利用TALEN技术编辑水稻光温敏核不育基因PMS3

TALEN-mediated Editing of Photoperiod-temperature-sensitive Male Sterility PMS3 Gene in Rice

【目的】利用TALEN技术定向突变水稻pms3的原始突变区域,创制不同类型osa-smR5864小RNA编码序列突变体,鉴定不同突变体的雄性育性光温反应变化规律,为基于定向编辑水稻PMS3基因的方法培育水稻两系不育系材料提供科学依据。【方法】构建基因编辑载体TALEN-PMS3,转化日本晴和明恢86,通过测序鉴定pms3突变体,利用福州夏季自然长日高温鉴定T2代pms3突变体的雄性育性及自交结实率。【结果】转化获得25个转基因T0代克隆,其中日本晴有4个克隆产生了突变,明恢86有5个克隆产生了突变,突变率分别为40.0%和33.3%。在T1代非转基因材料中,日本晴背景中有3种纯合突变类型,明恢86中有5种纯合突变类型。在福州夏季自然长日高温条件下,T2代pms3突变体能够产生正常可育的花粉,自交结实率正常,并未表现出类似培矮64S的光温敏雄性不育特征。【结论】本研究通过基因编辑获得的多种类型水稻pms3突变体并不能产生光温敏雄性核不育表型,说明了pms3位点调控水稻光温敏核不育性分子机制的复杂性。

【Objective】TALEN technology was employed to alter the original mutant region in pms3 gene of rice for creating different genotypes of small RNA osa-smR5864 mutants in studying the photoperiod-temperature-sensitive male sterility (PTGMS) phenotypes of pms3 mutants to provide information for breeding two-line sterile rice by means of targeted editing of PMS3 .【Method】The TALEN- PMS3 vector was constructed and transformed into Nipponbare and Minghui 86 with the pms3 mutants identified by sequencing. The T 2 generation of the mutants were planted under the natural long-day-high-temperature conditions in Fuzhou. The male fertility and seed setting rates of the mutants were determined.【Result】 Among the 25 transgenic plants regenerated,4 clones of Nipponbare and 5 of Minghui 86 contained the target mutation with the mutation rates of 40% and 33.3%,respectively. The non-transgenic mutant plants were obtained in T 1 generations. The sequencing showed 3 homozygous mutation genotypes in Nipponbare and 5 in Minghui 86 background. Under the natural long-day-high-temperature,the T 2 pms3 mutants developed normal fertile pollens with a normal seed setting rate. But the pms3 mutants did not show a PTGMS similar to P64S.【Conclusion】The multiple genotypes of pms3 mutants were successfully obtained. However,no PTGMS phenotype could be generated. It suggested that the molecular mechanism of pms3 locus regulating the rice PTGMS was highly complex.