寡聚核苷酸介导的基因突变技术创制抗除草剂烟草新种质

Creating new tobacco germplasm with herbicide-resistance based on oligonucleotide-mediated mutagenesis(OMM)technology

关键词:  烟草, ALS基因, 除草剂抗性, 氯磺隆, OMM技术

Key words:  tobacco, ALS gene, herbicide resistance, chlorsulfuron, OMM technology

摘要:

为进一步优化烟草(Nicotiana tabacum)品种‘K326'的种质,该研究采用寡聚核苷酸介导的基因突变(oligonucleotide-mediated mutagenesis,OMM)技术,利用植物中支链氨基酸合成途径中第一个关键酶——乙酰乳酸合成酶(acetolactate synthase,ALS)突变后烟草对氯磺隆除草剂不敏感且产生抗性的特征,以及NCBI报道的ALS基因序列同源克隆了烟草品种‘K326'中的ALS基因,并根据ALS基因序列设计用于定点突变的RNA/DNA嵌合体,导入烟草品种‘K326'创制对氯磺隆除草剂具有抗性的烟草新种质。结果表明:(1)烟草品种‘K326'具有2条ALS基因,即ALS SuRA和ALS SuRB,大小分别为2 004 bp和2 010 bp。(2)根据2个基因的保守位点ALS SuRA 588脯氨酸位点和ALS SuRB 1719色氨酸位点设计用于ALS基因核苷酸第588位点的Chl-588嵌合体和第1719位点的Chl-1719嵌合体。(3)利用基因枪成功将这2个片段导入烟草愈伤组织,愈伤组织依次经抗性芽分化和生根,共获得氯磺隆抗性植株22株。(4)抗性植株ALS酶活性测定显示,8株抗性植株具有较强的活性,进一步对抗性植株中跨突变位点保守扩增、测序,最终确定有2株(f11和b18)分别在588、1719位点产生定点突变。综上认为,该研究在获得烟草品种‘K326'抗氯磺隆新种质同时,也为培育抗性烟草新种质提供了理想的亲本材料。

Abstract:

To further optimize the germplasm of tobacco(Nicotiana tabacum)‘K326', oligonucleotide-mediated mutagenesis(OMM)technique was used in this study. The ALS gene in tobacco ‘K326' was cloned by using the ALS gene sequence reported by NCBI based on the insensitivity and resistance of tobacco to the chlorsulfuron herbicide after the mutation of acetolactate synthase(ALS), the first key enzyme in the branched-chain amino acid synthesis pathway in plants. According to ALS gene sequence, RNA/DNA chimeras were designed for site-directed mutagenesis and introduced into tobacco ‘K326' to create a new tobacco germplasm resistant to chlorsulfuron herbicide. The results were as follows:(1)‘K326'had two ALS genes, namely ALS SuRA and ALS SuRB, with the sizes of 2 004 bp and 2 010 bp, respectively.(2)The chimeras of Chl-588 and Chl-1719 at site 588(Pro site)and site 1719(Try site)of ALS gene were designed according to the conserved sites, ALS SuRA 588 proline site and ALS SuRB 1719 tryptophan site.(3)The two fragments were successfully introduced into tobacco callus by gene gun, and the callus were successively differentiated and rooted by resistant bud, and a total of 22 chlorsulfuron-resistant plants were obtained.(4)The activity of ALS enzyme in resistant plants showed that eight resistant tobacco plants had strong activity, and in the further antagonistic plants, conservative amplification and sequencing of cross-mutation loci resulted in site-directed mutations in two lines(line f11 and line b18)at loci 588 and 1719, respectively. In conclusion, this study provides an ideal parental material for cultivating new germplasm of resistant tobacco while obtaining the new germplasm of ‘K326' resistant to chlorsulfuron.