| Longan(Dimocarpus longan Lour.),also known as Gui Yuan,is an important subtropical woody fruit tree,rich in sugar,iron and protein,with edible and medicinal value.Longan embryo development is closely related to its fruit yield and quality.Alternative splicing is an important factor in producing protein diversity.Sm protein is an important RNA binding protein,which mainly includes Sm and LSm proteins.Sm core proteins are able to form a variety of heptacyclic complexes with sn RNPs binding to participate in the assembly of variable spliceosomes and play a role in pre-m RNA splicing,while LSm proteins binding are not permanent and therefore have a broader role than Sm proteins.Studies have shown that LSm proteins can improve the drought resistance of plants,increase the sensitivity of plants to salt stress,and participate in the biological clock regulation of plants.However,the current functional studies of the Sm protein family are mainly focused on model plants such as Arabidopsis,which have not been reported in woody fruit trees,especially in longan somatic embryos.Thus,the present study carried out genome-wide identification of longan Sm proteins and bioinformatics analysis;analysis of alternative splicing(AS),single nucleotide polymorphism(SNP)and insertion-deletion(In Del)of the family using transcriptome data.With reference to the longan genome,the full lengths of the Dl LSm14 s were verified by RT-PCR and p CAMBIA1302-Dl LSm14s-e GFP vectors were constructed for sub-cellular localization.The real-time quantitative PCR(q RT-PCR)was used to analyze the expression of Dl LSm14 s under different hormones and temperature stress to provide scientific basis for the functional study of the Dl Sm family.And we also predicted and verified the mi RNAs of the Dl Sm family.The main findings are as follows:1.Genome Identification and Expression Analysis of Longan Dl SmThe amino acid sequences of Arabidopsis thaliana were used as probe sequences,and 29 Dl Sm genes were identified by local blast using longan genome database.Promoter analysis showed that the Dl Sm genes contained a large number of response elements related to light,anaerobic induction,physiological circadian rhythm control,endosperm development,and multiple hormones,suggesting that the family members are not only associated with longan stress resistance,but also able to participate in plant growth and development.AS analysis of the Dl Sm family showed that the longan NEC stage had the most events,and the AS events in each stage were different,which may be related to longan somatic embryo differentiation.The large occurrence of intron retention(IR)events in the NEC stage may affect longan SE,while Dl LSm14 c may promote the differentiation of longan EC into globular embryo(GE).Whether in SE stages or in different tissues,Dl LSm14 s occured more AS events than other members.It is speculated that this subgroup members played an important role in somatic embryo differentiation and organ development.Only AS events of Dl LSm14 c changed under different hormone treatments.More specific splicing were produced under blue light,and the AS events occured in Dl LSm14 c were the most under any treatment,and,this member may be involved in certain pathways of hormones and light.The analysis of base polymorphism in Dl Sm family showed that the base polymorphism of Dl Sm family was different in different longan varieties.To analyze the expression of Dl Sm genes in longan nonembryogenic callus(NEC)and the early stage of somatic embryogenesis(SE),and to synthesize the FPKM value analysis of different tissue sites,it was found that four Dl LSm14 members were highly expressed at the embryonic callus(EC)stage and three members were highly expressed in seeds.It was speculated that this subgroup member might be related to longan SE and embryogenesis maintenance.FPKM-'q RT-PCR'comparative analysis found that 25 genes expressed decreased from EC to GE stages,speculated that Dl Sm genes expressions were affected by longan somatic embryo growth and differentiation,and decreased with the increase of somatic embryo differentiation.Under different hormone and light qulity treatments,Dl LSm4 b,Dl LSm14 c,Dl LSm14 e and Dl LSm8 were found to be highly expressed only under kinetin(KT)treatment.It was speculated that the above genes may be related to the promotion of somatic embryo differentiation.White light can promote the Sm genes expression of longan EC,while blue light can inhibit the expression of Sm genes.2.Gene cloning,bioinformatics analysis and subcellular localization of Dl LSm14sTo explore the function of Dl LSm14 s,we cloned Dl LSm14 a,Dl LSm14 d and Dl LSm14 e c DNA full-length for 1791 bp(Gen Bank accession number: MT326637),1929bp(Gen Bank accession number:MT326638)and 1707bp(Gen Bank accession number: MT326639),respectively.and encoding 596,642 and 568 amino acids,respectively.The phosphorylation modification sites prediction indicateed that three proteins were rich in the phosphorylation modification sites of serine and threonine,but the number and distribution of sites were different,which may make them produce functional differences.Phylogenetic analysis showed that Dl LSm14 s had a close relationship with Acer yangbiense and Citrus sinensis.A p CAMBIA1302-Dl LSm14s-e GFP fusion vector was constructed using one step cloning technology.The results showed that all three genes were nuclear localization genes3.Expression analysis of Dl LSm14 s under hormone and abiotic stressUsing q RT-PCR technique to analyze the expression of abscisic acid(ABA),methyl jasmonate(Me JA),salicylic acid(SA)and gibberellin A(GA)under different time and KT under diffierent concentration treatment.The results showed that Dl LSm14 a,Dl LSm14 c,Dl LSm14 d and Dl LSm14 e responded to all five hormones,and the response expression patterns of different members were different.ABA and Me JA transient treatments(24 h),which have positive regulatory effects on four Dl LSm14 genes,may be involved in related stress responses by promoting their expression.GA treatment for 24 h negatively regulates the expression of Dl LSm14 a,Dl LSm14 c and Dl LSm14 d.SA treatment(24h)has a positive regulatory effect on Dl LSm14 a and a negative regulatory effect on Dl LSm14 c and Dl LSm14 d.Dl LSm14 s differences in response expression patterns to exogenous hormones may reflect differences in gene function.The results of q RT-PCR analysis under low temperature stress and heat stress showed that low temperature treatment(24 h)could promote the expression of Dl LSm14 a and Dl LSm14 e and inhibit the expression of Dl LSm14 c and Dl LSm14 d,indicating that the response of the four genes to low temperature is not the same.At heat stress(24 h),the expression of the four genes was higher than that of the control,this suggests that four genes may be involved in some regulatory pathways under longan somatic embryo heat stress.4.Dl Sm family mi RNAs prediction and validationTo further understand the possible regulatory relationship between Dl Sm genes and mi RNAs,we predicted the mi RNAs of Dl Sm family,and the result revealed that six members contained conserved mi RNA inhibition sites,of which Dl LSm14 s contained mi R156 and mi R393 cleavage sites,while the Dl LSm2 contained only translation sites.Dl LSm14 s was likely to have a regulatory relationship with these mi RNAs,which in turn affects longan embryogenesis.The experimental results show that mi R156 a can crack Dl LSm14 a,speculate that there may be some regulatory relationship with mi R156 a.Taken together,the Dl Sm protein family is a highly conserved protein family whose promoters contain a large number of elements associated with longan embryogenesis and plant growth and development.Among them,Dl LSm14 s members had a large number of AS,SNP and In Del,whose expression changes with the period of SE and was regulated by different hormones and temperature,which may play an important role in the regulation of longan SE. |
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