Structural Analysis Of The Coding Region Of The Sugar Beet BvNLP2-like Gene And Establishment Of A Prokaryotic Expression System

Sugar beet,as the main sugar crop in North China,is still lack of genetic resources.In the preliminary work of the research group,a molecular marker site related to the multi grain trait of sugar beet was obtained from the sugar beet genome,and the national invention patent（ZL 2018 1 1319502.5）was obtained.The gene site near the marker was cloned from the sugar beet genome,predicted to encode an NLP gene,and the gene was named BvNLP2-like.In this study,the complete and exact coding sequence（CDS）was obtained by gene cloning with the gene site as the starting material and c DNA as the template.The length of the coding region was determined to be 2877BP.The gene belongs to the gene family of NIN like protein（NLP）.Analyze the structure of the coding region in the gene site,predict the gene splicing site combined with the obtained sequence and the previous analysis results,and the design scheme is verified by experiments;The accurate nucleotide and amino acid sequences were used for bioinformatics analysis,the appropriate enzyme digestion sites and fusion code reading frame were designed,and the prokaryotic expression vector of the gene was constructed;The identified prokaryotic expression vector was transformed into E.coli,and the reaction conditions such as culture,induction and SDS-PAGE electrophoresis were optimized to establish a prokaryotic expression system suitable for the target gene.Through this study,we will have a further understanding of the sequence and structural characteristics of BvNLP2-like gene,and lay a foundation for the functional analysis of BvNLP2-like gene.The main results are as follows:（1）The coding region of BvNLP2-like gene was cloned,and the complete ORF sequence of the gene was obtained,with a length of 2877 bp,a coding product length of958aa.（2）After obtaining the CDS of sugar beet BvNLP2-like gene,the structure of the CDS was analyzed and verified via different strategies.According to the exact sequence,the structural analysis results and the PCR experiments,it is found that the target sequence contains two splicing sites,including three exons and two introns in the genome locos.（3）The amino acid sequences translated by the coding region were analyzed by bioinformatics.The results showed that the protein encoded by sugar beet BvNLP2-like gene BvNLP2-like had a relative molecular weight of 104.463 k D and a theoretical isoelectric point of 10.48.Its molecular formula was C₄₇₅₈H₇₇₅₇N₁₃₂₁O₁₂₁₆S₄₈.At the amino acid level,it was inferred that the protein encoded by this gene had no obvious transmembrane domain,might be a hydrophilic protein,and might be located in the nucleus.The protein function was consistent with that of NLP gene family.（4）The prokaryotic expression vector PET21a-NLP1 was constructed,the prokaryotic expression system was established,and the experimental parameters of BvNLP2-like gene expression were optimized.Through SDS-PAGE electrophoresis,it was found that the optimal induction conditions of protein were 37℃,24h,and the final concentration of IPTG was 0.5mM.