Expression Vector Construction Of TaCYP78A3/TaCYP78A5 And Wheat Transformation

Wheat is one of the major food crops in the world, whose yield and quality is directly related to food security. The main purpose of wheat breeding in China is to developed wheat varieties with high yield. With the development of biotechnology, wheat improvement by genetical modification will gradually become an important supplementary way in wheat breeding program.. Cytochrome P450(CYP) form one of the largest family of plant proteins(http://drnelson.uthsc.edu/cytochromeP450.html) and have been regarded as genes for crop improvement. In Arabidopsis and rice, several members of CYP78 A gene family, such as CYP78A5,CYP78A9 and CYP78A13, have been identified to control the process of seed development. TaCYP78A3 and TaCYP78A5 in wheat were cloned and their silencing leads to a decrease in wheat seed size according to our primary study. In order to investigate the effects of these two genes on wheat seed size, plant expression vector of TaCYP78A3 and TaCYP78A5 were developed, and immature or mature embryo callus of spring wheat cultivar Ningchun16 were used metarials to make wheat transfermation in this study. The main results were as follows:(1) Development of TaCYP78A3 expression vector and wheat transformationTwo expression vectors of TaCYP78A3, pTaCYP78A3::TaCYP78A3 and pINO::TaCYP78A3 driven by its own promoter(pTaCYP78A3) or ovule specific INNER NO OUTER promoter(pINO) were developed. Mature embryo callus of Ning chun16 were genetically transformed with pINO::TaCYP78A3 by biolistic particle. Through screening with PPT and differentiation, 6 regenerated plants were gained. 3 regenerated plants were identified by PCR analysis and only 1 was checked to be transgenic positive plant, it selfseeding and getting 11 seeds. The other 3 regenerated plants are still very small and not be identified yet.(2) Development of TaCYP78A5 expression vector and wheat transformationTwo expression vectors of TaCYP78A5, pTaCYP78A5::TaCYP78A5 and pINO::TaCYP78A5 driven by its own promoter(pTaCYP78A5) or ovule specific INNER NO OUTER promoter(pINO) were developed. Immature embryo callus of Ningchun16 were genetically transformed with pTaCYP78A5::TaCYP78A5 by biolistic particle. Through screening with PPT and differentiation, 6 regenerated plants were gained. 1 of 6 was checked to be transgenic positive plant by PCR analysis in the T0 plants with selfseeding and getting 5 seeds. In addition, immature embryo callus of Ningchun16 were genetically transformed with pINO::TaCYP78A5 by biolistic particle. Through screening with PPT and differentiation, 7 regenerated plants were gained. 4 regenerated plants were identified by PCR analysis and 2 was checked to be transgenic positive plant, it selfseeding and getting 17 seeds. The other 3 regenerated plants are still very small and not be identified yet.(3) optimazation of regeneration system of diploid wheat(T. Urartu)mature embryoThree different concentrations(1.0mg/L,2.0mg/Land3.0mg/L)of 2,4-D differentiation culture medium were set according to previous research. The result showed that the best callus statue and the highest rate of callus induced was in the condition of 2 mg/L 2,4-D. In addition, four differentiation mediums with different ratio of hormone were to compare, including A:NAA0.3mg/L,KT4mg/L; B:6-BA1mg/L,NAA0.3mg/L,KT2mg/L; C, 6-BA 1mg/L, NAA0.1mg/L, KT 4mg/L; D: 6-BA1mg/L,NAA0.3mg/L,KT4mg/L. The results showed that the mature embryo callus of T. Urartu showed higher regeneration rate in two differentiation media B and D, The regeneration rate was 36.36±3.4% and 35.38±2.8%, respectively.