| The soybean (Glycine max), which belongs to dicolyedons. is now the world’s most important legume and economic crop. With the excessive use of pesticides and change of global climate, occurrence of pests and diseases are increasingly in soybean production. In order to identify ideal disease resistance genes from soybean, and breed new cultivars which can resist pests and diseases, we launched into exploring the potential resistant genes to pests and disease related to secondary metabolism. Glucosinolate is a secondary metabolic product found in crucifer. And its hydrolysis products function in inhibiting the proliferation of bacteria, fungi, and insects. However, there are a few reports on disease resistance about Glucosinolate in soybean. In our lab, we found the existence of glucosinolates metabolic pathways in the nodules of soybean, and cloned the key gene GmSOT1 related to glucosinolates metabolism for the first time. Then we want to know whether overexpression of GmSOT1 in soybean could increase the glucosinolate content in soybean plants and promote pest and disease resistance.Although transgenic soybean had been cultivated globally, genetic transformation is still the difficulty to do the research. Agrobacterium-mediated exogenesis transformation system can accelerate progress of soybean strains improvement theoretically. However, soybean genetic transformation efficiency is still low. The main reasons are below:1) difficulties of soybean in vitro regeneration,2) poor manipulating reproducibility,3) strong reliability on soybean strains and exogenesis,4) complicated culture conditions. Theses reasons limited genetic improvement to soybean with gene engineering methods to a large extent. The establishment of highly efficient soybean regeneration system on the basis of optimizing Agrobacterium-mediated genetic transformation is a problem to be solved in soybean genetic transformation technique nowadays.In this study, we used soybean K06-82 embryonic tips as starting explants. We optimized highly efficient regeneration system, improved transformation technique and did the genetic transformation of GmSOT1.Then we conducted molecular analysis including GUS staining, PCR verification and Southern blot of resistant plants overexpressing GmSOT1.Finally we preliminarily identified insect resistance effects in transgenic plants and measured enzyme activity in GmSOT1 transgenic plants.The results are below:1. Establishment of soybean K06-82 efficient and stable regeneration systemWe got explants embryos tip by chlorine sterilization.MSB5 were used as basic culture medium.To induce adventitious shoots, explants were cultured in medium added 3.0 mg/L 6-Benzylaminopurine (6-BA) under light for 5 days, then were transferred to medium added 0.5 mg/L 6-BA and 0.05 mg/L naphthaleneacetic acid (NAA).And they were cultured successively biweekly. The composition of medium for adventitious shoots elongation is MSB5 added 0.2 mg/L 6-BA and 0.3 mg/L gibberellin (GA3) and cultured successively biweekly. The composition of medium for adventitious shoots rooting is 1/2MSB5 added 1.0mg/L indoleacetic acid (IBA). This method is suitable for rooting of adventitious shoots with or without hypocotyl and the rooting efficiency in both of them is up to 96.7%.2. Optimization of soybean genetic transformation systemWhen K06-82 embryonic tips were used for genetic transformation, preculture time should be 24 hours. The best infection condition were infection for 1 hour at 28 ℃,200 r/min.The concentration of acetosyringone (AS) for coculture is 200 μmol/L, and the concentration of IBA for K06-82 transgenic resistant adventitious shoots rooting were 1.2 mg/L.The rooting rate of adventitious shoots with no hypocotyls were higher than those with hypocotyls. Then the optimized genetic transformation method was applied to transfer GmSOT1 into K06-82 embryo tips, and finally transformation efficiency were up to 25.1%.3. Molecular verification and Insect-resistance assay of GmSOTl transgenic soybeanUsing GUS staining, PCR amplification and Southern blot, it had been proved that GmSOT1 gene were transferred to the soybean genome and mostly inserted as single copy. The results showed that compared with the control group, hazard classes of transgenic plant leaves were decreased and so was the average weight of larval with increased mortality. However, there was a big difference of insect-resistant ability among different lines of transgenic plants. |
