| Rapeseed(Brassica napus L.)is the largest oil crop in China,which always regard as weed control a technical problem in production.Developing herbicide-resistant germplasm and breeding resistant varieties with chemical weed control,is one of the enconomical and effective means of weed extinguishing.PB(pyrimidyl-benzoates)herbicide can effectively control most weeds in rice fields with the advantage of a shorter half-life and no harm to subsequent crops,comparing to other ALS herbicides.However,rapeseed is sensitive to PB herbicides and the development of resistant germplasms is the precondition for the application of such herbicides in rapeseed.In this study,the identification of RP-1 resistance and cross resistance to other ALS herbicides has been performed by the germplasms of the PB herbicides-resistant mutant RP-1,the SU resistant mutant EM28 and the wild type N131.The biochemical and molecular mechanism of the RP-1 resistance to PB herbicides were revealed and molecular markers were also developed to detect functional genes of resistance.Effects of amino acid combinations at different mutation sites on resistance were further investigated in order to provide technical support for the selection and breeding of resistant varieties.The main results are as follows:1.Study of resistant mechanism in rapeseed mutant to PB herbicidesCompared with the control,RP-1 exhibited high resistance to PB herbicides at the dosage of 1-12 times recommended concentration(RC),moderate resistance at the dosage of 16 times RC,and high levels of cross-resistance to three other ALS herbicides.In vitro enzyme activity assays showed that the mutant enzyme in RP-1 was significantly less sensitive to bispyribac in comparison with N131 and EM28,being presumed as target resistance.Genetic study suggested that the resistance is controlled by one semi-dominant nuclear gene.The gene cloning and expression analysis of ALS showed that respective amino acid substitutions of Trp556 Leu and Ser635 Asn were due to a double-site mutation at the +1667(G/T)and +1904(G/A)of the ALS3 gene in RP-1.And the ALS3 gene expression level was significantly higher in RP-1 than that in N131 and EM28 plants,suggesting that the mutation and expression difference of ALS gene may be associated with the production of PB resistance in rapeseed.The ALS3 gene of RP-1 was transferred into Arabidopsis thaliana,and the obtained transgenic progeny were treated with bispyribac.The results showed that the transgenic plants grew well,indicating that the mutate gene in RP-1 was expressed in Arabidopsis with the fuction of resistance to PB.CAPS and KASP markers were further developed and obtained for rapid detection of resistance functional gene sites,based on the base differences in mutant sites.2.Study of prokaryotic expression and enzymatic in mutant gene resistance to PB in rapeseedThe prokaryotic expression vectors were constructed to express ALS3 gene with subsequent purification for vitro enzyme activity analysis in N131,EM28 and RP-1,respectively.Then the inhibitory effect of different ALS enzymes were determined by bispyribac,bensulone and imazethapyr.The results showed that the ALS of RP-1 was significantly less sensitive to bispyribac than that in N131 and EM28,to further demonstrate the molecular mechanism of resistance of RP-1 to PB herbicides.3.Study on the function of different amino acid combinations at the resistance mutation sitesBased on the mutation sites in ALS3 gene of the mutant RP-1,new mutant ALS3 genes with 5 different amino acid combinations were respectively created and transformed to obtain Arabidopsis thaliana transgenic progeny plants.After bispyribac treatments the results showed that the transgenic plants with amino acid mutation types of W556L+S635T and W556L+S635I survived,while others died.It was speculated that the resistance of mutant RP-1 to bispyribac was due to a combination of mutations at W556 L and S635 N sites,moreover the mutation at the W556 L site is more critical for the resistance. |
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