Cloning And Expression Analysis Of Cadmium-Responsive Gene CibZIP43 And Its Promoter Of Chrysanthemum Indicium

Chrysanthemum indicum is is a kind of perennial plant of Compositae,which has high medicinal value,strong cold and drought tolerance.It is one of the important ornamental flowers in autumn in north China.In addition,Chrysanthemum indicum has the capability of remediation of heavy metal contaminated soil At present,the research on Chrysanthemum indicum mainly focused on its medicinal value,volatile substance composition,chemical composition,etc.However,there are few studies on the mining of stress resistance genes of Chrysanthemum indicum through genetic engineering technology to improve the stress resistance of plants.bZIP transcription factor is one of the largest transcription factor regulatory family,which plays an important role in plant growth and development,biotic and abiotic stress responses,and the synthesis of secondary metabolites.In this study,the transcriptome of Chrysanthemum indicum under cadmium stress was analyzed,and the response genes to cadmium stress were analyzed to explore the resistance function The main results are as follows:1.The transcriptome of Chrysanthemum indicum cadmium stress was analyzed,41 CibZIP genes were screened,and a phylogenetic tree of Chrysanthemum indicum and Arabidopsis thaliana bZIP genes was constructed.41 bZIP genes of Chrysanthemum indicum were divided into 12 sub-families,and they were classified with Arabidopsis thaliana similarly.Analysis of bZIP differentially expressed genes under cadmium stress revealed that 4 genes were up-regulated in roots,2 genes were down-regulated in roots,and only 1 gene was down-regulated in leaves.Based on the analysis results of the transcriptome,TRINITY_DN155816_c0_g1 was selected for cloning and renamed CibZIP43.2.CibZIP43 was cloned by RT-PCR.The bioinformatics analysis of CibZIP43 showed that its ORF is 516 bp in length,encoding 171 amino acids,which is an unstable protein,a hydrophilic protein,no transmembrane structure,no signal peptide,and a non-secreted protein.The protein structure is dominated by alpha helix and random coils,with 4 glycosylation sites and 19 phosphorylation sites.The multi-sequence comparison of CibZIP43 protein with other homologous species showed that the protein has high homology with the bZIP protein of Compositae,and is closely related to Artemisia annua.Under the treatment of 20 mg/L CdCl2,the expression of CibZIP43 showed a downward trend,with the lowest expression in leaves at 1 h and the lowest expression in roots at 8 h.3.A plant expression vector pBI 121-CibZIP43-GFP was constructed and transformed into Arabidopsis thaliana by inflorescence transfection.The T1 generation seeds were screened in a selection medium,and the transgenic plants were obtained by DNA identification.4.A 1900 bp promoter sequence of CibZIP43 was cloned by Genome Walking.Cis-regulatory element analysis showed that the promoter region had plant hormone-like response elements(abscisic acid,methyl jasmonate,ethylene,salicylic acid),stress-responsive cis-regulatory element(drought,anaerobic,pathogenic bacteria),photoresponsive elements,and plant growth and development regulatory elements.5.According to the position of the cis-acting element,the promoter 1900bp was divided into 5 segments with 5’-end deletion,named P(1900bp),P1(1504bp),P2(1046bp),P3(585bp),P4(246bp),and replaced the original CaMV35s promoter on the PBI121-GUS vector.The five constructed vectors and PBI121-GUS vector were used to transform Nicotiana benthamiana by injection and stained with GUS.The results showed that the PBI121-GUS,P,and P3 stained darker,and P1,P2,P4 stained lightly,indicating that the 5’-end deleted fragments of the promoter were all active,and the P and P3 activities were strong.