Cloning And Functional Analysis Of CgbHLH113 Gene From Chrysanthemum×grandiflora

Chysanthemum × Grandiflora is a kind of garden flower widely used in Northeast and North China.It has the characteristics of strong adaptability,early flowering,short plant,round crown,dense flowering and strong cold resistance.At present,most of the researches on open field chrysanthemum are focused on the physiological response to abiotic stress and cross breeding,while few researches on the molecular level breeding and resistance gene mining of open field chrysanthemum.BHLH transcription factors are a very important family of transcription factors in plants,which play a very important role in plants against the adverse environment.Under adverse environmental stress,bHLH transcription factors in plants can regulate their functions by regulating related resistance genes,which can enhance the tolerance of plants to adversity.In order to further explore the specific molecular regulation mechanism of Chrysanthemum indicum under salt stress and screen out the functional genes with stress resistance,the transcriptome data were screened,analyzed and cloned,and the successfully constructed plant expression vector was transferred into the model plant tobacco to explore the resistance function of transgenic tobacco under salt stress.The main results are as follows:1.A total of 25 bHLH transcription factor related sequences were found by transcriptome analysis and screening under salt stress.Among them,19 sequences with complete domain,11 sequences located in the nucleus and 8 sequences located outside the nucleus.The phylogenetic tree of bHLH gene in Chysanthemum×grandiflora and Arabidopsis thaliana showed that 12 sequences were clustered in 6 subfamilies of Arabidopsis thaliana.Analysis of bHLH differentially expressed genes under salt stress showed that 4 related sequences were up-regulated and 5 related sequences were down regulated.Based on the analysis of transcriptome data,c51361.graph_c0 was used as the target gene for subsequent experiments.named CgbHLH113,and the GenBank accession number is MW792182.2.qRT-PCR was used to explore the expression pattern of CgbHLH113 gene under Abiotic stress.The results showed that salt stress,drought stress and ABA stress could induce the expression of CgbHLH113 gene,and the expression of CgbHLHl13 gene under drought stress was the highest,followed by salt stress and ABA stress.The expression levels of genes in root and leaf tissues of different tissues and organs of Chysanthemum indicum seedlings were different,and the expression levels in root tissues were higher.3.The target fragment was amplified by gradient PCR using the cDNA of Chrysanthemum ×grandiflora.The 450bp CgbHLH113 gene was successfully cloned by RT-PCR,which encodes 149 amino acids with a molecular weight of 16429.80.It is an unstable hydrophilic protein.The protein peptide chain is located outside the membrane and belongs to non-transmembrane protein.It was predicted to be a non-secreted protein by the gene signal peptide,and the homology analysis showed that the homology between the gene and Artemisia annua was up to 99%.4.Through double enzyme digestion of PUC-T-CgbHLH113,the target fragment was ligated overnight with T4 ligase to construct plant overexpression vector pBI121-CgbHLH113-GFP.The recombinant plasmid pBI 121-CgbHLH 113-GFP was transferred into tobacco,and the positive tobacco plants were obtained through verification.Through salt stress treatment of tobacco seedlings,the results showed that the root length and germination rate of transgenic tobacco seedlings with CgbHLH113 gene were higher than those of wild-type plants.Tobacco plants were irrigated with 3000 mmol·L-1 NaCl solution,and the phenotypic changes of tobacco plants were observed at 0 d and 100 d.The results showed that with the extension of NaCl solution irrigation time,tobacco leaves appeared yellow wilting phenomenon,while the damage degree of transgenic tobacco plants with CgbHLH113 gene was less.The chlorophyll content of tobacco plants overexpressing CgbHLH113 gene was higher than that of non transgenic plants,which indicated that overexpression of CgbHLH113 gene could improve the photosynthetic capacity of tobacco plants.The MDA content of non transgenic plants was higher.The activities of SOD and pod in CK plants were higher than those in over expression of CgbHLH113.The plant overexpression vector pBI121-CgbHLH 113-GFP was constructed,and the recombinant plasmid was transferred into tobacco.The salt resistance of transgenic tobacco was analyzed.The results showed that the root length and germination rate of transgenic tobacco seedlings were higher than those of wild-type plants.Tobacco plants were irrigated with 300 mmol·L-1 NaCl solution,and the changes of phenotypic and physiological indexes of tobacco plants were observed at 0 d and 10 d.The results showed that with the prolongation of NaCl solution irrigation time,tobacco leaves appeared yellowing and wilting phenomenon,while the leaves of transgenic tobacco plants with CgbHLH113 gene were less damaged.The chlorophyll content of tobacco plants overexpressing CgbHLH113 gene was higher than that of non transgenic plants,which indicated that overexpression of CgbHLH113 gene could improve the photosynthetic capacity of tobacco plants.The MDA content of non transgenic plants was higher...