we found that it formed a PLE subclass with TAG1 of Solanum lycopersicum and PLE of Antirrhinum majus . The results of real-time PCR showed that CjPLE gene had high expression in the carpel of wild Camellia japonica . In peony type double-flower varieties ‘Hongluzhen’ and ‘Zhuangyuanhong’, the highest expression of CjPLE gene was in stamens, while in ‘Rongqiu’, the expression of CjPLE gene in inner petals and stamens were both high. The above results indicate that CjPLE is a homologous gene of the PLE branch in which belongs to C function gene in Camellia japonica , and might play a role in regulating the development of floral organs.

All regulatory genes in "ABCDE" model belong to MADS-box gene family except A gene APALATA2. MADS-box gene is a very important transcriptional regulatory factor. In angiosperm, C function gene AGAMOUS (AG) is the largest branch of MADS-box. However, in eudicotyledon Arabidopsis thaliana and Antirrhinum majus, two core branches were proved to exist at the same time: euAG and PLENA (PLE), and functional studies had found that these branches were also involved in the development of internafloral whorls of organs (Fourquin and FerrNdiz, 2012). After a long period of artificial domestication, the ornamental variety of camellia contains abundant changes of flower patterns. Double-flower camellia includes 5 types: semidouble type, peony type, rose type, amenone form and complete double type. It is still not clear about how ABCDE genes regulate the formation of double flower. The study on Camellia japonica gene CjAPL1 and CjAPL2 found that the expression level of A function genes were correlated with the degree of double flower, but overexpressed Arabidopsis plants didn't have the double flower phenotypes (Sun et al., 2003a). The study on C gene CjAG of Camellia japonica found that two irreversible evolution paths existed in the expression changes of C function genes during the formation of double flower in Camellia japonica: (1) The expression level of C functional gene in complete double Camellia japonica 'Shibaxueshi' was entirely lost which was the same as the other double flowers species; (2) In amenone type ('Jinpanlizhi'), instead of being inhibited, the expression level of C function genes increased, and ectopic expressed in the inner petals. In this study, focusing on the PLE branch of C function genes, gene cloning and evolution analysis were carried out in Camellia japonica. Combined with the gene expression pattern in wild single and double flower varieties, the regulation of CjPLE in flower development and double flower formation was discussed.

Molecular cloning of the full-length CjPLE gene
Through the local BLAST comparison, a full length of 1 019 bp gene numbered c56140.graph_c0, homologous with PLE\, was discovered in transcriptome data of Camellia japonica bud (Li et al., 2017), of which the open reading frame (ORF) was 738 bp and encoded a total of 245 amino acids. The 5' non-coding region was 107 bp and 3' non-coding region was 174 bp. Based on this sequence information, PCR primer was designed outside the ORF of the gene, and a 901 bp sequence was obtained through specific PCR amplification. Using ORF Finder to analyze CjPLE gene sequence, the result showed that the gene ORF was 780 bp and could encoded a complete protein. The 5' non-coding region was 60 bp and 3' non-coding region was 61 bp. Using BioEdit to compare the protein sequences of these two genes, the result showed that c56140.graph_c0 had more 14 amino acids than cloned CjPLE while other amino acid was identical. The above results indicated that the cloned CjPLE gene was a full-length gene and had been submitted to NCBI, the accession numbered was MF278983.
Through the analysis of protein structure encoded by CjPLE gene by CD-Search on NCBI, we found that the encoded protein contained a K-box (E-value: 9.74e-31) domain and a highly conservative region in the specific MADS-box (E-value: 9.42e-46) domain ( Figure 1).

Sequence alignment and phylogenetic analysis of CjPLE and related genes in different plants
To investigate the evolutionary relationship between CjPLE and other species, 18 protein sequences, which had high homology with CjPLE (referring to the phylogenetic tree of Alice Tadiello et al.), were selected to construct NJ (Neighbor-joining) phylogenetic tree using MEGA 7.0, (Figure 2). The result showed that CjPLE had the closest evolutionary relationship with TAG1 from tomato, and formed a PLE sub branch with Antirrhinum majus AmPLE (Alice et al., 2009).

Gene structure
Through the specific PCR amplification, the cDNA sequence of CjPLE was obtained. To study the complete structure of the gene, we constructed 4 libraries using Universal GenomeWalkerTM 2.0 kit by taking wild Camellia japonica as a template. Besides, two pairs of specific primers were designed to amplify target gene. However, only one pair of primers successfully amplified (Table 1). The primers were directed towards the 5' terminal and 3' terminal of the gene, respectively, and contained a repeating area, which ensured the acquisition of whole sequence.
Genome walking result showed that the full length of the gene was 1 195 bp with two small introns of which were 33 bp and 118 bp, respectively (Figure 3).