| Strawberry(Fragaria x ananassa Duch.)is a major berry crop around the world, and its production is increasing steadily.Many cultivars of strawberry have already been bred,thereby allowing this crop to be cultivated under various conditions.However, there are still many factors which limit plant yield significantly,the narrow genetic base of the cultivated strawberry,combined with the polyploidy nature of the crop constrain traditional breeding methods.One problem in strawberry production is liable to a post-harvest deterioration that impairs their preservation and is a cause of great losses.In this case,Biotechnological approaches,especially genetic engineering,are an alternative efficient strategy to implement strawberry improvement and to produce novel breeding materials.Fruit ripening is a complicated biochemical and physiological process and ethylene is a major factor regulating the ripening process.It's very import to study the biosynthesis of endogenesis of ethylene and ethylene signal transduction.Ethylene action can be regulated at the level of both hormone synthesis and sensitivity.Although the regulation of ethylene biosynthesis has been studied extensively,much less is known about the regulation of ethylene perception.Much of what is known regarding the steps involved in ethylene perception and signal transduction has been realized through studies of the model plant species Arabidopsis.Strawberry is non-climacteric fruit,Strawberries can produce ethylene,although in limited amounts.In some cases it has been seen that ethylene can hasten the post-harvest deterioration.Up to now little is known about the mechanisms that regulate their ripening.No results have been obtained that can demonstrate a clear relation between ethylene and the ripening of these fruits.The ethylene receptors are responsible for sensing ethylene changes in the growth.Three ethylene receptors have been isolated in strawberry.We used antisense inhibition of strawberry ethylene receptor gene to investigate the role of ethylene receptor in ripening strawberry fruit and determine its mode of action.This experiment carried on systematic research to strawberry tissue culture system; optimized Agrobacterium tumefaciens-mediated genetic transformation system of strawberry.Four antisense ethylene receptors expression vector were constructed and used in the genetic transformation of strawberry.The transgenic plants were obtained and carried on relevant molecular biology analysis.The antisense strawberry plants might benefit for further study on the physiological function of ethylene receptors and the mechanism of ethylene in maturation of nonclimacteric fruits such as strawberry.Aslo the anti-transgenic strawberry provide precious materials for the further research.1.By using the specific primers designed from the FaEtrl cDNA,the FaErs1 cDNA and the FaEtr2 cDNA,580 bp fragment of FaEtr1,445bp fragment of FaErs1 and 345 bp fragment of FaEtr2 were cloned.The FaEtr1,FaErs1 and FaEtr2 cDNA were inserted in reverse orientation between the CaMV 35s promoter and Nos terminator into the expression vector pBI121,respectively,and three antisense expression vectors called pBI121Etr1,pBI121Ers1 and pBI121Etr2 were obtained.FaEtr1 and FaErs1 antisense gene with 35s promoter and Nos terminater were inserted into expression vector pCAMBIA2301 together to generate double genes plant expression vector pFRS.Vectors of pBI121Etr1,pBI121Ers1,pBI121Etr2 and pFRS were checked by PCR and restriction enzymes analysis and transformed into Agrobactrium tumefaciens EHA 105. The correct construction and transformation were confirmed by PCR and restriction enzymes analysis.2.Strawberry,Fengxiang,Guilugan,Quanmingxiang,Zhangji,Hali,Xingxiang,was investigated for factors affecting the regeneration of adventitious bud.A highly efficient in vitro regeneration system from leaf explants was established.The results indicated that the genotype,hormones and seedling ages were the main factors affecting regeneration. The optimum shoot inducing medium of the leaves from Guilugan was MS+6-BA 2.0 mg·L-1+IBA 0.1mg·L-1,the better medium for the induction of callus from the leaves of Zhangji was MS+6-BA 3mg·L-1+2,4-D 0.2 mg·L-1;the optimum medium for shoot elongation was MS+6-BA 0.5 mg·L-1+IBA 0.5 mg·L-1,the optimum root inducing media were MS+IBA 0.2 mg·L-1.The survival rate of the plantlets transplanted was 87%.Dark incubation for 1 week can reduce the browning of cultures.3.Several factors affecting Agrobacterium tumefaciens-mediated gene transfor -mation of strawberry were studied by using the transient expression of gus gene with introns as criteria.The results were as follow:The explants of strawberry leaves were rather sensitive to kanamycin,and the corresponding concentration of 20 mg·L-1that could substantially inhibit the growth of non-transformed tisse and be used to screen the leaves of strawberry after transformation;Timentin at the concentration of 300 mg·L-1 could be used to eliminate Agrobacterium tumefaciens and had minor impact on the growth of the explants;The preculture time for 2-4 days was beneficial to improve transformation frequency;Co-culture for 2-3days not only had higher gus transient expression but also could avoid the over-growth of Agrobacterium;The bacterial concentration of OD600nm=0.4 was the best inoculum density and 10 minutes was the best inoculation time.The PCR analysis showed that the target gene gus was successfully integrated into the genome of strawberry.4.The antigene FaEtr1 and FaErs1 were introduced into leaves of stawberrywith Agrobacterium tumefaciens,and regenerated plants were obtained by kanamycin selection.15 plants were confirmed by PCR and Southern blot analysis that the antigene FaEtr1 and FaErs1 gene were transferred and integrated into the genome of strawberry. Northern blot analysis indicated that FaEtr1 and FaErs1 mRNA abundance were decresased in antisense strawberry plants.The antigene FaEtr2 was introduced into leaves of strawberry with Agrobacterium tumefaciens,and regenerated plants were obtained by kanamycin selection.26 plants were confirmed by PCR and Southern blot analysis that the antigene FaEtr2 gene was transferred and integrated into the genome of strawberry.Northern blot analysis indicated that FaEtr2 mRNA abundance was decresased in antisense strawberry plants.5.Physiological changes of the transgenic lines,including ethylene production, abscission of petiole,were investigated or analyzed.The ethylene production in transgenic leaf significantly decreased.Upon exposure toexogenous ethylene,the abscission of transgenic petiole was slowerred. |
PDF Full Text Request