Screening Of The Key Candidate Genes Of Rice Heat-tolerance At Milky Stage And CRISPR Vector Construction

Global warming,with frequent high temperature（especially high night temperature）in summer reduce the rice crop and decrease the quality of rice which is threatening the food-safety of our county.Therefore,it is urgent to study on the molecular mechanism behind rice in response to high temperature stresses and breed excellent rice varieties with heat resistant trait.In this study,the patterns on gene expression at early milky stage of rice in response to high night temperature were analyzed on transcriptic by RNA-seq and full-length transcript by PacBio technique,and was analyzed on proteomics by iTRAQ and liquid mass spectrometry.The results showed that the gene Os05g0585500（LOC_Os05g50810）which was involve to signal transduction is the key gene involved in regulating rice responding to high night temperatrue stress during milky stage,according to the gene expressed patterns between the transcription and protein levels.By sequence alignment we designed targeting site for the key gene,and constructed its specific CRISPR/Cas9 vector.The main findings are as follows.1)By the RNA-seq and Pacbio full-length mRNA sequence,the sequences and gene expressed patterns of 26,327 full-length transcripts were obtained.expressed volume for the 3,130 proteins were obtained by iTRAQ and liquid chromatographic series mass spectrometry.The analysis on differential expressed genes showed that 10,996 transcripts showed over 1.5 fold-change differential expression,70 proteins showed over 1.5fold-change differential expression in proteomics level,between the heat-resistant and heat-sensitive rice.2)Among the 70 differential expressed proteins obtained from proteomics,these proteins may be involved in photosynthesis,biosynthesis,transcription regulation,defense response,stress response,energy metabolism,materials transport,oxidation,signal transduction.The analysis on the acciation between transcriptics and proteomics showed that the gene LOC_Os05g50810 which was involve to signal transduction showed only0.97 fold-change in transcriptics level while 1.93 fold-change in proteomics level between heat-resistant and heat-sensitive rice.The small changes in transcriptics lead to significant changes in proteomics,the gene LOC_Os05g50810,meanwhile in the calcium-dependent signal transduction proteins,the gene showed highest differential expressed proteins between heat-resistant and heat-sensitive rice which was suspected to be involved in signal transduction dependent Ca²⁺,is the key gene for regulating the heat resistance of rice during milky stage.3)Based on the full-length transcription sequence obtained from sequencing and database matching,the gene target sit for the key gene LOC_Os05g50810 was designed and its specific CRISPR/Cas9 expression vector was constructed which had laid the foundation for further genetic transformation,detection of target efficiency and biological function verification of candidate genes.