【作者】 张磊；

【导师】 刘克德；

【作者基本信息】 华中农业大学 ， 作物生物技术， 2006， 硕士

【摘要】 苯达松除草剂用于水稻田阔叶类杂草和莎草的防治已超过20年。利用苯达松敏感突变体bel，可以清除杂交水稻制种过程中混杂的自交种。在7356株F₂代单株中，筛选到1776株对苯达松敏感的植株。利用这些敏感植株对苯达松抗性基因Bel进行精细定位，最终将Bel基因定位在两个RFLP标记L5和P17之间一个36kb的区域内。序列分析表明，这个区域共有5个可能的候选基因，它们分别是乙烯反应基因OsER33和四个重复的P450相关基因CYP81A5、CYP81A6、CYP81A7和CYP81A8。通过重组分析，发现RFLP标记P17位于候选基因CYP81A8的编码区域且与Bel基因之间有一个重组单株，因此，剔除了CYP81A8这个候选基因。在野生型和突变体之间对这四个候选基因进行比较测序，在候选基因ER33、CYP81A5和CYP81A7的编码区域内没有发现任何差异，但在候选基因CYP81A6的编码区域发现了一个单碱基G的缺失，对CYP81A6进行全长cDNA分析，发现这个单碱基G的缺失发生在CYP81A6起始密码子下游1332bp的地方。这个G的缺失使候选基因CYP81A6的编码区域发生了移码突变，产生了一个提前终止子，使P450基因CYP81A6编码的氨基酸序列发生了改变，造成了亚铁血红素绑定模体的丢失，而这个模体是细胞色素P450所共有的最具特征性的结构，因此，可以推测发生了移码突变的候选基因CYP81A6就是水稻中的抗性基因Bel。Bel基因的精细定位对于将bel等位基因转入光温敏雄性不育系是非常有用的，Bel基因的克隆也必将促进具有苯达松抗性的转基因作物的发展。更多还原

【Abstract】 Bentazon have been used for selective control of broadleaf weeds and sedges in ricefields for more than 20 years. A bentazon susceptible mutant, bel, was previouslyidentified for the purpose of allowing these herbicides to be used for removing falsehybrids from hybrid rice. Using 1,776 susceptible plants from a population ofapproximately 8000 F₂ individuals, we constructed a fine map for the Bel locus anddelimited it to a 36kb DNA fragment between two RFLP makers, L5 and P17.Bioinformatic analysis indicated that there are five genes within this interval, anethylene-responsive OsER33 gene and four tandem repeats of cytochrome P450 genesdesignated as CYP81A5, CYP81A6, CYP81A7 and CYP81A8, respectively. Comparativesequencing could not find any differences in the coding regions of the OsER33、CYP81A5、CYP81A7 and CYP81A8 genes between the mutant bel and its wild-typeprogenitor W6154S, but did identify a single base G deletion at position+1,332 bpdownstream from the translation start codon of CYP81A6. This deletion introduces apremature stop codon and leads to the loss of the heme-binding motif, which is essentialfor cytochrome P450 function because it contain an absolutely conserved cysteine thatserves as the fifth ligand to the heme iron. CYP81A6 presumably functions as ahydroxylase for the detoxification of bentazon herbicides in rice. A genespecific cleavedamplified polymorphic sequence marker and tightly linked flanking marker weredeveloped that will be very useful for selection of the bel allele transferred tophotoperiod-/thermo-sensitive genic male sterility and CMS lines in hybrid rice breedingprograms.更多还原