Joint Sequencing Analysis Of Transcriptome In Chrysanthemum Leaves Under Low Temperature Stress And Cloning And Functional Identification Of DgMYB1 Gene

Chrysanthemum?Dendranthema morifolium?Ramat.?Tzvel.?is one of the top ten traditional flowers in China.It has a cultivation history of more than 3,000 years in China and has a high ornamental,medicinal,edible and economic value.Chrysanthemum is susceptible to low temperature damage during flowering,which has a serious impact on its ornamental and economic value.The MYB transcription factor is one of the largest family of transcription factors in plants,and the MYB transcription factor plays an important role in plant response to external stress.In this study,the"Jinba"chrysanthemum leaves under low temperature stress were used as experimental materials,and the single-molecule real-time sequencing technology and Illumina HiSeqTM 4000 double-end sequencing technology was used to analyze the transcriptome data of chrysanthemum.A MYB gene was cloned and transformed into"Jinba"chrysanthemum,which provided a theoretical basis for improving the cold tolerance of cultivated chrysanthemum by molecular biology.The main findings are as follows:1.Using single molecule real-time sequencing technology and Illumina HiSeqTM 4000double-terminal sequencing technology,the transcriptome data of chrysanthemum leaves under low temperature stress were analyzed.Six cDNA libraries were established.A total of8.34 GB data were obtained.Finally,3,717,516 Subreads were obtained,with an average length of 2,243 bp.305,231 CCS sequences were obtained,and 257,144 full-length reads were repaired by site mismatching.After modification,141,445 consensus sequences were obtained.After correction and redundancy removal,418,153 Unigenes were collected.A total of 283,566 unigenes were annotated in seven databases.In addition,18,592 genes were differentially expressed,including 10,021 up-regulated genes and 8,571 down-regulated genes.2.A MYB gene sequence was screened from the chrysanthemum transcriptome database under low temperature stress,and the full-length primer was designed and cloned to obtain the full-length gene sequence of the MYB gene,which was named DgMYB1.Sequence analysis showed that DgMYB1 has a full length of 1,240 bp and an open reading frame of 990 bp,encoding 328 amino acids.The molecular weight of the encoded product is 36.47 kDa,and the theoretical isoelectric point is PI=7.74.Sequence alignment revealed that DgMYB1 has high homology with AtMYB73,NtMYB44,ZmMYB44,GmMYB29,MdMYB7 and HvMYB6.Phylogenetic tree analysis showed that DgMYB1 was closely related to Raymond's cotton GrMYB44,Arabidopsis AtMYB73,and Brassica napus BnMYB44.3.The plant cloning vector pEASY-MYB1 was constructed.The plasmids containing pEASY-MYB1 and pCAMBIA2300 expression vectors were digested by Sac I and Xba I.Finally,DgMYB1 was transformed into chrysanthemum by Agrobacterium-mediated method.The DNA of transgenic chrysanthemum was extracted as template for PCR detection,and the expression level of DgMYB1 in chrysanthemum was quantitatively detected by fluorescence.4.Tolerance of transgenic DgMYB1 gene chrysanthemum to low temperature stress was identified.The results showed that the expression level of DgMYB1 gene in chrysanthemum leaves was higher than that in stems and roots.Under normal growth conditions,there was no significant difference in the phenotype between transgenic chrysanthemum and wild type chrysanthemum.Under low temperature stress,wild type chrysanthemum leaves gradually wilted,dehydrated,and even the whole plant died,while transgenic chrysanthemums grew well.The physiological index showed that the survival rate of transgenic DgMYB1chrysanthemum was much higher than that of wild type under low temperature stress;the relative electrolyte permeability,malondialdehyde?MDA?content,H₂O₂ content and O₂-content of transgenic chrysanthemum were lower than wild type.DAB and NBT staining were also lighter than wild type,while antioxidant enzymes?SOD,POD,CAT?activity,proline content,soluble sugar content and soluble protein content were higher than wild type,which proved that transgenic chrysanthemum was more than wild type.Chrysanthemums show greater cold resistance.5.Real-time fluorescence quantitative PCR was used to study the expression of cold-tolerant genes CuZnSOD,CAT,APX,DREB1A,DREB2A,P5CS,CSD1 and CSD2.The results showed that the expression levels of these genes in transgenic chrysanthemum were significantly higher than those in wild-type.In summary,this study improved the transcriptome information of chrysanthemum leaves under low temperature stress,and obtained the transgenic"Shenma"chrysanthemum plant by overexpressing DgMYB1 gene,and found that the transgenic plants had improved cold tolerance by low temperature stress.The new low temperature resistant varieties provide an effective gene reserve.