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

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. The purpose of this research was to construct of a binary vector pMSH1-Lys carrying chicken Lysozyme (Lis) gene and introduce pMSH1-Lis on K. alvarezii. The binary vector expression was transformed into Agrobacterium tumefaciens LBA4404 by triparental 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-Lys was established. Percentage of transformation of pMSH1-Lis on K. alvarezii was 23.56%, while the efficiency of regeneration 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.