The Establishment Of CRISPR Activation System And Activation The SYM Pathway In Rice

Symbiosis pathway(SYM)is a common pathway of rhizobia and mycorrhizal fungi invading plants.Studies have revealed that the SYM pathway of leguminous plants is activated during the symbiosis with rhizobium,but the SYM pathway of monocotyledons such as rice and maize that can be symbiotic with mycorrhiza fungal is silenced during interaction with rhizobium.The expression of three receptor kinases(symbiotic receptor kinase SYMRK and nod factor receptor kinases NFR1,NFR5)in the SYM pathway plays a decisive role in the recognition of plants and rhizobium.The CRISPR activation system activates the expression of endogenous genes,but activation of three types of receptor kinases in the SYM pathway of rice has not been reported;A.caulinodans ORS571 can colonize rice and induce to produce root nodules under the action of 2,4-D,while slightly increasing the dry weight and total nitrogen of rice,but whether to activate the SYM pathway at the same time remains to be explored.Therefore,this experiment used the CRISPR system to activate the expression of OsCERK1,OsNFR5 and OsSYMRK.On the other hand,2,4-D and A.caulinodans ORS571 were combined to induce nodulation in rice seedlings to detect the expression of related genes on the SYM pathway.This study laid the foundation for the preliminary experiment based on the combination of CRISPR activation system and 2,4-D induced rice to symbiotic nodulation.The results of this study are as follows:1.The full-length sequences of OsCERK1,OsNFR5 and OsSYMRK were successfully cloned.Bioinformatics software analysis showed that the amino acids of OsCERK1,OsNFR5 and OsSYMRK proteins were 625,624 and 576 respectively.Each has a transmembrane domain;The phylogenetic tree and amino acid sequence alignment showed that they were closely related to legumes and the sequence similarity was high.2.Based on the p35S-e GFP vector,OsCERK1,OsNFR5 and OsSYMRK subcellular localization vectors were constructed and transiently transformed into rice protoplast.The results showed that OsCERK1,OsNFR5 and OsSYMRK were all located on the cell membrane in accordance with the bioinformatics analysis results.3.Two key nuclease splicing sites of Cas9 protein were mutated by point mutation to obtain d Cas9.4.CRISPRa vector for agrobacterium-mediated genetic transformation and CRISPRa vector for instantaneous transformation of rice protoplasts were constructed.Meanwhile CRISPRa vectors targeting three receptor kinase genes of OsCERK1,OsNFR5 and OsSYMRK was constructed.The instantaneous transformation of rice protoplasts indicated that the expression of OsCERK1,OsNFR5,and OsSYMRK were up-regulated.5.Rice seedlings were inoculated with 2,4-D and A.caulinodans ORS 571 at the same time.The expression of OsCERK1,OsNFR5 and OsSYMRK in the SYM pathway were up-regulated.