Functional Analysis Of Grape Transcription Factor VvMADS45 In Seed Development

Grape is a mainly economical fruit tree around the world,and a commonly cultivated fruit tree in China.The breeding of new seedless grape cultivars is one of the main objectives in the grape breeding.At present,seedless grape cultivars are few.It's long to breed a new grape,especially seedless grape.Therefore,it is very important to research on genetic mechanism of seedless grape to cultivate new seedless grape cultivars through transgenic technology.In the previous study,genome and transcriptome analysis of ovules at different stages showed that VvMADS45 gene expression in seeded grape was significantly higher than that in seedless grape,so it's hypothesized that VvMADS45 gene played a important role in seed development.Based on this,the relative expression,gene sequence and the cis-acting elements of promoter of VvMADS45 were analyzed in this study.In addition,the function of VvMADS45 gene was firstly demonstrated by ectopic expression of VvMADS45 in tomato and genome editing of tomato homologue SlAGL104.The main results are as follows:1.The expression level of VvMADS45 gene in every organ and six ovule development stages of grape was analyzed.It was found that the expression level of VvMADS45 gene was lowest in fruits and significantly increased in the late stage of ovule development in seeded grape ‘Red Globe';however,in seedless grape ‘Thompson seedless',the expression level of flowers was the highest,and was relatively low in ovule at each development stage.2.The cloning and sequencing of the VvMADS45 gene found that the VvMADS45 gene sequence had a total length of 969 bp in ‘Red Globe' and ‘Thompson Seedless',encoding 322 amino acids,but there was a difference in the individual bases.Analysis of the VvMADS45 gene promoter sequence discovered that the sequence in ‘Red Globe' was significantly different from that in ‘Thompson Seedless',and most of the cis-acting elements existed in ‘Red Globe' and ‘Thompson Seedless'.But some of the cis-acting elements only existed in ‘Red Globe' or ‘Thompson Seedless',such as the Box III in ‘Red Globe';the AT-rich element,the AT-rich sequence and the I-box in ‘Thompson Seedless'.3.Function of VvMADS45 gene was analyzed through constructing VvMADS45 overexpressing vector and using CRISPR/Cas9-mediated targeted mutagenesis of tomato homologous gene SlAGL104 to transform Micro-Tom.The flowers,fruits and seed phenotypes of wild-type and transgenic plants were observed.It was found that the flowers,fruits and seeds of VvMADS45 overexpressing plants were larger than these of wild-type plants,while the flowers,fruits and seeds of tomato homologue SlAGL104 knock-out plants were smaller than these of wild-type plants.Furthermore,the number of seeds in VvMADS45 overexpressing plants and SlAGL104 knock-out plants decreased.4.The pollen grains of wild type and transgenic plants were stained with FDA(fluorescein diacetic acid)and germinated in vitro.The results showed that there was no significant difference in pollen activity and pollen germination ability between wild type,VvMADS45 overexpression and tomato homologue SlAGL104 knock-out plants.Furthermore,the hydration of pollen grains on stigma was observed by scanning electron microscopy(SEM).It was found that more abnormal pollen grains failed to hydrate appeared in VvMADS45 overexpressing plants,while less abnormal pollen grains appeared in SlAGL104 knock-out plants.5.The expression analysis of tomato genes related to flower,fruit and seed development showed that expression level of these genes were changed in VvMADS45 overexpression plants and SlAGL104 knock-out plants.