Cloning and functional analysis of JnCYCD3;1 in Jatropha nigroviensrugosus CURRENT

Background: As a new variety of Jatropha the female male ratio and yield of nigroviensrugosus are higher than the common J. curcas . Using pre-transcriptome data, the full-length gene sequence, subcellular function localization, and verification of the transgenic function were obtained in order to understand the specific functions of differentially expressed genes (DEGs). Results: Results revealed that the open reading frame (ORF) of J. nigroviensrugosus CYCD3;1 ( JnCYCD3;1 ) was 414 bp long, encoding 137 amino acids (aa). Compared to J. curcas , the presence of intron retention led to early termination of the coding frame. JnCYCD3;1 had the highest expression in new leaves, which was 68.42 times root expression, followed by inflorescence buds. The JnMYC2 ORF was 2025 bp, encoding 674 aa. JnMYC2 had the highest expression levels in inflorescence buds. JnCYCD3;1 functioned in the nucleus, while JnMYC2 was distributed in both the nucleus and cytoplasm and may possess transmembrane membrane behavior. Bimolecular fluorescence complementation (BIFC) experiments indicated that JnMYC2 interacted with JnCYCD3;1 . JnCYCD3;1 transgenic tobacco considerably advanced the reproductive cycle and may promote flower formation and transformation. Conclusions: The related experiments obtained new CYCD3;1 transcript, verified that CYCD3;1 is related to flower bud differentiation, proved the interaction between hormones-related genes. The study provides a new research direction for gene function of CYCD3;1 .

flowers [8]. In order to investigate the regulatory mechanism of transition from monoecious to gynoecious plants, a comparative transcriptome analysis between gynoecious and monoecious inflorescences was conducted. Six homologous genes, including knotted1-like homeobox gene 6 (KNAT6) and MYC2, may be candidate genes for sex differentiation [9]. During the flower bud stage in Rhododendron, transcripts of MYC2, TIR1, CYCD3, COL-1, and EIN3 peak [10]. Additionally, upregulation of CYCD3 is thought to be associated with increased inflorescence meristems of J. curcas [11].
CYCD3;1 is an unstable protein (half-life, 7 min) that is involved in cell cycle regulation. As a plant hormone signal, it changes in-cell cycle regulator expression and has an effect on the development of leaves and other organs [12]. Cytokinins can increase the rate of cell division by inducing CYCD3 expression [13]. Overexpression of CYCD3;1 can replace the cytokinins required to induce callus in Arabidopsis leaves; on cytokinin-free medium, the number of tissues formed by callus was 5-times greater than the control [14]. Moreover, CYCD3;1 overexpressing Arabidopsis leaves significantly increased smaller cells and cell division replaced cell expansion as the primary mechanism for leaf growth [15]. initiates transcription [16]. Given the important role of MYC2, it has been proposed as the primary mediator of JA signaling and crosstalk along with abscisic acid, ethylene, and optical signaling pathways [17]. The myc2 mutant has a similar flowering time as wild-type Arabidopsis, while the triple mutant of myc2/3/4 exhibits an early flowering phenotype, and MYC2 is considered a negative regulator of the FT gene [18]. As the core regulator of JA, MYC2 may play an important role in multihormone crosstalk.
The question remains, however, as to why MYC2 and CYCD3;1 are up-regulated during flower differentiation in J. nigroviensrugosus. Is there an interaction between the two genes? The sequence cloning and expression analysis of differentially expressed genes (DEGs) are helpful for understanding the function of these genes and provide preliminary data on the underlying mechanism of flowering in J. nigroviensrugosus. This study focused on the cloning of MYC2 and CYCD3;1 gene expression and the gene expression of different tissues. Subcellular functional localization and bimolecular fluorescence complementation (BIFC) experiments were conducted in order to identify the location where JnCYCD3;1 functions and discovers complex proteins. The findings of this study will provide a basic resource for future flowering regulation and flower and fruit management.

Expression correlation analysis
Results of the correlation analysis revealed that the expression of MYC2, CYCD3, and DELLA was positively correlated with different parts of J. nigroviensrugosus. CYCD3 expression was negatively correlated with JAZ (Fig. 1A). MYC2 and CYCD3 expression were positively correlated. MYC2 (gene11878) exhibited higher transcript expression levels in the inflorescence buds of cluster1 ( Fig. 1B), while DELLA (gene13701) and CYCD3 (gene38) were in cluster3 and exhibited higher transcript expression levels in new leaves, followed by inflorescence buds.

