Molecular Cloning And Structural Analysis Of NaD1 In Nicotiana Alata

The NaD1(Nicotiana alata Defensin 1)protein is a plant defensin extracted from the flowers of Nicotiana alata.As a cationic antibacterial protein,NaD1 protein not only has significant resistance to various plant pathogens and human pathogenic fungi,but also has the function of tearing cancer cells.Therefore,NaD1 protein has important research value in botany and medical research.In this study,NaD1 gene was cloned from Nicotiana alata,and the results are as follows:1.Molecular cloning and structural analysis of NaD1geneThe homologous cloning technology was used to clone the coding sequence of NaD1 gene from Nicotiana alata.The analysis showed that the sequence length was318 bp.The homology with the known NaD1 gene coding sequence was 100%.According to the bioinformatics analysis,NaD1 gene encoded a hydrophobic protein of 105 amino acids,whose relative molecular mass was 11.7kDa and isoelectric point was 6.56.It was predicted that this protein contains signal peptide,transmembrane region and 5 phosphorylation sites.The secondary structure of NaD1 protein included 3 ?-helix,1 ?-sheet,which confirmed the characteristics of the plant defensin.Using homologous cloning technology,the NaD1 gene DNA sequence was successfully cloned from the genome of Nicotiana alata.The DNA sequence is514 bp in length and contains two introns and one intron.The cleavage of exons and introns conforms to the GT-AG principle.The intron of NaD1 gene was analyzed and it was found that the intron belongs to U12-type intron.Its sequence is rich in ATand its content is 76.02%,which is consistent with the high content of AT in non-coding region.Using thermal asymmetric interlaced PCR,the 5'-end regulatory sequence of the full-length 644 bp NaD1 gene was successfully cloned.Analysis shows that,In this sequence,the position of 538-588 is the basal promoter region.The transcription initiation site is A.The analysis showed that there are multiple conserved regions associated with the promoter in this sequence.It contains the promoter basic cis-acting elements TATA-box and CAAT-box.It also contains a 5'UTR py-rich stretch,a highly efficient transcriptional element,and cis-acting elements that respond to factors such as light,methyl jasmonate,and ethylene.2.Transcriptional activity analysis of prompter of NaD1 geneThe prompter fragments A(644 bp)and the prompter fragments B(310 bp)of NaD1 gene were each ligated to the plasmid pCAMBIA1391 z toconstruct the plant expression vectors pCAMBIA1391z-PNaD1-644::GUS and pCAMBIA1391z-PNaD1-310::GUS,which drive the expression of the GUS gene.The Agrobacterium-mediated callus genetic transformation method was used to transform Nicotiana alata with this tow recombinant plasmids,respectively.The positive transgenic plants containing GUS gene have been detected by GUS histochemical assay.The results showed that the fragment B had transcriptional activity.3.Expression of NaD1 gene in different tissues of Nicotiana alataThe expression of NaD1 in Nicotiana alata(roots,shoots,leaves and buds)was analyzed by fluorescent quantitative PCR.It was found that the expression level of NaD1 gene in flowers was 988 times that in roots,15 times that in the top shoots,and 10 times that in the leaves.