Genome-wide identification and expression analysis of TMK gene family in Dimocarpus longan Lour.

Abstract:【Objective】Transmembrane kinases (TMK) belong to a small subfamily of receptor-like pro- tein kinases (RLKs). They participate in auxin signal transduction pathway on cell membrance and mainly control the cell expansion and proliferation and the downstream signals of auxin. Dimocarpus longan Lour. is one of the important economic crops in Sapindaceae, and its embryo development is closely related to fruit quality. Endogenous hormones play a vital regulatory role in the embryo develop- mental process, and auxin can promote the early somatic embryo development of longan. Longan em- bryos have a highly heterozygous genetic background and are difficult to obtain. Therefore, it is not easy to use molecular biology methods for researching. Based on the high similarity of the developmen- tal process between longan somatic embryos and zygotic embryos, Lai established a stable, efficient and capable of regeneration longan somatic embryogenesis system for researching the molecular biolo- gy in longan. At present, the TMK genes have been studied only in a few plants , and there is no report in longan. The research was carried out to comprehensively analyze the biological characteristics of lon- gan TMK gene family, the expression patterns during the early embryogenesis of longan and when treat- ed with different kinds and concentrations of hormones, and provide a reference for the study of auxin signaling pathway during the somatic embryogenesis of longan.【Methods】Four AtTMK amino acid se-quences were used as probe sequences, and a variety of bioinformatics software were used for screen- ing. In the end, we obtained 6 DlTMK genes with complete conservative domains. A variety of bioinfor- matics related software were used to analyze and predict the gene structure, basic physicochemical prop- erty, transmembrane domain, subcellular location, chromosome location, phosphorylation site, phyloge- netic tree, cis-acting elements and the transcriptome data of the DlTMK gene family members. The pos- sibility of DlTMK family members responding to multiple hormones were explored by analyzing the ex- pression patterns of the genes under different hormone treatments.【Results】A total of 6 DlTMK family members were screened, all of them have a transmembrane structure with no signal peptide, and they are macromolecular hydrophilic proteins. According to the subcellular localization prediction results, the DlTMK gene family are mainly located in the cytoplasm, plasma membrane and nucleus. Phosphor- ylation site prediction analysis showed that phosphorylation mainly occurre on the serine and threonine sites, and serine was the main phosphorylation site. Each member has a simple structure with one intron and two exons, as well as 13 highly conserved motifs and 4-7 LRR repeats. Chromosome mapping results showed that 6 DlTMK members are randomly distributed on 5 chromosomes, and the DlTMK1-1 and DlTMK1-2 are tandem repeats. The homologous relationship between DlTMK gene family andArabidopsis thaliana and poplar were closed, we speculated that they would have similar evolutionary mode. The promoter cis-acting elements of DlTMK family members mainly contain three hormone re- sponse elements: auxin (IAA), abscisic acid (ABA) and gibberellin (GA3) responsive elements. All members contain light responsive elements, 5 members have anaerobic induction responsive elements, and a few members have methyl jasmonate (MeJA) response, salicylic acid (SA) response, low tempera- ture stress, circadian control, zein metabolism and meristem responsive elements. The differences types and numbers of cis-acting elements among different members indicated that their biological functions are different. The expression trends of DlTMK family members during the early somatic embryogenesis of longan was basically consistent with the results of transcriptome analysis. The transcriptome change trend of DlTMK1-1, DlTMK1-3 and DlTMK4 was the same as the actual detection results. The expres- sion of Incomplete embryotic compact structure (ICpEC) of DlTMK1-2 and DlTMK3-1 in transcrip- tome was higher than that of embryogenic callus (EC), but it was found that the expression in this stage was slightly lower than that in EC stage. The expression of DlTMK3-2 in ICpEC stage was the lowest among the three stages, which was opposite to that in transcriptome data and might be caused by se- quencing error. All DlTMK family members responded to IAA, ABA and GA3 hormones. Under the IAA treatment, the DlTMK1-1 and DlTMK1-3 showed a“W”shape expression trend. DlTMK1-2, DlT- MK3-1 and DlTMK4 expressed highly first and then decreased, and reached the peak on the 6th day. DlTMK3-2 showed a down-regulation trend at first and then up-regulation during the 0th day to the 6th day, and a significant downward trend from the 6th day to the 12th day. Under the ABA treatment, the expression of every two DlTMK members had the same trend. For example, DlTMK1-1 and DlTMK1- 2 showed a“M”expression shape and reached peak on the 9th day, while DlTMK1-3 and DlTMK3-2 were significantly decreased on the 3rd day, then increased gradually, and reached the peak on the 9th day, and decreased significantly again on the 12th day. The expression of DlTMK3-1 and DlTMK4 in- creased at first and then decreased, DlTMK3-1 reached the peak on the 6th day and DlTMK4 reached the peak on the 9th day. Under the GA3 treatment, the expression of DlTMK1-1 and DlTMK3-2 had no significant change from the 0th day to the 6th day, but the expression of DlTMK1-1 decreased gradually from the 6th day to the 12th day, and the expression of DlTMK3-2 decreased significantly and then in- creased significantly from the 6th day to the 12th day. Both DlTMK3-1 and DlTMK4 reached the ex-pression peak on the 3rd day. The expression of DlTMK1-2 and DlTMK1-3 showed opposite expres- sion trends: DlTMK1-2 was significantly up-regulated on the 3rd day, down-regulated from the 3rd day to the 9th day, and increased on the 12th day, but not significantly different on the 9th day. DlTMK1-3 was significantly down-regulated on the 3rd day, and up-regulated from the 3rd day to the 9th day, and significantly down-regulated on the 12th day. In addition, the expression trends of the same gene with different treatments were similar.【Conclusion】This study showed that the DlTMK family members are highly conservative in evolutionary characteristics and they might participate in a variety of hormone re- sponses. According to the mode of action of RLKs and TMK gene families in other plants, we speculat- ed that DlTMK gene families would be also involved in hormone signal transduction through reversible phosphorylation and regulate longan somatic embryogenesis.