Bioinformatics And Expression Assay Of Mulberry MLX56 Gene Family

Mulberry tree is a dicotyledonous plant,also as one of an important economic crops.It is widely distributed in tropical and temperate zones.Mulberry leaves played an important material base of sericulture industry for it is the optimal nature feed for domesticated silkworm(Bombyx mori L.) Mulberry also attracts people for its fruit that is nutritious and the taste is delicious,not only can be used as the functional food raw materials, but also can be used as a Chinese traditional medicine.The application prospects is promising.Latex have the function of antimicrobial and resistant to insects,are the plant secondary metabolites that secret by the mulberry tree.Mulberry latex gene plays an vital role in determining anti-insect and defense. Identification the MLX56 gene family from the mulberry genome database, the genetic evolution,gene structure and gene expression was analysised in mulberry will be helpful to study the function identification of plant latex genes. According to mulberry genome database and bioinformatics method, analyzing the gene structure and evolution of mulberry MLX56 gene family, a phylogenetic tree was created using the MEGA4.1 program. The MLX56 gene expression level in different mulberry populations and different tissures was analyzed using differential expression analysis and semi-quantitative. A recombinant plasmid pET28a-MLX56-6 was constructed.Then transferred pET28a-MLX56-6 into Escherichia coli BL21 (DE3), inducing MLX56-6 protein expression with 0.5 mmol·L-1IPTG Next, samples induced at different times were collected and SDS-PAGE was used to analyze the protein expressed by E. coli BL21 (DE3). Using ultrasonic wave to break the efficiently expressed bacterium liquid, then SDS-PAGE was used to analyze the solubility of MLX56-6 protein. Western Blot confirmed the successful expression of MLX56-6 in E. coli BL21 (DE3). The main results of this study were showed as followed:1.Bioinformatics analysis and cloning of mulberry MLX56 gene familyA total of six MLX56 genes were identified from mulberry genome database, they are tandem repeat sequences,the length of six MLX56 gene coding sequence is about 2000 bp,among these genes,four MLX56 gene include one intron number,the last two MLX56 gene include two intron numbers.Moreover,we have cloned a new MLX56 gene,named MLX56-7(GeneBank accession number:KJ496133). Structure domain analysis show that the MLX56 gene family conclude two chitin binding domain,between them,it have an extension domain,all the MLX56 gene have signal peptide, belongs to secretory protein. Phylogenetic analysis revealed that the highest degree of identity (66%) was between mulberry MLX56 gene and Sambucus nigra hevein-like protein, and a lower value (49%,48%) between mulberry MLX56 and Camellia sinensis chitinase and Vitis vinifera chitinase.2.The expression level in eighteen mulberry materies and M. atropurpurea ’Guiyou62’ tissues of MLX56 gene familywe designed universal primers based on the seven MLX56 gene extension domain diversity and use different mulberry materies young leaves cDNA as a template to PCR amplification.Nucleic acid PAGE electrophoresis analysis indicated that MLX56 gene expression patterns in different mulberry populations are different, Tissue specificity analysis showed MLX56 gene in M. atropurpurea Roxb. ’Guiyou62’ expression patterns of different organization is different. The MLX56 gene in different mulberry populations was analyzed using semi-quantitative. The result showed that MLX56-2, and MLX56-4 were expressed in every mulberry meteries; MLX56-3 gene was not detected in any mulberry meteries. MLX56-1,MLX56-5,MLX56-6 and MLX56-7 was detected in partial mulberry materies.Tissue specific expression analysis revealed MLX56-2, MLX56-4, MLX56-5,MLX56-6 and MLX56-7 were expressed in all tissues of M. atropurpurea ’Guiyou62’,MLX56-1 only expressed in petioles and stems, MLX56-3 gene was not detected in any tissues in M. atropurpurea ’Guiyou62’.3. MLX56-6 gene prokaryotic expression analysis and determination of growth rateProkaryotic expression analysis suggested that the fusion protein was successfully expressed with 0.5 mmol·L-1 IPTG induction. Solubility analysis showed that the fused protein mainly existed as inclusion bodies. Western Blot confirmed that the molecular weight of the recombinant MLX56-6 was about 56 kDa. But the Escherichia coli growth rate was inhibited by MLX56-6 protein.