Functional Identification And Analysis Of Salt Tolerance Of Rice RNA Helicase RH6 And RH8

Soil salinization caused by global climate change has seriously affected the safety of grain production and economic development in China.As an important economic crop,the growth and development of rice are not only inhibited,but also the expression of many important genes are affected under salt stress.In this study,genes related to salt stress in rice were excavated to reveal their mechanisms by means of biochemical physiology and molecular biology,which provided a theoretical basis for improving salt tolerance of rice varieties and improving rice yield and quality.In this study,genes related to salt stress in rice were excavated to reveal their mechanisms by means of biochemical physiology and molecular biology,which provided a theoretical basis for improving salt tolerance of rice varieties and improving rice yield and quality.RNA helicase is involved in all aspects of RNA metabolism and function,and plays an important role in regulating plant growth,development and response to environmental stress.In the degradation of eukaryotic cells,the poly(A)tail of most m RNAs in the cytoplasm usually initiates the 5’-3’m RNA degradation pathway by deadenylation,followed by the uncaping reaction of m RNAs,which requires the participation of A variety of uncaping factors.DEAD(Asp-Glu-Ala-Asp)-box RNA Helicase 6(DHH1/DDX6)is a capping factor involved in the 5’-3’m RNA degradation pathway.So far,the above-mentioned DHH1/DDX6-like RNA helicase response to abiotic stress(salt and drought)mediated by Arabidopsis thaliana has been reported sporadically,but the related biological functions and regulatory mechanisms in rice have not been reported yet.In this paper,the regulation of rice DHH1/DDX6-like RNA helicase RH6 and its homologous gene RH8 in salt stress tolerance response and related mechanism were studied.The results are as follows:First,the sequence similarity between OsRH6 and Arabidopsis At RH6 and At RH8respectively was 73.48%and 80.59%.The sequence similarity between OsRH8 and At RH8and At RH6 was 80.80%and 74.48%.The results showed that RH6/RH8 had high homology between rice and Arabidopsis thaliana.Further analysis showed that OsRH6 and OsRH8 also had high homology(89.59%).OsRH6 and OsRH8 both contain DEXDc and HELICc conserved domains.RT-PCR analysis of whole fertility showed that OsRH6 and OsRH8 were mainly expressed in roots and leaf sheaths during vegetative reproductive and booting stages of rice.At flowering stage,it was mainly expressed at nodes.It was mainly expressed in roots and seed coat at maturity stage.During the whole physiological period of rice,OsRH6 and OsRH8 had low expression levels in leaf composition.Under drought stress,the transcriptional expressions of OsRH6 and OsRH8 were not significantly different in roots and leaves,while under salt stress,the transcriptional expressions of OsRH6 and OsRH8 were induced and upregulated in roots,but there was no significant difference in leaves.The results of subcellular localization in tobacco showed that OsRH6 and OsRH8were both located in the nucleus and the Processing body.In order to further study the function of OsRH6 and OsRH8 under salt stress,OsRH6-Cas9 and OsRH8-Cas9 single knockout lines,OsRH6/8-Cas9 double knockout lines,OsRH6 and OsRH8 overexpressed lines(OE)were constructed,and corresponding homozygous(T3)rice transgenic materials were obtained.Compared with wild-type(WT),OsRH6-Cas9 plants were insensitive to salt stress,with increased dry weight,decreased electrical conductivity and decreased Malondialdehyde(MDA)content.There was no significant difference in the growth phenotype of OsRH8-Cas9 plants,but the electrical conductivity and MDA content were decreased.OsRH6/8-Cas9 plants were insensitive to salt,which were mainly characterized by lower leaf burning degree,higher plant height,higher dry weight,higher chlorophyll content,lower electrical conductivity and lower MDA content,and less cell damage.On the contrary,OsRH6 and OsRH8 overexpressed plants were more sensitive to salt stress,which mainly showed yellow leaves,decreased plant height,decreased dry weight,decreased chlorophyll content,increased electrical conductivity and MDA content,and greater degree of cell damage.By ICP-MS determination of Na~+and K~+in rice of accumulation,the results show that under salt stress,compared with WT,OsRH6-Cas9 and OsRH8-Cas9 knockout strain accumulated in the stem leaf less Na~+,K~+/Na~+value increases,OsRH6-Cas9 knockout strain also accumulate less Na~+in roots,but no difference between the content of K~+,OsRH8-Cas9 less knockout strain accumulation in the root of K~+,Na~+content has no obvious difference,the root values of K~+/Na~+increased.OsRH6/8-Cas9 knockout lines accumulated less Na~+in both stems,leaves and roots,and K~+/Na~+values increased,but K~+content had no significant difference.However,OsRH6 and OsRH8 overexpressed plants had the opposite results.In conclusion,OsRH6 and OsRH8 knockout lines can improve the tolerance of rice to salt stress,and OsRH6 knockout lines have a greater tolerance to salt stress than OsRH8 knockout lines.Compared with single gene knockout material,double gene knockout could improve the tolerance of rice to salt stress.Under normal conditions,OsRH6-Cas9,OsRH8-Cas9 and OsRH6/8-Cas9 knockout lines had a certain delay effect on seed germination time,but under ABA treatment,compared with WT at the same time,OsRH6-Cas9,OsRH8-Cas9 and OsRH6/8-Cas9knockout lines could increase the germination rate of seeds.Through Bimolecular Fluorescence Complementation(BIFC),it was found that OsRH6,OsRH8 and OsRH12 proteins can interact with each other in living plant cells.However,OsRH6 and OsRH8 have a different localization in the cell.Originally,OsRH6and OsRH8 were located in the nucleus and the p-body,but after the interaction,they could only be located in the p-body.OsRH6 and OsRH8 can interact with Arabidopsis thaliana.At DCP5(Decapping 5)in plant living cells,respectively,and the process is dynamic.Transcriptome sequencing results showed that OsRH6/8-Cas9 knockout lines differed in the expression of some salt-related genes involved in the metabolism of antioxidant enzymes,cell protective proteins(such as osmotic proteins)and response to salt stress compared with wild-type rice.GO enrichment analysis showed that the differentially expressed genes were mainly related to stress response and metabolic process in biological process.In terms of molecular function,the main functions are kinase activity,catalytic activity and transferase activity.Among the cellular components,it is mainly related to the membrane components.KEGG significant enrichment analysis showed that differentially expressed genes were mainly involved in plant-pathogen interaction,photosynthesis,plant hormone signal transduction and MAPK signaling pathways.