Cloning And Functional Analysis Of SlASA1Gene In Tomato

Auxins play a pivotal role in various aspects of plant growth and developmentalprocesses such as cell division, extension, and differentiation, apical dominance,tropism movement, embryonic development, laterals formation, and vasculardifferentiation, via regulating the expression of early auxin responsive genes.Tomato（Solanum lycopersicum） is a ideal model plant for fruit development research, whichhave many specific characteristics such as: small genome size, Gene homozygote fromself-pollination, short growth cycle, easy plant in green house and existing genetictransformation system. Therefore, in order to research the function of Auxin in plant andfruit development, we utilize tomato as raw material.ASA1gene expresses a rate limit enzyme element （Anthranilate synthasecomponent I）, which catalyze the reaction of Anthranilate （auxin precursor） synthesis.Meanwhile, as already confirmed in Arabidopsis, ASA1gene is the interaction node ofAuxin and Ethylene. It play an important role in plant，via regulating the Auxinsynthesis.Aiming at reveal the role of SlASA1by auxin signaling regulation in plant, thestudy utilizes tomato as material clone and identified the two genes, individually. Then,by expression pattern analysis, plant hormonal stimulation, and promoter-GUS analysis,the study further understand the expression specific of the two genes. Meanwhile, thestudy constructs the gene overexpression vector and suppress vector of SlASA1. Bytransforming these vector into tomato, the gene overexpression and suppress lines wasobtained, and then was used to observe the phenotype that was caused by geneexpression diversity. Finally, we have some results as follow:1)Based on the AtASA1gene sequence （NM₀01203302.1）, an AtASA1-like gene（SGN-U573188） was isolated from SGN （http://solgenomics.net） and designated asSlASA1. According to the sequence of SlASA1, the length is1929bp contain a1347bpopen read frame （ORF） that encodes a polypeptide of448amino acids. By promoterprediction, a2123bp promoter was isolated from upstream of SlASA1. We found thatthe promoter contain a lot of Ethylene and Auxin response elements and seeddevelopment and protein storage elements. Few Gibberellin and Abscisic acid responseelements were also be found in the promoter region.2)By expression pattern analysis, we found that SlASA1highly express in Bud and Fruit. In three different fruit stages （green fruit（GF）,yellow fruit（YF）,red fruit（RF））,ASA1express highest in YF about twofold higher than the expression level in bud Withthe relatively lower expression in GF and RF, they also show about threefold higherthan the expression levels in other tissues （Root, Stem, leaf and flower）. Indicate thatSlASA1gene may play a pivotal role in flower, fruit, and seed development.3)The SlASA1gene fragment was cloned by PCR and was build into expressionvector pLP100with CaMV35S as its promoter, the final vector is an overexpressionvector named pLP100-35s-ASA1. We isolated a400bp fragment from the middle regionof SlASA1gene. Using RNA interference strategy, we build the fragment into specificRNA interference vector pCambia-HP1, the final vector named pCambia-HP1-ASAi.Clone the SlASA1gene without the termination codon, and build it into expressionvector pGreen-GFP, the final vector named pGreen-ASA1-GFP. The isolated ASA1promoter was build into expression vector pLP100with GUS reporter gene, the finalvector named pLP100-ProASA1-GUS. All the vectors were transformed intoAgrobacterium for future application.4)Via Agrobacterium-mediated transformation, we transform pLP100-35s-ASA1,pCambia-HP1-ASAi, pLP100-ProASA1-GUS into tomato and obtained the transgenicplants. The Positive transformation plants were identified by PCR and GUS staining.The expression quantity of SlASA1was detected by RT-PCR.5)Through paraffin section and Scanning Electron Microscope, we found that theoverexpression of SlASA1can promote the cell division, increase the number oftrichomes and reduce the number of stoma. The change of the leaf morphology suggeststhat SlASA1may contribute to the stress resistance of plant. Meanwhile, we found thatSlASA1overexpression can decrease the weight of seed and SlASA1RNAi plants appearparthenocarpy, suggest that SlASA1positively regulate the seed development.6) The result of Ethylene and Auxin signal genes detection, in SlASA1overexpression plants, indicate that SlASA1gene positively regulate the Auxin signalingand negatively regulate the Ethylene signaling. Meanwhile, the genes detection resultsproper proof some phenotype of the transgenic plants.In conclusion, SlASA1is an important Auxin synthesis gene, through Ethyleneresponse regulate the Auxin signaling. It positively regulates the Auxin synthesis,vascular bundle formation, plant stress resistance and seed development.