【作者】 郇志博；

【导师】 王金信；

【作者基本信息】 山东农业大学 ， 农药学， 2011， 博士

【摘要】 黑龙江省是我国大豆主产区,也是我国最早使用除草剂的地区之一,稗草是大豆田主要的禾本科杂草,芳氧苯氧基丙酸酯类(APP)和环己烯酮类(CHD)除草剂在该地区广泛应用,尤其是APP类除草剂精喹禾灵在某些农场使用年限达到10年以上,在一些农场的大豆田由于精喹禾灵的累年连续使用,稗草产生了抗药性。为探究黑龙江省几个主要农场抗性稗草的发生情况,从11个农场大豆田采集稗草种子,通过整株生物测定法和种子生物测定法测定稗草对精喹禾灵的抗性水平;为探明抗药性机理,采用分子生物学和酶学等方法对抗性稗草的靶标酶基因、靶标酶活性和几类非靶标酶活性进行分析。主要结果如下:(1)种子生物测定法测定结果:格球山农场R型稗草的GR50值为2847.0 mg L-1,S型GR50值为22.7 mg L-1,相对抗性指数RI值为125.5;853农场R型稗草GR50值为63.6 mg L-1,S型GR50值为22.8 mg L-1,相对抗性指数为2.8;红星R型稗草的相对抗性指数为2.6;其他R型稗草的GR50值为26.5-50.1 mg L-1,S型的GR50值为21.3-32.1 mgL-1,相对抗性指数小于2.0。(2)整株生物测定法测定结果:格球山农场R型稗草的抗性水平仍为最高,GR50值为1066.4 g ai ha-1,S型GR50值为12.2 g ai ha-1,抗性指数为87.3倍;853农场R型稗草GR50值为92.5 g ai ha-1,S型GR50值为15.3 g ai ha-1,相对抗性指数为6.0;其他采集地R型生物型的GR50值为29.5-61.2 g ai ha-1,S型生物型的GR50值为13.5-20.8 g aiha-1,相对抗性指数在1.5-3.6之间。结合种子生物测定法测定结果表明格球山农场R型稗草为高抗生物型,其他十个农场的稗草抗性水平较低。(3)选取格球山农场R型稗草,853农场R型稗草,五大连池农场R型稗草分别作为不同抗性水平的抗性生物型研究对象,分别测定三种生物型在喷药60 g ai ha-1的精喹禾灵0d,2d,4d,6d,8d后的SOD,POD,CAT,GSTs的活性变化趋势,结果表明853农场R型稗草和五大连池农场R型稗草的GSTs在施药后较同期对照活性大大增加,格球山农场R型稗草GSTs活性几乎无变化,三种保护酶中高水平抗性和低水平抗性生物型酶活差异最大的是POD酶。结果表明两种低水平抗性与GSTs的活性增强相关,高抗生物型的POD酶活性被大大激活。(4)利用HPLC测定出精喹禾灵对高抗生物型格球山农场R型稗草(RR)和敏感对照格球山农场S(SS)型稗草质体型ACCase的IC50,结果表明精喹禾灵对RR型稗草的IC50为145.38μM,SS的IC50为0.69μM,相对抗性指数为210.7倍。结果表明高抗生物型的靶标酶较敏感对照对精喹禾灵的敏感性大大降低。(5)扩增出格球山农场R型稗草(RR)和格球山农场S(SS)型稗草质体型ACCasecDNA全长各约7527 bp,开放性阅读框(ORF)6951bp,5’非翻译区(UTR)193 bp,3’非翻译区(UTR)383bp,各编码2316个氨基酸,相对分子量256 KD,PI=5.97。经BLAST、系统进化树分析和转导肽序列分析证明扩增得到的序列为稗草质体型ACCase cDNA序列,此二序列已在GenBank上注册,登录号为HQ395759和HQ395758。经比对RR和SS型稗草的质体型ACCase cDNA全长序列,发现相对于大穗看麦娘核苷酸位置5341位A突变为T,相应的1781位的氨基酸Ile突变为Leu,分析表明该点突变与格球山农场R型稗草对精喹禾灵的高抗性相关。(6)通过SYBR Green I实时荧光定量PCR试验,分别测定高抗生物型RR和敏感生物型SS在喷洒60 g ai ha-1的精喹禾灵后,质体型ACCase基因的表达情况,结果表明在喷药0d,2d,4d,6d,8d后RR型稗草的质体型ACCase基因表达先被抑制然后逐渐恢复,SS的趋势相同。结果表明高抗生物型的靶标酶基因表达与抗性机理无关。综上所述,在被检测的十一个农场中仅格球山农场出现高抗生物型,其他农场抗性水平较低。抗药性机理研究结果表明稗草对精喹禾灵的高水平抗性与靶标酶基因突变相关,低水平抗性与解毒酶GSTs的活性增强相关。更多还原

