Cloning And Functional Analysis Of Soybean GmAOS Gene

Soybean(Glycine max(L).Merr) leaf-feeding insects affect the yield and quality of soybean.In this study,the 58 accessions were tested for the antibiosis to cotton worm (Prodenia litura,Fabricius) under inoculation condition according to the larval and pupal weight.The result indicated that both of the larval weight and pupal weight were all significantly different among varieties.Low larval and pupal weight indicates the high level of antibiosis of material.Larval weight was positively correlated with pupal weight. According to the results of 2004 and 2005,three genotypes,Huangpixiaoqingdou (HPXQD),shandongdadou and PI229358 were identified to be high resistant to cotton worm;Three genotypes,Xutuandadou(XTDD),mianyangbaimaodou and zhongyanghouzimao were identified to be high susceptible to cotton worm.These genotypes can be used to clone gene and functional analysis and inheritance study.GmAOS and its promoter was isolated from soybean use RACE.The full length sequence of GmAOS were 1789 bp which containing an ORF,encoding for 519 amino acids polypeptide with a calculated molecular mass of 58.3 kDa and an pI of 8.97.GmAOS had one exon and the protein possesses serine,threonine and tyrosine.The N-terminal region of GmAOS displayed features of a typical chloroplast targeting peptide.GmAOS has two copies in soybean and promoter of GmAOS contained two TATA-box,one CAAT-box, one GA inducing elements(TAACAA),one W-box element,two salt and pathogen inducing elements(GAAAAA) and one G-box(CACGTG) can be induced by JA.The different expression patterns of GmAOS and GmAOS1(ABB917763) were analyzed in different organs and induced by various treatments in two soybean accessions (XTDD,HS and HPXQD,HR).GmAOS and GmAOS1 transcript levels correlated with materials resistance grades.GmAOS mRNA and GmAOS1 mRNA were widely distributed among all organs examined.GmAOS and GmAOS1 had higher transcript levels in HPXQD (HR) than that in XTDD(HS).GmAOS and GmAOS1 gene had responded to cool(4℃)/heat(45℃)/JA/SA/CuSO4/Cu/SMV treatments.Cotton worm showed a strong inducement of the GmAOS and GmAOS1 transcript.In XTDD(HS),GmAOS reached the maximum mRNA level after induced by 24 h in XTDD(HS) while after induced by 3 h in HPXQD(HR).GmAOS1 reached the maximum mRNA level after induced by 12 h in XTDD(HS) while after induced by 3 h in HPXQD(HR).The activity of AOS in HPXQD (HR) was 4 times than that of XTDD(HS).These results showed that GmAOS and GmAOS1 in XTDD(HS) reached the maximum mRNA level significantly later than that of HPXQD(HR).This result also showed that the GmAOS and GmAOS1 transcript levels were correlated with materials resistance grade to cotton worm and overexpression of GmAOS might be one way to improve defense abilities in higher plants.To further examine the effect of GmAOS gene,it was overexpressed in tobacco and genomic southern blot showed that it had 2～4 copy in transgenic tobacco.Insect bioassay was performed to test the insecticidal activity of the GmAOS overexpressed in transgenic tobacco plants.Host selection experiment showed after 15 h,16 cotton worms had migrated controls,while 4 cotton worms chosed transgenic tobacco plants.The result indicated that cotton worms showed more preference to controls than transgenic tobacco,transgenic tobacco leaves were significantly less damaged compared to the control and the transgenic tobacco plant decreased the RGR of cotton worms.Scanning electron micrographs revealed that the average number of trichomes on transgenic tobacco plant leaves was 60.2 and the average number of trichomes on control tobacco plant leaves was 10.2 in the same field of vision.Analysis of enzyme activities showed that,in compared to the control,90% transgenic tobacco plants enhanced AOS activity,70%transgenic tobacco plants enhanced PPO and POD activity respectively and 85%transgenic tobacco plants enhanced CI activity.The hormone assay indicated that,in contrast to the control,85%transgenic tobacco plants enhanced the contents of JA and GA respectively,all the transgenic tobacco plants enhanced the contents of ABA and the content of IAA also increased in transgenic tobacco.The results presented in this work contribute to reveal the importance of GmAOS in plant defense.and overexpression of GmAOS might be a useful tool for improving resistance against cotton worms in high plants.