Isolation And Analysis Of Genes Associated With Mutant About Pilose Fruit In Banan(Musa Spp.)

Banana (Musa spp.) is very important fruits around the world. For a long time, the principal means for banana breeding were selected mutants because of its triploid. However, the molecular mechanism of banana mutations poorly understood. The law about mutation would be known better and provide more effective guidance for breeding through research mutation mechanism. The pilose fruit mutant was from Brazil banana(Musa spp."Cavendish" AAA), it is characterized with the rachis, fruit and brach were combined by hairy phenotypes, and early flowering. In this research, suppression subtractive hybridization library (SSH) had been built by the mutant and wild and some genes about mutant were selected. To lay the foundation for better validation banana gene function, conditions of embryogenic suspension cells transformation were further optimized. The main results were following:The pros and cons of two subtractive cDNA libraries were built with banana pilose fruit mutant and wild type as "tester" and "driver" by suppression subtractive hybridization (SSH) technology and a total of591gene fragments were obtained, including325from the forward library and266from the reverse library. The results of BLAST analysis showed that the forward library which have206fragments with high homology to known functional genes from NCB1database,7fragments with homology but unknown function,112fragments failed to find the homology gene; the forward library which have266fragments with high homology to known functional genes from NCBI database,9fragments with homology but unknown function,19fragments failed to find the homology gene. These genes can be divided into21categories by gene ontology (GO).According to the results of previous research on the regulation of plant trichome and genes function prediction from subtractive cDNA libraries,4groups including22genes that may be associated with trichome were detected by RT-PCR and fluorescence quantitative Real-time PCR about their differences expression in wild and mutant during fruit development. The results were as follows:(1)3key transcription factors:①In the process of trichomes fast development, MYB was significantly higher expression level in wild than the mutant;②WD40was higher expression level in mutant in the initial stage of the trichome development;③B-ZIP was slightly higher expression levels in mutant in the maturity and late stage of the trichome development;(2)9genes about hormone-related:①ARP (auxin repressed protein) was higher expression in mutant than wild in the process of trichomes fast development;②ILR was similar expression trend but some differences level in the two phenotypes of fruit development;③The two ABAR (abscisic acid8’-hydroxylase) genes had different expression patterns, the expression of No. M49was little difference in mutant and wild, the expression of No. M286was significantly higher in wild than mutant:④4ARF (auxin response factor) were different expression, they were all higher expression in wild than mutant in the process of trichomes fast development;⑤AP2/ERF was always higher expressionn wild than mutant;(3)1gene of4MAPK were high expression in the wild type, the expression of the other3genes were little difference;(4)6genes related to the cell division were similar expression in two phenotypes, the more development of fruit, the lower expression.So, a series of genes expression had been changed in mutation.MaaERF is always higher expression in the wild than the mutant during fruit development. Full-length cDNA of MaERFwas obtained by RACE technology. The analysis of the amino acid sequence of this gene showed it belongs ERF subfamily B2subsets contains one AP2/ERF conserve domain, some hydrophilic and hydrophobic regions, but no transmembrane domain structure; MaERF gene contains one intron and low-copys in the genome. RT-PCR analysis showed that MaERF was expressed in all organizations of banana, but the expression was higher in reproductive organs. Overexpression MaERF in Arabidopsis, there was no significantly diversification of phenotype, indicating that the gene were little effect in the process of trichome.One AYB gene named MaMYB was obtained by RACE technology for better understood its role in the banana peel trichome process. The analysis of the amino acid sequence of this gene showed it belongs R2R3MYB subgenotype family, contains two conserve domains, some hydrophilic and hydrophobic regions, but no transmembrane domain structure; MaMYB gene contains one intron and low-copys in the genome. RT-PCR analysis showed that MaMYB was expressed in all organizations of banana, but the expression was higher in reproductive organs. Overexpression MaMYB in Arabidopsis, the trichomes reduced in transgenic plants leaves and missing in their flower organs, roots were longer, the fruit clip shorter than the wild, so that MaMYB not only participated in the development of trichomes but also related to fruit development. In the process of transformation about banana embryogenic suspension cells, the suspension cells had some resistance to kanamycin, hygromycin and herbicide (Basta) were suitable as a selecting agent antibiotic, and the concentrations were10mg/L and5mg/L; Banana embryogenic suspension cells were subculture5-6days, and co-culture with Agrobacterium6h and then direct selected on selective medium, bar gene as a selection marker,75%regenerated plants were positive plants, and their herbicide-resistant capacity has significantly improved.