The Study On Transformation Of Tomato With Fruit Ripening-specific Gene CsPEMI Promoter From Citrus

The separation of citrus fruit ripening-specific promoter and analysis its functions have greatsignificance in researching the molecular mechanisms of fruit mature and improving fruit quality withbiotechnology. The separation of citrus fruit ripening-specific promoter and analysis have greatsignificance which can reveal the fruit mature molecular mechanisms and improve fruit quality.However, citrus fruit ripening-specific promoter hasn’t been reported at present. We have alreadyseparated the5’upstream promoter region1137bp of citrus fruit ripening-specific genes CsPMEI withGenome Walking technique and built three-binary expression vector contained different promotersregion to driven intron-GUS. On the basis, this study established a GUS staining system forMicro-Tom tomato different tissues and organs, and analyzed the regulation and expression ofdifferent promoter region in different tissues and organs of tomato by the transient expressiontechnology. By optimizing hygromycin Micro-Tom tomato genetic transformation system, weobtained the transgenic tomato plants with test promoter and the promoter function was identified byfurther GUS staining. The main results are summarized as follows:(1) Establishment of GUS staining systemIn order to eliminate the interference of endogenous GUS’s activity in Micro-Tom tomato, pH7.2is the best choice of phosphate buffer after compared different pH conditions for Micro-Tom tomatoorgans staining.(2) Transient expression analysisUsing agrobacterium mediated transient expression, different tissues of Mrcro-Tom were used tostudy the test promoter regulation. According to the staining results, it is indicate that-334~-1137area of the CsPMEI promoter region has fruit-specific function. The result need further verification bytransgenic.(3) Optimization of genetic transformation system for Micro-Tom resistant to HygIn order to optimize the genetic transformation system, we tried to find an optium selectingconcentration of Hyg. The cotyledon and hypocotyl of10-day-old Micro-Tom plants were used asexplants, co-cultured with MS solution containing Agrobacterium (OD600=0.1) for15min and thentransferred the explants onto the medium without the Hyg for36h. After, the explants were transferred to the medium with10mg.L-1Hyg to select resistant buds. We obtained18transgenic plants，and thetransformation rate was10.8%.(4) Identification of stable expression of the target promotor functionBy use of PCR technology, transgenic resistant plants were detected. The results showed that allthe resistant plants had the target amplification bands. GUS staining system was used to analyse theexpression and regulation of promoters in various tissues and organs of transgenic tomato. The resultsshowed that-334to-1137region of CsPMEI promoter can drive the GUS reporter gene expression inripening tomato fruits specificly, which is consistent with transient expression analysis.