Construction Of Mutants Of SRPK And AFC Gene Family In Arabidopsis And Study On The Mechanism Of Response To Salt Stress

Pre-mRNA splicing can participate in the regulation of a variety of physiological processes in plants and is a universal regulatory mechanism for eukaryotic gene expression.The occurrence of splicing requires the participation of splicing factors.The SR protein family is an important class of splicing factors.The function of this type of protein requires SR protein kinase to regulate its phosphorylation.In Arabidopsis,SR protein kinases are divided into two types: SRPK and AFC.SRPK includes two families: SRPK1(SRPK1a,SRPK1b)and SRPK2(SRPK2a,SRPK2 b,and SRPK2c).AFC includes three members: AFC1,AFC2,and AFC3.In this experiment,the SRPK and AFC families of Arabidopsis were studied.Based on the previous research,SRPK-related mutants were constructed using CRISPR/Cas9 technology,and the phenotypes were observed and analyzed.Under salt stress,RNA-seq method was used to analyze the changes of SRPK mutant gene expression and alternative splicing;some SR related proteins were obtained through yeast two-hybrid screening library and the corresponding mutants were successfully constructed;Arabidopsis mutants-afc3,afc2/3 were obtained;experimental results revealing the Arabidopsis SRPK2 regulation of salt stress response pathways,and have important theoretical significance.The thesis work is mainly divided into the following aspects:1.Identification and phenotypic analysis of Arabidopsis SRPK mutantsThe mutants srpk1a1b and srpk2a2b2c obtained in the early stage of the laboratory were identified and the SRPK-related mutants were subjected to stress treatment.Under the condition of 0.25 μM ABA,the greening rate of cotyledons of srpk2a2b2 c was significantly lower than that of wild-type and srpk1a1 b,indicating that srpk2a2b2 c was more sensitive to ABA.The GUS strains driven by the SRPK2s promoter obtained in the earlier stage were treated with different concentrations of salt and stained with GUS.The results showed that salt stress can induce the expression of SRPK2 s.Srpk1a1b,srpk2a2b2c and wild-type Arabidopsis were treated with salt stress.Under the condition of 150 m M Na Cl,the growth of the three mutants of srpk2a2b2 c was more strongly inhibited,which showed that the root length became shorter and the fresh weight became lighter.Compared with SRPK1,the SRPK2 subfamily is more important in response to salt stress.2.RNA-seq analysis of change in srpk2a2b2c gene expression and alternative splicing under salt stress conditionsIn order to further explore the molecular mechanism of SRPK2 in response to salt stress,we performed transcriptome sequencing on salt-treated srpk2a2b2 c.The analysis found that the expression levels of 524 genes in the mutants were up-regulated,and the expression levels of 819 genes were down-regulated.Significantly different alternative splicing events occurred in 1381 genes.The reliability of the transcriptome sequencing data was verified by q PCR,and GO functional enrichment analysis was performed on differentially expressed genes and alternatively spliced genes with significant changes in differences.It was found that most genes were enriched in salt stress response pathways and ABA response pathways.Further analysis revealed that the genes enriched in the salt stress response pathway included SOS1,SOS2,AKT1 and the genes enriched in the ABA response pathway included PYL8,ALDH3I1,TOR.This shows that Arabidopsis SRPK2 plays an important role in response to salt stress pathways.3.Exploration of SRPK2 interaction proteins in ArabidopsisIn order to understand the regulatory pathways of SRPK,the yeast two-hybrid screening library method was used to screen the interaction proteins of SRPK2 c,and nine candidate proteins were obtained,including AT1G16610(SR45),AT1G51510(Y14),AT1G55310(SCL33),AT3G53500(RS2Z32),AT4G36980,AT5G13340,AT5G42820(U2AF35B),AT3G13570(SCL30A)and AT3G63400.Among them,SR45,SCL33,RS2Z32 and SCL30 A are all SR proteins,which play an important role in the process of m RNA splicing;Y14 is one of the core components of EJC,and EJC can detect RNA quality;AT4G36980 is a serine/arginine protein related to CLK4;AT5G13340 It is a type of protein rich in arginine/glutamic acid;AT3G63400 is a cyclophilin-like peptidyl-prolyl cis-trans isomerase family protein;U2AF35B is a small subunit of U2 cofactor,which is used in the assembly process of spliceosome play a role.Based on the results of the yeast two-hybrid screening library of SRPK2 a and SRPK2 b in the early stage of the laboratory,the yeast two-hybrid screening library was performed on the common interacting protein and SRPK2 c.The results showed that both AT3G63400 and AT5G13340 interact with SRPK2 c.Combining the preliminary quantitative phosphorylation proteomics analysis and the structural analysis of several candidate proteins,we selected four SR-related proteins: AT3G63400,AT4G36980,AT5G13340,and AT1G10890.To further explain the functions of these proteins and explore the pathways of SRPK2,Using CRISPR/Cas9 technology,the corresponding genes of these four proteins were edited,and homozygous mutants of at3g63400,at4g36980,and at5g13340/at1g10890 were obtained.Preliminary observations found that these mutants have no obvious phenotype,and maybe in plants.Due to the redundant functions of SR-related proteins,these mutants will be subjected to stress treatment in the later stage to further explore the functions of these proteins and the regulation pathway of Arabidopsis SRPK2.4.Construction of Arabidopsis AFC-related mutantsTo explore the function of Arabidopsis SR protein kinase AFC,we obtained afc3 mutants through CRISPR/Cas9 technology,and obtained afc2/3 double mutants through hybridization with afc2,and used CRISPR/Cas9 technology to analyze AFC1 on this basis.And on this basis,using CRISPR/Cas9 technology to edit AFC1,lay the foundation for obtaining afc1/2/3 triple mutants and exploring the functions of AFC protein family.Innovation points of this experiment:1.The response of plants to various adversity stresses in nature cannot be separated from the regulation of alternative splicing.At present,there are many studies on the splicing factor SR protein,but there are very few studies on the plant SR protein kinase SRPK and AFC.In this paper,based on obtaining Arabidopsis SRPK-related mutants,by applying stress treatment and performing transcriptome sequencing analysis,the molecular mechanism of Arabidopsis SRPK2 in regulating stress response has been preliminarily explored;and through The yeast two-hybrid screening library method has conducted a preliminary study on the interaction proteins of SRPK2,which has laid a foundation for revealing the function of plant SR protein kinases,and has important innovative significance.2.A mutant of the Arabidopsis SR protein kinase AFC3 gene was constructed by CRISPR/Cas9 technology and hybridization methods to obtain an afc2/3 double mutant,which laid the foundation for obtaining an afc1/2/3 triple mutant and exploring the function of Arabidopsis AFC.