| Plant regeneration is an important way for plants to cope with damage,which is closely related to plant growth and development.Plant tissue culture and adventitious bud induction are key to the construction of transgenic plants.Through research on model plants such as Arabidopsis,the key processes of plant regeneration and the functions of some key genes controlling plant regeneration have been revealed.Mulberry trees are widely planted and widely used,with good application prospects in medicine,health care,environmental protection,livestock and poultry feed,etc.Mulberry tissue culture is an important means of mulberry breeding and gene function research.Currently,mulberry tissue culture technology is relatively complete,but research on regeneration related genes in mulberry trees is scarce,and there is a lack of understanding of mulberry regeneration mechanism.Cotyledons from Morus alba.Var Fengchi were used as materials to investigate the effects of hormone types and concentrations,culture conditions,and explant status on the induction rate of adventitious buds in mulberry cotyledons.Combined with morphological and histological analysis,the key periods and locations that affect the formation of adventitious buds in mulberry cotyledons were identified.Transcriptome analysis was used to reveal the changes of gene expression during the development of adventitious buds in mulberry cotyledons,screen and analyze transcription factors and hormone signal regulation pathways closely related to adventitious bud induction,and explore the role of some genes in the formation of adventitious buds.Based on Transcriptome data and other databases,AP2/ERF transcription factor families in mulberry were screened and their potential functions were analyzed.Some AP2/ERF family genes were verified by fluorescent quantitative PCR,and the Homeotic gene Mafc-DREB-A6-2 of Arabidopsis WIND1 in mulberry was screened for subsequent analysis.The results of gene analysis and Functional verification through expression profile,promoter region analysis and tobacco overexpression showed that MafcDREB-A6-2 could promote adventitious bud regeneration of tobacco leaves independent of exogenous Cytokinin.The above results provide a theoretical basis for elucidating the molecular mechanism of mulberry tree regeneration in vitro,and provide reference and data support for mulberry tree regeneration research.The main results and conclusions are as follows:1.The induction of adventitious buds from mulberry cotyledons has strong tissue specificity.Only the base of young cotyledons can induce the production of adventitious buds,while explants from cotyledons in other states or positions can only induce the production of callus.This indicates that the key to the formation of adventitious buds from mulberry cotyledons is not cell proliferation but the acquisition of cell pluripotency.2.According to changes in morphology and gene expression,the development of adventitious buds in mulberry cotyledons can be divided into three stages.The first stage is called the PC stage,which lasts from the beginning of induction to 4 days after induction;The second stage is called the bud primordium formation stage(BF stage),which lasts from 4days after induction to 6 days after induction;The third stage is called the bud primordial development stage(BD stage),which lasts from 6 days after induction to 10 days after induction.In the second stage,a portion of the proliferating cells acquire pluripotency,which is the key to affecting the formation of adventitious buds in mulberry cotyledons.3.A total of 11116 differentially expressed genes were screened during the development of adventitious buds in mulberry cotyledons.The trend analysis of gene expression indicates that there is a significant stage variation in gene expression during the development of adventitious buds in mulberry cotyledons.According to different expression trends,all differentially expressed genes screened were classified into three categories: controlling cotyledon growth and development(Class I),controlling adventitious bud formation(Class II),and assisting in various physiological activities(Class III).The genes that control the formation of adventitious buds in mulberry cotyledons showed significant changes in expression on the 4th and 6th days of adventitious bud induction,mainly involved in RNA modification and RNA binding,controlling the formation of adventitious buds in mulberry cotyledons.During the development of adventitious buds in mulberry cotyledons,the expression of genes involved in cytokinin and auxin signal transduction pathway significantly increased.CRE1,HP4,ARR and other genes are responsible for the intracellular Cytokinin signal transport to maintain the level of cytokinin and promote cell differentiation.A large number of AP2/ERF family genes have been screened participating in the formation of adventitious buds in mulberry cotyledons.4.A total of 94 AP2/ERF family genes were screened in mulberry trees,and their distribution among different subfamilies is similar to that in other plants.Most genes begin to express or significantly increase in expression after D4,participating in the process of adventitious bud formation.Mafc AP2-8,Mafc ERF-B1-4,Mafc ERF-B2-2,and Mafc ERF-B6-1 play regulatory roles at D4,D3,D4,and D5,respectively;Mafc AP2-7 has the highest expression level at D0 and does not participate in adventitious bud formation;The expression levels of Mafc AP2-3 and Mafc RAV-1 were significantly increased in D6 and D3,respectively,but returned to similar levels as D0 in D10;Mafc DREB-A6-2 and Mafc ERF-B1-6 are the homeotic gene of Arabidopsis WIND1 and ESR1,respectively.The expression mode shows that Mafc DREB-A6-2 activates the expression of Mafc ERF-B1-6.5.Mafc DREB-A6-2 has a total length of 1071 bp,encodes 356 amino acids,contains one AP2 domain,and belongs to the DREB subfamily of AP2/ERF family species.A motif involved in stress signal response,a variety of Plant hormone signal response motifs and a large number of light signal response motifs were screened in the upstream 1000 bp region,but no motif responding to Cytokinin signals was screened,indicating that Cytokinin is not required for transcriptional activation of this gene.The expression level of Mafc DREB-A6-2is significantly higher in the early stages of leaf and fruit development than in other tissues and developmental stages,actively participating in plant growth and development mediated by light signals.6.In the process of mulberry cotyledon adventitious bud induction,Mafc DREB-A6-2was first induced by wound stimulation,and then participated in the transcription activation pathway of adventitious bud regeneration mediated by plant hormone and other signals,which is the key to connect the two pathways.Overexpression of Mafc DREB-A6-2 can promote the formation of adventitious buds in tobacco leaves without foreign plant hormone,indicating that the function of this gene is conservative in Solanaceae and Mulberry families.The sensitivity of the plant hormone signal pathway participated by Mafc DREB-A6-2 may be the key to the difference in the regeneration ability of tobacco and mulberry. |
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