Cloning, Expression And Mutation Detection Of Acetyl-coenzyme A Carboxylase Gene In Wild Oat (Avena Fatua L.)

Wild oat?Avean fatua?,a malignant crop weed in the word,is mainly controlled by chemicalherbicides,which has produced serious resistance because of excess and single use of herbicides.To explore the resistant mechanism of wild oat to herbicides,the susceptibility of wild oat from 24 populations conducted by whole-plant pot bioassays and in vitro acetyl-coenzyme A carboxylase?ACCase?conducted by enzyme-linked immunosorbent assay kit?ELISA?to fenoxaprop-p-ethyl,then the differential expressionconducted using RNA-seq,digital gene expression?DGE?profling and mutation detection ofACCase gene from some relatively resistant and susceptible populations were evaluated in the study.The results are as follows:1.The susceptibility of 24 wild oat populations to fenoxaprop-p-ethyl was conducted by wholeplant deteimination assay.In terms of the stalk,the wild oat population from Xihua?Zhoukou?was the most unsusceptible to fenoxaprop-p-ethyl,the value of IC₅₀0 was 643.790 g.a.i/hm².The wild oat population from Xiangxian?Xuchang?was the most susceptible to fenoxaprop-p-ethyl,the value of IC₅₀0 is 4.23g.a.i/hm².The difference between Xihua and Xiangxian was 15.21 times in the value of IC₅₀.In terms of the root,the wild oat population from Wuzhi?Jiaozuo?was the most unsusceptible to fenoxaprop-p-ethyl,the value of IC₅₀0 was 2977.041 g.a.i/hm².The wild oat population from Yanling?Xuchang?was the most susceptible to fenoxaprop-p-ethyl,the value of IC₅₀0 was 793.75 g.a.i/hm².The difference between Wuzhi and Yanling was 31.6 times in the value of IC₅₀.Though there was a great difference in the susceptibility of root and stalk to fenoxaprop-p-ethyl,it had developed varying degrees of resistance to fenoxaprop-p-ethyl.2.In virto the susceptibility of ACCase conducted by ELISA from 24 wild oat populations tofenoxaprop-p-ethyl were deteimined,respectively.The results showed that the wild oat population from Heshan?Hebi?was the most susceptibility to fenoxaprop-p-ethyl,it's value of IC₅₀0 is 11.334 mg/L;The wild oat population from Sheqi?Nanyang?is the most unsusceptible population,the value of IC₅₀0 was17720.677 mg/L.It indicated a strong relationship between the resistance of wild oat to fenoxaprop-p-ethyl and ACCase.3.The differential expression was conducted using RNA-seq,digital gene expression?DGE?profling in wild oat treated by the IC₁₀fenoxaprop-p-ethyl concentration?6.9 mg/L?for 24 hours and quantitative real-time PCR?qRT-PCR?validation.The results showed that 8 unigenes were annotated as ACCase,5 up-regulaed expression and 3 down-regulated expression were observed.To further clarify the relationship between ACCase and the wild oat resistance to fenoxaprop-p-ethyl,three resistant populations Sheqi?Y21?,Xihua?Y3?,Zhaoling?Y24?and a susceptible population Heshan?Y11?were selected and treated by fenoxaprop-p-ethyl for 24 hours.The result indicated that relative expression level of ACCase gene from three resistant populations and a susceptible population had been suppressed by fenoxaprop-p-ethyl,but three resistant populations were significantly less suppressed than susceptible populations.This proved that the resistance of wild oat to fenoxaprop-p-ethyl is related to the expression level of ACCase gene.we also found that the relative expression level of ACCase gene from Sheqi?Y21?was he higest,followed by Xihua?Y3?and Zhaoling?Y24?.This conclusion was in accord with the result of susceptibility of ACCase to fenoxaprop-p-ethyl absolutely.4.To validate whether the resistance of wild oat to fenoxaprop-p-ethyl was caused by themutation of ACCase gene.The ACCase genes of four resistant populations,Suiping?Y19?,Sheqi?Y21?,Xihua?Y3?,Zhaoling?Y24?and four susceptible populations,Heshan?Y11?,Wenxian?Y7?,Wuyang?Y9?,Huaiyang?Y16?were cloned and sequenced.Then ACCase sequences cloned were made multiple sequence alignment with a model complete ACCase sequence of Alopecurus myosuroides?GeneBank accession No.AJ310767?.The results indicated that mutations point of amino acid,Ile-1781-Leu,Trp-1999-Cys,Trp-2027-Cys,Ile-2041-Asn,Asp-2078-Gly,Cys-2088-Arg published were not found.It suggested that the resistanceof wild oat to fenoxaprop-p-ethyl was not caused by the point mutation of the target site published.