Construction Of LeHB-1Sense And Antisense Fruit Specific Expression Vector And Its Genetic Transformation To Tomato

LeHB-1is the transcription factor of tomato gene ACO1. So by studying the activity oftranscription factor LeHB-1, we can understand the regulation mechanism of ethylenesynthesis. In this study, the sequences of fruit-specific promoter2A11and LeHB-1gene werecloned from tomato by PCR.Then the sense and antisense fruit-specific expression vector ofLeHB-1were constructed and transformed into Agrobacterium GV3101, then the planttransformation project bacteria was obtained and transformed into by Agrobacterium. Theresults were as follows:(1)The cloning of2A11promoter. Tomato genomic DNA was extracted by the method ofCTAB. According to the2A11promoter sequence (No M87659.1) registered in GeneBank todesign a pair of primers, it cloned a DNA fragment of1134bp in length by using PCRtechnology. It comapired with the2A11promoter sequence (No: M87659.1)that has publishedand the corresponding nucleotide sequence homology is as high as91%.(2)Establish the transient expression system of2A11. The pMD19T-2A11and pBI121cloningvector digested with HindⅢ and BamHⅠto construct pBI121-2A11expression vector. GUSis as a reporter gene as for detecting the a transient expression system by Agrobacteriuminjection.GUS staining showed that the dyed signal is apparent in the peel pulp and leavesafter the injection did not find GUS staining signal. The fruit in control that were injeced byPBI121plasmid are stained obvious in various parts. It showed that fruit-specific promoter2A11only express in fruit, not express in other parts.(3)The cloning of LeHB-1gene. Total RNA was extracted from tomato leaves.According tothe HB-1sequence registered in GeneBank (No: AM902288.1) to design specific primers, theRNA was reverse transcribed into cDNA for cloning LeHB-1gene. We obtained a DNAfragment length of858bp which compared with HB-1sequence that has published (No:AM902288.1), the homologythe of corresponding nucleotide sequence is99%.(4)The construction of sense and antisense pBI121-2A11-HB-1-specific expression vectors.The pMD19T-HB-1and pBI121-2A11specific expression vector was digested withSac Ⅰw ith SmaⅠ s eparatelyto construct pBI121-2A11-HB-1specific expression vectorsuccessfully, then it was transformed into Agrobacteriu by freeze-thaw method to obtainransformants. (5)The establishment of tomato genetic transformation system by. Tomato leaves is asexplants. The pBI121-2A11-HB-1specific sense and antisense expression vector weretransformed into a tomato by using Agrobacterium-mediated to amplify PCR. It can beamplified with the same size strips to lasmid DNA. So it has preliminary describe thatexogenous gene has been successfully integrated into the chromosome regenerated DNA.