The Carrot (daucus Carota) Antifreeze Protein Gene Cloning And Transformation Of Tomato (lycopersicon Esculentum)

Cloning of a carrot gene encoding antifreeze protein and its transformation to tomato Doctor Yin Mingan Director Cui Hongwen Fan Daiming Abstract Antifreeze protein gene (afk) in carrot Daucus carota var. autumn King from British and three native carrot cultivars (Daucus carota var. sat ivus Hoffm Deutschl), Wuzhong carrot in Ningxia, Huaxian carrot in shaanxi and Hanzhong carrot in shaanxi, was cloned by PCR (polymerase chain reaction). It was found that af~ existed in all tested materials. Afp seguence of Wuzhong carrat was compared with that of Daucus carota var. autumn King. There were 35 different bases between two varieties in 1004 seguenced nucleotides, among which there were 20 monsense mutations and 15 sense mutations. Based on sense mutations homology was 98.5%. Cloning vector pTAF of afp from carrot var. autumn King was constructed with pUCmT vector. pTAF was digested with EcoR I and became linear. Its ends were filled with DNA Polymerase I Klenow fragment. Then it was digested with Xba I and a designed fragment( afr )with a cohesive end and a blunt end was released. Plant expression vector pBI 121 was digested with Xba I and Sma I and a linear plasmid with a cohesive end and a blunt end was obtained. The linear plasmid and the designed fragment (afp) were directively ligated with T4 DNA ligase, and the plant expression vector of carrot aTh was constructed. Then the expression vector was transferred into agrobacterium tumefacien LBA4404 by direct DNA tranfer. Cotyledon and hypocotyl from tomato 慫F?were used as explant, and 7 combinations between cytomin and auxin were tested and compared. It was determined that MS+BA 1 .OmgIL+IAA 0.2mg/L was the best shooting medium. The best rooting medium was MS+IAA 0.O5mgIL. When tomato seedings grew 5? days after germination, cotyledons were cut into 0.5cmX 0.5cm discs and hypocotyls into 1cm segments. The discs and segments were pre-cultured on shooting medium for 24h (26 C, 2600 Lux). Agrobacterium LBA4404 were cultured overnight and diluted 1 0?0 times with MS medium, in which explants were soaked for 5 minutes. Then the exptants were co-cultured for 48h (28 慍, dark). After that, the explants were cultured on medium MS+BA 1 .Omg/L 盜AA 0.2mg/L+Kan 25mgIL+cef 200mg/L for selection with fransfer every two weeks. When shoots were 2cm high, they were cut and transferred on medium MS+IAA 0.O5mglL+Kan 12.5 mg/L+eef I OOmgIL for rooting. Up to now, 221 shoots were obtained from 877 explants. 80 shoots were transferred on rooting medium and 12 plants were transferred in field. 10 plants were assayed by PCR and 2 of them were positive, which preliminarily proved that carrot atj gene had been transferred into the tomatoes.