Rational Nomenclature And Expression Analysis Of SR Protein Family In Manihot Essulenta Crantz

Alternative splicing(AS)is an important regulation mechanism in eukaryotic gene expression.The pre-mRNA of genes can form a variety of different mature mRNA through alternative splicing,which then lead to generate a lot of different structures and functions of the protein.Therefore,it is an important way for produce diversity proteins with multiple functions in eukaryotic.Splicing factor is an alternative splicing participant and a direct regulatory element.There were two kinds of basic splicing factors which is involved in regulating AS,and serine/arginine-rich proteins(SR proteins)are one of these most important splice factors.In recent years,much works have been greatly increased on plant SR protein family.Most of these works were focused in the model plants,such as Arabidopsis and rice.However,less relevant works were reported on Cassava,which is an important economic crops in tropical and subtropical area.In this study,we identified 18 genes which can encode a potential SR proteins according to the Cassava genomic database(https://phytozome.jgi.doe.gov/)through Blast program.We then build the phylogenetic tree through MEGA program and systematically annotate those proteins.Moreover,some of biological roles were analyzed in these 18 cassava SR proteins.The results are followed:Firstly,according to multiple amino acid sequence alignment,cassava SR proteins were classified into six subfamilies of plant SR protein according to the structural characteristics and homology,respectively.The protein physical and chemical properties were then analyzed.The results showed that all SR proteins are hydrophilic basic proteins due to the abundance of positively charged Arg residues.Gene structure analysis shows that members of the same subfamily have a certain degree of conservation in exon/intron numbers,while exons and intron lengths are different.The analysis and prediction of the cis-acting elements contained in the promoter region of the gene showed that the specific elements including the photo-responsive elements,various hormone response elements and stress response elements preliminarily reflected the close relationship between the SR protein and stress stress.Secondly,we extracted RNA samples from different tissues and stress treatments in cassava and analyzed their splicing patterns and expression patterns by RT-PCR and qRT-PCR.The results showed that 14(78%)of the 18 SR genes showed tissue-specific patterns.Moreover,the AS patterns of these SR proteins were increased in response to abiotic stress treatment,and the expression and splicing patterns of some cassava SR genes were affected by one or more stresses,which is consistent with the relevant results in Arabidopsis.Besides,SR proteins appear the diversity of AS patterns under ABA treatment,while only three genes have different splicing diversity in Arabidopsis.Lastly,in order to better understand the role of SR proteins,we extracted RNA samples from different tissues and stress treatments in cassava,and then 18 cassava SR genes were cloned into pDONR207 by Gateway BP clonase.These entry vectors were then cloned into the destination vectors which contained N or C-terminal GFP by Gateway LR clonase.We then transiently expressed those SR proteins contain GFP fluorescent tag in tobacco.The results showed that all 18 genes have fluorescent signals located in the nucleus,indicating that cassava SR proteins are nuclear localization,which is consistent with the involvement of SR protein as an important splicing factor in the regulation of the physiological function of post-transcriptional splicing in planta.