EMS诱变水稻创制抗咪唑啉酮除草剂种质

为了创制水稻非转基因抗除草剂种质,丰富我国水稻轻简化栽培杂草防治所需的配套种质资源,本研究以0.5%(w/v)甲磺酸乙酯(EMS)水溶液诱变水稻种子14 h,然后以咪唑啉酮类除草剂筛选3~4叶期的M₂水稻幼苗。结果发现,M₂幼苗茎叶喷雾160 mg·L^-1甲咪唑烟酸15 d后,非抗性苗黄化、生长被抑制甚至枯死,抗性苗叶色正常、株高增长明显。M₃抗性水稻对甲咪唑烟酸的抗性能稳定遗传且其抗性水平不低于1 200 mg·L^-1甲咪唑烟酸,其抗性水平是野生型对照的100倍以上。分别克隆M₃抗除草剂突变体和野生型的乙酰乳酸合成酶基因(ALS),序列比对分析发现M₃抗性水稻的ALS基因开放阅读框在第1 879、1 880 nt由AG碱基突变为CT碱基,导致编码的第627位氨基酸由丝氨酸(AGT)突变为亮氨酸(CTT),或在第1 880 nt由G碱基突变为A碱基,导致编码的第627位氨基酸由丝氨酸(AGT)突变为天冬酰胺(AAT)。间接比色法测定M₃抗除草剂突变体与野生型的ALS对甲咪唑烟酸的敏感性,结果表明抗性水稻对甲咪唑烟酸的耐受性是野生型的109倍。本研究获得了水稻ALS第627位丝氨酸突变为亮氨酸或天冬酰胺的抗咪唑啉酮新种质材料,获得的抗咪唑啉酮水稻材料的抗性倍数高,能稳定遗传,具有生产应用价值。

Screening of Imidazolinone-Resistant Rice From EMS-Mutated Populations

In order to create non-transgenic herbicide-tolerant rice germplasm which would control the weed easily in rice simplified cultivation in China, rice seeds were treated with 0.5% (w/v) ethyl methanesulfonate for 14 h. The resulted M₂ generation at 3-4 leaf stage were sprayed with 160 mg·L^-1 of imazapic. The non-resistant seedlings turned into yellow, stunted or even died, while the resistant seedlings were normal and the plant height increased evidently. The resistance of M₂ to imazapic was stably inherited to their progeny M₃, reached up to 1 200 mg·L^-1 imazapic, and 100 times as high as the wild type did. The acetolactate synthase(ALS) genes in the imidazolinone-resistant M₃ mutants and the wild-type were cloned by PCR. Sequence analysis revealed that the open reading frame of ALS gene in the imidazolidone-resistant M₃ plants carry two points mutations A1879C and G1880T, which results in a substitution of serine(AGT) to leucine(CTT) at amino acid 627, or a point mutation G1880A, which results in a substitution of serine(AGT) to asparagine(AAT) at amino acid 627. A colorimetry method was used to compare the enzyme sensitivity of ALS to imazapic between the herbicide resistant M₃ mutant and the wild type. The tolerance of ALS enzyme to imazapic in the imidazolinone resistant rice was 109-fold of that in the wild type. Taken together, imidazolidone- resistant rice mutants with substitution of serine to leucine at amino acid 627 was a novel mutation and was first reported. The imidazolidone-resistant rice obtained in this study showed stable and high resistance to imazapic, make it potential in the application of weed control.