The Biological Function Of Specific RNA Splicing And The Corresponding Transcripts In SbWIN1 Gene

By 2017,Sorghum field has reached 15 million acres in China.And among them,grain Sorghum is the main type of varieties planted.There are lots of visible pruina at the Sorghum leaves and leaf sheaths surface after heading stage,called as wax powder.Sorghum has thick wax layer on the inner and surface of plants,that provides a primary waterproof barrier and protection against different environmental stresses.Then it strengthens the resistance abilities of Sorghum facing drought,aridity and waterlogging etc.and increases production of Sorghum.In order to fully excavate and use the genetic potentiality,this research analyzed the mechanism of waxy genes functions in Sorghum,linking drought resistance genes with their biological functions.According to the wax powder contents of different materials in the field,Sorghum maintainer lines BTX622 was selected for transcriptome analyses.Meanwhile full-length transcript(SbWIN1-204)and specific RNA splicing transcript(SbWIN1-27)of wax-related gene SbWIN1 were cloned to construct overexpression vectors in transgenic Arabidopsis plants for functional analysis.The main results are as follows:1?Variation patterns of wax powder content in different types of Sorghum materialsColorimeter method was used to measure the wax powder content.There were significant differences among different types of Sorghum varieties,the wax powder content of grain Sorghum BTx622 was the highest.The top,middle and bottom stems of Forage Sorghum contain high,low and high content of wax powder,respectively.The starting time and duration of the different types of Sorghum varieties in each growth stage varied significantly,and the content of accumulated wax powder in the same growth stage was also different.The tendency of wax powder contents among Sorghum growth stages and varieties are slightly different: in Forage Sorghum,heading stage>flowering stage>filling stage>wax ripeness stage>complete ripeness stage,while in Sweet Sorghum,filling stage>heading stage>flowering stage>wax ripeness stage>complete ripeness stage.The diurnal changes of wax powder content followed a monopeak curve,reached the the peak value around 13:00.2?Transcriptomic analysis of different Sorghum organsThe transcriptomic profiles and bioinformatics of roots,stems,leaves,sheaths,spikelets and seedling in Sorghum were clarified and analysed by Illumina Hi Seq 4000 platform.Theresults showed that a total of 321 790 986 raw reads were obtained with 311 985 466 clean reads.469 601 alternative splicing events were observed from Sorghum genome corresponding to 37 310 genes.In the process of alternative splicing analysis,seven alternatively spliced variants of wax-related gene SbWIN1 was identified,all of them belong to transcription start site(TSS)and transcription terminal site(TTS)splicing variant,beyond that of full-length.Among them,we found the full-length transcript could encode 204 amino acids(named SbWIN1-204),but the shortest transcript only encoded 27 amino acids(named SbWIN1-27).3?Cloning and preliminary characterization of different alternative splicing transcripts of WIN1 gene in SorghumIn order to clarify the differences and similarities between the two transcripts of SbWIN1 gene,we cloned and constructed overexpression vector with EGFP reporter gene by the Gateway cloning method.Then,the Arabidopsis plants were transformed by these genes through Agrobacterium mediated method,respectively,and functional identification of the wax-related genes.SbWIN1-204 and SbWIN1-27 proteins contain the AP2 conserved domains and are members of AP2 family.They could be detected from six organs of Sorghum on different expression levels with the highest one in stems.When we observed EGFP transgenic Arabidopsis plants under the fluorescence microscope,SbWIN1-204-EGFP distributed exclusively in the nucleus but SbWIN1-27-EGFP can be detected on various subcellular areas.Ultrastructures of glossy leaves uncovered by Scanning Electron Microscope clearly showed the wax layer of transgenic plants was much thicker and rougher than those of control plants.Consequently the drought resistance of transgenic Arabidopsis plants were improved though SbWIN1-204 plants was little more tolerant than SbWIN1-27 plants.SbWIN1 gene might positively regulate the wax synthesis in Sorghum.Finally,in order to establish Sorghum transformation system,we used the immature spikes and embryos as explants to conduct tissue culture and Agrobacterium mediated transformation.The results demonstrated that the differentiation rate of XinLiang 52 and SanChiSan genotypes were comparatively higher and small amounts of the resistance callus have been obtained using them.However,all of them can't generate transgenic seedlings,and become brown to die.