Cloning Of CaM-likeprotein Gene(CML38-like) And Functional Verification In Soybean

As an important oil crop and food crop,the drought of soybean planting area has become an important factor affecting soybean yield and quality.Therefore,to discover the soybean drought resistance gene is an important way to solve the problem of soybean drought resistance.The study found that the crop calmodulin-like?CML?gene has a certain effect of drought resistance and salt tolerance,and the soybean CML38-like gene studied in this experiment is a kind of calmodulin-like gene.The study found that this gene can be combined with calcium in cells,which cause the idiosyncrasy of downstream of the excitant,make the intracellular calcium ion concentration increase or decrease,thus under abiotic environmental stresses in plants caused by stress response,have a certain effect on drought stress.The CML38-like gene studied in this research is one of the calmodulin-like genes.Using soybean JN38 genome as template,CML38-like gene with fragment size of 264bp was obtained by PCR,and it was connected to pMD18T cloning vector.After a series of bioinformatics analysis,the function of the gene was preliminarily speculated.Subsequently,the over-expression vector pCAMBIA-3301-CML38-like and the interfering expression vector pCAMBIA-3301-RNAi-CML38-like were constructed by seamless cloning.The expression vector was transformed into the soybean receptor JN38 by agrobacterium-mediated method and pollen tube channel method,and the positive transformed plants were detected by PCR.Subsequently,PCR,Southern blotting and fluorescence quantitative PCR were used to detect the integration of this gene and its expression in the root,stem and leaf of soybean.Finally,the indoor drought resistance of positive plants was analyzed to identify the drought resistance function of CML38-like gene in soybean.The test results are as follows:1.The soybean CML38-like gene was cloned by PCR,and the cloned vector pMD18T-CML38-like was constructed.Domain analysis of the gene revealed that the soybean CML38-like gene belongs to the Ef-hand family and has two Ef-hand structures,each of which has four calcium binding sites.The prediction of secondary structure and tertiary structure of proteins confirmed that this gene family can bind Ca²⁺and regulate the physiological mechanism of crops under drought.The homology similarity of this gene with multiple drought-resistant genes analized in phylogenetic tree were high.2.Through seamless cloning kit,pCAMBIA-3301-CML38-like gene over-expression vector of soybean CML38-like gene and RNA interference expression vector pCAMBIA-3301-RNAi-CML38-like were constructed by cloning vector pMD18T-CML38-like.3.Agrobacterium-mediated method was used to transfer the constructed recombinant plasmid into the recipient soybean JN 38.After PCR detection,3 plants containing the soybean CML38-like drought resistance gene T₀ generation transduction expression vector positive plant were obtained,1 plant interfering expression vector plant were transferred,7 plants were T₁generation over-expression vector positive plant,and 3 plants were T₁ generation transgenic expression vector positive plant.Four plants with positive T₁ over-expression vector and 3plants with positive T₁ interference expression vector were obtained by the pollen tube passage method.All T₁ generation transgene positive plants were supergenerated indoors,and a total of23 T₂ generation expression vector positive plants and 11 interference expression vector plants were obtained.4.Promoter 35s,terminating Nos,and screening marker Bar of the transformed target gene were detected in all T₂ transformed strains,indicating the target gene was inherited stably.5.The results of Southern hybridization of T₂-generation transgenic plants showed that the genes of the transgenic soybean CML38-like over-expression vector and the genes of the transgenic soybean CML38-like interfering expression vector were integrated into the soybean genome in the form of single copy and multiple copy,and the integration sites were different.6.The transgenic plants with signals from Southern hybridization were subjected to repeated drought,and the plants were detected by fluorescence quantitative PCR after 7day-drought.The results showed that the genes of CML38-like over-expression vector were all expressed in the roots,stems and leaves of soybean plants,among which the highest expression was found in the leaves with a relative expression of 3.059,and the lower expression was found in the roots and stems with an average expression of 2.502 and 1.647.The gene of transgenic soybean CML38-like interfered expression vector inhibited the expression of soybean endogenous genes and reduced the expression of roots,stems and leaves,among which the expression in leaves decreased the most,the average value is 0.224.The expression levels in roots and stems decreased less,and the relative expression levels were 0.271 and 0.297,respectively.7.The drought resistance of T₂ generation transgenic positive plants was identified.After 7day-drought treatment,the physiological and biochemical indexes of the plants were detected.In summary,the results indicated that CML38-like gene could play a positive role in drought resistance during soybean growth.