Application Of MIGS Technology In Petunia Hybrida

Gene silencing technology as one of the important tool to study gene function was widely used in plant molecular biology research and genetic improvement. Since the discovery of gene silencing phenomenon, a variety of gene silencing technology have established, mechanism and principle are also different, including cosuppression, antisense RNA, hpRNAi and VIGS, however, all these methods have their limitations. MIGS silencing technology as a new plant gene silencing technology, has the new breakthrough in terms of accuracy and efficiency of gene silencing. MIGS silencing technology using Arabidopsis thaliana miR173 mediated produces a series of tasiRNA, The number of tasiRNA is very great, and only complete complementary with target genes will give play to effect of silence, so MIGS silencing technology regulation has significant advantages in such aspects as the efficiency and accuracy.At present, the technology of the MIGS silencing on the petunias application has not yet been reported. The silencing experiment of phytoene desaturase gene (PDS) and chalcone synthase gene (CHS) of Petunia hybrida to analyze the effect of technology on Petunia hybrid. With green callus and flower color changes as a tag, phenotypic changes are easy to observe, and can rapid evaluation of the effects of gene silencing carrier, save time, improve work efficiency, and can lay the foundation for using MIGS silencing technology research the function of Petunia hybrida related genes. Petunia hybrida is one of the most important pot and bedding plants, we can use the method of gene engineering transform traits of Petunia hybrida, providing theoretical and practical guidance for the improvement of Petunia hybrida varieties.therefore, the study has important significance in the experiment.The results obtained are as follows:1 MIGS silencing technology research on Petunia hybridaWe make the pGREEN binary vector system convert to MIGS silent carrier, the MIGS silencing carriers including:miR173 genes, with miRl 73 targeted gene fragment, the purpose of the report gene. In the design of PCR primers to amplify the gene sequence, the upstream primer 5’ end additional plus miR173 complementary sequence, plus additional amplification of gene fragments with miR173 gumming points, So that miR173 can be complementary with objective gene sequence and function in transgenic plant.2 TA cloning in the application of plant expression vector to constructIn the construction of expression vector pMIGS-T, the expression of frame ends by adding Xcml enzyme site. When the construction of plant expression vector, we directly use Xcml single enzyme digestion the pMIGS-T vector,and then the PCR amplified products can be linked with pMIGS-T vector, completed the construction of plant expression vector. Consider the sequence of connected, only need to select the connection can be positive, greatly shorten the build process, save manpower and material resources. The T vector applied to the process of building a expression vector by TA cloning process, rapid, Directly to the PCR product of target gene was inserted into the expression vector, make the vector construction more convenient, Truly achieve a step of constructing vector.3 cloning of PhPDS gene from PetuniaAccording to the analysis of EST sequence of PDS gene from Petunia, then designed primers. By RACE cDNA cloning technology Get the cDNA full length sequence of PDS gene from Petunia, named PhPDS (GenBank accession number: KP677483). The length of 1749 bp, the gene encoding 583 amino acids. Cloning of PDS gene in Petunia can be applied to the validation of MIGS silencing in Petunia, provide a directly and rapidly endogenous test report gene, and provides a basis for the study of the evolution of PDS gene of Petunia hybrida. Detection of PhPDS gene of Petunia hybrida by semi quantitative RT-PCR reaction expression in different tissue of petunia MD, PhPDS gene is specifically expressed in tissues with high, expressed in 6 leaf stage of stem and leaf quantity highest, expressed in the root volume is low, in the mature period of flower, PhPDS gene is also have a small amount of expression, the prediction of Petunia hybrida PhPDS gene may be involved in the regulation of plant growth and development.4 the construction of plant expression vectorSelection of Petunia hybrida phytoene desaturase gene (PDS) and chalcone synthase gene (CHS) as the target gene, using miR173 induced gene silencing (MIGS) technology system change of Petunia hybrida color character, make the phenotype change of transgenic plants are more likely to be observed.the purpose gene sequence was inserted into the expression vector pMIGS-T, constructing plant MIGS silencing vector. The recombination plasmid mediated respectively into the Petunia MD strains and carpet strains by Agrobacterium, using a series of resistance screening and DNA level detection, and then selecting transgenic petunia hybrida plants. The transgenic Petunia hybrida were phenotypically observed with PDS gene silencing vector inoculated Petunia after about 2 weeks, began to appear white callus; Transformation of Petunia silencing vector containing CHS gene, transgenic plants obtained appear faded petals, from purple flowers into white flowers, thus proving that can be applied in Petunia MIGS silencing system.5 MIGS technology of petunias silence effectExtract RNA of transgenic plants that phenotypic changes obviously, detection of expression level after transcription, the results showed that CHS and PDS gene respectively in transgenic plants, the expression level was decreased significantly; Determination of cleavage sites of CHS, PDS gene by RACE Technology, the results revealed multiple sites of CHS gene was cut off, and This result is consistent with the miR173 can mediate produce a series of play the silencing effect of tasiRNA; We performed transcriptome sequencing, and then analyze MIGS technology efficiency of silencing on the petunias.