Effects Of PhTCP14 And PhTCP15 On The Regeneration Of Adventitious Buds From Petunia Hybrida Explants

The names of TCP family genes are derived from the acronyms of maize,snapdragon,and rice,the TB1 gene extracted from maize,the CYC gene isolated from snapdragon,and the PCF1 and PCF2 genes of rice.As a domesticated gene,one of its functions is to inhibit the meristem of axillary buds and promote tissue growth;the CYC gene isolated from snapdragon regulates the symmetrical development of flowers and leaves;and the PCF1 and PCF2 of rice are involved in cell cycle regulation and DNA replication.As a plant-specific transcription factor,the secondary structure of TCP gene protein has an atypical b HLH conserved region called TCP domain.Due to the differences in TCP domains,the TCP gene family can be divided into Class I and Class II genes.The difference between these two types of genes is that,compared with Class Ⅰ gene and Class Ⅱ gene,the basic domain of Class Ⅰ gene has a four-base deletion;Class Ⅰ gene and Class Ⅱ gene antagonize each other in terms of their functional expression.Class Ⅰ gene Promote plant growth,Class Ⅱ genes inhibit plant growth.In TCP related reports,the TCP gene family mainly regulates plant morphology,flower and leaf development,and cell proliferation.TCP14 and PhTCP belong to Class I of the TCP gene family.The functional expression of TCP14 and TCP15 in plants are highly similar.In previous studies,it was found that the pair of genes mainly regulates the cell proliferation of the flower and leaf of the plant,thereby affecting the growth of the plant.status.In the process of adventitious bud regeneration of petunia leaf explants,cytokinin explants induce callus formation and adventitious bud regeneration;TCP14 and TCP15 directly regulate the upstream genes of cytokinin.Express or indirectly promote the increase of cytokinin signal to affect the regeneration of adventitious buds of petunia leaf explants.In this paper,PhTCP14 and PhTCP15 genes were overexpressed and knocked out,and PhTCP14 and PhTCP15 overexpression plants,PhTCP15 single gene knockout plants and PhTCP14,PhTCP15 double gene knockout plants were obtained.Through overexpression and knockout of PhTCP14 and PhTCP15 genes,the effect of the pair of genes on the regeneration of adventitious buds from petunia leaf explants was studied.The results obtained in the research of this paper are as follows:1.Construction of petunia PhTCP14 and PhTCP15 overexpression vectorsSearch for the cDNA coding frame sequences of the Petunia PhTCP14 and PhTCP15 transcription factors.The corresponding specific primers PhTCP14-F1,PhTCP14-R1,PhTCP15-F,PhTCP15-R were designed and synthesized,and the total DNA of Petunia MD genome was used as a PCR amplification template to obtain the DNA sequences of PhTCP14 and PhTCP15 genes.The overexpression vectors of PhTCP14 and PhTCP15 were constructed,and Agrobacterium-mediated leaf disc method was used to transform petunia to obtain overexpression plants.2.Quantitative analysis of PhTCP14 and PhTCP15 genes in Petunia hybridaThrough fluorescence quantitative PCR analysis,the relative expression of PhTCP14 and PhTCP15 genes in petunia leaf explants at the level of 0.5μmol/L 6-BA hormone during the regeneration of adventitious buds of petunia leaf explants.In the petunia K5610 plant,knocking out Class Ⅱ genes that inhibit cell proliferation,the relative expression levels of PhTCP14 and PhTCP15 genes have been significantly increased,proving that Class Ⅰ and Class Ⅱ genes are functionally antagonistic to each other.3.Construction of Petunia PhTCP14 and PhTCP15 double knockout and PhTCP15 single knockout vectorIn the CRISPR-GE genome editing tool,the primers PhTCP14-sgf,PhTCP14-sgr,PhTCP15-sgf,PhTCP15-sgr were designed;using the p YLCRISPR/Cas9P35s-B vector of Liu Yaoguang’s research group at South China Agricultural University,PhTCP14 and PhTCP15 double knockouts were constructed respectively Vector,and PhTCP15 single knockout vector.4.PhTCP15 gene is not significant for adventitious shoot regeneration from MD leaf explants of Petunia hybridaRegeneration experiments were performed on the PhTCP15-overexpressing petunia resistant plants.Among the 31 samples,7 samples were significantly different from the wild-type MD,and 5 of them were significantly improved.The results showed that PhTCP15 gene expression The regeneration of petunia leaf explants was not significant.