Effects Of Overexpression Of PhMIR156g And PhSPL13 On The Growth And Development Of Petunia Hybrida

Plant microRNAs(miRNAs)are a kind of endogenous noncoding small molecular RNAs with a length of 21-25 nucleotides(nt).They inhibit the function of target genes by binding to target gene mRNA.Research shows that miRNA target genes in plants mostly belong to transcription factors,which play an important role in the early differentiation and development of plant cells,the growth and development of the whole life course of plants,stress,signal transduction and metabolism.miR156 plays a regulatory role by combining SPL(SQUAMOSA PROMOTER BINDING PROTEIN-like)family genes.Among the 16 SPL genes in Arabidopsis,10 are mi R156 target genes,among which AtSPL9,13 and 15 play a key role in the growth and nutrition reproduction phase transition of growth period;AtSPL6 can increase the stress resistance of plants;AtSPL3,4 and 5 can promote the transformation of meristem.Among 21 PhSPL genes of Petunia hybrida,there are 14 potential target genes to predict miR156 and mi R157,which are mainly expressed in axillary buds and inflorescences.Among them,PhSPL9 a and PhSPL9 b have been proved to promote the phase transition from nutrition to reproduction.In this research,the effect of miR156 on the growth and development of Petunia hybrida was analyzed by over-expression of its own gene PhMIR156 g,and the regulatory effect on the growth and development of Petunia hybrida was analyzed by over-expression of its miR156 target gene PhSPL13.The main research results are as follows:1.Cloning of PhMIR156 g of Petunia hybridaBased on the predicted MIR156 gene sequence of Petunia hybrida genome sequencing results,blast the pre-miR156 sequence in Petunia hybrida EST database.After sequence analysis,we selected the sequence of stem ring structure region encoding miR156 in PhMIR156 g,designed PCR specific primers,used MD cDNA as template,cloned the sequence,and finally obtained 325 bp stem ring structure region sequence for this experiment.2.Construction and genetic transformation of PhMIR156 g over-expression vectorThe over-expression vector pG156 was constructed by connecting the stem ring structural region sequence of PhMIR156 g with the vector pG319 and placing it between the 35 S promoter and 35 S terminator.pG156 was transformed into Petunia hybrida MD by Agrobacterium mediated method,genomic DNA was extracted and identified by PCR,and 12 transgenic plants were obtained.3.Phenotypic observation of over-expression of PhMIR156gThe expression of PhMIR156 g in Petunia hybrida was identified by qRT-PCR,and the following phenotypic changes were found in Petunia hybrida:(1)Morphological development characteristics:the growth of young plants is prolonged,flowering is delayed,and branches are increased in clusters;the production of flower organs is intermittent,the leaves,flowers and fruits are smaller,and the number of seeds is multiplicative decrease;at the same time,The length difference between stamens and pistils in the same flower is reduced.(2)Regeneration ability:the ability to regenerate adventitious roots was enhanced in the explants of transgenic plants at young stage;The leaves of tissue cultured plantlets were placed on 0.5 ?mol / L-6BA medium,and it was found that the ability to regenerate adventitious buds of transgenic plants with high expression was also improved.(3)Drought resistance ability:after drought treatment,the number of wilted-leaves of over-expression plants was less,and the drought resistance ability was enhanced.4.Analysis of miR156 target gene expression in Petunia hybridaThe results of quantitative PCR showed that the expression of miR156 target genes PhSPL2,6,9 and 13 in PhMIR156 g over-expressed plants was significantly down regulated,indicating that miR156 may play a role by negatively regulating these target genes.5.Study on the over-expression of PhSPL13 mutant gene resistant to miR156We use PCR technology to make synonymous mutation on the site which is complementary to mi R156 in PhSPL13 sequence,and obtain the mPhSPL13 which can't be complementary to mi R156,and connect it to pGMF500 vector to obtain the mPhSPL13 over-expression vector pG531(35S:mPhSPL13);then we use InFusion technology to connect the EGFP gene to the 3 'end of pGMF531 vector to obtain the pG532(35S:mPhSPL13-EGFP).It was found that the transgenic plants with high expression of mPhSPL13 were short and slow growing.Other characters need further observation and analysis.