Transformation Of Callus With Maize Phytochrome Gene

The growth and development process of plants is greatly affected by environment, light is one of the most important environmental factor which controls the growth and development of plants. On one hand, light is the most fundamental energy source for plants growing and developing, on the other hand, as a light signal regulates the growth and development process of plants, including seed germination, seedling to yellow, phototropism, and gravitropism, shade-avoidance, stem elongation, leaf opening, stomatal opening, chloroplast movement and the flowering period,etc. From this, we know the light signaling pathway controls the whole growth and development process of plants. If we transduce the relevant critical genes of light signal pathway into plants, it will inevitably lead to changes in plants growth and development process, especially in traits of yield and improving resistance for crop breeding.Plants feel the light changes of environment through the light receptor. In plants, light receptors including four kinds:Phytochrome, UV-B receptor, and blue/UV-A receptors, the light pigment. Of the four light receptors, the phytochrome is the initial light receptor. It controls the whole process of plant growth and development. In recent years, studies have shown that PhyA promotes flowering and PhyB inhibits of flowering start. PhyA over-expression of Arabidopsis thaliana may have some morphological changes of plants. For example, inhibition of hypocotyl elongation, seedlings growing shorter, leaf turning color, reducing apical dominance and leaf aged laler, etc. The transgenic plants of PhyA overexpression all flower earlier than the wild-type in both short day (SD) and long photoperiod (LD). PhyB overexpression may have seedlings dwarfed. PhyB mutation of Arabidopsis thaliana also maded hypocotyl abnormal elongation.Maize is an important food and feed crop in our country, although the maximum yield reached 20142.75 kg/hm2, the average is very low, so the yield of maize can not meet the demand of social development. How to improve the yield, especially under the high density planting is still one of the main objectives of corn breeding. Using traditional breeding methods to improve maize production is very difficult now. because the high-yielding germplasm resources which can be made use of is limited. By way of transgenic methods, moving the maize phytochrome gene into maize, and then making maize phytochrome overexpression in vivo. This may change the light signaling pathways of maize, reduce shade avoidance response and improve their production under high density planting.In this study, I respectively connected the maize phytochrome gene PhyA1, PhyA2, PhyB1 to the plant expression vector pCUbi1390. Choosed 18-599 red callus as receptor, respectively transformed maize phytochrome genes PhyA1, PhyA2, PhyB1 into it by agrobacterium-mediated method, for the purpose of obtaining transgenic positive plants and exploring the use of plant light signaling pathways to improve crop based on identification of materials. The results are as follows,1)Maize phytochrome gene PhyA1, PhyA2, PhyB1 respectively connected into plant expression vector pCUbi1390 successfully. Cloned maize phytochrome gene PhyA1, PhyA2, PhyB1 respectively as a template, added BamHI and SpeI restriction sites full length primers PCR amplification. recycled target band and connected the maize phytochrome gene PhyA1, PhyA2, PhyB1 target band to the plant expression vector pCUbi1390, then transformed them into E. coli, selecting positive clones on medium with kanamycin. Last, use BamHI and Spel restriction enzyme digesting. The result showed maize phytochrome gene PhyA1, PhyA2, PhyB1 respectively connected into plant expression vector pCUbi 1390 successfully.2)Through PCRamplification, it preliminarily confirmed hygromycin phosphor-transferase marker gene and CaMV35S promoter has been transferred to receptor maize inbred 18-599red, but whether maize phytochrome genes PhyA1 or PhyA2 or PhyB1 into maize inbred 18-599 red genome is yet not to be confirmed. I respectively transformed maize phytochrome gene PhyA1, PhyA2, PhyB1 into embryogenic callus of inbred lines 18-599 red through agrobacterium tumefaciens. After three selected rounds of hygromycin 5,8,12 mg/L, the number of resistant seedlings were respectively 107,123,48. Designing specific hygromycin phosphotransferase gene primers, using genome DNA of transformed plants as a template, PCR amplification showed that the positive plants of transformated maize phytochrome genes PhyA1, PhyA2, PhyB1 were respectively 5,5,19. Designing CaMV35S specific gene primers, using genome DNA of transformed plants as a template, PCR amplification showed that the positive plants of transformated maize phytochrome genes PhyA1, PhyA2, PhyB1 were respectively2,2,0. The four regenerated plant which CaMV35S promoter tested positive. However, hygromycin phosphotransferase gene test was also positive.