Study Of Strawberry Ethylene Receptor FaEtr2 Gene RNAi Plant Expression Vector Construction And Genetic Transformation

Cultivated strawberry (Fragaria×ananassa Duch.) is a fruit crop species with high values, but its fleshy fruits, which are getting less firm during progressive ripening, are liable to a post-harvest deterioration that impairs their preservation and causes great economic losses. Further research on gene function of FaEtr2 may demonstrate a relation between ethylene and the ripening of non-climacteric fruits and pay the way for improving storage performance of strawberry fruit through biological techniques. Using genomic DNA as template, a FaEtr2 gene fragment was amplified by Polymerase Chain Reaction (PCR). FaEtr2 gene antisense and RNAi expression vectors were constructed. Using All-Star in vitro plantlet leaf as explants, the effects of basic medium, hormones, physiological state of the explants, seedling ages and dark-culture time on strawberry leaf regeneration were studied to optimize the regeneration conditions. Efficient regeneration system of All-Star strawberry was obtained. Using All-Star strawberry leaf discs as explants, genetic transformation system mediated by Agrobacterium tumefacien was established. Kanamycin resistant plantlets were obtained. Gus chemical staining and PCR detection showed that FaEtr2-RNAi gene was transformed into All-Star strawberry plantlets. The main results were as follows:ⅠCloning of FaEtr2 and its plant expression vector construction1. A pair of specific primers were designed according to the published Chandlar strawberry ethylene receptors FaEtr2 gene conservative amino acid sequence. A 1049bp fragment was amplified. Sequence analysis showed that the nucleotide sequence of the cloned fragment was 100% identity with Chandler-Etr2 in GenBank, and no intron sequence, which demonstrated that the fragment was All-Star strawberry FaEtr2 fragment.2. Double digested by XbaⅠand BamHⅠ, All-Star strawberry FaEtr2 nucleotide sequence was subcloned into XbaⅠ- BamHⅠsite of pBI221 vector in reverse orientation, which resulted in plasmid pBI221-anti-Etr2. A fragment including CaMV 35S promoter and FaEtr2 antisense sequence was cut from pBI221-anti-Etr2 vector by BamHⅠand HindⅢ. The fragment was directionally inserted into the same restriction enzymes digested pBI121 plasmid, which resulted in pBI121-anti-Etr2.3. Double digested by BamHⅠand SmaⅠ, All-Star strawberry FaEtr2 nucleotide sequence was subcloned into BamHⅠ-SmaⅠsite of pBI221 in sense orientation, which resulted in plasmid pBI221-sense-Etr2. The plasmid pBI121-anti-Etr2 and pBI221-sense-Etr2 were double digested by BamHⅠand SmaⅠ, respectively. The sequence with FaEtr2 sense fragment was inserted into the double digested pBI121-anti-Etr2 vector, which result in pBI121-Etr2-RNAi plasmid.4. The vector of pBI121-anti-Etr2 and pBI121-Etr2-RNAi was transferred into A. fumefeciens strain LBA4404 by the freezing-thaw method. The transformation was confirmed by PCR amplification with specific primers.ⅡEstablishment of leaf regeneration system1. Compared with MS and 1/2MS medium, MS (1/2N) medium was more suitable for All-Star strawberry leaf regeneration, in which differenciation rate of 75.76 % was obtained. Leaf explants of 30 to 35 days were suitable for regeneration, of which the highest differentiation rate of 58.62 % and 58.86 % were gained. The differentiation rate and the average number of adventitious buds of young leaves explants were 35.71% and 0.46, respectively, significantly higher than the old leaves which were 19.23% and 0.23, respectively.2. With the increase in TDZ concentration, leaf differentiation rate, average sites of adventitious buds were both in upward trend, but the vitrification rate was also on the rise at the same time. The leaf differentiation rate (36.67 % and 32.98 %, respectively) and average sites of adventitious buds (0.42 and 0.47, respectively) of TDZ 1.0 mg·L-1 and 1.5 mg·L-1 are higher than other treatments. However, the vitrification rate of the latter was significantly higher than that of the former. Comprehensive consideration of the effects, TDZ 1.0 mg·L-1 was more suitable for All-Star Strawberry leaf regeneration.3. The appropriate concentration of IBA and NAA were both of 0.05 mg·L-1 in which leaf differentiation rates were 68.99% and 38.93%, respectively. IBA 0.05 mg·L-1 and TDZ 1.0mg·L-1 were the suitable homone combination for All-Star leaf regeneration. Regeneration buds in higher concentrations of NAA (0.2 mg·L-1 ~ 0.3 mg·L-1) were prone to vitrification. In the medium of IBA combination with TDZ, differentiation rate of leaves, average sites of adventitious buds and average number of adventitious buds were significantly higher than that of NAA combination with TDZ at the same level.4. Regeneration rate was low for the leaves without dark-culture, and the differentiation rate and the average sites of adventitious buds were in upward trend with the dark time prolong. The differentiation rate of dark-culture 9 and 12 days obtained the highest differentiation rate of 40.54% and 34.29%, respectively, significantly different with other treatment.ⅢEstablishment of genetic transformation system1. The in vitro plantlets and lateral buds grew normally in medium with Cef 200 mg·L-1. When Cef concentration reached or exceeded 300 mg·L-1, plantlets growth and lateral buds differentiation were inhibited. 200 mg·L-1 Cef could inhibit most Agrobacterium tumefaciens growth. Km 20 mg·L-1 could inhibit non-transformed leaf differentiation, and could be used for resistance buds screening.2. Agrobacterium tumefaciens LBA4404 proliferated the fastest at the concentration of about OD600=0.5; 3 days of pre-culture generated the highest differentiation rate of 18.9%; Adding 10 or 20 mg·L-1 of AS, differentiation rate of resistant buds was significantly promoted from 7.53 % to 15.09 % and14.55 %, respectively.3. Infection leaves with re-suspended bacteria liquid OD600 of 1.0 was better than direct infection with the bacterium liquid of OD600=0.5, differentiation rate was promoted from 6.15% to 15.8%. Of the different concentrations of re-suspended bacteria liquid, OD600 of 1.0 was suitable, which generated the highest differentiation rate of 18.55 %. Of the different infection time treatment, 10 min was most appropriate, which obtained the highest differentiation rate of 21.46%.4. Among the different time of co-cultured and de-culture combinations, co-cultured 3d, de-culture 3d obtained the highest differentiation rate, average sites of resistant buds and average number of resistant buds of 13.49%, 0.18, 0.22, respectively, which was the optimal combination for All-Star strawberry genetic transformation. Screened by Km, seven lines of transgenic plantlets were confirmed by GUS histochemical staining and PCR amplification.