Study On The Response Of EES-related GhIRE1 To Salinity Stress In Cotton

As an important raw material of textile industry,cotton inhabits an important position in the national economy of China.A variety of environmental stresses,such as drought,high temperature,and salinity often affects the growth of cotton,severely limiting the cotton yield.Endoplasmic Reticulum Stress?ERS?is a widely existed stress mechanism to external stimuli in plants and animals.This pathway maintains the ER homeostasis and alleviates stress damage through regulation of a series of gene expression.Therefore,the study of ER stress mechanism is of great significance for improving plant stress resistance mechanism and screening of multi resistance genes.Soil salinization is a worldwide problem affecting crop yield,so it is very important for stable yield of crop to study the salt tolerance mechanism and related genes.In this study,structure changes and gene expression of endoplasmic reticulum under salt stress were explored,followed by screening of ER stress related genes which harbor a key role under salt stress.Finally,the mechanism of the ER stress related gene participating in the regulation of salt stress were preliminarily discussed.1. Structural changes and gene expression of endoplasmic reticulum under salt stress in cottonThe relationship between salt stress and endoplasmic reticulum?ER?was analyzed using transmission electron microscope and RNA-seq data at cellular ultrastructure and molecular levels respectively.The observation of cell ultrastructure indicated that salt stress induced the expansion or deformation of the ER lumen followed by degradation of the subcellular organelles present in cytoplasm including destruction of ER structure.Finally,organelles and most of the cytoplasm were degraded.Stress process of leaf cells was observed slightly slower than that of apical cells.At molecular level,salt stress induced differential expression pattern of endoplasmic reticulum related genes as well as genes related to protein synthesis,endoplasmic reticulum associated degradation and unfolded folded protein response.2. Functional Analysis of ER stress related key genes in upland cottonFive bZIP genes,two from subgroup B?GHbZIP92,GHbZIP197?and three from subgroup K?GHbZIP40,GHbZIP129,GHbZIP145?identified by gene family analysis were found directly involved in ER stress.Subgroup B bZIP genes were homologous to AtbZIP17 and may participate in the AtbZIP17/28-S1P pathway.Subgroup K bZIP gene which were homologous with AtbZIP60 were possible involved in IRE1 mediated bZIP m RNA splicing pathway.Among them,the proteins of GHbZIP92 and GHbZIP129located in the nucleus,while GHbZIP197,GHbZIP40 and GHbZIP145 located in both endoplasmic reticulum and the nucleus.The promoters of these five genes are rich in hormone response elements,such as Me JA,SA,GA,ABA,and so on.These five genes are significantly differentially expressed under the conditions of high temperature,low temperature,salt and PEG stress.It is suggested that the endoplasmic reticulum stress-related bZIP genes were involved in responses to a variety of severe stresses.By comparing with IRE1 genes of Arabidopsis thaliana and rice,four upland cotton IRE1genes were identified,among which GH?A02G2057 and GH?D03G0007 were closely related to Arabidopsis thaliana IRE1a,and GH?A01G1004,GH?D01G1034 and were homologous with At IRE1b.Protein interaction analysis showed that bZIP proteins in subgroups B and K interacted with Gh IRE1 proteins as well as with GhbZIP proteins in other subgroups.Therefore,different pathways of ER stress in upland cotton are not completely independent,and they may work together to regulate the expression of stress-related genes.3. Mechanism of ER stress gene Gh IRE1 participating in salt stress regulationGene silencing experiments showed that Gh IRE1 genes plays a more critical role in regulation of salt stress as compared to B subgroup bZIP genes and Gh IRE1a and Gh IRE1b are may be functionally complementary to each other.In order to further explore the function of Gh IRE1 gene,we carried out the overexpression analysis of Gh IRE1 gene in Arabidopsis thaliana.s1 and s2,two splicing variants of ER protein Gh IRE1a?GH?A02G2057?,can respectively express the complete IRE1 protein containing the ribonuclease domain and the kinase domain and the protein containing only the kinase domain.Two splicing variants s1 and s2 were overexpressed in Arabidopsis thaliana,respectively.The salinity tolerance of s1 overexpressed plants was lower than that of the wild type,and the salinity tolerance of s2 overexpressed plants was not significantly different from that of the wild type.It indicates that the ribonuclease domain of IRE1 plays an important role in the regulation of salt stress.Treating cotton with ribonuclease inhibitors can alleviate the damage caused by salt stress and reduce the accumulation of H₂O₂ in cotton leaves,indicating that the ribonuclease domain of Gh IRE1 may participate in the response to salt stress by regulating the accumulation of H2O2.4. Gene editing and molecular detection of Gh IRE1 in cottonFor further functional studies,we performed gene editing of Gh IRE1 using the CRISPR/Cas9 system.We constructed a recombinant vector containing four sg RNA target sequences of Gh IRE1 genes.The enzyme digestion verification results showed that the double-enzyme digestion of recombinant vector can produce a short band with the size of 2000?3000bp,which was consistent with the expectation,indicating the successful construction of the vector.Sixteen transgenic candidate plants were obtained by gene gun-mediated pollen transformation technology.Using primers of Cas9 gene for PCR detection,three cottons that could be amplified to the target band were obtained,which were preliminarily judged to be positive cotton.The acquisition of positive plants laid a foundation for the further study of Gh IRE1 function and was also of great significance for the creation of new salt-tolerant germplasm.