| Insect pests are one of the important factors that affect crop yield and quality. Soybean leaf-feeding insects seriously affect the yield of soybean. Cotton worm (Prodenia litura) is a major pest of soybean in southern China. Now, to improve the soybean resistance to cotton worm is one of the main breeding objectives. The study of induced resistance is important to improve plant resistance to pests and reduce pesticide use and improve the environmental protection.In this study, cotton worm is induced resistance factor. Cotton worms were used on leaves of cultivars Nannong 99-10 for 48h to induce resistance and were then removed from plants. The induced resistance of soybean was measured by assessing the feeding preference indices (PI) and relative growth rates (RGR) of cotton worms. We collected leaves from both control and previously treated plants at five sampling times (0 h,6 h,12 h, 24 h, and 72 h) following the end of induced treatment. There were five replications in this study. In one replication leaves from the damaged plants had not received any direct damage. Dual-choice feeding preference test showed that most of cotton worms chosed to eat control leaves and PI<1 at 0 h,6 h,12 h,24 h. The RGRs of cotton worms feeding control leaves were higher than that those feeding treatment leaves at 0 h,12 h,24 h,72 h. Our results showed that cotton worm can induce systemic resistance in soybean.Proteomics, a major tool in functional genomics research, has been widely used in genetic, physiologic and pathologic studys. To evaluate the induced resistance of soybean induced by cotton worm, the related changes in protein expression were investigated using the proteomic approach. Proteins from leaves of control and treatment with cotton worm at five sampling times were extracted and separated by two-demensional polyacrylamide gel electrophoresis. After induced by cotton worm,11 protein spots were detected, most of which were up-regulated. A total of 12 unique proteins from these 11 spots were identified by mass spectrometry, including serine/threonine kinase-related protein, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, enzymatic resistance protein, cytosolic phosphoglycerate kinase, ATP synthase beta subunit, malate dehydrogenase, phenylalanine ammonia-lyase, S-adenosylmethionine synthetase, ascorbate peroxidase,20S proteasome alpha subunit, vegetative storage protein A and predicted protein. The majority of them are energy metabolism-related proteins.2-DE maps of leaves from the damaged plants had not received any direct damage are similar to the maps of damaged leaves. This result also demonstrated that cotton worm can induce systemic resistance in soybean.Quantitative RT-PCR analysis of mRNA expression was performed as a paralleled experiment in contrast with 2-DE. The mRNA and protein relative expression levels were not totally consistent. The mRNA levels of most differentially expressed proteins were also up-regulated after induced by cotton worm.S-adenosylmethionine synthetase (SAMS) is a key enzyme that catalyzes the formation of S-adenosylmethionine (SAM) from methionine and ATP. The protein levels of SAMS were up-regulated at five replicates, while mRNA relative expression levels were up-regulated at 6 h,12 h,24 h, and 72 h. SAMS was involved in the cotton worm induced resistance response in soybean.Full length cDNA of GmSAMSl was isolated from soybean (Nannong 99-10), containing an 1188 bp open reading frame which was deduced to encode a peptide of 395 amino acids. SAMS protein contains 3 relatively conservative domains (N-terminal, C terminal and middle domain). Sequence alignment based on soybean, chickpea, wild soybean, arabidopsis, tobacco etc., showed that they are highly homologous. SAMS proteins are a highly conserved family.To further investigate function of GmSAMSl,35S::GmSAMS1 transgenic tobacco was obtained by Agrobacterium-mediated transformation. Insect bioassay was performd to test the insecticidal activity of transgenic tobacco which overexpressed GmSAMS1. Dual-choice feeding preference test showed that most of cotton worms chosed to eat control leaves, and transgenic tobacco leaves were significantly less damaged compared to the control, PI<1. Cotton worms were force-fed with control and transgenic tobacco leaves respectively. After 24 hours consumed transgenic tobacco leaves were significantly less than control, and the size and RGR of insects feeding transgenic tobacco leaves were significantly less than those fed by control leaves.The results suggested that GmSAMSl could enhance insect resistance of tobacco. Overexpression of GmSAMSl might improve resistance against cotton worms in other high plants. |
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