Cloning And Expression Analysis Of A Homologue AP3 Gene In Salix L.

AP3 plays important roles in floral development. As a B class gene of "ABC" model, AP3 or PI and AG control the development of stamen together. A pair of primers were designed according to published Populus trichocarpa gene (PTD), and an 2.1K fragment was amplified (PCR) by using the genomic DNA of Salix. Babylonica , Salix.caprea, Salix.albaf,Salix.integra, Salix.erioclada and Salix. suchowensis respectively.By using 5'RACE and 3'RACE technique, the homologue AP3 gene was obtained in the S.suchowensis. The completed cDNA sequence was 826 bp and encoded for a polypeptide of 241 amino acids, 28KD molecular weight.According to the the full-length cDNA of S.suchowensis and the genomic DNA sequences, the full-length cDNA sequences of homologue AP3 in S. babylonica , S.caprea, S.albaf,S.integra and S.erioclada were predicted. The result indicated that the homologue AP3 genes of Salix were high conservative, with 95%-98.7% identity of nucleotides and 95.2%-99.6% of proteins.To determine the expression pattern of SSMADS during the plant development in S. suchowensis, real time RT-PCR technique was applied. The results showed that the expression of SSMADS in transcript levels, in different organs and at different develop -ment stages, was significantly different, with very low expression in the leaf, shoot and root and high expression in floral organ, and increased at the early development stage but decreased at the late stage of floral organs.We introduced the plant expression vector with the OsMADS16 gene to Agroba -cterium tumefaciens EHA105. Then the tobacco was transformed by the method of the leaf disc transformation, and 95 hygromycin-resistant buds were obtained. When the bud was more than 1.5 centimeters, it was cut and transferred into MS medium supplemented with hygromycin 50mg/L. After 20 days, the majority of the young shoots could root normally. 20 individuals which could resist hygromycin was tested by PCR, and 18 of them were positive.We established the transgenetic system of S. suchowensis. The influence of elementary medium and the hormone combination on shoots induction was studied, but elementary medium had little influence on it. The best hormone combination was BA 1.0mg/L and NAA 0.01mg/L, from which the rate of shoots-regenerated was 100% and the average number of shoot induction was 3. When regenerated shoot was as high as 3 centimeters, it was cut and transferred into White or B5 elementary medium with no hormone and the rooting rate was more than 90%.