| After years of research,it is found that GF677 is a peach rootstock resistant to continuous cropping,which is of great significance to solve the problem of peach garden regeneration.This excellent trait can be better preserved only through asexual reproduction,and the prerequisite for asexual reproduction is the occurrence and growth of adventitious root,but GF677 is difficult to take root when asexual reproduction is carried out.Exploring the influencing factors of GF677 adventitious root occurrence is of great significance for realizing GF677 asexual reproduction and solving the problem of continuous cropping resistance in peach orchards.With the continuous discovery of miRNA functions,multiple miRNA families have been found to be involved in the occurrence of plant roots.In this study,GF677 rootstock was used as the test material,and Ppe-miR166 with obvious expression changes during the formation of GF677 adventitious root was screened by exploring the asexual reproduction system of GF677 and the expression changes of multiple miRNA families during the development of adventitious root.The transgenic experiment of Arabidopsis showed that Ppe-miR166 regulated the growth and development of adventitious root.The main research findings are as follows:1.Exploring a tissue culture system using the new shoots of peach resistant rootstock GF677 as explants,the optimal proliferation medium was NN69+0.6 mg/L 6-BA+0.1 mg/L IBA,with a proliferation coefficient of 3.55 and good growth of tissue culture seedlings;The optimal rooting culture medium is 1/2MS+1 mg/L IBA+0.1 mg/L NAA,with a rooting rate of up to 15% and lateral roots occurring.The transplant survival rate is high,but the rooting rate is low.2.The expression pattern of multiple miRNAs in the process of cutting adventitious root development was analyzed by RT-qPCR with the new slightly lignified cuttings of GF677 in autumn.Ppe-miR166 changed significantly.The 5’RACE showed that PpHB8 and PpHB15 were the target genes of Ppe-miR166,both belonging to the HD-Zip family.23 HD-Zip genes were identified from peach by whole genome analysis.Phylogenetic tree analysis showed that the HD-Zip family could be divided into four subfamilies(I-IV),and the 23 HD-Zip genes had a homeomorphic domain(HD)and a Leucine zipper domain(LZ).Among them,PpHB8 and PpHB15 are members of subfamily III and have a unique MEKHLA domain compared to other subfamily members.23 HD-Zip genes have different expression patterns in different tissues,and PpHB8 and PpHB15 are mainly expressed in stems and leaves.3.Exogenous application of IBA induced the formation of GF677 adventitious root.It was found that adventitious root not only differentiated from callus,but also formed directly,which was a mixed rooting type.In the process of adventitious root formation,exogenous application of IBA can induce the rapid synthesis and accumulation of endogenous auxin,and the content of auxin at 6 h of rooting induction is 11 times that at 0 h.While auxin can inhibit the expression of Ppe-miR166.When treated with auxin transport inhibitor,Ppe-miR166 expression was up-regulated by 7.9 times,and PpHB8 and PpHB15 expression were significantly inhibited.4.Ppe-miR166 was expressed in Arabidopsis Thaliana.The formation of adventitious roots was inhibited in transgenic Arabidopsis thaliana strains overexpressing Ppe-miR166,the process of adventitious root formation slowed down and the number of adventitious root also decreased.The expression of At WOX4,At WOX11,At WOX12 and At LBD29,four positive regulators related to adventitious root formation,was down regulated after overexpression of Ppe-miR166,while the expression of At IAA20,a negative regulator,was up regulated.The phenotypes of Arabidopsis lines with Ppe-miR166 silencing and PpHB15 overexpression are exactly the opposite.In conclusion,this study found that Ppe-miR166 targets PpHB8 and PpHB15,and negatively regulates the formation of GF677 adventitious root by influencing the auxin signaling pathway. |
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