Obtention Of Transgenic Phytase Gene Maize And Progeny Test Analysis

Maize is an important economic crop,which is widely used in every field.It has a huge role in developing. One of the important content of corn breeding research now is improvement the maize quality by genetic engineering technology. Application of this technology not only shorten the breeding cycle, but also can avoid the incompatibility of distant hybridization between species, it is a very effective breeding way.Phytase is a kind of enzymea which has special space structure which can hydrolysis phytic acid and its salts into phosphoric acid(salt) and inositol the floorboard, it can effectively hydrolysis of phytic acid and phospholipid states organophosphorus, and release the inorganic phosphorus. Plants grow needed to a large number of elements, soil can meet the supply of plant required elements. As a necessary element of phosphorus in the soil, for the most part, exists in the form of state organic phosphorus, accounted for more than 50%, phytic acid salt plant cannot directly on the uptake, so a specific enzyme(phytase) must be used organophosphate hydrolysis for inorganic phosphorus plants can directly absorb and make use of it. Phytase can make phytic acid salt in the soil solution for inorganic phosphorus, inositol and some trace elements, used the genetic engineering means make the phytase gene import into maize, so this can not only improve the efficiency of maize by using soil phosphorus, but improve the growth of maize state, and can improve the output of maize, promote the development of China’s maize crop.This research is mainly to transformed the plant expression vector whitch contained root specific promoter and phytase gene(phyA) into maize inbred lines, and made it specific expression in the maize root, utilizing the roots secrete phytase, degradating organic phosphorus in the soil and promoting the ability to absorb phosphorusabsorption of corn in the soil, create a new germplasm of corn which can absorb and utilize phosphorus efficiently. The study transformed the plant expression vector pCAMBIA1301-ZmGLU1P-phyA-Nos-BADH into maize H99 inbred lines, identified and analyzed the T3 generation, T4 generation of maize that transformed the plant expression vector pCAMBIA1301-ZmGLU1P-phyA-Nos-bar. The results obtained are as follows:1. Using agrobacterium mediated transformed phyA gene in the pCAMBIA1301-ZmGLU1P-phyA-Nos-BADH into maize H99 inbred lines. By determining the NaCl selective pressure on callus, access transformation of maize plants. Total DNA extraction from leaves of transgenic plants, and used it as a template, use the specific primers for phyA gene sequence and detected T0 generation plant by PCR. The results had 7 T0 generation were positive plants, and harvest the selfing T1 generation seeds;2.Planted the phyA gene and herbicide resistant gene T3 generation seed(provided by the laboratory) in plots, through the detection of T3 generation obtained 147 positive plants by PCR, then selfed T3 generation and obtained 147 panicle.3. The T4 generation phytase maize(Bar filtering) seeds were planted, real-time quantitative PCR detection in the T4 generation plants and quantitative analysis of the test, to obtain positive T4 generation plants 160 strains(from the T3 generation of 97 positive plants). The PCR Southern blot analysis detected the positive T4 generation plants proved the phytase gene had been integrated in the genome of T4-generation plants. Utilizing metavanadate method by Southern blot analysis that the positive T4 generation plant roots phytase activity was measured, the measurement results show that phytase maize root phytase activity compared with the control plants were highly significant increased(P> 0.05), the average increase over control plants by 10.9 times, the highest activity reached 5.432 U / g.4. Using the method of Bowman-Cole, tested the positive T4 generation phytase maize(Bar filtering) of plants rhizosphere soil organic phosphorus determination of components, and non-transformed plants rhizosphere soil as control. According to the results, the T4 generation plants rhizosphere soil organic phosphorus content compared with the controlled plant rhizosphere soil organic phosphorus content were significantly decreased, the highest reduce 5.21 mg·kg-1, 7.8 times that of control.5. Counter rotating phytase gene analysis of agronomic traits and plant laboratory test from the plant, the results showed that phytase gene can improve the corn grain weight, compared with controls up to 25.8 g, but for other the physiological and biochemical indexes of corn did not significantly affect.