Cloning Of ALS Gene From Cyperus Rotundus L.and Sensitivity Study Of Different Populations To Two Sulfonylurea Herbicides

Cyperus rotundus L.,perennial grass,is classified as cyperaceae and is one of the main weeds to be controlled in sugarcane,corn,peanut and other crop fields in China.Halosulfuron-methyl and trifloxysulfuron are effective sulfonylurea herbicides to control C.rotundus,however,in recent years,the control effect of the two herbicides has decreased significantly in many areas.ALS target enzyme is the only target of sulfonylurea herbicides.In order to determine the status of resistance to halosulfuron-methyl and trifloxysulfuron in different populations of C.rotundus,the cDNA and DNA sequence of ALS gene of C.rotundus were cloned by RACE and PCR techniques,and analyzed the target mutations in different populations.The differences of sensitivity to halosulfuron-methyl and trifloxysulfuron were studied by determined ALS activity in vitro.Analyzed and compared the differences in ALS m RNA expression levels between the most susceptible and unsusceptible C.rotundus populations.The effects of sulfonylurea herbicides on the activities of target enzyme（ALS）,protective enzymes（SOD,POD,CAT）,and the contents of amino acids and malondialdehyde（MDA）in C.rotundus were systematically studied from the physiological and biochemical mechanisms,in order to provide basic data for rational use of sulfonylurea herbicides in different geographical areas.The main results are as follows:1.The full-length of C.rotundus ALS gene is 2239 bp,including 54bp 5′UTR,250 bp 3′UTR,1935 bp open reading frame,and encoding 645amino acids.The DNA length of ALS gene is 3339 bp,including an intron of 1359 bp.2.There was no mutation of target sites in the populations of C.rotundus collected from 31 different crop fields in China,and no target resistance of C.rotundus to sulfonylurea herbicides.3.In vitro ALS activity showed that there were significant differences in the sensitivity of different geographic populations to halosulfuron-methyl and trifloxysulfuron.The sensitivity of C.rotundus in Anshun,Guizhou XFZ27 population to halosulfuron-methyl is the lowest one,and its I₅₀ was 3.330 mmol/L.the following ones were Ruzhou XFZ01 in Henan,Chongzuo XFZ17 and Hezhou XFZ16populations in Guangxi,with I₅₀ values of 3.244,2.944 and 2.800mmol/L,respectively.Xuchang XFZ29 C.rotundus in Henan was the most sensitive one,its I₅₀ was 0.438 mmol/L.The XFZ08 C.rotundus in Hangzhou,Zhejiang was next,with an I₅₀ value of 0.460 mmol/L.The most unsusceptible Anshun,Guizhou population showed 7.60-fold more insensitive than the most susceptible Weidu,Xuchang.The population of C.rotundus in Hezhou,Guangxi was the most unsusceptible to trifloxysulfuron,I₅₀ was 5.463 mmol/L.C.rotundus from Zhejiang,Hangzhou was the most susceptible to trifloxysulfuron with I₅₀ of 1.035mmol/L.The most unsusceptible XFZ16 population showed 5.28-fold more insensitive than the most susceptible XFZ08 population.Compared comprehensively,the XFZ16 population in Hezhou,Guangxi was unsusceptible to both herbicides,while the XFZ08 population in Hangzhou,Zhejiang was susceptible to both herbicides.The ALS activity in vivo to the herbicides showed that susceptible XFZ08 C.rotundus was lower than unsceptible XFZ16 population and the inhibition of ALS activity was higher than unsceptible XFZ16 C.rotundus.4.The expression of ALS gene in XFZ08 and 16 C.rotundus populations were determined by qRT-PCR.Under normal condition,the ALS gene expression level of XFZ08 and XFZ16 population was significantly different at the 3d and 10d.After the treatment of halosulfuron-methyl and trifloxysulfuron at 1d and 5d,the ALS expression of XFZ16 C.rotundus was significantly higher than XFZ08population.The ALS-m RNA expression of XFZ16 population reached the peak value at 1 day after the spray treatment of halosulfuron-methyl and trifloxysulfuron,which were 5.71-fold and 9.80 times higher than the control group,respectively.The difference in the expression of ALS gene may be one of the reasons for the difference in the sensitivity of C.rotundus to sulfonylurea herbicides.The amino acid content determination showed that:2 hours and 7 days after the treatment of herbicides,the contents of leucine,isoleucine and valine in XFZ16 C.rotundus were all higher than XFZ08 population,but the differences were not significant.5.After treatment of halosulfuron-methyl and trifloxysulfuron,the activities of SOD,POD,CAT in XFZ16 C.rotundus were higher than most susceptible XFZ08 population,which indicated that the ability of scavenging active oxygen and free radicals was strong,and quickly restore the normal metabolism.The content of MDA in the susceptible XFZ08 population was higher than susceptible XFZ16 C.rotundus,indicating that the susceptible population of C.rotundus had a weak resistance to beibicides and the damage to cell membrane system was more serious.The enhancement of protective enzyme activity in different populations of C.rotundus may be one of the reasons for the difference in sensitivity to sulfonylureas.In this study,the sensitivity of different geographic C.rotundus populations to halosulfuron-methyl and trifloxysulfuron herbicides was first reported,and clarified the mechanism of the difference in sensitivity from physiological,biochemical and molecular biological levels,provided basic data for the control and rational use of sulfonylurea herbicides on C.rotundus.