Abstract
Longan (Dimocarpus longan Lour.) is an important commercial fruit tree in southern China. The embryogenesis of longan affects the quality and yield of fruit. A large number of alternative splicing events occurs during somatic embryogenesis (SE), which is regulated by serine/arginine-rich (SR) proteins. However, the functions of SR proteins in longan are poorly understood. In this study, 21 Dlo-SR gene family members belonging to six subfamilies were identified, among which Dlo-RSZ20a, Dlo-SR30, Dlo-SR17, Dlo-SR53 and Dlo-SR32 were localized in the nucleus, Dlo-RSZ20b, Dlo-RSZ20c, Dlo-RSZ20d, Dlo-SC18, Dlo-RS2Z29, Dlo-SCL41, and Dlo-SR33 were localized in chloroplasts, and Dlo-RS43, Dlo-SC33, Dlo-SC37, Dlo-RS2Z33, Dlo-RS2Z16, Dlo-RS2Z24, Dlo-SCL43, Dlo-SR112, and Dlo-SR59 were localized in the nucleus and chloroplasts. The Dlo-SR genes exhibited differential expression patterns in different tissues of longan. The transcript levels of Dlo-RSZ20a, Dlo-SC18, Dlo-RS2Z29, DLo-SR59, Dlo-SR53, and Dlo-SR17 were low in all analyzed tissues, whereas Dlo-RS43, Dlo-RS2Z16, Dlo-RS2Z24, and Dlo-SR30 were highly expressed in all tissues. To clarify their function during SE, the transcript levels of Dlo-SR genes were analyzed at different four stages of SE, comprising non-embryonic callus (NEC), friable-embryogenic callus (EC), incomplete compact pro-embryogenic culture (ICpEC) and globular embryo (GE). Interestingly, the transcript levels of Dlo-RS2Z29 and Dlo-SR112 were increased in embryogenic cells compared with the NEC stage, whereas transcript levels of Dlo-RSZ20a, Dlo-RS43, Dlo-SC37, and Dlo-RS2Z16 were especially increased at the GE stage compared with the other stages. Alternative splicing events of Dlo-SR mRNA precursors (pre-mRNAs) was detected during SE, with totals of 41, 29, 35, and 44 events detected during NEC, EC, ICpEC, and GE respectively. Protein–protein interaction analysis showed that SR proteins were capable of interaction with each other. The results indicate that the alternative splicing of Dlo-SR pre-mRNAs occurs during SE and that Dlo-SR proteins may interact to regulate embryogenesis of longan.
Highlights
Alternative splicing (AS) allows the production of a large number of protein isoforms from a small number of genes
Analysis of the physicochemical properties of the Dlo-SRs protein with ExPASy Protparam revealed that the length of Dlo-SR proteins ranged from 141 amino acids (Aa) (Dlo-RS2Z16) to 1,013 Aa (Dlo-SR112), the molecular weight varied from 16.16 kDa (Dlo-RS2Z16) to 112.05 kDa (Dlo-SR112), and the isoelectric point ranged from 6.19 (Dlo-SR53) to 12.28 (Dlo-SCL43) (Table 2)
Consistent with the nomenclature for Arabidopsis SR proteins [7], Dlo-SR gene family members were named systematically according to the phylogenetic tree and the molecular weight (Table 2)
Summary
Alternative splicing (AS) allows the production of a large number of protein isoforms from a small number of genes. Alternative splicing is generally regulated by splicing factors, of which one category is highly conserved serine/arginine-rich (SR) proteins. These SR proteins play vital roles in spliceosome assembly and mRNA splicing, and influence the selection of AS sites through changes in concentration of SR proteins [2]. (1) Classical SR proteins contain one or two RBDs at the N-terminus and one RS domain at the C-terminus. Classical SR proteins can be recognized by the monoclonal antibody mb104 and participate in constitutive and alternative RNA splicing. The SC subfamily consists of SR proteins containing one RBD and one RS domain. Proteins of the RSZ, SCL, and RS2Z subfamilies contain a single RBD and a typical RS domain. The differences among the RSZ, SCL, and RS2Z subfamilies are that one zinc knuckle is present in RSZ proteins, a N-terminal charged extension is present in SCL proteins and two zinc knuckles are contained in RS2Z proteins [7]
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