Cold Tolerance Function Of CmFADs/CmTLP Genes In Mutant Model

Chelidonium majus L.is a perennial herb of Chelidonium L.in Papaveraceae family.It is rich in various alkaloids and has clear pharmacological activities such as anti-inflammatory and cough.In terms of plant physiology,it has strong cold resistance and cold resistance.It is of great significance and value for cold resistance research and related gene mining.The laboratory previously constructed a transcriptome database of Chelidonium sativa response to low temperature stress,and found the Fatty acid desaturase gene by screening.FAD2,FAD3)and Thanmatin like protein(TLP)were significantly up-regulated under low temperature stress,showing physiological functions highly related to cold resistance and cold resistance.In this study,transgenic mutants At Cmtlp,At Cmfad2 and At Cmfad3 were constructed by allogeneic transformation of Arabidopsis Thaliana.The aim is to further study and elucidate the functions of FAD2,FAD3 and TLP genes in Chelidonium Chelidonium under the background of model plants,especially the gene expression characteristics and cold tolerance function under freezing stress.(Arabidopsis thaliana,(Col).)Is a transgenic receptor,the target genes Cm FAD2,Cm FAD3 and Cm FAD3 were derived from Chelidonium,and the ecotype wild-type Arabidopsis thaliana(Wt)was the control.Four aspects were studied: analysis of gene expression characteristics of At Cmtlp,At Cmfad2 and At Cmfad3 mutant(T3)plants under cryogenic freezing stress,physiological study,study of cold resistance mechanism of mutant based on transcriptome,and analysis of cold resistance mechanism of mutant based on target gene mapping.The physiological functions of FAD2,FAD3 and TLP genes in Chelidonium japonica were analyzed.The following results were obtained:1.Based on fluorescence quantitative PCR method,the expression characteristics of target genes under different temperature stresses were analyzed.Compared with wild-type Wt,At Cmtlp,At Cmfad2 and At Cmfad3 mutant plants were significantly expressed at various temperature points,especially at the freezing stage.The relative expression ratio was At Cmtlp> At Cmfad2> At Cmfad3.2.Physiological indices of wild-type Wt and At Cmtlp,At Cmfad2 and At Cmfad3 mutant plants under different temperature stress showed that relative electrical conductivity,malondialdehyde(MDA)and soluble protein(SP)contents of wild-type mutant plants increased gradually under low temperature freezing stress,while decreased under deacclimation stress.Both NBT and DAB staining increased the staining area and deepened the color in the freezing stage.In the deacclimation stage,the staining area decreases and the color becomes lighter.The contents of chlorophyll a,chlorophyll b and total chlorophyll decreased at low temperature and freezing stage,and returned to normal at deacclimation stage.The contents of carotenoid,soluble sugar(SS)and free proline(Pro)increased in cold acclimation stage,decreased in cold acclimation stage,and recovered to normal in deacclimation stage.The activities of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)were the highest in the freezing stage,while the activities of glutathione peroxidase(GPX),glutathione reductase(GR),polyphenol oxidase(PPO)and ascorbate enzyme(APX)were the highest in the cold acclimation stage,and decreased in the deacclimation stage.The contents of auxin(IAA)and abscisic acid(ABA)were the highest in freezing stage,and returned to normal in deacclimation stage.The contents of gibberellin(GA)decreased gradually with decreasing temperature,reached the lowest value in freezing stage,and increased in deacclimation stage.It can be seen that physiological hormones of Wt,At Cmtlp,At Cmfad2 and At Cmfad3 are significantly expressed at low temperature and freezing stage,and the physiological and hormonal response at low temperature and freezing stage is At Cmtlp,At Cmfads>Wt,At Cmtlp >At Cmfads.3.Transcriptome sequencing of Wt and At Cmtlp seedlings treated at 20℃,4℃ and-40℃ showed that,compared with Wt,the differentially expressed genes of At Cmtlp increased significantly at each temperature point,and the most differentially expressed genes were found at the freezing stage,with the highest expression rate.GO enrichment analysis showed that differential genes were mainly enriched in photosynthesis,response to cold,polysaccharide metabolism,etc.KEGG enrichment pathway showed that differential genes were mainly enriched in plant hormone signal transduction,circadian rhythm,starch and sucrose metabolism,etc.Screening of differentially expressed genes at the cryogenic freezing stage showed that significantly up-regulated genes were cell membrane,plant hormones,coldresistance-related enzymes and proteins,sugar metabolism and enzyme-related pathways,which were consistent with the physiological and hormone indexes measured above.4.The insertion sites of target genes in At Cmtlp,At Cmfad2 and At Cmfad3 mutants were detected by chromosome step method.The results showed that At Cmtlp was inserted into three sites,which were significantly different from TLP in situ.The insertion sites were FKBP-like protein,TBF-related factor 13 and myosin binding protein,respectively.Upstream regulatory genes were GT2,F-box and DUF1685protein;The downstream regulatory genes were RING/U-box,α isotype inducible protein 6 and MYB60.At Cmfad2 was inserted into one site,which was significantly different from FAD2 in situ.The insertion site was a histidine kinase member.The upstream regulatory gene was RING/U-box;The downstream regulatory gene was SCP1.At Cmfad3 located two loci,which were significantly different from FAD3 in situ.The loci were CDF5 and ATMFP2,respectively.The upstream regulatory gene was MYB domain protein,DIN3 protein;Downstream regulatory genes are homologous domain proteins rich in amino acid family proteins.The above genes are widely involved in and regulate cold resistance,physiological hormone response,morphological development and life cycle extension of plants.In conclusion,At Cmtlp,At Cmfad2 and At Cmfad3 plants have strong cold resistance,significant morphological differences and life cycle characteristics,which are closely related to the insertion position of Arabidopsis thaliana and the regulation of upstream and downstream genes.