Transgenic Sugar Beet Harbouring High-affinity NH₄⁺ Transporter Fusion Gene By Agrobacterium-mediated

Sugar beet(Beta vulgaris L.) is one of the important sugar crops in the world.But over the past 10 years,beet production was slow growth and tuber quality appeared downward trend.In addition to variety and seed quality,low utilization rate of nitrogen is one of the key factors.Therefore,efficient utilization of nitrogen to cultivate new varieties of sugar beet can reduce the amount of nitrogen fertilizer,production costs and water pollution,and can increase yield as well improve quality.The development of genetic engineering technology offers the possibility of new varieties.In the nitrogen stress conditions,the parts of NH4+ transporter protein play an important role on the improvement of plant nitrogen uptake and use efficiency in plant species.One research has shown that the high-affinity ammonium transporter fusion gene-specific over-expression can significantly enhance the resistance of transgenic plants with low nitrogen stress,the same time,effectively improve the capacity of roots to absorb nitrogen of transgenic plants.The research can be used to obtain resistant to low nitrogen new varieties.In this study,high-affinity NH4+transporter fusion gene was successfully transformed into the cotyledon node explant of suger beet by using A grobacterium- mediated transformation methord,and established the high efficient genetic transformation system of suger beet.Transgenic plants were obtained and done molecular detection,functional identification.The major results studied as follows:1.Established genetic transformation system:cotyledon node 24h for pre-culture, infection transformation(bacterium concentration OD600=0.4-0.5,infection 8-10min),pH 5.84 of co-culture media,48h and 25℃for co-culture,Cef concentration 400mg/L for MS liquid medium washed bacteria and bacteriostatic culture,hygromycin selection 9mg/L of differentiation culture,and hygromycin 7mg/L of rooting culture.Resistance seedlings were obtained,after hardening-seedlingand transplanting.2.This study realized the genetic transformation on a high-affinity ammonium transporter fusion gene of sugar beet,As the experimental result,140 resistant seedlings were obtained and differentiation rate was10.14%,PCR assaying of plant DNA confirmed 45 transgenic plants;The 4 transgenic plants were selected to RT-PCR detecte,and we found that the fusion gene had different gene induction expression in roots and leaves.3.In the low-nitrogen treatment,the chlorophyll and total nitrogen of the above mentioned transgenic plants and wild-type were determined.With the nitrogen levels decreased,the results showed that the chlorophyll and total nitrogen contents of transgenic sugerbeet all improved to some extent compared with control one.Forethemore,they had significant difference between transgenic sugerbeet and wild-type.