The Cloning And Analysis Of ARF3Gene From Banana And Function Features

ARF3gene is a member of the ARF gene family, it is relatively conservative, the main function is to control the intracellar substances transportation. In this study, the full cDNA of the ARF3was cloned from banana, conducted bio-informatics analysis, and constructed Cam35s-gfp-gus-ARF3expression vector, which contains the green fluorescent protein and the GUS reporter gene, it can be more accurate detection of transgenic plants. ARF3gene was transferred to tobacco genome by agrobacterium-mediated, checked transgenic plants and identified its function. The rests are as follows:1. The banana RNA extraction method optimization. Based on the previous research, the banana RNA extraction method was improved, it widely applied for different organs, and the extracted RNA which had clear bands, pollution-free and good integrity, can better meet the needs of later experiments.2. The full cDNA cloning and bio-informatics analysis of ARF3gene. The full cDNA of ARF3gene was got in the method of RACE for the first time. This gene is1427bp, encodes338amino acids, with an estimated protein molecular weight and an isoelectric point of38.24KD and5.36respectively. The open reading frame is1166bp, start codon is located at174bp, stop codon at1340bp. The nucleotide sequence of Banana ARF3gene has69%-74%similarities to the ARF gene nucleotide sequence of the other plants as well as81%-93%similarities in amino acid sequence.3. Expression characteristics analysis of banana ARF3gene in the different organs. Expression characteristics were analyzed in the method of RT-PCR, the rests showed that expression of banana ARF3gene was the highest in the leaves, followed by roots, stems was the lowest.4. Optimization of tissue culture system for banana. Explant materials used to tissue culture had three kinds of banana male inflorescence, shoot tips growing point and stem segments crosscutting flakes, after comprehensive comparison, the rests showed that the method of using stem segments crosscutting flakes had some advantages of simple operation, low explants pollution rate and short cultivating cycle in reproducing sterile banana seedlings, it can be faster to reproduce a large number of banana seedlings and meet the needs of the experiment.5. Construction of expression vector and genetic transformation system optimization. The target gene was inserted into the multiple cloning region of Cam35s-gfp-gus vector which carries two restriction enzymes Sac I and BamH I, thereby completed construction of the vector. Kanamycin lethal concentration was selected to be400mg/L as well as200mg/L with the median lethal concentration in the process of genetic transformation system optimization, carbenicillin concentration was500mg/L in resistance medium which can control the growth of agrobacterium well, the conversion efficiency was the highest when agrobacterium infection time was15min and OD600of the agrobacterium suspension was0.8.6. Detection and functional analysis of transgenic plants. DNA and RNA were extracted from tobacco, positive plants were detected to in methods of specific primers amplification and green fluorescent protein, transient expression of banana ARF3gene was detected to take advantage of the GUS reporter gene in tobacco. The rests showed that the positive conversion rate reached nearly80%, in the process of dyeing reaction, the tobacco leaves appeared blue plaques, fluorescence microscopy can be clearly seen tobacco partial mesophyll cells have a strong green fluorescence issued. Transgenic tobaccoes showed fast-growing, tall plants, dark green leaves, leaf area significantly increased features was compared with the non-transgenic control.