Genetic Transformation Of Gene Shys Of The Key Enzyme For Starch Synthase On Maize

Maize （Zea Mays L.） is the second largest cereal crop and fodder crop in the world. Withthe continuous development of processing industry, the demand of maize is also increasing.It is one of the important ways to corn breeding for high yield that transfer good genes intoexcellent inbred lines with the genetic engineering. In recent years, the research of this fieldhas made a lot of progress. AGPase is a key enzyme in starch biosynthesis and is regulatedby Pi negatively. Two of the negative regulatory sites are on the regulatory subunit Sh2, andthe mutated gene is named the shys. It improves the activity of ADP glucosepyrophosphorylase and then increases crop starch synthesis efficiency. In our study, maizeinbred lines A188, Qi319, and18-599were used as test materials. The Agrobacterium dipstrength were optimized and the factors that affect biolistic transformation efficiency werestudied. Then the corn starch biosynthesis gene shys was transformed byAgrobacterium-mediated technique and gene gun bombardment. The main results of thisstudy were as follows:1. The dipping strength of Agrobacterium has great effect on the transformationefficiency and plants regeneration. In this study, we explored the optimum dip strength ofA188and18-599. A188has the lowest browning rate and highest differentiation rate whenOD₆₀₀=0.4and t=15min, and so does18-599when OD₆₀₀=0.6and t=20min.2. The transformation rate of callus is largely affected by bombardment distance （BD）,bombardment times （BT） and plasmid concentration （PC）. The optimum bombardmentparameters of18-599were BD=9.5cm, BT=2, PC=1.0μg, and BD=9.5cm, BT=1, PC=1.0μg for that of Qi319.3. Rooting and growth-promoting medium is good for rooting of resistant plants. Therooting of A188and Qi319is well when the concentration of ABT was0.5mg·L^-1. For18-599, the number of root was3, and the average root length can reach2.6cm when the concentration of ABT was1.5mg·L^-1.4.15transgenic plants of A188and19transgenic plants of18-599byAgrobacterium-mediated with PCR were obtainted, and positive seedlings rate were8.5%. Inthe PCR-positive transgenic plants,3plants of A188and6plants of18-599wereRT-PCR-positive and the positive seedlings were3.5%.13PCR-positive strains by gene gunwere got, and positive seedlings rate were6.7%. There were two positive plants of Qi319and2strains of the18-599after RT-PCR detection, and the positive rate was3.2%. Through thecomparison of two transformation methods, the results showed that the positive seedlings byAgrobacterium mediated technique were3.5%, while gene gun seedlings of positive is3.2%,It is explained that transformation of Agrobacterium-mediated efficiency is higher than thegene gun slightly. Part of the result of Southern blotting showed obvious hybrid strip.5. After genetically modified seedlings transplanted to field, they were isolated by bagsand self-percentages. The seeds of T₀generation of18-599and Qi319were obtained.