| Paulownia（Paulownia Sp.）is a kind of fast-growing wood-producing tree species with high environmental and economic value,which is native to China and has been introduced to many other countries and regions.However,Paulownia Witches’ Broom disease（Pa WB）caused by phytoplasma infection,which has got highly infectious and a wide harming range,affects the growth and development of Paulownia,and even leads to death.It has got tremendous affections on the economic and ecological profit of Paulownia,and caused serious harm to the development of Paulownia industry.However,as far as we known,the molecular mechanism of phytoplasma infection,which inducing Pa WB,remains unclear.In order to further revealing the PaulowniaPa WB resistance molecular mechanism,improving and perfecting the theoretical basics of studies on paulownia biotic stress resistance.In this study,we choose health,Pa WB phytoplasma-infected and different concentrations(15 mg,mg L-1/30,L-1)methyl methane sulfonate treated Paulownia tomentosa × Paulownia fortunei tissue culture seedlings as experimental materials,using Illuminate sequencing platform for the detection of gene expression variations during the Pa WB occurrence.And the results were mapped to Paulownia genome,for quality control,alternative splicing detection and analyzing,and filtering Pa WB related genes for functional annotation analysis.Some of the sequenced genes were randomly selected for q RT-PCR verification.The results are summarized as follows:（1）In this study,the Illumina sequencing platform were adopted to provide a transcriptome sequencing of P.tomentosa × P.fortunei samples.Totally,335,278,558 raw reads were obtained,and after data filtering,the libraries of 4 samples respectively get 64,075,714（PTF）,75,308,384（PTFI）,92,383,062（PTFI-15）and 88,500,118（PTFI-30）clean reads,in which Q30 base percentage were all above 96.3%,and the GC base content were all greater than 46.3%.（2）The clean reads sequencing results were mapped to the reference genome for identifying the alternative splicing.And totally,four types of alternative splicing event were detected,the introns retention,exons skipping,alternative 5’ slice site and alternative 3’slice site,in which the introns retention event was the most frequently occurrence event type.And this type of AS event was not been detected in the healty samples,but highly occurred in all PAWB samples,including both MMS treated and comparisons,in which the PTFI-15 sample has got most amount of ocurrance.Revealing that the IR event may have a protential conection to the Pa WB and MMS treatment.（3）The sequencing results were mapped to the Paulownia reference gene and genome,for quality control,and FPKM calculating,in order to identifying differentially expressed genes（DEGs）.After filtration,there were 2,671 DEGs between PTF vs.PTFI,3,791 DEGs between PTF vs.PTFI-15,2,443 DEGs between PTFI vs.PTFI-15,and 4,257 DEGs between PTFI vs.PTFI-30.（4）The filtering scheme were designed to excluded the reagents,growth and development variations,and other factors affections,and find out 152 DEGs that related to the Pa WB occurrence.Functional annotations and pathway enrichment analysis were performed on these DEGs,and showed that these genes were involved in biological processes such as stress response,cell membrane,catalytic activity and metal ion binding,and were also closely related to the metabolic pathways,biosynthesis of secondary metabolites,plant hormones signal transduction of,and plant-pathogen interaction.（5）8 differentially expressed genes were randomly selected from the transcriptome sequencing results for Quantitative real-time PCR verification.The q RT-PCR results were consistent with the trend of the transcriptome sequencing results. |
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