Studies On Physiological And Molecular Bases Of Cold Response And Preliminary Research On Editing Mutation Of TCP Gene

Tobacco is the main cash crop in China and the tobacco growth has been restricted by many stress factors in recent years,the effects of low temperature stress was the most serious factors in the product and quality of tobacco.Therefore,learn the response mechanism of tobacco to low temperature stress and improve their defense ability has become the important basic part in tobacco research.To study the physiological and molecular basis of cold response and genetic transformation of the recombinant gene-editing vector of the cold-related TCP gene in tobacco,We detected respectively these physiological and biochemical indexes of Superoxide Dismutase(SOD),Peroxidase(POD),Catalase(CAT),and Proline(PRO),Malondialdehyde(MDA)and Abscisic acid(ABA)of NC567 and taiyan8 in cold stress treatment,analyze and validate the resultsof miRNAs in two cultivars by molecular experiments such as physiological and biochemicaltests,highthroughputsequencing technology,qRT-PCR.Subsequently we structure CRISPR/Cas9 vector,acquire the transgenic plants by genetic transformation.Conduct the following results:1.After 24 hours under cold stress,the activity of SOD and CAT declined significantly,the activity of POD,and the content of PRO,MDA andABAall increased.Only the content of MDA in Taiyan8 is more than that in NC567,the level of other indicators in NC567 is higher than that in Taiyan8 significantly.the results of variance analysis show that the activity level of SOD,POD,CAT and the content of PRO,MDA,ABA have significant difference around the cold stress treatment.2.More than 22 M raw reads were obtained for each sample.The alignment against the miRbase identified 164 conserved miRNAs.A total of 25 cold-regulated(COR)conserved miRNAs in NC567 and 13 in Taiyan8 were discovered.Further analysis revealed the important roles of down-regulation of miR169 family members in the cold-tolerance ability for NC567,and of up-regulation of nta-miR399 expression and the target genes related to catalysis and cell wall modification in the cold-sensitivity of Taiyan8.The contrasting expression patterns of most COR miRNAs in two cultivars suggest genotype-specific miRNA-mediated cold response mechanism in tobacco.GO analysis showed that the target genes for the down-regulated miRNAs are mainly involved in transcription,and reproduction regulation in both cultivars.3.Construct the CRISPR/Cas9 directional knockout plasmid,detected 8 transgenic materials by random selection.The detection results and the sequencing showed that all of these materials was positive clones and both produced site-directed gene mutations.