Isolation Of HPPD-inhibitor Herbicides Resistant Strain And Cloning And Resistance Evaluation Of The HPPD Gene

4-hydroxyphenylpyruvate dioxygenase(HPPD)is an important herbicide target.In recent years,a series of novel contact inhibitors targeting HPPD,such as mesotrione,tembotrione and isoxaflutole,have been discovered,which are mainly used for corn,soybean,sugarcane and other crops to control broad-leaved weeds and gramineous weeds.High-intensity use of glyphosate has resulted in serious environmental hazards and resistant weeds.Therefore,developing novel herbicides and cultivating corresponding herbicide-resistant crops are necessary.HPPD-inhibitor herbicides have many advantages such as high efficiency,low toxicity,crop safety and good environmental compatibility,thus are considered as promising target herbicides for the engineering of genetically modified(GM)herbicide-resistant crops.At present,foreign research shows that two transgenic HPPD crop products showing tolerance to the mesotrione or isoxaflutole have been developed.However,a few of resistant HPPD genes were reported in china,and none of them was used for commercial application.This article aims to isolate and screen strains with highly resistant to HPPD inhibitors and clone its resistant HPPD gene.This study provides valuable genetic resources for the genetic engineering of GM herbicide-resistant crops,which is significant for promoting the innovation and application of agricultural biotechnology in China.1、Isolation and identification of HPPD-inhibitor herbicides resistant strainsIn this research,the soil samples and sludge samples were collected from Jiangsu and Anhui province,and serially diluted and spread on TMSM plate(using L-tyrosine as the sole carbon source)containing mesotrione.Finally,12 strains which could tolerate 100-500μM mesotrione were isolated.Phylogenetic analysis based on 16S rRNA gene sequences indicated these strains belonged to Pseudomonas sp.,Ensifer sp.,Ochrobactrum sp.,Agrobacterium sp.and Acinetobacter sp.Specially,a strain Peq-28 with the highest resistance to 500 μM mesotrione was screened.Based on the 16S rRNA gene sequence analysis and phylogenetic evidence,strain Peq-28 was identified as Ensifer sp.Strain Peq-28 was Gram-negative,the optimal temperature,pH and NaCl concentration for growth was 30℃,7.0 and 0.5%,respectively.In addition,an isolate HX2-24 which could tolerate 100 μM mesotrione was also screened.Phylogenetic analyses based on 16S rRNA gene sequence indicated that strain HX2-24 was most closely related to Extensimonas vulgaris S4T(98.1%similarity),and shared less than 97%similarities with other type strains.In the phylogenetic tree based on NJ algorithm,strain HX2-24 formed a subclade with E.vulgaris S4T.Chemotaxonomicanalysis revealed that HX2-24 possessed ubiquinone 8(Q-8)as the isoprenoid quinone,the major polar lipids are phosphatidylethanolamine(PE),phosphatidylglycerol(PG),diphosphatidylglycerol(DPG),aminophospholipid(APL),glycophospholipid(GPL)and aminoglycolipid(AGL),the major cellular fatty acids are C16:0,summed feature 3(C16:1ω7c and/or C16:1ω6c)and C17:0 cyclo,the DNA G+C content is 64.4 mol%.The ANI and dDDH values between HX2-24T and E.vulgaris S4T were 92%and 41%,respectively,which were below the 95%(ANI)and 70%(dDDH)threshold values suggested for the description of bacterial species.Thus,based on the phenotypic,chemotaxonomic,and genotypic characteristics,strain HX2-24T represents a novel species in the genus Extensimonas,for which the name Extensimonas perlucida HX2-24T sp.nov.is proposed.The type strain is HX2-24T(=KCTC 72472T=CCTCC AB 2019178T)2、Research on the resistant of strain Peq-28 and cloning the resistant gene EnhppdStrain Peq-28 was highly-resistant to HPPD herbicides and could tolerate saturated concentrations of mesotrione,tembotrione and isoxaflutole.The HPPD gene Enhppd,was successfully cloned from the genomic DNA of strain Peq-28.Enhppd was 1,113 bp with the G+C content of 61%.The deduced EnHPPD protein sequence contained 370 amino acids and had a calculated molecular mass of 41.1 kDa.Multiple alignments of amino acid sequence showed that EnHPPD belonged to 4-hydroxyphenylpyruvate dioxygenase,and the C-terminal sequence was relatively conservative and had high homology with the reported HPPDs,while the N-terminal sequence was quite different from the reported HPPDs.The homology between EnHPPD and the reported resistant HPPDs was from 33.9%to 54.6%and EnHPPD showed the highest similarity(54.6%)with HPPD(006695)from Vibrio vulnificus CMCP6.3、Characteristics and resistant research of EnHPPDThe Enhppd gene was heterologously expressed in E.coli.and EnHPPD was purified by Co2--chelate affinity chromatography.The purified EnHPPD was a dimer with a subunit molecular weight of 40 kDa.The specific enzyme activity of EnHPPD was 10.2 U/mg.The IC50 values of mesotrione,tembotrione and isoxaflutole against EnHPPD were 14.2 μM,10.5μM and 8.2 μM,respectively.The optimal reaction temperature and pH of EnHPPD was 30℃ and 7.0,respectively.The enzyme activity of EnHPPD was significantly increased by Fe2+,Fe3+,Ca2+,Mg2+,but seriously inhibited by Al3+,Hg2+,Zn2+,Co2+,Ni2+,Cu2+,Mn2+.In addition,the enzyme activity of EnHPPD was completely inhibited by EDTA,DEPC and sightly inhibited by Ba2+.