Characterize Of Cassava Sr Protein On The Role Of Alternative Splicing And Function In Response To Salt Resistance In Arabidopsis

Cassava,the important source of staple food in the tropic area,is consumed by nearly 600 million populations in the world.The main development direction of cassava industry is edible,feed and industrial raw materials.Cassava is divided into edible cassava and industrial cassava by its application.The motif of cassava industrial developmental goal is to develop the different varieties of cassava in order to apply in different purpose.With the development of genomics,transcriptomics and proteomics,the molecular biology is used to guide the development of new varieties of cassava.It is the basic of increasing the yield of cassava and expanding the suitable area to study the mechanism in response to biotic and abiotic stress.Previous studies of molecular biology at the transcriptional level are focused on the steady state m RNA accumulation,and less information is known about the splicing regulation of pre-m RNA in response to stress and hormone treatment,which is a post-transcription regulation.Alternative splicing refers to the process of producing different m RNAs from a pre-m RNA through a complex and controlled process.Alternative splicing occurs mainly on the spliceosome.The main process of alternative splicing is two trans-esterification reactions performed by five small nuclear ribonucleoprotein particles(sn RNPs)and a series of non-sn RNP splicing factors.Arginine/ serine-rich protein,SR protein,is a kind of important splicing factor that regulates the alternative splicing.It mainly recognizes the splicing enhancer on pre-m RNA and assists sn RNP in completing the splicing.The SR protein is characterized by one or two RNA recognition motifs(RRM)at the N-terminus and multiple serine and arginine repeats at the C-terminus.The main function of RRM domain and SR motif are respectively binding to pre-m RNA and interacting with other proteins.The SR proteins in animals are mainly divided into three subfamilies,and the plants have other three unique subfamilies in addition to those three subfamilies.The research on alternative splicing in cassava has not been reported in details.This project was systematically named the genes encoding SR protein in cassava and preliminarily studied their functions.The main research work and results were summarized as follows:1.In this paper,18 possible SR genes were found through blast,and their evolutionary relationships with Arabidopsis and rice were analyzed.The result indicated that cassava was closer with Arabidopsis than rice in evolutionary relationship.2.The basic information of 18 SR genes was analyzed,including the length of CDS,isoelectric point,protein size,gene structure,protein structure,etc.;3.Real-time PCR(RT-PCR)experiment was analyzed the expression pattern and alternative splicing pattern of 18 SR genes in different tissues.The results showed that SR genes were constitutive genes,and expressed in all tested tissues and had different splicing transcripts in different tissues.4.RT-PCR experiment was analyzed the alternative splicing of 18 SR genes in response to abiotic stresses and hormones.It was found that SR genes had different splicing patterns in response to tested abiotic stresses and hormones,and salt stress had the greatest impact on the splicing patterns of SR genes.5.Quantitative real time PCR(q PCR)experiment was analyzed the expression levels of 18 SR genes in different tissues,under different abiotic stress and under hormone treatment.The results indicated that the one stress might have different effects on the same gene in different tissues.6.Me SR34 was selected as the experimental object and then it was overexpressed into Arabidopsis to study its function.It was found that the overexpressors of Me SR34 in Arabidopsis were more tolerant to salt than wild-type.The results suggested that Me SR34 might be related with calcineurin B-like proteins(CBLs)-CBL-interacting protein kinases(CIPKs)pathway.7.The results through RT-PCR were found that most of the 36 genes of CBL-CIPK changed in expression level,but there was little effect on alternative splicing compared with the wild type.