Studyd On Genetic Transformation Of Isa-H1 Gene In Maize

Maize is one of the most important food crops. Pre-harvest sprouting (PHS) in maize refers to germination of grains in a physiologically mature maize spike before harvest when prolonged wet weather occurs. The pre-harvest sprouting can lead to quality decrease. The barleyα-amylase inhibit protein (BASI) can inhibit theα-amylase activity effectively, improve the starch quality and express well in wheat genetic background.In this study, theα-amylase inhibitor gene Isa-H1 and the barley endosperm specific promoter Horp were cloned and plant effective expression vector pHorp-Isa-H1 were constructed. Stable genetic transformation systems were established and transgenic materials of maize elite inbred lines were obtained by using Agrobacterium Tumefaciens and pollen tube methods.The full length of Horp and Isa-H1gene were amplified by PCR method. The plant expression vector pHorp- Isa-H1was obtained by ligation of Horp, Isa-H1and fragment of pCAMBIA3301 with T4 ligase.In maize genetic transformation, there are few maize inbred lines can be used as recipient materials for the limit of genotype. In this study, immature embryos of elite maize inbred lines Y412 and Y423 were screened from 40 maize inbred lines for recipient materials. It was found that the immature embryos of these two materials can efficiently induce a large number of somatic embryos of maize. The induced somatic embryos could be sub-cultured for a long time and multiply indefinitely. Without the exogenous hormone, the somatic embryos could also differentiate on two polar at the same time and have the characters of very high differentiation rate and survival rate. Somatic embryos (<1cm) were infected with A. tumefaciens strain EHA105 containing vector pHorp-Isa-H1. After infection, Somatic embryos were transferred to the surface of cocultivation medium and excess A. tumefaciens suspension was pipetted off the medium surface. Then selected resistant callus to differentiation and transplanting.A total of 13 plants were positive by PCR molecular detection, transformation ratio was 5%.There are some problems of callus used as recipient materials for transformation system of maize, such as long cycle time to induced callus and the limit of genotype. In this experiment, we used internodes which from 5 maize inbred lines used in the Northeast as recipient material for the maize transformaition mediaed by Agrobac- terium tumef aciens. Established a new stable genetic transformation system.In order to improve conversion efficiency, the transformation systems of different receptors are compared. We used immature embryos as recipient material also.In the experiments, the vector pHorp-Isa-H1 was transferred into maize inbred line by pollen tube pathway. A total of 12 plants (T0) were positive by PCR molecular detection. The seeds of transgenic plants were harvested to spike as the unit. The DNA extracted from endosperm of transgenic plants (T0), PCR identification show that the seeds containing exogenous gene were 55% of the total of T1.