| Objective : It was found in the production practice of korla pear,the low temperature of the flowering period can significantly increase the ratio of calyx,but the mechanism is unclear,Preliminary research results preliminarily indicate,KfpSPL may be involved in sepals development and regulation.Therefore,cloning kfpSPL gene and its promoter sequences of DNA,to predict the role of components and functions of flowering on its expression in low temperature and detection,to clarify flowering low temperature is advantageous to the shedding of sepals molecular mechanism lay a certain foundation.Method:In korla fragrant pear flower organ for material,in full bloom(50% full tree bloom),collected tag "korla fragrant pear tree all to spend(more than pedicel part)30,remove from the sepals and petals ovary border divided into two parts.(1)design primers based on the cDNA sequence of kfpSPL gene and the pear genome information.The sequence of kfpSPL genomic DNA was amplified by the DNA sequence of korla pear genome.The total length of cDNA of kfpSPL gene was compared with the pear genome database,and the reference sequence of upstream promoter was obtained.Using the reference sequence design primers,the upstream promoter sequence of korla pear kfpSPL gene was obtained by PCR amplification.(2)Based on the full length of kfpSPL gene obtained,the primer5.0 design was used to design real-time fluorescence quantitative PCR primers,extracts of korla pear total RNA were extracted using EASYspin plant RNA for rapid extraction of kit.Genomic DNA in total RNA is removed DNase I,RNase-free(1 U/μL),total RNA reverse transcription useing PrimeScriptTMRT reagent Kit(Perfect Real Time)(TaKaRa,Japan).Sample extracted cDNA is template to use CFX manager(Bio-Rad USA)Real-time quantitative PCR analysis of kfpSPL gene was performed.(3)Using the Illumina sequencing and bioinformatics methods to 4 ℃ low temperature processing of korla fragrant pear flower organ for differences in gene screening and functional annotation.Results :(1)the kfpSPL genomic DNA with a length of 3320 bp was amplified from the genome DNA of korla.From korla fragrant pear genomic DNA clones have been upstream promoter sequences,by sequencing start smaller for 2332 bp,promoter sequences through bioinformatics analysis,forecasting cis function components and their functions.(2)Comparing at 4 ℃ for 24 hours,12 hours 4 ℃ cold treatment can more effectively improve kfpSPL relative expression of genes in korla pears early flowering.Compared with the other two,the treatment of 6 h after the spraying of cold water can effectively improve the relative expression of kfpSPL gene in the earlyflowering period of korla pear.(3)by Illumina high-throughput sequencing establishes the korla fragrant pear flower organs in 4 ℃ low temperature treatment under the condition of 8 hours and 24 hours processing samples and the control sample data,after data Mosaic,96662 Unigenes were obtained and the functional annotation was made in the 7 database.Functional comments Unigenes were obtained in 67769 of the 7 large databases.A total of 154020 transcript sequences were obtained by sequencing.Conclusion: the test by PCR technology won the korla fragrant pear promoter for the genomic DNA and upstream regulation sequence,the second series of bioinformatics analyses,got the translation initiation codon,and prediction of the cis control components and function of promoter sequences.By real-time fluorescent quantitative PCR for low temperature processing under the condition of korla fragrant pear flower organ kfpSPL gene expression in quantity for testing,that gene expression is obtained by data analysis to improve the processing time.Illumina high-throughput sequencing was used to screen the differentially expressed genes in korla pear flower organs under low temperature treatment,and the functional annotation was carried out in 7 large databases. |
