Studies On The Transformation Of Antisense ACS Gene Mediated By Agrobacterium Tumefaciens And Cloning Of ACS Gene From The Leaves Of Banana

In this experiment, a systematic transgenic procedure mediated by Agrobacterium tumefaciens with antisense ACS gene was developed in Musa spp. cv. Tianbaojiao (AAA group) and Bengalese plantain (ABB group), and cloning of ACS gene from the leaves of'Tianbaojiao'were also conducted, which laid the foundation for self anti-sense expressing vector construction and self-transformation in Musa. The main contents included the optimization of the plant regeneration system of banana, the introduction of antisense ACS gene into banana mediated by Agrobacterium tumefaciens, a whole technical system of selection of banana resistant buds, transgenic plant regeneration and transgenic assay; subculture multiplication and transplant of transgenic plantlets at early stage, the cloning of the ACS conservation region from the leaves of'Tianbaojiao'was carried out. The main results were described as follows:1. The plant regeneration system of banana was optimized.Suckers of new line of'Tianbaojiao'and'Bengalese plantain'were used as explants to carry out the studies on optimizing conditions of the regeneration in vitro. In the vigorous growth period (from April to May), after drawing materials 0.1 % mercuric chloride added with 3-5 drops of Tween-80 was the preferable sterilization way for the explants. The explants were cultured on the liquid medium of MS for 1-2 weeks, and then they were transferred on the solid medium, which was the suitable for sterilization; it is the preferable sterilization effect on the low-salt medium and adding combination of AC, PVP and Vc .2. The introduction of antisense ACS gene to banana was conducted, and the optimizedparameters used in Agrobacterium-mediated transformation were obtained. The best transient expression of GUS was obtained under the conditions as follows: the thin cross-sections were pretreated with 0.15 mol﹒L-1 mannitol for 5 h; and the Agrobacterium suspension was with OD600 value of 0.8- 1.0 and supplemented with 100 g﹒L-1 glucose; the samples were infected for 10~15 min, and then they were transferred onto the non-selective medium (pH 5.6) for co-culture for 4 days at 26℃in the dark.3. A whole technical system of selection of banana resistant buds, transgenic plant regeneration and transgenic assay was established.After co-culture, the thin cross-sections of a new line of'Tianbaojiao'were selected on MS medium supplemented with 100 mg﹒L-1 kanamycin,1 mg﹒L-1 BA and 0.1 mg﹒L-1NAA. At last, 2 resistant bud lines were maintained for further studies;however, when the thin cross-sections of'Bengalese plantain'were selected on MS medium supplemented with 120 mg﹒L-1 kanamycin, 1 mg﹒L-1 BA and 0.1 mg﹒L-1NAA, no normal resistant bud lines were obtained. The PCR assays proved that the gus and ACS gene had integrated into the genome of the 2 resistant bud lines.4. Subculture multiplication and transplant of initial transgenic plantlet were condected.The initial transgenic plantlets were maintained on MS medium supplemented with 4.0 mg﹒L-1 BA and 0.3 mg﹒L-1 NAA for 20 days and The multiplication rate was the highest; plantlets with roots were transplanted into perlite in spring , and survival rate was about 95 %.5. The ACS conservation region from the leaves of'Tianbaojiao'was cloned.The modified CTAB for extraction and purification of total RNA from the leaves of'Tianbaojiao'was developed. The ACS conservation region in'Tianbaojiao'was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR). Conservation region amplified by PCR was reclaimed, connected, transformed and sequenced. The results showed that the part of ACS cDNA of'Tianbaojiao'was 569bp and encoded a protein of 189 amino acids. The sequence had high identities with that of ACS gene which had been cloned.The studies on the plant regeneration system of banana, the introduction of antisense ACS gene to banana, the cloning of the ACS conservation region from the leaves of'Tianbaojiao'were of great importance for genetic improvement in Musa, and provided technical plate for the studying banana transformation.