Cloning Of SlARF6 Gene, Research It's Expression Character And Genetic Transformation In Tomato

Auxin plays an important role in plant development and growth. ARF(Auxin Response Factors) is one of the key factors in auxin signal transcription pathways. ARFs encode transcriptional factors that bind with specificity to TGTCTC-containing auxin response elements (AuxREs) found in promoters of primary/early auxin response genes and are involved in many important processes during plant development. Therefore, the research of ARF is important on parthenocarpy and improving the character of tomato fruit.In our study, we forcus on the spatial and temporal expression character of SlARFs and several auxin response genes during tomato fruit set and development. Meanwhile, we also treated the tomato ovaries with different hormones, and then study the expression character of ARFs genes. The purpose of this study is to provide the helpful tools for understanding moleculer mechanism of the regulation of fruits growth with auxin. The major study results are as following:1) Firstly, we found a Expressed Sequence Tag, which is similar to AtARF6 with 78%. Then using homologous gene amplification, a new ARF gene, SIARF6( Solanum lycopersicum Auxin Response Factor 6) was identified from tomato fruits. The total cDNA longth is 3036 bp and and the largest open reading frame (ORF) was composed of 2607 bp with a deduced 868 amino acids. The sequence was submitted to NCBI (HM594684). Sequence analysis revealed the typical structure of an ARF:a N-terminal DNA binding domain (DBD, amino acids 117-219) and a C-terminal dimerization domain (CTD, amino acids 735-817), which are implicated in protein dimerisation. Moreover, SlARF6 contain a middle region rich in glutamine(444-557) We also got the DNA sequence which was composed of 5960bp and included 12 extrons and 11 introns.2) The expression patterns of SIARF6 and some other auxin response genes, including SIARF2, SlARF4, SlARF7, SIARF8, IAA1, IAA9, SAUR and GH3 in sepal, petal, stamen, ovary, leaf, stem and root of tomato plant., were characterized using quantitative real-time RT-PCR. All of the auxin response genes were detectable in diverse organs in tomato except the GH3. The transcript levels of SIARF2, SlARF6, SlARF7 and SIARF8 increased during flower development, remained constantly high in mature flowers, and was down-regulated significantly within 72 hours after pollination; while SlARF4 showed a different expression pattern:high in bud but low in mature flowers. 3) When the flower buds were treated with exogenous GA3,IAA, NAA and 2,4-D, the transcript levels of SlARF2, SIARF6 and SIARF8 were down-regulated in tomato embryo and young fruits, while SIARF4 was up-regulated. It is concluded that the auxin response factors may play different roles during fruit set and development based on their different expression patterns.4) The plant plant expression vector of SLARF6 was constructed with the constitutive promoter CaMV35S, named pCAMBIA1301-35S-SlAR.F6. Then the expression vector was introduced into tomato by by the leaf discs method of Agrobacterium-mediate teansforomation.. At last, 5 kanamycin resistant shoots were obtained successfully so far. The job is still going on.