Cloning, subcellular localization and expression analysis of RNA binding protein LSm14 gene from embryogenic calli of Dimocarpus longan Lour.

Abstract:【Objective】The LSm14 protein is an important RNA-binding protein with conserved N-termi- nal and extended C-terminal, which can participate in the metabolism of RNA and affect embryonic de- velopment. The LSm14 has been shown to be involved in cell division and post-transcriptional regulation in animals. Longan is an important tropical and subtropical woody fruit tree in China. Its embryogenic state significantly affects the quality and yield of the fruits. At present, the effect of the DlLSm14 on non- embryonic callus (NEC) and different embryogenic cultures of longan has not been reported. To investi- gate the effect of the LSm14 on somatic embryogenesis of longan, we cloned RNA- binding protein DlLSm14e, observed its subcellular localization, predicted the physicochemical properties, structure and functional sites of the protein, and analyzed its expression characteristics under different concentrations of kinin(KT) treatments.【Methods】In this study, firstly, the five longan LSm14 genes (Dlo_026784.1, Dlo_006168.1, Dlo_011575.1, Dlo_021757.1, Dlo_029021.1) were selected by using the amino acid se- quence of Arabidopsis thaliana LSm14 as the probe sequences and local blast with the longan genome da- tabase (NCBI accession number: BioProject PRJNA305337). The Longan LSm14 genes were named as the DlLSm14a, DlLSm14b, DlLSm14c, DlLSm14d and DlLSm14e according to the search annotation of the Arabidopsis LSm14 in the longan genome and its chromosomal location. Through the previous data analysis, we selected the DlLSm14e for cDNA full-length validation and overexpression vector construc- tion. DNAMAN was used to design specific primers DlLSm14e-CF and DlLSm14e-CR at both ends of the CDS sequence. RT-PCR was used toclone the full-length. The homologous sequences of 15bp linear cloned vectors were introduced into the 5 and 3 ends of the target fragment by RT-PCR, respectively. Hieff Clone TM Plus One Step Cloning Kit was used to recombine the target fragment with the terminal ho- mologous sequence of the linearized clone vector with the pCAMBIA1302-GFP linearized vector，and the target fragment and the GFP sequence of recombinant vector were verified by PCR. The constructed pCAMBIA1302- DlLSm14e vector was transferred into agrobacterium and injected into tobacco after three days of culture and then a fluorescence confocal microscope was employed to observe subcellular localization. The basic physicochemical properties of amino acids were predicted by ExPASy; the protein domains and functional sites were predicted using NCBI Conserved domains search and ScanProsite on- line software, respectively; the protein transmembrane domain was predicted using TMHMM 2.0.; pro- tein phosphorylation sites were predicted using NetPhos 3.1 Server. SOPMA and SWISS-MODEL were used to predict the secondary and tertiary structure of the protein, respectively; WOLF PSORT was used to predict the subcellular localization PlantCARE was used to predict the promoter cis-acting elements. The FPKM values of longan NEC, different embryogenic cultures [including loose type embryogenic cal- lus (EC), incomplete embryo compact structure (ICpEC), and globular embryoand (GE)] and longon EC with different hormone treatments were analyzed. To investigate the effect of KT on longan somatic em- bryogenesis, longan EC was treated with different concentrations of kinin (0、0.25、0.5、0.75、1和2 mg·L-1) and the DlLSm14e was detected by qRT-PCR.【Results】The results showed that the total length of CDS of the gene was 1 707 bp, encoding 568 amino acids, and the protein did not contain signal peptides and transmembrane structures, so it was not a secretory protein or transmembrane protein. In addition, the protein contained an N-terminal conserved domain and an extended C-terminal, and contained 74 phos- phorylation sites, among them, 46 were serine (Ser) modified sites, 20 were threonine (Thr) modified sites and 8 were tyrosine (Tyr) modified sites. Phylogenetic analysis showed that DlLSm14e protein had a higher similarity with Acer yangbiense (74.5%), Pistacia vera (63.45%), Citrus clementina (58.82%) and Citrus sinensis (58.27%), and was mostly distant to monocotyledonous plants such as Zea maize, Sor- ghum bicolor, Oryza sativa and Musa acuminat. The cis-acting element analysis showed that the gene contained a large number of light response elements and a variety of hormone (abscisic acid, gibberellin, methyl jasmonate and ethylene) response elements. The results of subcellular localization showed that the gene was a nuclear loci gene. Expression profile and fluorescence quantitative analysis showed that the FPKM value of the DlLSm14e was the lowest in the NEC stage and the highest in the globular em- bryo stage. It is suggested that the accumulation of the DlLSm14e might be beneficial to the early mor- phology of longan somatic embryo., 2, 4-D inhibited the expression of the gene, KT promoted its expres- sion of the gene, , and the concentration of KT could affect the expression of the gene.【Conclusion】TheLSm14e gene of longan was located in the nucleus, and the FPKM value of this gene increased with the increase of the somatic embryogenesis of longan, and the gene responded to various hormones related to the somatic embryogenesis of longan. The results of qPCR under different concentrations of KT showed that the concentration of KT could affect the expression of the DlLSm14e. It is speculated that this gene may be involved in the early morphogenesis of the somatic embryo of longan.