Functional Analysis Of The RcCMLs Salt-Responsive Genes In Ricinus Communis L.

Soil salinity is one of the most important abiotic stressors affecting plant growth,and molecular breeding to create new salt-tolerant crop varieties is an effective means of exploiting and improving saline soils.Based on the transcriptomic data of castor obtained by our group,the differentially expressed Calmodulin-like proteins(CMLs),RcCML13 and RcCML42,were compared between "untreated castor and salt-treated ZheBi 3 "genes.Using ZheBi 3 as the material,RcCML13 and RcCML42 were cloned to analyse the expression characteristics of castor under salt stress at seedling stage,and the transgenic material was obtained by root cancer Agrobacterium-mediated transformation of arabidopsis and castor to overexpress the two genes and analyse their role in salt stress tolerance in castor.The RcCML42 gene with high up-regulation ploidy was selected for editing and knocking out to further analyze the salt tolerance function and to lay the foundation for obtaining salt tolerance tool genes and salt tolerance germplasm resources.The following research findings were obtained:1.The RcCML13 and RcCML42 genes were obtained by cloning in ZheBi3.The Open Reading Frames(ORFs)of the two genes were 444 bp and 597 bp respectively,and the number of amino acid residues encoded was 147 and 198.Both genes do not contain transmembrane structures and are typical of non-transmembrane proteins.Secondary structure prediction showed that the alpha-helix was the most abundant structure,containing 87 in the RcCML13 gene and 98 in the RcCML42 gene.The results of protein conserved domain prediction for both showed that three EF-hand calcium-binding structural domains were present in both genes,but there were differences in the location of each domain in the gene.2.The spatio-temporal specific expression of the two genes in the roots,stems and leaves of castor seedlings under salt treatment was determined using quantitative real-time fluorescence PCR(qRT-PCR),and the RcCML13 and RcCML42 genes were found to be expressed in castor roots,stems and leaves,and the expression of these two genes was highest in castor leaves and second highest in stems and roots.Positive arabidopsis and castor plants containing overexpression of the RcCML13 and RcCML42 genes were identified by PCR,respectively,after the cotyledonary nodes of castors were infiltrated by Agrobacterium rhizogenes-mediated,DNA was extracted after culture and positive plants containing the CRISPR/Cas9-RcCML42 gene were identified.3.The results of the physiological indicators showed that under salt stress,the leaves of castor overexpressing the RcCML13 and RcCML42 genes generally had lower Na+content,higher K+content,higher Ca2+ content,higher superoxide dismutase(SOD)and catalase(CAT)activities and lower malondialdehyde(MDA)content than the wild type,and combined with the role of each substance in the plant body it can be concluded that the transgenic plants showed greater salt tolerance.The CRISPR/Cas9 knockdown of the RcCML42 gene revealed that the ion profiles in castor leaves under salt stress were opposite to those of the overexpressed RcCML42 gene,that MAD content was higher in the gene edited plants than it was in the wild-type plants,and that SOD and CAT in the gene edited plants showed lower activity than in the wild-type castor.It can be inferred that gene editing to knock out the RcCML42 gene reduced salt tolerance in castor.