The Evolution, Expression And Silencing Study Of Endo-β-Mannanase Gene Family In Tomato

Compared to other species,cell wall is the special structure in plants.It is divided into primary cell walls and secondary cell walls,and composed of cellulose, hemicellulose and pectin and so on.The cell wall plays an important role in growth and development of plants,such as cell shaping,signal transmission,stress response and wound defence.Mannan, which is classified into four kinds of polysaccharides(pure mannan, galactomannan, glucomannan and galactoglucomannan),is a very important component of hemicellulose.Mannan extensively exists in cell walls of various tissues and organs.For example,endersperm,pericarp and lignin in perennials.Endo-?-1,4-mannosidase(endo-?-mannanase) is a polysaccharide hydrolyase, it can hydrolyse mannan ?-1,4-linked mannose backbone.So far, its activity have been detected in many plants.These enzymes present high similarity in biochemical function,but they could play various roles in different tissues,organs and development stages of plants.In this study,we identified endo-?-mannanase genes in genome-wide of tomato and soybean,and analyzed the structure,and evolution of these genes from Arabidopsis thaliana,tomato and soybean by comparative genomics strategy. Expression pattern of endo-?-mannanase genes in tomato was researched by RT-PCR. RNAi expression vector for endo-?-mannanase genes was successfully constructed,and was transformed into tomato plants by Agrobacterium tumefaciens mediated method.Some transplants were detected though PCR and RT-PCR. Our study provides some essential evidences for getting deeper insight into biological function of endo-?-mannanase genes in growth and development of plants and may contribute to transgenic breeding.The results of this study are as follows:(1) Using bioinformatic methods,11 genes and 20 genes were identified from tomato and soybean genomes.Some specific amino acid residues and conserved motifs were found through protein sequence alignment. Genetic structure and phylogenic analysis showed that intron gain and loss may play an important role in gene evolution.Evolution analysis of endo-?-mannanase genes from many species revealed that plant endo-?-mannanases divergency may occur prior to the divergency of the lower and higher plant.(2) By researching promoters and tissues-specific expression pattern of these genes,we found that endo-?-mannanases not only are involved in growth and development process of tomato,but also associatied with the stress response in plants.(3) By sequence alignment of the four cloned endo-?-mannanase genes,we artificially selected the RNAi target fragment,and inserted it into RNAi expression vector forward and reverse. RNA interference expression vector pTCK303-LeMANs was constructed successfully.(4) MicroTom plants were transformed by Agrobacterium tumefaciens mediated method,then 5 plants were obtained from hygromycin resistance screening system.These plants presents positive in PCR and RT-PCR detection.