The Selection Of Long Shelf-Life Strains Of Cucumis Melo L. Cv Hetao Transformed With A Vector Backbone And Selecting Marker-Free Antisense ACS1 Gene

Melon(Cucumis melo L.) belongs to the Cucurbitaceae family,and is an annual herbaceous rambler.Melon fruits are the essential components fo human and animal diets.This research used a melon(Cucumis melon L.cv Hetao) variety as material, which is cultivated crop in He Tao area in the west Inner Mongolia.It is the typical respiratory climacteric fruit which had post-harvest peak and turned soft rapidly.One of the most important target of breeding research of hetao melon is improving the fruit storability.In this research,we used the plant binary expression vector pPZP221 (GenBank Sign No.:U10491) for the initial vector,pPZP221 was digested by restriction endonuclease Mssl and VspI to remove the gentamycin acetyltransferase gene aacC1 from T-DNA,and the CaMV 35S promoter and nos terminator sequence was inserted into this site.The antisense fragment of a 0.65 kb cDNA coding region of 1-aminocyclopropane-1-carboxylic acid synthase 1(ACS1) was constructd into the site of Xba1 and Sac1 of the vector.Finally,we used MssI and VspI to digest both sides of the border of T-DNA and got the antisense ACS1 gene expression cassette which had no selecting marker gene and no vector backbone sequence.We recovered the fragment by electrophoresis and dissolved it in the 0.1×SSC solution(pH 7.0).Its (?)nal concentration is 10ng/μL.The DNA fragment was transformed into hetao melon by the pollen-tube pathway transgenic technology.The T₁ generation transgenic strains with improved shelf-life were selected.The results were as followed:1.30 flowers were transformed and 24 fruits were harvest.10 seeds were selected from each fruit for PCR detection.The result show that 6 fruit were with high positive rate.2.30 seeds were selected from each positive fruits and planted in the field.Then 5 plants with improved storage ability were chosen.3.30 seeds were chosen from each of the 5 fruits with high storage ability and planted in the tray.After PCR detection,positive plants were tranferred to greenhouse:4.The fruit of the T₀ and T₁ generation can be stored for 40 days at room temperature.The content of soluble solid was the same as the control fruit which was stored 10 days.While untransgenic control hetao melon were rotten in 20 days after harvest and the content of Soluble solid could not be measured.5.When the untransgenic control fruit were stored 10 days,the hardness was about 3.1kg/cm² and decreased gradually.While the hardness of transgenic fruit was 4.6kg/cm² when it was stored 40 days. 6.After 40 days,the endogenous ethylene concentration of transgenic fruit was lower than that of control and was about 3%-5%of the control one.