Binary Vector Construction and Transformation of Lysozyme Gene in Seaweed Kappaphycus alvarezii Using Agrobacterium tumefaciens-mediated Transfer

Abstract

Ice-ice disease is the biggest problem in the cultivation of seaweed Kappaphycus alvarezii. The disease is caused by bacterial infection and induced by drastic changes of water quality. Lysozyme has the ability to break down bacterial cell wall. Lysozymes are well-characterized hydrolases, which cleave beta-1,4 linkages of N-acetylglucosamine (GlcNAc) homopolymers and beta-1,4 linkages of the bacterial cell wall component GlcNAc-N-acetylmuramic acid peptidoglycan. Chicken lysozyme had lytic activities against Micrococcus lysodeikticus, Flavobacterium columnare, Aeromonas hydrophilla and Vibrio anguillarum. The purpose of this research was to construct of a binary vector pMSH1-Lis carrying chicken lysozyme (Lis) gene and introduce pMSH1-Lis on K. alvarezii. Lysozyme gene was amplified from pJfKer-Lis using PCR with specific primers for chicken lysozyme gene. Plasmid pMSH-1 and PCR product of lysozyme gene was cut with enzymes NotI and SpeI. Lysozyme gene was ligated into the expression vector pMSH1 to design a binary expression vector of pMSH1-Lis. The pMSH1-Lis was transformed to Escherichia coli DH5α by heat shock, cultured and then the plasmid was isolated. The binary vector expression was transformed into Agrobacterium tumefaciens LBA4404 by tri-parental mating. Thallus was inoculated with A. tumefaciens carrying pMSH1-Lis and then the transformed thallus was selected by adding 20 mg/L hygromycin to the culture medium.PCR analysis showed that the construction of the binary plasmid pMSH1- Lis was established. Percentage of transformation of pMSH1-Lis on K. alvarezii was 23.56%, while the efficiency of putative bud was 11.32%. PCR analysis showed that three of the regenerated thallus contained lysozyme gene. Thus, transgenic K. alvarezii was produced successfully and this can be useful for studying the mechanisms of seaweed defense against bacterial infection. Based on the percentage of the transformation, the transformation protocols need to be established for increasing transformants and regenaration thallus. Test bacterial resistance and environmental stress is necessary to examine the transformants tolerance.