Functional Analysis Of Tomato Transcription Factor BES1/BZR1-7 Regulating Fruit Elongation Gene SUN

Tomato(Solanum lyccopersicum)formed various fruit shapes during the process of domestication and selection.The shape of tomato fruit has an important biological significance and commercial value.SUN is a key QTL that contributes to regulating the long fruit shape of tomato.It is located on the chromosome 7 short arm of tomato and encodes calmodulin binding protein.It is shown that SUN regulates fruit shape by changing the cell division and hormone distribution.However,little is known about the mechanism of transcriptional regulation.Therefore,further exploration of the relevant regulatory factors is of great significance to analyze the genetic basis of SUN controling fruit shape and promoting the selection of fruit shape excellent varieties.In this study,the transcription factor BES1/BZR1-7 was found by using yeast one hybrid.The genetic function and regulation mechanism were confirmed by transgenic verification.Meanwhile,we systematically analyzed the transcription factor family BES1/BZR1 in tomato and verified the transgenic function of other members of BES1/BZR1 transcription factor family to explain the functional redundancy of BES1/BZR1-7.The main results are as follows:1.BES1/BZR1-7 promotes tomato fruit elongation.The trans acting factor BES1/BZR1-7 was obtained from tomato c DNA library by yeast one hybrid(Y1H)using the promoter sequence of SUN,a positive regulatory factor of tomato elongated fruit shape,as bait.BES1/BZR1 is a key transcription factor in BR signaling pathway.Furthermore,yeast one hybrid assay,transient expression analysis report and electrophoretic mobility experiment all proved that BES1/BZR1-7 could bind to the cis-element on the upstream promoter of SUN.Transgenic functional verification showed that the shape of tomato fruit was significantly longer when the BES1/BZR1-7 gene was overexpressed.But the fruit shape of the knockout line was similar to that of the wild type.Then,functional verification of other BES1/BZR1 members proved that there was functional redundancy.In addition,overexpressing BES1/BZR1-7 changed the leaves shape and leaves angle.2.BES1/BZR1-7 has a constitutive expression pattern.The expression level was high in fruit though low in root,stem,leaf and flower.The expression level gradually increased with the development process,reached the highest level at the color breaker stage,and then decreased.The expression of GUS was driven by BES1/BZR1-7 promoter found that GUS was mainly expressed in fruit.It was low expressed in stem,leaf and flower.Subcellular localization showed that the protein encoded by BES1/BZR1-7 was located in the nucleus.Tissue section analysis showed that the number of pericarp cell layers of overexpressed plants was less than that of wild type.This suggests that it may change the polarity of cell division to elongate fruit shape.3.BES1/BZR1-7 interacts with MAP65-1a protein,which makes microtubules bundle.Co immunoprecipitation showed that BES1/BZR1-7 interacted with MAP65-1a protein,which suggested that it might regulate microtubule polymerization through MAP65-1a protein to make fruit longer.4.BES1/BZR1 family is functional redundancy in tomato.Seven members of BES1/BZR1 gene are located on chromosomes 3,4,7,10 and 12 of tomato.The gene structure is two exons and one intron.Subcellular localization showed that most of the BES1/BZR1 proteins were located in the nucleus and a few in the cytoplasm.The spatiotemporal expression pattern is constitutive.Their promoter is rich in plant hormones and cis-elements related to stress response.Transgenic experiments showed that BES1/BZR1-5 and BES1/BZR1-6 overexpressed plants also showed the phenotype of long fruit.This indicates that there is functional redundancy in BES1/BZR1 family in tomato.