Preliminary Study On EgMIXTA Associated With Petals Texture Of Eustoma Grandiflorum

Eustoma grandiflorum(Gentianaceae)is popular for its ornamental value due to velvet luster texture of its petals along conical inflorescences.This burnishing texture regulated by MIXTA family is forming the shape characteristics and optical properties of conical epidermal petal cells which change the biological features.It is conical cells that have effects on surface texture,color,wettability,temperature and volatility of petal surfaces to establish superiority to pollinators and to make favorable conditions for breeding.In this study,I detected copy number,expression pattern and function of MIXTA in plants by molecular biological methods and bio informatics approaches with a target on reforming the epidermal cell shape and texture of petals according to researches on velvet luster texture of Antirrhinum majus.The overall results are summarized as follows:1.The petals and leaves of Eustoma grandiflorum with or without velvet luster texture during various developmental period were investigated by scanning electron microscope.The materials from velvet texture petals presented cone-shape in different degree,especially from mature petals.However,in non-velvet texture petals and leaves the photos demonstrated just flat epidermal cells from surface.2.The whole sequence of EgMIXTA1 and EgMIXTA23 cloned by RACE technology was 1128 bp and 1134 bp,respectively,which showed the similarity to AmMIXTA-like2 from Antirrhinum majus by 86%.Their ORFs encoded 375 and 377 amino acid polypeptide chains.EgMIXTA protein,a MYB transcription factor,had two SANT domains of highly conserved R2R3-MYBs.It had high homology with AmMYBML3 at the rate of 82%.NJ showed EgMIXTA protein had the highest relativity to MYB gene from Petunia.3.To confirm the copy number of MIXTA in Eustoma grandiflorum genome,Southern Blotting was performed with DNA isolated from leaves.Three copies were acquired to provide evidence for MIXTA family pattern.Taking advantage of the Eustoma grandiflorum genome DNA to clone MIXTA,I acquired two sequences named as EgMIXTAD3 and EgMIXTAD5,and the latter was the same as EgMIXTA23.4.pGEX-EgMIXTA for prokaryotic expression was constructed to identify the feasibility of MIXTA expressing in lower organisms.Both protein sequences were located in 41 KDa.Making use of petals developing in four stages to perform Real-time PCR,there is high expression quantity of EgMIXTA in flower buds existing in the early stage of petal development.5.pH7WG2D-EgMIXTA and pK7GWIWG2D-EgMIXTA were constructed,respectively,as plant expression vector and interference vector harboring EgMIXTA gene for transformations of Eustoma grandiflorum by Agrobacterium-mediated method.Using electron microscope and polymerase chain reaction(PCR),I identified positive regeneration plants.