The Research Of Chloroplast Transgenic Sugar Beet Resistant To Herbicide With Bar Gene

Phosphinothricin (PPT) is a non-selective herbicide. As an irreversible inhibitor of glutamine synthase, it can lead to several immediate metabolic dysfunctions and plant death. Bar gene encodes phosphinothricin acetyl transferase (PAT), which can inactivate PPT by acetylation. Sugar beet is a commercially important sucrose-producing crop in our country, but it is extremely sensitive to herbicide, including PPT. Due to such reasons, we constructed chloroplast transformation vector of sugar beet, optimized chloroplast transformation system of sugar beet and introduced bar gene to sugar beet chloroplast genome through biolistic gun which has many advantages.This research can provide female parent material for developing sugar beet resistant to PPT in future.Large numbers of sugar beet tissue culture plants for subsequent experiments were obtained by researches on differentiation and regeneration of explants and optimization of sugar beet tissue culture system. The research about sensitivity to antibiotics of sugar beet petioles indicated that the appropriate selection concentration of kanamycin and spectinomycin were 50 mg/L and 40 mg/L. Statistical analysis indicated the toxicity of spectinomycin was more serious than that of kanamycin. In addition, spectinomycin is a kind of most common used selection maker of chloroplast transformation, so we chose spectinomycin as our selection maker and its selection concentration was 40 mg/L.We constructed vector PSARBar containing bar gene expression cassette, aadA expression cassette and homologous fragments (rbcL and atpB), which had been attested by enzyme digestion. After chloroplast transformation with biolistic gun and screening of plants resistant to antibiotic, we have obtained 26 putative plants. Through extraction of chloroplast genome DNA of some ones and PCR assay, the electrophoresis denoted 3 plants have been introduced into bar gene. PCR-southern with bar gene segments as probes was performed to attest PCR reaction, which indicated that the chloroplast genome of 2 plants indeed recombined bar gene. The result of this research indicated bar gene has been...