TALEN-mediated Editing of Photoperiod-temperature-sensitive Male Sterility PMS3 Gene in Rice
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摘要:
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。 Abstract: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 T2 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 T1 generations. The sequencing showed 3 homozygous mutation genotypes in Nipponbare and 5 in Minghui 86 background. Under the natural long-day-high-temperature, the T2 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. -
Key words:
- rice /
- PTGMS /
- PMS3 /
- gene editing
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图 1 T1代pms3纯合突变体序列
注：WT表示野生型序列; N-pms3-#和M-pms3-#分别代表日本晴和明恢86背景中的突变植株。在野生型序列中，osa-smR5864的编码序列标注为灰底字母，其中带下划线的碱基G为农垦58S的原始突变位点。黑色三角形所处位置为缺失突变发生的位置，空心三角形处为插入突变发生的位置。
Figure 1. Sequencing chromatograms of mutations in T1 homozygous pms3 mutant lines
Note: WT indicates wild-type sequence; N-pms3-# and M-pms3-# represent the mutant plants in Nipponbare and MH86 backgrounds, respectively. In the wild type sequence, the osa-smR5864 sequence is marked in gray letters, the underlined G is the original mutation site of Nongken 58S.The deletion and insertion sites are indicated by black triangle and hollow triangle, respectively.
图 2 福州自然长日高温下pms3突变体花粉育性正常
注：标尺长度上4图为1 mm，下4图为100 μm。
Figure 2. Anther morphology and pollen fertility of pms3 mutants showing similarities to those of wild types under natural long-day-high-temperature conditions in Fuzhou
Note: Scale length for the upper four graphs is 1 mm, for the lower four graphs is 100 μm.
表 1 试验所用引物
Table 1. Primers employed
引物
Primer序列(5′- 3′)
Sequence(5′- 3′)试验目的
ApplicationHptF AAGCTGCATCATCGAAATTG 转基因阳性株检测 HptR TCGTTATGTTTATCGGCACT 305F CTCCCCTTCAGCTGGACAC TALEN质粒验证 306R AGCTGGGCCACGATTGAC LDF1 TAAGAACTGCTGCTCCAAAT PMS3基因扩增、测序 LDR1 CGGCTCCGTTATAGATAGAC 
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表 2 T1代pms3纯合突变类型
Table 2. Homozygous mutation types of T1 pms3 lines
品种背景
Background株系号
Line突变序列
Mutation sequences突变类型
Mutation types日本晴
NipponbareN-pms3-#1 ATGGATGGTAGACAA-CAATG -1 N-pms3-#2 ATGGATGGTAGA----CAATG -4 N-pms3-#3 ATTGGATGGTAGACAAACAATG +1 N-pms3-#4 ATGGATGGTAGACAA-CAATG -1 明恢86
MH86M-pms3-#1 ATGGATGGATCTAGAGTATCTGAA +12 M-pms3-#2 ATGGATGGTAGA-----------… -82 & +23 M-pms3-#3 ATGGATGGTAGAC---CAATG -3 M-pms3-#4 ATGGATGGTAGA----CAATG -4 M-pms3-#5 …--------------------TG -62 注：短划线和灰底字母分别表示缺失和插入突变，…表示已省略的序列。带下划线的G碱基为农垦58S的原始突变位点。+：插入，-：缺失，具体数字表示所涉及的核苷酸数。
Note:Deletion, insertion and omitted sequences are indicated by dashes, gray letters and dots, respectively. The underlined G is original mutation site of Nongken 58S. +: insertion, -: deletion. Data indicate number of nucleotides involved.
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表 3 T2代pms3纯合突变体花粉育性及结实率
Table 3. Pollen fertility and seed setting rates of T2 pms3 mutant lines
材料
Lines花粉黑染率
Stained pollens rate/%每穗结实率
Seed setting rates of individual panicles/%平均结实率
Average seed setting rates/%明恢86MH86 90.3±6.2 a 95.0 86.1 93.6 94.1 89.7 78.9 83.6 82.3 76.8 91.2 87.1±6.6 a M-pms3-#1 89.9±5.1 a 88.1 92.7 91.0 89.5 85.3 90.2 77.5 81.6 80.8 85.4 86.2±5.0 a M-pms3-#2 91.5±6.1 a 91.2 89.3 90.6 88.5 79.9 91.4 87.3 82.2 78.4 86.7 86.6±4.8 a M-pms3-#5 90.5±5.8 a 90.8 83.4 84.5 78.8 91.3 92.8 76.3 87.2 80.3 87.6 85.3±5.6 a 日本晴Nipponbare 92.1±6.8 a 97.8 87.6 85.5 93.0 91.4 82.4 88.9 89.6 88.2 88.6 89.3±4.2 a N-pms3-#1 91.8±5.5 a 95.3 89.2 86.3 91.1 93.9 81.5 87.8 86.5 90.1 84.3 88.6±4.2 a N-pms3-#2 92.0±4.8 a 86.2 90.2 93.1 86.9 90.8 89 92.3 89.6 93.5 86.0 89.8±2.8 a N-pms3-#3 91.9±6.0 a 89.0 91.5 88.7 92.1 85.2 90.8 87.4 90.1 90.2 91.0 89.6±2.1 a 培矮64S P64S 0 0 0 0 0 0 0 0 0 0 0 0 注：平均花粉黑染率为10穗黑染率的平均值±标准差。同列数据后无相同小写字母表示在0.05水平差异显著(P < 0.05)。
Note:The average pollen stained rates were the mean ± standard deviation calculated from 10 panicles. The average seed setting rates were the mean ± standard deviation calculated from 10 panicles and different lowercase letters behind the number in the sane column indicated significant difterences(P < 0.05).
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