Study On Salt Tolerance Of Chrysanthemum Based On Transcriptome Sequencing

Salt stress seriously affects the growth and production of chrysanthemums,so it is particularly important to find the key genes in response to salt stress.We selected Chrysanthemum moliflium'Jinba'as the test material.After selecting the appropriate treatment time and concentration,transcriptome sequencing was performed on chrysanthemum roots under normal culture and salt stress to screen for differentially expressed genes in important pathways.And then the expression of candidate genes was verified by qRT-PCR.At the same time,changes in physiological indicators such as ion content,osmotic adjustment substance content,and antioxidant enzyme activity of chrysanthemum roots before and after salt stress were measured.The main research conclusions are as follows:1.After treatment with 100,200,and 300 mM NaCl for 0,3,6,12,24,36,48 h,the changes in REC,MDA content,chlorophyll content,and root activity were measured.The measurement results show that under 100 mM salt treatment,the changes of indicators were not much different from those of the control group;under 300 mM salt treatment,all indicators showed a significant upward or downward trend;but under 200 mM salt treatment,especially after 24 h of processing,the change appeared a turning point.Therefore,we speculated that chrysanthemum would activate the regulatory mechanism to adapt to stress environment at this time,and determined that 24 h and 200 mM were suitable values for transcriptome sequencing.2.Illumina Hiseq sequencing platform was used to perform transcriptome sequencing on the chrysanthemum root system,and 6 cDNA libraries?CK1,CK2,CK3,SS1,SS2,SS3?were created.A total of 68.30 Gb effective sequences were obtained.70,764 unigenes were assembled by de novo and compared with the public databases TrEMBL,NR,Pfam,KOG,GO,and KEGG to obtain gene annotations,which covering almost all functional and metabolic pathways.Using|log2?fold change?|>1 and P value?0.05 as the screening criteria,2117 differentially expressed genes were identified,including 1347 up-regulated genes and770 down-regulated genes.A large number of differentially expressed genes are enriched to regulate various physiological and biochemical processes in cells,which provides a wealth of data for studying the salt tolerance regulation mechanism of chrysanthemum roots.3.We based on gene function annotations,enrichment results,and previous studies to screen differentially expressed genes related to signal transduction,plant hormones,ion transport,osmotic regulation,antioxidant regulation,important functional proteins,and transcription factors.And we chose select 20 genes with large gene multiples from them as candidate genes.We used qRT-PCR to determine the expression patterns of candidate genes at 0,3,6,12,24,36,and 48 h after salt stress.The expression patterns of different DEGs are different,suggesting that these genes may participate in chrysanthemum root stress through different regulatory pathways.The response also verified the reliability of the sequencing results.4.The determination of ion content showed that the Ca²⁺content increased first and then decreased.The Na⁺content decreased and the K⁺content increased during the initial stress,indicating that Na⁺outflow and K⁺inflow increased significantly.Both of them were inhibited with the increase of stress time.5.The determination of osmotic adjustment substance content showed that the proline content increased first and then decreased,and the soluble sugar content was always increasing.6.The measurement of antioxidant enzyme activity showed that the POD activity first increased and then decreased,and the SOD activity was always inhibited,the CAT activity was initially inhibited and then activated again.In summary,this study was based on transcriptome sequencing to screen salt-tolerant genes in chrysanthemum roots in response to salt stress.We explained the mechanism of salt tolerance of chrysanthemum roots by identifying candidate genes,comparing their differences in expression patterns,combining gene expression with physiological data.