Function Analysis Of BpTCP3 Gene In Betula Platyphylla

TCP is a plant-specific transcription factor with helix-ring-helix bHLH domain.It participates in cell proliferation and differentiation,organ morphogenesis,photomorphogenesis and senescence by mediating hormone biosynthesis pathway.In this study,the expression characteristics of BpTCP3 gene were explored in different tissues,hormones,abiotic stress and nitrogen stress response,phenotypic observations and nitrogen utilizationof BpTCP3 transgenic lines were carried out,the research results are as follows:(1)The expression patterns of BpTCP3 and BpTCP12 genes in different tissues exogenous phytohormones,abiotic stress and nitrogen stress were analyzed using Real-time quantitative PCR.The results showed that BpTCP3 and BpTCP12 genes were both highly expressed in axillary buds,shoots and leaves,and shared similar expression patterns during axillary bud and leaf development.And these two genes are involved in multiple hormones and abiotic stress responses.(2)The plant expression vector of proBpTCP3::GUS was constructed,genetically transformed into Betula platyphylla genome,and the tissue expression specificity and the response of plant hormone treatment(BR,JA,SA,ABA,IAA,6-BA,Me-JA),abiotic stress treatment(CdCl2,NaCl,NaHCO3,PEG)and nitrate-ammonium nitrogen,nitrate nitrogen,ammonium nitrogen and nitrogen starvation analyzed.The results showed that BpTCP3 gene was expressed in the top young leaves,the edge of mature leaves,epidermal hair and roots of Betula platyphylla,especially in roots.It is further demonstrated that BpTCP3 gene responds to most hormone and abiotic stresses and nitrogen stresses.(3)The BpTCP3 gene was cloned using RT-PCR mothed,and the overexpression vector of BpTCP3 gene and the expression vector containing a conserved EAR motif at the C-terminus were constructed by GATEWAY technology.The Agrobacterium-mediated transformation of Betula platyphylla was used to genetically transform Betula platyphylla,and finally 5 overexpression transgenic lines and 8 suppressing expression transgenic lines were successfully obtained.The seedling height,ground diameter,pitch,leaf length to width ratio and root length of the transgenic BpTCP3 gene and the non-trangenic lines were measured.The results showed that the the seedling height,the pitch of the leaves,the leaf length to width ratio and the root length of overexpression trangenic lineswere lower than those of non-trangenic line.(4)Root growth and biomass accumulation of transgenic lines under N stress and the expression of key genes in nitrate and ammonium nitrogen metabolism pathways were analyzed.The following results were obtained:the length of roots,the number of lateral roots,and the aboveground part and total bioaccumulation of transgenic BpTCP3 gene inhibiting expression lower than non-trangenic line under nitrate-ammonium nitrogen,nitrate nitrogen,ammonium nitrogen and nitrogen starvation.The number of lateral roots of transgenic BpTCP3 gene overexpressing was significantly higher than non-trangenic line under except ammonium nitrogen treatment conditions.The bioaccumulation of overexpression trangenic lineswere was significantly lower than non-trangenic line under ammonium nitrogen and nitrogen starvation,and other significant changes were not observed.Compared with the control,the expression levels of four key nitrogen metabolism genes NiAl,NiR,NRT3 and NRT3.1 under nitrated N and N starvation treatments were lower than those of transgenic BpTCP3 overexpression,but higher than that of BpTCP3.At the same time,the expression levels of Gln1 and Glu,the key genes of ammonium nitrogen metabolism,were increased in BpTCP3 overexpressing and inhibiting expression plants,but the expression level in overexpressing plants was higher than that in inhibiting expressed plants.(5)The interaction between 11 TCP proteins was verified by yeast two-hybrid method.It was demonstrated that TCP proteins are biased to form heterodimers.