Pitaya DREB1D Gene Is Involved In The Molecular Regulation Of Stress Respons

DREB(Dehydration responsive element binding)transcription factor is widely present in plants,mainly involved in various abiotic stress responses.Pitaya(Hylocereus monacanthus)belongs to the cactus family,because its outer skin resembles the outer scales of the dragon and is named,showing strong stress resistance in drought,high temperature and low temperature.At present,there are few studies on DREB transcription factors in pitaya,so our team screened out pitaya DREB transcription factors from previous work,and used the pitaya variety as experimental materials to study the characteristics of HmDREB1 D transcription(HU02G01866)factors and related stress resistance,and the main results are as follows:1)Gene cloning and stress response analysis of HmDREB1 D.The DREB1 D gene was successfully cloned from pitaya,and the ORF total length was 723 bp.Multiple sequence ratio comparison shows that HmDREB1 D protein has a highly conserved AP2 domain.Phylogenetic analysis showed that pitaya HmDREB1 D protein sequence had high homology with Arabidopsis thaliana At DRE1 D and Oryza sativa Os DRE1 D.The expression pattern of HmDREB1 D gene under different stress sources of pitaya was analyzed by q RT-PCR technique.It was found that the expression of HmDREB1 D was up-regulated after 12 h of drought and high temperature stress,but the low temperature stress was up-regulated 6 h,indicating that it could respond to the induction of drought,high,low temperature.2)HmDREB1 D gene promoter cloning and sequence analysisThe full-length sequence of HmDREB1 D gene was found from pitaya genome database,the 2000 bp sequence upstream of HmDREB1 D gene starting code was selected,and primers were designed.The results show that in addition to the necessary promoter core components TATA-BOX and CAAT-BOX,2drought-inducing elements with MYB participation,1 ABRE drought response element,6 STRE core elements related to heat stress,1 cryogenic response element,8light response elements,1 cis-action regulating element involved in methyl jasmonate response,are included.4 MYC binding sites and 1 meristem expressing relevant elements.3)Subcellular localization and genetic transformation of Arabidopsis thaliana and Nicotiana tabacum.Subcellular localization analysis using agrobacterium impregnated with the epidermis of Nicotiana tabacum showed that the HmDREB1 D protein was localized to the nucleus.A total of 6 transgenic Arabidopsis thaliana strains were obtained by dipping flower,super-expressing transgenic Arabidopsis thaliana strains(OE3,OE4,OE5)were screened by qRT-PCR,a total of 7 transgenic tobacco strains were obtained by leaf disc method,and super-expression transgenic Nicotiana tabacum strains(OE1,OE5,OE6)were screened out by q RT-PCR technology.4)Analysis of stress resistance of HmDREB1 D gene transgenic Arabidopsis thaliana.Under drought treatment,the germination rate,root length and fresh weight of transgenic Arabidopsis thaliana were significantly higher than those of wild types.Through the physiological and biochemical indexes of transgenic Arabidopsis thaliana,it was found that the relative conductivity of transgenic Arabidopsis thaliana was lower than wild type,while the activities of antioxidant enzymes(POD,SOD and CAT)were significantly higher than those wild type.At the same time,the expression patterns of HmDREB1 D gene overexpression lines in different stress response genes were analyzed by q RT-PCR technique.The results showed that the expression levels of stress response genes in overexpression lines were higher than those wild type.5)Analysis of stress resistance of HmDREB1 D gene transgenic Nicotiana tabacum.Under drought treatment,the germination rate,root length and fresh weight of transgenic Nicotiana tabacum were significantly higher than wild types.The expression level of stress genes in transgenic lines was analyzed by q RT-PCR.The results showed that the expression level of stress genes in overexpressed lines was higher than wild type.The above results showed that HmDREB1 D gene enhanced the stress resistance of transgenic lines under abiotic stress by improving the antioxidant capacity and stress gene expression level of transgenic lines under abiotic stress.This study lays a foundation for the analysis of the biological function of HmDREB1 D transcription factors in resisting various stresses.