| Cell totipotency can be interpreted from protoplast culture and regeneration into complete plantlets,which is an important theoretical basis of tissue culture technology of fruit trees and other crops.Protoplast is a good system to explore the theory of life,which can improve crop traits through various cellular and genetic manipulation,and has great potential for production and application.However,a major obstacle to the widespread application of protoplast technology is recalcitrance of protoplasts of many fruit trees.To date,the molecular mechanism involved in regulating protoplast regeneration remains unclear.Therefore,in this study,the main materials in the process of protoplast isolation and culture of citrus sinensis were selected for transcriptome sequencing,and key regeneration genes were mined.Bioinformatics analysis of heat shock transcription factor family of citrus sinensis was carried out.Callus of overexpressed CsllsfA2 and silenced CsllsfA2 were obtained by agrobacterium tumefaciens-mediated transformation.The changes of protoplast yield,viability and other physiological markers were analyzed.Transcriptome sequencing was performed to identify the DEGs.The full-length and three fragments of CsllsfA2 promoter were cloned and GUS vector was constructed.The effects of H2O2 and DPI treatment on the expression level of CsHsfA2 in freshly isolated callus protoplasts were observed.Finally,the leaves and callus of citrus sinensis were treated with heat treatment,protoplasts were isolated,and the changes of yield and viability were measured.The main results are as follows:1.A large number of DEGs were detected after the callus and mesophyll protoplast isolation of citrus sinensis,but the number of DEGs decreased significantly after 6 days of culture.During the isolation and culture of citrus sinensis protoplasts,GO analysis showed that transcriptional regulation activity and DNA transcription factor activity of molecular processes were significantly enriched together,and carbon metabolism,amino acid biosynthesis and protein processing in endoplasmic reticulum in KEGG metabolic pathway were significantly enriched together.There were 564 differentially expressed transcription factors,including AP2,MYB,WRKY,GRAS,GRAM and HSF.A protein-protein interactional network analysis showed the transcription factor CsHsfA2（Cs4g18310）is one of the key regulatory factors of protoplast regeneration in citrus sinensis.2.There were 18 Hsf family members in the citrus sinensis genome,which distributed on 8 chromosomes and unknown chromosomes and were predicted to be located in the nucleus.A total of 1-2 exons,4-9 motifs;Phylogenetic analysis was divided into three subgroups.Promoter region contains a large number of cis-acting elements,such as hormone response elements,stress response elements,etc.CsHsfA2 expression was the highest in mesophyll protoplast and callus protoplast of citrus sinensis.3.The content of soluble protein,H2O2 and antioxidant enzyme activity in the callus overexpressing CsHsfA2 was significantly higher than that in wild-type callus,and the yield and activity of protoplasts isolated from the callus overexpressing CsHsfA2 were also significantly increased,while the silencing of CsHsfA2 showed the opposite effects.Overexpression or silencing of CsHsfA2 significantly changed the gene expression profile,and many redox-related genes were found after the analysis of co-expressed differential genes.H2O2 significantly up-regulated the expression levels of CsHsfA2.Heat shock treatment could increase the yield of citrus sinensis protoplasts.The results of this study provide a theoretical reference for further revealing the expression characteristics,biological functions,response to stress,especially the role in protoplast regeneration of heat shock transcription factors in citrus,and provide a theoretical and technical basis for germplasm innovation in citrus and other fruit trees involved in the use of protoplasts,also enrich the theory of plant cell totipotency. |
PDF Full Text Request