Sequence structure analysis
The JnCYCD3;1 sequence was obtained by cloning, and the ORF finder predicted to obtain 534 bp. The predicted analysis revealed that the JnCYCD3;1 protein structure has a Cyclin_N conserved domain at positions 292-513. NCBI blastp alignment revealed that JnCYCD3;1 and J. curcas CYCD3 (XM_012210210.2) had 95% sequence coverage identity compared to its genomic sequence (NW_012124049.1) and XM_012210210.2. JnCYCD3;1 had an intron retention, resulting in the early termination of its coding frame (Fig. 2). The JnMYC2 sequence was also obtained by cloning. The

Expression analysis during flowering
The quantitative expression level of fluorescence indicated that JnCYCD3;1 was expressed in all parts of the plant (Fig. 3). The highest expression levels were observed in new leaves, which were 68.42 times greater than root expression, followed by inflorescence buds; expression levels were the lowest during the pollen formation period. JnMYC2 exhibited the highest expression levels in parts of the inflorescence buds, which was 2.6 times greater than root expression. There was no difference between the expression of new leaves and female or male flowers (Fig. 4).

Genetically modified function verification
After transgenic tobacco was differentiated and rooted ( Fig. 8), it was transferred to a substrate for culturing. During the transfer process, the root medium was cleaned and placed indoors for refining.
By conducting PCR (detection primers were hygromycin primers and JnCYCD3; 1 specific primers) (Table 1), transgenic tobacco was obtained for further phenotypic and functional identification. Table 1 List of related primers.

Primer name
Primer

Discussion
In previous studies, the PD-NINJA complex was found to control the initial shape of the leaf and inhibit CYCD3, thereby controlling leaf flatness in angiosperms [18]. Boucheron et al. [19] transferred CYCD3 to tobacco, which resulted in increased leaf growth rates and changes in the structure of the shoot apical meristem. Dewitte et al. [15] found that Arabidopsis leaves transfected with CYCD3 curled toward the medial axis. The A. thaliana AtCYCD3;1 gene was also found to wrinkle and curl leaves in transgenic tobacco [20]. CYCD3 overexpression caused delayed morphological differences in SAM and leaf senescence [14]. Additionally, CYCD3 was also found to be expressed at its highest levels during no Cyclin_C domain; thus, the Cyclin_C domain may not be critical for its function [28]. Intron retention is the most common alternative splicing form in plants [29]. In JnCYCD3;1, retention of the first intron leads to its coding frame in advance. However, the termination or loss of the Cyclin_C domain, whose structural differences may lead to changes in traits, requires further investigation. The expression pattern of CYCD3 in different tissues of 2 varieties of J. curcas was essentially the same, and both were the highest expressed in young leaves, followed by inflorescence buds during the differentiation process. Transcript abundance of CYCD3;1 increased more than 5-fold during the early

Conclusion
In this study, comparative transcriptomics data, using ene cloning, relative fluorescence quantification, and subcellular functional localization, were used to explore the reproductive advantages of J. nigroviensrugosus. The JnCYCD3;1 ORF was found to be 414 bp, encoding 137 aa.
Compared to J. curcas, the presence of intron retention led to early termination of the coding frame.
RT-qPCR indicated that JnCYCD3;1 exhibited the highest expression levels in new leaves, which was 68.42 times greater than root expression, followed by inflorescence buds. JnCYCD3;1 was also found to play a role in the nucleus. BIFC experiments demonstrated that JnMYC2 interacts with JnCYCD3;1.

Experimental methods Correlation analysis of transcript expression
Focusing on the differential gene set in the plant hormone signaling pathway of the buds of 2 varieties, expression correlations and trends in different parts of the plant were analyzed [8]. A correlation circle map was created using the R circlize package [32]. The clustering of expression trends was performed using Python DP_GP [33].

RNA extraction and cDNA synthesis
Extraction of total RNA from the samples was conducted using an EASYSPIN plus plant RNA extraction kit following the manufacturer's instructions. After RNA extraction, the quality was verified. Firststrand synthesis of the cDNA was conducted using an EasyScript First-Strand cDNA Synthesis SuperMix kit following the manufacturer's instructions.

Cloning the gene of interest
Specific primers were designed using the J. curcas genome ( Table 1). The PCR reaction system

Subcellular functional localization and BIFC experiments
According to JnCYCD3;1, due to JnMYC2's gene sequence and vector characteristics, the pBWA(V)HSccdb-GLosgfp vector was selected. The vector was constructed by the golden gate technique [35].
The vector was ligated, and 5-10 µL of the ligation product was transformed into Escherichia coli.

Consent for publication
Not applicable.

Availability of data and materials
Amplification curves and melting peaks of RT-qPCR can be found in the Additional file 1; The negative control of BIFC experiment can be found in the Additional file 2. The datasets used and analyzed during the current study could be available from the corresponding author on request.

Competing interests
The authors declare that they have no competing interests.

Funding
The work was supported by National Natural Science Foundation of China-Study on flower bud differentiation and flowering mechanism of Jatropha nigroviensrugosus CV Yang. (31360165). The funding body was not involved in the design of the study, analysis, and interpretation of data in the manuscript.

Authors' contributions
X.F and Z.Y designed and executed the experiment; W.X.R conductedadditional analyses; X.F wrote the manuscript. All authors have read and approved the manuscript.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.