The Construction Of Expression Vector Of Anthurium Laccase Gene And Transformation Of Agave Americana Linnaeus

Sisal is an important fiber crops planted in subtropical and tropical regions,has wide development foreground. Sisal is hazarded by physiological diseases, insect-borne diseases and pathogenic diseases, especially the sisal zebra diseases, and the production reduced significantly.In this study, the culture mediums of sisal callus induction were optimized by using the material of sisal leaves. Anthurium RNA was extracted and reverse transcripted into cDNA. The laccase gene amplified from Anthurium cDNA replaced the GUS gene, and formed the plant expression vector pBI121-Lac. The laccase gene was inducted to sisal genome and the transgenic plants were analyzed by PCR and RT-PCR.The main results were as follows:(1) The culture medium of sisal regeneration was optimized. The culture medium were: MS + 6-BA 2.0 mg/L + NAA 0.1 mg/L + 30 g/L sucrose + 7 g/L agar powder, pH 5.8; the culture medium for bud differentiation was:MS + 6-BA 1.0 mg/L + NAA 0.1 mg/L + 30 g/L sucrose + 7 g/L agar powder, pH5.8; the culture medium for bud elongation was:MS + 6-BA0.1 mg/L + NAA 0.01 mg/L + 30 g/L sucrose + 7 g/L agar powder, pH 5.8; the root inducible medium was:MS + IAA 0.1 mg/L + 30 g/L sucrose + 7 g/L agar powder, pH 5.8.(2) Exploring several factors of genetic transformation of the sisal mediated by Agrobac-terium. The median lethal concentration and lethal concentration of Hyg were 15 mg/L and 25 mg/L, respectively; the transient expression was highest when OD600 of the Agrobacterium suspension was 0.6; soaking time in Agrobacterium suspension was 10-15 min; co-culture medium with the concentration of the hygromycin 200μmol/L; selective medium with the concentration of the carbenicillin 400 mg/L.(3) Anthurium RNA was reverse transcripted into cDNA and the full length of laccase gene cDNA was amplified.(4) Replace the GUS gene with laccase gene, and constructed the plant expression vector pBI121-Lac.(5) The laccase gene was inducted to sisal genome by Agrobacterium-mediated and the laccase gene had been integrated into the sisal genome and expressed analyzed by PCR and RT-PCR. Sisal leaves appeared red spots.