Cloning And Functional Analysis Of Moso Bamboo Functional Unknown Genes Regulated By Brassinosteroids

Moso bamboo(Phyllostachys edulis)has great economic,ecological and cultural value,as well as specific biological characteristics.The bamboo shoot grows rapidly,which can elongate about 1 meter within the 24 hours and is able to grow to mature height in three months.The rapid growth mechanism of bamboo shoots has been attracted more and more attention by researchers now.The coordinated regulation of multiple hormones is essential for the rapid growth of bamboo shoots.As a group of plant steroid hormones,brassinosteroids(BRs)play important roles in promoting cell elongation,as well as other various growth and development processes and responses to biotic and abiotic stress.Transcriptome analysis has revealed that BR responsive genes were highly enriched in fast-growing internodes.The detailed molecular mechanism of BR involved in the fast-growth process is yet to be uncovered.Our previous work identified thousands of differentially expressed genes upon BR and PPZ(a specific BR biosynthetic inhibitor)by transcriptome sequencing.Based on genomic annotation and bioinformatics analysis,22 functional genes unknown BBRGs(Bamboo BR regulate genes)were selected for further analysis.BBRG genes were cloned and overexpressed in the model plant Arabidopsis.Multiple transgenic plants showed significant morphological differences compared with wild type.Then one of them,the BBRG14 gene were focused on and continued with the expression and functional analysis.Bioinformatics analysis suggested that the BBRG14 homologs were only identified from wheat(Triticum aestivum),rice(Oryza sativa),Brachypodium distachyon.Since all of these species are from poaceae,BBRG14 renamed PSBR1(Poaceae Specific and BR response gene 1).quantitative RT-PCR showed that PSBR1 was expressed in the leaves of bamboo seedlings.PSBR1 gene were detected from different tissues of elongating bamboo shoot(35 cm above ground)including the shoot apical meristem,un-elongated and elongating internode,un-elongated and elongating node and scale leaves.PSBR1 was highly expressed in the nodes,especially in the elongating node,but less expressed in the un-elongated and elongating internodes.Transcriptome data and qRT-PCR showed that the expression of PSBR1 was up-regulated by PPZ but down-regulated by subsequent BR treatment.Subcellular localization analysis showed that PSBR1-YFP fluorescence signals co-localized with mitochondrial localization signals in tobacco leaf epidermal cells,Arabidopsis transgenic plants and moso bamboo protoplasts.Although most mitochondrial proteins destined for import into the mitochondria bear mitochondrial targeting sequences(MTSs),which are commonly 20-60 amino acids at N-terminal.The first 20,40,60 amino acids of the PSBR1 protein are not sufficient to target YFP protein to the mitochondria.PSBR1 overexpressing plants showed growth inhibition at multiple growth and development stages.Transgenic plant showed growth defects of quiescent center(QC)and meristem zone in root apex.During the vegetative growth stage,the leaves are light green,cracked,and the rosette leaf number is reduced.During reproductive growth stage,smaller flower organs,shorter and twisted silique and shorter seeds are found.In this study,we performed bioinformatic and expression pattern analyses of BRRG genes and observed phenotype of overexpression transgenic plants in Arabidopsis thaliana.We planned to generate the overexpression and loss of function transgenic lines in moso bamboo in the future studies,which will provide an experimental basis for further studies how BR regulates bamboo growth and development.