Cloning、Evolution And Functional Study Of Soybean GmAOC Gene Family

Soybean (Glycine max (L.) Merrill) is one of the most important economic crops which provide quality protein and oil for human. However, various insect pests significantly affect the yield and quality of soybean. As a signaling molecule, Jasmonate has a crucial in orchestrating plant defense responses to several kinds of stresses, especially to biotic stresses. Allene oxide cyclase (AOC, EC5.3.99.6) catalyzes the most important step in the jasmonate biosythesis pathway and its specificity determines the correct stereochemical structure of jasmonate.In this study, six AOC genes were isolated from soybean, randomly distributing on chromosomes 1,2,8,13,18 and 19. The ORFs ranged from 759 to 771 bp which encoded proteins containing 253 to 257 amino acids. The calculated molecular masses were about 28 kDa and the alkaline PI (isoelectric point) ranged from 8.75 to 9.23. The six AOC proteins were clustered into three groups with overall similarity values ranging from 78% to 94%. The amino acid sequences of all six AOC proteins comprised eight highly conserved β-strand residues and contained an N-terminal choroplast transit peptide (cTP) with the most likely cleavage site located between the amino acids 56 to 75. The results of subcellular localization showed that GmAOCs were all localized to chloroplasts. The non-synonymous to synonymous substitution rate ration indicated that GmAOC1, GmAOC2, GmAOC3 and GmAOC4 were under positive selection, while GmAOC5 and GmAOC6 were under neutral evolution. Besides the basic transcription elements TATA-box and CAAT-box, some motifs responsive to the external environment were found in the six GmAOC gene promoters, including elements responsive to salt, heat stress, ethylene and salicylic acid as well as TC-reach repeats. The real-time PCR results revealed the six AOC genes have distinctive and complex expression patterns in multiple organs and under adversity stresses. Of the six AOC gene, only GmAOCl expressed slightly in the seeds. GmAOC2 exhibited higher expression in roots, while GmAOC5 was only expressed in the stems. GmAOC3 was unique in showing highest expression in leaves. GmAOC4 and GmAOC6 were always expressed weakly in roots, stems, leaves and flowers. Further more, the GmAOC gene famliy showed different expression pattern under stress conditions. Following phytophagous insects predation, GmAOC3 transcripts were rapidly activated and attained the peak expression in leaves. Under salinity and drought stress, GmAOCl and GmAOC2 respectivey attained the highest expression level in the minimum time in roots. For oxidative stress, GmAOC5 was significantly induced by H2O2, whereas GmAOC1 was down-regulated and the other four genes showed no obvious change.It is an attention-worth question that how the relationship between AOC gene and plant insect-resistance. Transgenic tobacco plants that constitutively overexpressed GmAOC1, GmAOC3 and GmAOC5 were generated for evaluating their resistance to insect by insect bioassay. In free-feeding experiment showed cotton leafworm preferred to feed leaves from wild-type tobacco plants. Compared to wild-type tobacco, leaves of transgenic tobacco plants overexpressing GmAOCl and GmAOC3 were significantly less damged. Meanwhile, the RGR of cotton leafworm fed by were leaves from transgenic tobacco plants overexpressing GmAOC1 and GmAOC3 significantly decreased. Scanning electron micrographs reveales that the number of trichomes on transgenic tobacco plants overexpressing GmAOC1 and GmAOC3 were more than that of wild-type tobacco plants. QRT-PCR analysis revealed that the expression level of PI and PMT in transgenic tobacco plants overexpressing GmAOC1 and GmAOC3 were higher than of wild-type tobacco plants. Further more, PAL activity, phenolic and JA content in transgenic tobacco plants overexpressing GmAOC1 and GmAOC3 were significantly higher than of wild-type tobacco plants. The volatile components assay revealed that a total of 11 volatiles relatived to insect-resistance were identified and their content increased in transgenic tobacco plants overexpressing GmAOC1 and GmAOC3. The all above-mentioned changes of insect-resistance, physiological and morphological indexes were not apparent in tobacco plants overexpressing GmAOC5. In summary, overexpression of GmAOCl and GmAOC3 gene in transgenic tobacco plants improved tobacco resistance to insect and this work might provide appropriate genes and theoretical basis for plant molecular breeding for insect resistance.