Overexpressionof Arabidopsis MiR319α Affect The Morphological Development In Petunia Hybrid

Plant MicroRNAs (miRNAs) are endogenous 21-25 nt noncoding RNAs that can play important regulatory roles in plant developmental patterning, cell differentiation and stress response by targeting mRNAs for cleavage or translation repression. In Arobidopsis, the family miR319a contains three members:miR319a,miR319b and miR319c. Their sequences are very conservative, but the function are different.TCP genes are a class of transcription factor and contain a TCP domain in plant. The TCP genes are divided into two basic class:Class I (PCF) and Class II (TCP-C) according to the structure characteristics of the TCP domain.where Class II is divided into two subtypes CYC/TBl and CIN subclasses, the CIN subtype gene had been confirmed as a target of miR319. The TCP genes are mainly involved in controlling the branching and regulation of leaf development in plant.Petunia (Petunia hybrida Vilm.) is an important horticultural ornamental plant, it’s dicotyledon Petunia Solanaceae. Petunia has the advantages that they are simple cultivation, clear genetic background and susceptible to Agrobacterium-mediated transformation, which has become a model plant of transgenic research.Currently, Little is known about the regulation effect of miRNA on Petunia (Mitchell Diploid, MD), Our study material was Petunia (Mitchell Diploid, MD), we introduced this AtmiR319a gene into the genomes of Petunia hybrida, and more systemically analyzed the function of this gene expression on growth of plants from the morphological, molecular level and anatomical. its ectopic expression of effects on PhTCP5 and PhTCP6 gene of Petunia hybrid. The results are summarized as follows:1. Cloning of PhTCP5 and PhTCP6 in Petunia hybridSpecific primers design are based on s3 and c7-3 sequence of Petunia hybrid, "MD "genome DNA were used as template, using RACE and hi-TAIL cloned the full-length gene, and designated as PhTCP5 and PhTCP6 respectively, phylogenetic analysis showed that PhTCP5 and PhTCP6 belong to the CIN clade.2. The transcription expression analysis of PhTCP5 and PhTCP6 gene in Petunia hybridThe expression analysis of PhTCP5 and PhTCP6 gene in different tissues in Petunia by using real-time fluorescence quantitative PCR (qRT-PCR).the results showed that PhTCP5 gene was expressed mainly in leaves, besides expressing at high levels in axillary buds (<0.5cm), flower buds, roots, stems. However, while PhTCP6 gene was the highest expressed in axillary buds (<0.5cm), other tissues had a low expression level. We speculated that PhTCP5 and PhTCP6 genes would be involved in the regulation of leaf and axillary buds development.3. Overexpression of AtmiR319a in Petunia hybridThe target gene AtmiR319a was inserted into the expression vector PG523 to construct the plant expression vector (named:PG605). Total 24 AtmiR319a transgenic plants are obtained using the leaf disc method mediated by Agrobacterium tumefaciens in Petunia hybrid, all of this are positive plants to be confirmed by detection of DNA molecular. Transgenic plants have an important ornamental value due to the leaves and flower morphological changes.When the AtmiR319a gene was overexpression in Petunia hybrid, the transgenic plants exhibited the following abnormal features:(1) In leaf development, the transgenic plant leave showed a crinkled shape, un-flat and asymmetry, also showed shorted petiole, veins inconspicuous midrib, and venation change.(2)In flower development, the transgenic corolla was also observed a small, slightly curled and extreme cleft petals, tube color had became dark and tube diameter had showed more longer.(3)On the development of plant type, transgenic plants had a strong phenotype with a high degree of ranching and short internodes. another remarkable phenotypic trait was that lost apical dominance.4. The effects of miR319 on target gene PhTCP5 and PhTCP6Using bioinformatics software prediction results show that, the 1233:1253 base position of PhTCP5 is matching position in miR319, and the 1884:1904 base position of PhTCP6 is matching position in miR319; Using of RNA ligase mediated RACE (RLM-5’RACE 5’) show that PhTCP5 and PhTCP6 genes have the same the splice site is located10-11 base on the base of miR319. The expression analysis of PhTCP5 and PhTCP6 genes in different tissues by using of real time fluorescent quantitative PCR (qRT-PCR).We found that the expression level of PhTCP5 and PhTCP6 genes significantly down regulated in the transgenic plants. This indicates that AtmiR319a transgenic plants negative regulation of PhTCP5 and PhTCP6 gene.5. The mutation resarch of PhTCP5 and PhTCP6 genesAccording to the pairing sites in AmiR319 and the target gene PhTCP5 and PhTCP6, two pairs of specific primers were designed for mutation bases, The results show that PhTCP5 mutant 7 bases and PhTCP6 mutation of 6 bases, PhTCP5 and PhTCP6 connected to the pGMF500 vector, the difference is PhTCP6 is using own promoter, the mPhTCP5 mutation transgenic plants show that more axillary buds were compared with wild type, mPhTCP6 mutant transgenic plants with similar changes in mPhTCP5 transgenic plants, but the change is less than mPhTCP5 transgenic plants, other characters needs further observation and analysis.