Transcriptome Analysis In Chrysanthemum Roots Under Salt Stress And Cloning And Functional Identification Of DgMBF1 And DgJAZ1

Chrysanthemum is an ornamental plant widely cultivated all over the world because of its high economic value,but is often affected by the environment stresses such as salt,alkali,drought and low temperature,which resulting in a lower yield.High salinity can cause yellowing of chrysanthemum leaves,inhibit growth of plant,and even lead to plant death.The transcriptional coactivator MBF1 and the transcription factor TIFY family play important roles in the growth and development of plants and were involved in various environmental stresses.In this study,chrysanthemum var.'Jinba'were used as experimental materials.The combination of Illumina HiSeq^TM 4000 and single molec?le real time?SMRT?sequencing were used to analyze the transcriptome data of chrysanthemum,and the differentially expressed genes were screened from the results.A transcriptional coactivator MBF1 and a TIFY family transcription factor JAZ were screened and transformed into'Jinba'to further explore the salt-tolerant molec?lar mechanism of chrysanthemum.The main findings are as follows:1.In this study,the combination of Illumina HiSeq and SMRT sequencing technology was first used to sequence the transcriptome of chrysanthemum roots.The full-length transcriptome library based on the combined sequencing technology contains 550,823high-quality Unigene,of which 52,120 ones were longer than 3kbp.But only 19,464 Unigene were longer than 2 kbp after assemblied with Illumina HiSeq.The combined sequencing significantly improved the efficiency of identifying the full-length transcript.Many salt stress response genes were screened among 48,396 differentially expressed genes?screening thresholds P<0.05 and|log2FC|>3?,and mainly involved in signal transduction,ion transport,membrane stability,reactive oxygen scavenging,osmotic adjustment and other aspects.2.A MBF1 gene and a JAZ gene were screened from the full-length transcriptome database of chrysanthemum roots.The primers were designed and cloned to obtain the coding region gene sequences,which were named DgMBF1 and DgJAZ1.The open reading frame?ORF?of the two genes was obtained based on bioinformatics analysis,and the fragments were 438 bp and 594 bp,encoding 153 and 197 amino acids,respectively.DgMBF1 contained a MBF1 domain and a helix-turn-helix?HTH?domain and identified as a member of MBF1c group which has a high similarity to MBF1c in Artemisia annua.DgJAZ1 contained a TIFY domain and a Jas domain,and identified as a JAZ protein belong to TIFY transcription factor family and has a high similarity to the JAZ gene in Artemisia annua.3.Plant expression vector was constructed,and DgMBF1 and DgJAZ1 were transferred into chrysanthemum following the Agrobacterium-mediated method.The res?lts of DNA detection indicated that the target gene had been integrated into chrysanthemum var.'Jinba',and the transgenic plants were successf?lly obtained.The expression levels of the two genes in chrysanthemum were quantitatively detected by fluorescence.4.DgMBF1 transgenic plants OE-3 and OE-34,DgJAZ1 transgenic plants OE-7 and OE-31 and wild type?WT?were selected for salt stress experiments to observe the phenotype and survival rates.The transgenic plants were not significantly different from WT plants in phenotype under normal conditions.But under salt stress,the leaves of WT plants showed significant wilting and yellowing compared with the DgMBF1 transgenic plants.The survival rates of OE-3 and OE-34 were 78.65%and 80.92%,respectively,while WT was only 32.13%.However,the DgJAZ1 transgenic plants showed yellowing and withering earlier than the WT,and the survival rates of OE-7 and OE-34 were only 18.65%and 20.92%,respectively.5.Activities of superoxide dismutase?SOD?,peroxidase?POD?,ascorbate peroxidase?APX?and catalase?CAT?and the contents of proline and soluble sugar in DgMBF1transgenic plants were all significantly higher than those of WT under salt stress.Compared with WT,the contents of superoxide anion?O₂^-?and hydrogen peroxide?H₂O₂?were lower in transgenic chrysanthemum and the root length and fresh weight of transgenic chrysanthemum were better than WT.The indexes measured in the DgJAZ1 transgenic chrysanthemum were completely opposite.6.In order to study the molecular regulation mechanism of transgenic lines under salt stress,we determined the relative expression of six stress-related genes by Real Time Quantitative PCR?qRT-PCR?.The expression of these salt-responsive genes?DgCuZnSOD,DgAPX,DgCAT,DgP5CS,Dg6GDPH and DgMDH?in both transgenic and WT plants was up-reg?lated in different magnitudes.The results showed that the combined sequencing improved the accuracy and diversity of the transcriptome database and provided a more efficient gene reserve for breeding new salt-tolerant varieties.After functional verification of two transcription factors,it was found that the DgMBF1 transgenic chrysanthemum was more salt-tolerant than WT.DgMBF1 gene can regulate the activities of reactive oxygen scavenging enzymes,the contents of osmotic adjustment substances and the expression of related stress-responsive genes to enhance the salt tolerance of chrysanthemum.The DgJAZ1 gene cannot well-regulate the corresponding substances to respond to high salt stress,which increasing the sensitivity of chrysanthemum under salt stress,and reducing the salt tolerance of chrysanthemum.