| Chrysanthemum is one of the most important ornamental flowers in the world.The stress resistance of the plant and the hardness of the branches are the important factors affecting its quality.Lignin is one of the important components of plant secondary parietal cells.Its content and the expression of related enzymes are important factors affecting plant stress resistance,stem hardness and plant morphology.NAC transcription factors are plant specific transcription factors.Its members,such as NST,SND and VND,all act in the upstream of the regulatory network of plant secondary wall synthesis and play an important role in lignin synthesis.Regulation of NST,SND and VND can simultaneously affect multiple lateral metabolic pathways and produce more functional and phenotypic differences,which is of great significance for plant breeding.However,the role of NAC transcription factors related to lignin regulation in chrysanthemum is rarely reported.Chrysanthemum lavandulifolium is one of the important parents of modern chrysanthemum and also the model plant of Compositae.However,the research on lignin and related genes and the gene editing system of C.lavandulifolium have not been reported.Therefore,in this study,the NAC transcription factors related to the regulation of C.lavandulifolium lignin were studied to clarify the function of the NAC transcription factors.and the gene editing was used to control the breeding of C.lavandulifolium lignin.In order to provide a theoretical basis for the role of NAC transcription factor family in chrysanthemum,and provide a new way for the growth and development of modern chrysanthemum and the molecular breeding of stress resistance regulation.The main results are as follows:(1)A total of 153 putative NAC transcription factors were identified from the genome database of the C.nankingense,and a rootless phylogenetic tree was constructed,which was divided into 2 groups and 19 subfamilys,17 of which were known and 2 of which were unknown.Group 1 contained 111 members from 15 subfamilys and group 2 contained 42 members from 5 subfamilys.Based on the analysis of the functions of each subfamily,it was found that the members of Os NAC7 subfamily included 12 members of SND/NST/VND/BRN proteins,and all of its members had the function of regulating lignin accumulation in known studies.Therefore,the later studies mainly focused on the lignin-related Os NAC7 subfamily.(2)Twelve members of Os NAC7 subfamily in C.lavandulifolium were analyzed under osmotic stress induced by 20% PEG6000,salt stress induced by 200mmol/L Na Cl and different tissues during different growth periods.It was found that the 12 members were involved in the regulation of stem growth.Only one gene played a role in the aging process of 60-day-old stem,while the other 11 members played a major role in the rapid growth process of 44-day-old stem.5 members regulate root growth and development,5 members were involved in the regulation of leaf growth,and the periods with higher expression levels in leaves were all in the aging period of 60 days of growth.In addition,12 genes were involved in the regulation of drought and salt stress.Among them,5 genes might play a key role in the regulation of osmotic stress in C.lavandulifolium,and 8 genes might be important genes in response to salt stress.(3)Four members of Os NAC7 subfamily of C.lavandulifolium were cloned and their functions were validated in model plants.It was found that Cl SND1 gene could increase the secondary cell wall thickness and secondary xylem thickness of transgenic tobacco stem,and improve the tolerance of tobacco seedlings to salt or low temperature by regulating root growth.The transgenic tobacco also showed an early flowering phenotype.Cl SND1.1,Cl VND1,Cl BRN1 genes all promoted lateral root growth of Arabidopsis seedlings under stress conditions,improved the tolerance of Arabidopsis seedlings to salt or low temperature,and resulted in reduced growth and earlier flowering.The lignin switch transcription factor SND1 was cloned and evolutionary analysis was carried out in Ajania purpurea,Allardia glabra,C.lavandulifolium,Chrysanthemum mongolicum,Chrysanthemum arcticum,Hippolytia tomentosa,Hippolytia kaschgarica,Chrysanthemum bizarre.It was found that the differentiation time of Mikania micrantha,Helianthus annuus,Lactuca sativa and Cynara cardunculus was earlier than that of A.glabra,Tanacetum cinerariifolium,A.purpurea,C.bizarre and Artemisia annua and the differentiation time of H.tomentosa,H.kaschgarica,C.lavandulifolium,C.mongolicum and C.arcticum,was the latest.(4)A total of 43 C.lavandulifolium seedlings were cultured on the leaves of the same culture conditions,and it was found that there were differences in the response of the leaves of different Chamomile seedlings to hormones,with regeneration coefficients ranging from 0 to 3.8.Through the regeneration experiment,the efficient regeneration single plant was screened out.The direct regeneration culture system was MS+1.0mg/L 6-BA+0.5mg/L NAA+30g/L sucrose +6g/L Agar.The optimal concentration of hygromycin resistance for direct regeneration was 8mg/L,and the rooting medium was1/2MS+30g/L sucrose +6g/L AGAR +4mg/L hygromycin.(5)The expression efficiency of four actin gene promoters in C.lavandulifolium and the Arabidopsis At UBI promoter was verified,and the modified gene editing vector was used for single target editing and double target editing experiments of Cl SND1 gene.Five transgenic chamomile strains have been obtained,and their DNA was positive,but no vector sequence was detected in RNA,and there was no mutation at the target site.The cause of this result may be related to gene selection,or may be related to the concentration degree of the chromosome of C.lavandulifolium,which needs further study to confirm.In this study,the NAC family of C.nankingense was analyzed,and the expression pattern of ligninrelated Os NAC7 subfamily and the model plant function of its four members were studied in C.lavandulifolium.At the same time,an efficient regeneration system was established,and 4 actin promoters of C.lavandulifolium were used to modify the gene editing vector,and the verified SND1 gene gene editing test was conducted.The aim is to verify the function of SND1 gene in C.lavandulifolium,and to regulate the breeding of C.lavandulifolium lignin,so as to provide theoretical and technical basis for the research of C.lavandulifolium lignin and gene editing. |
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