Mechanism Of Rice(Oryza Sativa L.) Receptor-like Protein Kinase OsSRK Regulating Heavy Metal Cadmium Stress Response

With the increasing severity of soil heavy metal pollution,the problem of heavy metal pollution of food crops is imminent.It is extremely important to explore the molecular regulation mechanism of rice response to cadmium(Cd)for breeding low-Cd-accumulting crops and improving food safety production.Receptor-like protein kinases(RLKs)are a kind of important protein kinase in plants,which are involved in the regulation of plant growth and development,and a variety of abiotic stress responses.Taking the rice receptor-like protein kinase OsSRK as the research object,this paper studied the function of OsSRK in rice Cd accumulation and tolerance using CRISPR/Cas9 technology,analyzed the expression characteristics of OsSRK and its functional interaction with OsTDLIB ligand,and further excavated the downstream target proteins of OsSRK by transcriptome technique,in order to explore the Cd regulatory pathway which OsSRK was involved.1.Expression analysis of OsSRK gene and interaction between OsSRK and OsTDL1BThe length of OsSRK gene is 3951 bp,encoding 1316 amino acids.Bioinformatics analysis show-s that OsSRK protein has N-terminal signal peptide,and the extracellular domain include 11 LRR motifs and two transmembrane domains,one intracellular kinase domain,which is a typical LRR-RLK.3-week-old WT ZH11 plants were treated with 10μM CdCl2 for different time.The results of qPCR showed that the expression of OsSRK in the leaves increased after 3 h of Cd treatment;and the expression of OsSRK in the roots began to increase after Cd treatment for 36 h.We then analyzed the expression patterns of OsSRK and OsTDLIB together.We found that expression levels of OsSRK and OsTDLIB in leaves and roots had the same basic trend from 3 h to 72 h.Based on these results,we inferred the possible interaction between OsSRK and OsTDLIB.We then studied the interaction relationship between OsSRK and OsTDLIB by yeast two-hybrid system.Baits(pGBKT7-OSTDL1B)and capture(pGADT7OsSRK)vectors were constructed.The baits self-activation test showed no self-activation.The results of screening of fusant yeast defects showed that there was interaction between OsSRK and OsTDL1B.The interaction between OsSRK and OsTDL1B was further verified using bimolecular fluorescence complementary technique(BiFc).OsSRK and OsTDL1B was found to beinteracted on cell membrane by fluorescence confocal microscopy,which was consistent with yeast two-hybrid results.2.Response of rice ossrk mutant plants to Cd stress and Cd accumulation characteristics analysisWe used CRISPR/Cas9 to construct OsSRK knockout vectors,and successfully obtained homozygous ossrk mutants.The WT and ossrk mutants were treated with 10μM CdCl2,and the results were as follows:(1)Compared with WT under Cd stress,the ossrk mutants had lower growth vigor,shorter plant heights and root lengths;The chlorophyll contents also decreased compared with WT.The smaller the seedling age of rice,the more obvious the phenotype.(2)The contents of MDA and H2O2 were higher than WT,and the contents of POD and SOD were lower than WT.(3)The Cd contents in roots,stems and leaves of ossrk mutants were significantly higher than WT.(4)The expression levels of Cd uptake and transport related genes OsIRT1,OsNRAMP5,OsHMA2 and OsHMA3 in the mutants were higher than those in WT.These results indicated that ossrk mutation could increase rice sensitivity to Cd,and up-regulated the expression of Cd transporter genes to increase Cd uptake and transport in rice,thus enhancing the Cd toxicity of rice mutants.3.Exploration of the downstream genes of OsSRK by transcriptome sequencingThe WT ZH11 and ossrk mutants were used as materials to analyze differential genes and GO enrichment pathway by RNA-seq technique.It was found that rice Cd tolerance network might be regulated by HMP genes and MYB transcription factors.Based on qPCR analysis,the expression trends of 11 genes were consistent with RNA-seq results.,including six HMP genes(OsHMP,OsHMP15,OsHMP7/OsATX,OsHMP32,OsHMP31 and OsHMP6),a Cd uptake gene(OsIRT1),three MYB transcription factors(NIGT1,OsMYB4,OsMYB3)and a BHLH transcription factor gene(OsMBHLH091).Screened OsIRT1,OsATX(OsMHP7),MYB transcription factor OsMYB4 and NIGT1 may play roles in the OsSRK regulating Cd response pathway.