Functional Analysis Of Three Peanut UDP-glucosyltransferases In Plant Response To Florasulam Herbicide

Peanut is an important oil and economic crop.Compared with crops such as rice,corn and soybean,the research on herbicide resistance is relatively scarce.Therefore,it is urgent to improve and innovate its germplasm resources.In the early stage,our laboratory obtained a peanut mutant with high tolerence to florasulam and flumetsulam herbicides,and conducted a preliminary analysis of the molecular mechanism of its non-target resistance.Based on the RNA-seq technology,37 uridine diphosphate glucosyltransferase genes（UGT）were identified.Previous studies demonstrated that glycosyltransferases play a key role in eliminating endogenous and exogenous toxins.In order to clarify the specific role of glycosyltransferase genes in the formation of mutant non-target resistance,three uridine diphosphate glucosyltransferase（ENIS4M,E3VS3U and WIN7QF）genes with large changes in differential expression were screened.It is planned to analyze the effects of the herbicide-tolerant florasulam by means of bioinformatics and molecular biology.The results are as follows:1.In order to verify the stability of the M₈peanut mutants tolerant to herbicides,we conducted experiments on it under field and laboratory conditions.Firstly,the peanuts at the seedling stage were treated in the field by foliar spraying(33.2 mg.L^-1,herbicide concentration 4 times that of the field).The phenotypic investigation showed that the mutant strains H2-24-33 and H2-5-1-22 were superior to the wild type’Shanhua 15’in terms of plant height,fruit pod number and lateral root number.Moreover,physiological investigations showed that under herbicide stress,the SOD,POD and CAT enzyme activities of mutant strains H2-24-33 and H2-5-1-22 all increased at different times compared with the wild-type’Shanhua 15’.Secondly,using the seed soaking method(8.3 mg.L^-1,herbicide concentration 1times that of the field)under laboratory conditions,it was found that the emergence rate of the mutant was twice that of the wild type.At the same time,the root growth of the mutant strains H2-24-33 and H2-5-1-22 remained normal,the main roots were elongated and there were a large number of lateral roots,while only a few wild types grew main roots.2.Glycosyltransferases are an important class of multifunctional superfamily enzymes.We use bioinformatics to analyze three UGTs genes:ENIS4M,E3VS3U and WIN7QF.The results showed that the amino acids encoded by them have small differences in length,isoelectric point and molecular weight.They are all hydrophilic acidic proteins,without signal peptides and transmembrane domains,and are located in the cytoplasm.The secondary structure prediction shows that the proteins they encode only containα-helices,β-sheets and random coils.The tertiary structure shows that the proteins encoded by ENIS4M and WIN7QF genes belong to the GT-A type,while the proteins encoded by the E3VS3U gene belong to the GT-B type.In addition,the evolutionary relationship shows that the three genes belong to the L,D,and I subfamilies of the cultivated peanut UGT family,and the cis-acting element prediction shows that it contains more elements related to light,hormones and stress.All three UGTs genes were responsive to florasulam,and the expression levels in the mutants were lower than the wild-type 24 hours after treatment,which was consistent with the trend of the transcriptome through the q RT-PCR technology.In addition,their temporal and spatial expression characteristics in wild-type and mutant peanuts show that their expression levels in leaves,flowers and roots at different stages are higher,and their expression levels are low whether they are expressed in stems at the seedling or flowering stage and seeds.3.To further explore the functions of the three UGTs genes in the metabolic pathway of detoxification,we expressed them heterologously in the model plant Arabidopsis thaliana.First of all,three genes were cloned into a plant binary expression vector containing 35S promoter,and then were introduced into Arabidopsis by the way of Agrobacterium-mediated floral dipping.Finally,antibiotics are used to screen to obtain a homozygous single insertion site transgenic line of the target gene.The results showed that the cotyledon albino rate of the transgenic plants in the seedling stage was significantly lower than that of the wild type and the transgenic lines with empty vector under the treatment of low-concentration florasulam herbicide.Additionally,under high concentration treatment,the chlorosis degree of the above-ground leaves of the transgenic plants during the vegetative growth phase was slightly lower than that of the wild type and the transgenic lines with empty vector.The above results revealed the differences in the physical and chemical properties,secondary/tertiary structure and evolutionary relationship of the three UGTs gene-encoded proteins,as well as their response to herbicides and their temporal and spatial expression characteristics.Their heterologous expression in Arabidopsis has enhanced plant tolerance to herbicides to varying degrees,and this helps people understand the mechanism of herbicide non-target resistance deeply.