Cloning And Functional Analysis Of CibHLH1 Gene In Chrysanthemum Indicum Var. Aromaticum

Chrysanthemum indicum var.aromaticum grows in the area about 2600 meters above sea level in Shennongjia,Hubei.The whole plant has aroma,contains various compounds and has antibacterial effect.It is a good raw material for extraction of essential oils and pharmaceutical processing.It is Chinese unique precious flower varieties.Its living environment has sufficient and strong illumination,and it is affected by low temperature and high-intensity radiation.It is very significance to study the growth and photosynthesis of Chrysanthemum indicum var.aromaticum from the genetic level to improve the ornamental characteristics and to cultivate excellent varieties.The bHLH transcription factor plays an important role in the regulation of plant biological processes and stress responses,and it is involved in the regulation of flower morphogenesis,fruit formation and pigment synthesis.This study explored the role of bHLH transcription factor in the growth and development process of Chrysanthemum indicum var.aromaticum.The regulatory potential of the CibHLHl gene was studied by transgenically overexpressing the CibHLHl gene in model plant tobacco.In this study,we used the cDNA of reverse transcription from the RNA of Chrysanthemum indicum var.aromaticum’s leaves as the template and bHLH-F/bHLH-R as the specific primers to clone the bHLH transcription factor gene of Chrysanthemum indicum var.aromaticum by PCR and named it as CibHLH1 gene.Bioinformatics analysis of the CibHLHl gene showed that the ORF of the CibHLHl gene is 633bp,and encodes a total of 210 amino acids.It belongs to the HLH superfamily and regulates gene transcription,cell division,and cell cycle development.The process of chromatin and DNA recombination;through phylogenetic tree and amino acid sequence analysis,the homology and affinities of the CmbHLHl gene of chrysanthemum are higher.Finally,the expression level of CibHLHl gene in different organs was analyzed by fluorescence quantitative PCR method,and it is concluded that its expression level in roots is the lowest,under the treatment of 0.25%methyl jasmonate,the expression level of CibHLH1 gene was increased with the treatment time.The trend of high and then decreased showed that the CibHLHl gene is induced by methyl jasmonate.Based on the obtained CibHLH1 gene,a cloning vector was constructed with specific primers for CibHLH1-Kpn1/CibHLH1-SpeI,pBI 121-CiBHLHl plant expression vector was constructed using KpnI,SpeI and T4-DNA ligase,The pBI121-CibHLH1-GFP plant expression vector was located in the nucleus by gun-casting onion epidermal cells,the CibHLH1 gene and pBI121-GFP empty vector were transferred into model tobacco plants using Agrobacterium-mediated method,and the resistance was screened by PCR.Successfully,it is obtained two positive strains transgenic CibHLHl tobacco positive lines B1,B2 and pBI121-GFP empty vector EV.Through the determination of the appearance and photosynthetic characteristics of transgenic CibHLHl tobacco lines B1,B2 and control lines WT and EV,the function of CibHLHl gene was initially explored.The results showed that compared with the control group WT and EV,the CibHLHl transgenic tobacco lines B1 and B2 turned yellow,and the new leaves of the terminal buds shrank and slender.The buds emerged as abortions and the petal color appeared concentrated.Phenomenon;chlorophyll a,chlorophyll b,total chlorophyll:and carotenoid content were all significantly reduced,with values of 32.5%,33.1%,33.5%,43.1%,and 33.1%,37.7%,34.2%,and 39.1%of WT and EV,respectively.The chlorophyll a and chlorophyll b ratios did not change substantially;the maximum net photosynthetic rate(Pmax),apparent quantum efficiency(AQY),light saturation point(LSP)and water use efficiency(WUE)all decreased significantly;transpiration rate(Tr),stomata Both Gs and LCP have increased significantly.