林业科学Issue(4)：60-70,11.DOI:10.11707/j.1001-7488.20150408
桑树MLX56基因家族特性、克隆及表达分析
Characteristics,Cloning and Expression of the MLX56 Gene Family in Mulberry
摘要
Abstract
Objective]Mulberry( Morus) latex gene plays an important role in determining anti-insect and defense. Identification the MLX56 gene family from the mulberry genome database,and analysis of phylogeny,gene structure and gene expression in mulberry will be helpful to study the functions of plant latex genes.[Method]Based on mulberry genome database,bioinformatics approach was used to analyze the structure and evolution of mulberry MLX56 gene family, a phylogenetic tree was created using the MEGA4. 1 program. The expression of MLX56 gene in different mulberry species and different tissues were analyzed using semi-quantitative TR-PCR. A recombinant plasmid pET-28a-MLX56-6 was constructed and transferred into Escherichia coli BL21 ( DE3 ) . IPTG was used to induce MLX56-6 protein expression. Samples were collected from bacterial suspension at different times of induction and SDS-PAGE was used to analyze the protein expressed by E. coli BL21 ( DE3 ) . Ultrasonic wave was used to break the efficiently expressed bacterial suspension,and SDS-PAGE was used to detect the solubility of MLX56-6 protein. Western Blot confirmed the successful expression of MLX56-6 in E. coli BL21 (DE3),and the effect of the MLX56 gene on the growth rate of E. coli was also studied.[Result]A total of 6 MLX56 genes were identified from mulberry genome database,the mulberry MLX56 contains two chitin-binding domains,and they all have signal peptide,belongs to secretory protein. A new MLX56 gene MLX56-7 (GenBank number:KJ496133) was cloned. Phylogenetic analysis revealed that the highest homdogy (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. The semi-quantitative RT-PCR showed that MLX56-1, MLX56-2, MLX56-4, MLX56-5,MLX56-6 and MLX56-7 were expressed in all mulberry species. Tissue specific expression analysis revealed that MLX56-2,MLX56-4,MLX56-5,MLX56-6 and MLX56-7 were expressed in all tissues of M. atropurpurea‘Guiyou62’,MLX56-1 was expressed only in petioles and stems. MLX56-3 gene was not detected in mulberry species and tissues in M. atropurpurea‘Guiyou62’. Prokaryotic expression results showed 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 E. coli growth rate was inhibited by MLX56-6 gene.[Conclusion]In the process of mulberry evolution,gene duplication happened in MLX56 gene family,and differentiation occurred in the structure of the gene family. Accordiong to the protein and structure characteristics of MLX56 gene family,the gene family may belong to a chitinase with lectin activity. The diversity among different species and tissues in MLX56 gene expression reveals that the functions of these genes were different among different mulberry species and among different tissues of the same species.关键词
桑树/MLX56 基因家族/系统进化/基因表达Key words
Morus/MLX56 gene family/phylogeny analysis/gene expression分类
农业科技引用本文复制引用
韩淑梅,李军,吕蕊花,王晓红,刘长英,赵爱春,鲁成,余茂德..桑树MLX56基因家族特性、克隆及表达分析[J].林业科学,2015,(4):60-70,11.基金项目
国家农业部公益性行业(农业)科研专项“蚕桑资源高值化加工利用技术及设备”(201403064) （农业）
国家现代农业产业技术体系建设专项(CARS-22)。 （CARS-22）
