Construction Of Over-expression Vectors And Transformation Of Three Fertility Related Genes From Petunia Hybrida ’Fantasy’

Petunia is perennial herb plant that belongs to genus Petunia hybrida of nightshade family, often cultivated as an annual plant. It is widely cultivated in world and known as the king of flower bed, with a number of advantages like large, showy, variously colored flowers and strong adaptability. It is a model plant in molecular biology research of floral development.Our group found that petunia’Fantasy’produced mutant flower del under short-day. Differentially expressed genes in normal and abortive petunia flower buds were isolated and sequenced by Suppression subtractive hybridization (SSH). On this basis, regeneration system for genetic transformation for Petunia hybrida vs ’Fantasy’ was established, some over-expression vectors of high expression genes in abortive flower buds were constructed in this study. Over-expression vectors and interference vectors were transformed into tobacco and petunia in order to verify gene function. Main results and conclusions of research are listed as follows:1. Three Over-expression vectors were constructed.35S::PhGRP、35S::Phmfl and 35S::PhTs2 were obtained containing pCAMBIA2300s vector and cDNA fragment in Petunia hybrida’Fantasy’. The three vectors were all transformed into agrobacterium EHA105.2.35S::PhGRP,35S::Phmfl,35S::iPhGRP and 35S::iPhmf1 were transformed into tobacco and petunia respectively.35S::PhTs2 was only transformed into tobacco. Transgenic plants of different genes were obtained.3. Phenotypic characters of PhGRP transgenic tobacco were as follows:(1) Compared with wild type plants, morphology characters of pollen grains from transgenic plants were changed. Most pollen grains were aberrant and elliptical ball-shaped, while pollen grains from pollen grains were full and ball-shaped. There were three long and narrow pollen apertures which stretched to the two poles of pollen. There wasn’t the phenomena that pollen apertures became wider and protuberant, existed in pollen grains from wild type plants. Surface ornamentation of pollen grains was both netty, no obvious difference between transgenic and wild type plants. (2) Compared with wild-type plants, pollen viability of PhGRP transgenic tobacco plants was decreased, staining rate and germination rate of pollen dropped by 30% respectively.4. Phenotypic characters of PhTs2 transgenic tobacco were as follows:(1) Some anthers were aberrant, brown,long and narrow. They couldn’t dehisce and died at last. (2) Except line 13 and 22, germination rate of pollen from transgenic lines was significantly lower than staining rate of pollen from wild-type plants, respectively. (3) Except line 22, pollen number of transgenic tobacco lines were significantly less than pollen number of wild-type plants, respectively; (4) The expression level of PhTs2 gene in young flower bud was correlated to phenotype. There was low expression level was verified in line 22, so its phenotype was not obvious. Meanwhile, there was high expression level was verified in other lines whose phenotype was obvious.But, the expression level was not regularly from high to low among strong and weak phenotypic plants. PhTs2 gene was expressed both in anther and leaf, not only in specific tissues like anther.