Preliminary Function Analysis Of PMADS3and Its Interaction Candidate Genes In Petunia Hybrida

Petunia (Petunia hybrida), a kind of herbaceous plants in the Solanaceae family, is widely used in gardens. It is also a kind of model plants in studying the genetic mechanism of floral development. Since the1990s, researchers have carried out in-depth study of petunia floral organ development and proposed the ABCDE model on this basis. The PMADS3gene encodes a class C functional transcription factor in petunia. Its in situ expression is restricted in the third-and forth-whorl floral organs. It has the functions of determining the floral meristem and the floral organ at the same time. Excessive expression of PMADS3displayed a phenotype in which petals and sepals were transformed into anthers and carpels, while loss-of-function mutants exhibited abnormal flower morphology with an obvious stamen-petal on the third whorl. This shows that PMADS3has close relationship with the homeotic conversion of the second and third whorls. That is, the PMADS3gene is associated with double flower. It is very useful to study PMADS3and its interaction genes, which can provide guidance for the breeding of double flowers. And the character of petaloid stamens can also apply to create male sterile line.To in-depth explore the regulation path of PMADS3in floral organ development and find the interacting regulatory factors, we transformed the RN Ai vector of full-length of PMADS3and observed phenotype of transgenic plants. Further study of the expression of interacting factors in transgenic plants is adopted real-time quantitative PCR technique. Then we selected five gene fragments from the genes interacted with PMADS3screened by Yeast Two Hybrid System before. Then constructed RNAi vectors using gateway technique, and transformed petunia by Agrobacterium-mediated transformation method. We obtained transgenic plants and analyzed the morphological characteristics at last. The major contents and results are as follows:1. Introduced the RNAi vector of full-length of PMADS3into petunia and analyzed the phenotype of transgenic plants. We transformed the RNAi vector of PMADS3into petunia by Agrobacterium-mediated transformation method and then used PCR technique to detect the transgenic plants.67percent of PMADS3positive plants showed petaloid stamens on the third whorl to varying degrees. The ovary became bigger but shrinkinger than the control and the style became shorter than it. The stigma of transgenic plants closed and all of the transgenic plants did not have seeds. 2. The real-time quantitive PCR analysis of relevant genes of transgenic plants. Taking the GAPDH gene as internal reference, we detected the expression level of relevant genes by quantitive real-time PCR. The genes studied are differential expressed genes in petal flower and double flower screened by colleagues before. The results showed several genes have significant difference of the expression level in PMADS transgenic plants and non-transgenic plants. They are FBP6, FBP7, PMADS3, PMADS4, PMADS9and PMADS15. Among all of them, the expression level of FBP6, FBP7, PMADS3, PMADS4and PMADS9declined while the expression level of PMADS15is raised. Remarkably, the expression level of PMADS3in transgenic plants is only0.09, which showed highly significant difference.3. Construct five RNA interference expression vectors of gene fragments interacting with PMADS3by gateway. First, we extracted the total RNA of petunia, get the cDNA by RT-PCR, and then amplified the five target genes containing attB joints by PCR and recombined the attB-PCR products and Phellsgate2vectors by the BP reaction. At last we obtained five RNA interference expression vectors. They are Phellsgate2-AD23, Phellsgate2-AD30, Phellsgate2-AD35, Phellsgate2-AD63and Phellsgate2-AD66.4. Introduction of AD23, AD30, AD35, AD63, AD66into petunia. The five genes were introduced into petunia via Agrobacterium-mediated transformation method. Then use PCR technique to further confirm the transgenic plants. The number of positive transgenic plants is25,20,23,23and27respectively. For the TO generation of transforments integrated AD63, the leaves became curly, shrinking and thick. The whole plant became rosette and without apical meristem in vegetative period. Partial stamens transformed into petals and with bigger but shrinking ovary when flowering. The transgenic plants have less active seeds than control. While the phenotype of petaloid stamen became outstanding, that is, the top of anthers have petaloid structure. The transgenic plants of the other four genes did not show any difference compared with control.