Cloning, expression and functional analysis of the LcBES1 in Litchi

Abstract: 【Objective】Brassinosteroids (BRs) are plant steroid hormones known for promoting cell elongation. The BRs regulate diverse developmental and physiological processes including flowering,seed germination, vascular differentiation, senescence, photomorphogenesis, male fertility and respons-es to biotic and abiotic stresses in plants. In the recent decades, BR signaling network have been re-vealed and several key components in the BR signaling pathway have been identified. The BRI1-EMS-SUPPRESSOR1 (BES1) is one of the most important transcription factors acting as a positive regulator in the BR signal transduction pathway. In the presence of the BRs, the BIN2 is inactivated by the BSLs, and the BES1 is rapidly dephosphorylated by protein phosphatase 2A and accumulate in the nucleus where they can bind to the promoter of downstream genes. The BES1 has been well studied in model plants such as Arabidopsis and rice, but in Litchi the function of the BES1 has not been studied. To in- vestigate the BR signaling pathway in Litchi (Litchi chinensis Sonn.), we identified a BES1 homolog in Litchi and named it as LcBES1. In order to clarify the function of the LcBES1, the sequence characteris-tics, gene function, expression pattern, phylogenetic tree and subcellular localization were all analyzed.【Methods】The LcBES1 was obtained according to the RNA-seq data of panicle [National Center for Biotechnology Information (NCBI) Sequence Read Achieve accession number SRX2336010] and the full length ORF was predicted through Open Reading Frame Finder software (https://www.ncbi.nlm.nih.  gov/orffinder/). The total RNA was isolated from the different organs or tissues using EasyPure Plant RNA Kit (TransGen Biotech, China) according to the manufacturer’s protocol. To digest DNA, RNase-free DNase I (NEB, USA) was used before reverse transcription. First-strand cDNA was then synthe-sized from the total RNA using EasyScript First-Strand cDNA Synthesis SuperMix (Transgen Biotech, China). Semi-quantitative reverse transcriptase PCR (qRT-PCR) was used to analyze the expression pat-tern of the LcBES1 in the different tissues and organs in Litchi (including root, stem, leaf, panicle,flow-er, pulp and pericarp ). The relative expression level was calculated using the 2^-△△Ct method, and LcAC-TIN was used as internal reference. The multiple alignment analysis of LcBES1 and AtBES1 proteins was carried out using the AlignX program within the Vector NTI. The phylogenetic tree of BES1 pro-teins among Litchi, Arabidopsis and other fruit trees was constructed using the neighbor joining method in the MEGA 6.06 software. To investigate the function of the LcBES1, the entire coding region of the LcBES1 was transferred into pCAMBIA2300 vector, and then the fusion construct was transformed into the Agrobacterium strain GV3101 using the freeze-thaw method, and finally GV3101 was transformed into the Arabidopsis bri1 mutant. To monitor the subcellular localization of the LcBES1 protein, the ORF of the LcBES1 was inserted into the C-terminus of pCAMBIA2300-YFP vector with the Seamless Assembly Cloning Kit (TransGen Biotech). The construct was then transferred into Agrobacterium tu-mefaciens strain GV3101 using the freeze-thaw method and then introduced into tobacco (Nicotiana tabacum) leaves by infiltration.【Results】The BES1 homolog in Litchi was identified according to the annotation of the Litchi panicle transcriptome data (GenBank accession number SRX2336010), the iso-lated gene was named as LcBES1 based on the phylogenetic analysis. Molecular analysis indicated that the ORF of the LcBES1 was 984 bp in length and the deduced polypeptides was 327 amino acids. Multi-ple sequence alignment showed that the LcBES1 protein had 55.0% similarity with the AtBES1. Structural domain analysis showed that a BES1_N domain commonly associated with the BES1 function was identified at the N-terminus of the protein. qRT-PCR results demonstrated that the transcripts of the LcBES1 were detected in multiple tissues, but most highly detected in the pulps, relatively lowly in the roots, and the stems, and most lowly detected in the pericarps, the leaves, the flowers and panicles. The transgenic experiment showed that the LcBES1 significantly promoted plant growth. The height of Ara-bidopsis bri1 mutant was 6 cm (40 days seedling), while the height of the bri1 mutant overexpressed the LcBES1 could reach 15cm, the result demonstrated that the LcBES1 could partly rescue the dwarf phenotype of bri1 mutant. In the transient transformation assays in tobacco, fluorescence signals could be observed in the nucleus, indicating that the LcBES1 is a nuclear localized protein.The phylogenetic tree analysis showed that the the BES1 proteins of fruit trees were clustered into one group, while the BER1/BES1 family of Arabidopsis were clustered into the other group. In the fruit trees group, it could be found that the LcBES1 had the closest evolutionary relationship with the BES1 protein from durian.【Conclusion】In this study, the homolog of the BES1 transcription factors was identified in Litchi and denoted as LcBES1. The transient expression experiment in tobacco confirmed that LcBES1 was a nucle-us located protein. The function analysis showed that the LcBES1 had similar biological functions as the BES1. The expression analysis showed that the LcBES1 expressed in all of the tissues and organs detect-ed, indicating that the LcBES1 was a ubiquitous expression gene. The results demonstrated that the function of the LcBES1 was conserved and the LcBES1 was a positive regulator of the BR signal pathway.