Effects Of TCP3 And TCP4 Gene Knockout On Growth And Development In Petunia

TCP gene is a special transcription factor of bHLH family.BHLH functional domain consist of 50-60 amino acids,which can be devided into two distinct parts:a basic region of 10 to 15 amino acids and a ?-helix-loop-?-helix area(HLH area)of 40 amino acids.TCP gene family is divided into two subfamilies,class I and class II.According to the differences of TCP structural domain,Class II is subdivided into two branches:ECE and CIN,and the branch of ECE contains three subclasses:CYCl,CYC2 and CYC3.Transcription factor PhTCP3 and PhTCP4 belong to CYC1 class in petunia.During plant development and morphogenesis processes,TCP gene family functions in fioral symmetry,regulating plant architecture,leaf morphogenesis and senescence,embryo growth,and circadian rhythm.The CYC1 gene mainly regulating plant branching and the flowering time of branches.At present,most research of CYC1 comes from BRCl of Arabidopsis.It is involved in regulation of the growth of axillary bud and branch development in Arabidopsis;the excessive expression of SIBRClb gene in tomato significantly decreased plant branching,while the gene silencing lead to increased branching;PsBRCl gene mutants in pea significantly increased branch.This article study the TCP3 and TCP4 gene function in petunias through observing the phenotype of PhTCP3 and PhTCP4 gene over-expression or knock out plant(including collateral branch number,flowering time,cell size)observation analysis to study the petunias TCP3 and TCP4 gene function.Following achievements have been obtained:1.The phenotypic changes of PhTCP3 overexpression and CREST silent plant.PhTCP3 vector was used to transfect 431 slices of petunias semal explant,only four transgenic lines were obtained,fluorescence quantitative molecular assay showed that none of these strain have PhTCP3 excess expression.The analysis of two strains showed that the number of branches in the main stem was fewer than that of the wild ones,the flowering time was delayed,and the leaf area was smaller.Excessive expression of PhTCP3:SRDX lead to delayed flowering time and smaller leaf.The expression of genes that are related to cell division and flowering were reduced.It is speculated that PhTCP3 gene has the function of controlling branches.2.Phenotypic changes of PhTCP4 overexpression and CREST silent plant.The PhTCP4 gene expression in petunias T1 generation was detected by fluorescence quantitative PCR reaction.High and medium expression ones were choosed for outdoor phenotypic observationchoose express high quantity,express the amount of medium for phenotypic observation.We found that excessive expression of PhTCP4:SRDX lead to delayed flowering time.It is speculated that PhTCP4 gene may have the function of controlling branches.3.The knockout of PhTCP3 and PhTCP4 gene and its influence on phenotype.The target gene PhTCP3 and PhTCP4 were used to construct the plant knockout vector.Through Basta screening and PCR detection,the genetic transformation system with base mutation and large fragment deletion was obtained.Then we got the seeds and the T1 generation of transgenic plants for phenotype observation:We found that PhTCP3 knockout petunias showed increased main stem axillary bud,smaller leaf area and delayed flowering time compared with wild type.We speculate that PhTCP3 genes have the function of control branch,flower and leaf size as well as PhTCP4.