【Abstract】 Heilongjiang Province is the important soybean production in China and one of the earliestherbicide application regions. Echinochloa crusgalli (L.) Beauv. (barnyardgrass) is a maingrass weed in soybean fields. For controlling this grass weed, APP and CHD herbicides werewidely used in the region. Especially, quizalofop-p-ethyl was used more than 10 years onsome farms. Because of the continuous use of this herbicide for years, barnyardgrass evolvedresistance to this herbicide. In order to determine the popularization of the resistance,barnyardgrass seeds were collected from 11 farms where quizalofop-p-ethyl was used formany years and the resistance level was determined using whole-plant bioassay and seedbioassay. In order to clarify the mechanism of resistance, the resistance mechanism wasstudied using molecular biologymethods and enzymologymethods. Main results as follows:(1) Seeds bioassay results: GR50 of Geqiushan R was 2847.0 mg L-1, GR50 of S was 22.7mg L-1, RI was 125.5; GR50 of 853 R was 63.6 mg L-1, GR50 of S was 22.8 mg L-1, RI was 2.8;RI of Hongxing R was 2.6; GR50 of other Rs were 26.5-50.1 mg L-1, GR50 of Ss were21.3-32.1 mg L-1, RI <2.8.(2) Whole plant bioassay results: The resistance level of Geqiushan R was the highest.GR50 of Geqiushan R was 1066.4 g ai ha-1, GR50 of S was 12.2 g ai ha-1; RI was 87.3; GR50 of853 R was 92.5 g ai ha-1, GR50 of S was 15.3 g ai ha-1, RI was 6.0; GR50 of other Rs were29.5-61.2 g ai ha-1, GR50 of Ss were 13.5-20.8 g ai ha-1, RI were 1.5-3.6. Combined the dataof seeds bioassay, Geqiushan R was defined as the high resistance population, the resistancelevel of other Rs was low.(3) Geqiushan R, 853 R and Wudalianchi R were chosen as the study objects and theactivities tendency of SOD, POD, CAT, GSTs after spraying quizalofop-p-ethyl 60 g ai ha-1from 0d to 8d were determined. Results showed that: the activities of GSTs in 853 R andWudalianchi R increased and reached the peak at the eighth day. The activity of GSTs inGeqiushan R changed little compared the control. Among the three protective enzymes thevariation tendency of POD has significant difference between the high resistant biotype andthe low resistant biotypes. The results showed that the resistance mechanism of 853 R andWudalianchi R related to the enhanced detoxication of GSTs. POD activities inhigh-resistance biotype were significantly activated.(4) Using HPLC, the activities of plastid ACCase in Geqiushan R (RR) and Geqiushan S(SS) at series quizalofop-p-ethyl concentrations were determined. The quizalofop-p-ethylconcentration that inhibited the activity of plastid ACCase in RR and SS by 50% (IC50) wasdetermined. The results showed that IC50 of RR was 145.38μM, IC50 of SS was 0.69μM, RIwas 210.7. Results showed that sensitiveness of high-resistance biotype to quizalofop-p-ethylreduced greatly compared to the sensitive biotype. (5) Full-length cDNAs of barnyardgrass plastid ACCase in RR and SS were clonedsuccessfully. The plastid ACCase cDNA of RR is about 7 kb, includes an ORF of 6951bp,193 nucleotides of 5’ untranslated sequence (UTR) and 383 nucleotides of 3’ UTR. This geneis deduced to encode a protein of 2316 amino acids with a pI of 5.97 and molecular weight ca.256 KD. According to the results of BLAST, phylogenetic tree analysis and transit peptideanalysis, the cloned cDNA sequences were barnyardgrass plastid ACCase. The accessionnumbers in GenBank were HQ395758 and HQ395759 respectively. After compared thefull-length cDNA of barnyardgrass ACCase in RR and SS, one non-synonymous mutationoccurred at nucleotide position 5341, with codon 1781 (standardized to blackgrass), causingan ATA codon (isoleucine) to be changed into a TTA codon (leucine). After compared thesetwo sequences and other ACCase sequences which were resistant or sensitive toACCase-inhibiting herbicides, this point mutation was considered as an important reason ofthe resistance of plastid ACCase in RR to quizalofop-p-ethyl.(6) Using SYBR Green I Real-time PCR test, the expression of plastid ACCase gene in RRand SS was determined after spraying the quizalofop-p-ethyl at the dosage of 60 g ai ha-1.Theexpression of plastid ACCase gene in RR was inhibited at the beginning and then recovered;the tendency of SS was similar. So the expression of target gene was not the reason of thehigh resistance.To sum up, high resistant biotype only appeared in Geqiushan Farm and the resistancelevels in other ten farms were low. The resistance of RR to quizalofop-p-ethyl was induced bythe reduced sensitiveness of plastid ACCase. From the results of sequence analyse and geneexpression analyse, one point mutation was found compared the sensitive biotype. Theexpression of target gene was similar in RR and SS. So the reduced sensitiveness of plastidACCase in RR was induced mainly by this point mutation. In this study an enhanceddetoxication enzyme (GSTs) was considered as the main reason of the resistance in 853 R andWudalianchi R.更多还原