Development of Thermo-Sensitive Male Sterile Rice with CRISPR/Cas9 Technology
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摘要: 水稻光温敏核不育系的选育是两系法杂交稻选育的关键环节。温敏不育基因tms5在生产上应用最为广泛，通过CRISPR/Cas9基因组编辑技术突变可育水稻品种的TMS5基因，可以快速选育水稻温敏型两系不育系。利用CRISPR/Cas-9技术创制水稻温敏核不育系的试验结果表明：通过构建CRISPR/Cas-9基因编辑载体TMS502，转化优良中间材料GH89获得10株T0代转基因苗，有6株产生了插入或缺失突变，其中纯合突变株tms5-1和双等位突变株tms5-4在T0代就表现出温敏不育特征；T1代非转基因单株中共有4种纯合突变基因型，突变类型与T0代检测结果一致，并未产生新的突变；T2代tms5不育系材料的温敏不育起点温度约为24℃，符合水稻两系不育系选育要求。研究结果证明了利用基因编辑技术培育水稻两系不育系的有效性，所创制的不育系可供进一步选育利用。
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关键词:
- 杂交水稻 /
- 两系不育系 /
- 基因 /
- CRISPR/Cas9 /
- tms5
Abstract: Breeding of photoperiod or thermo-sensitive male sterile line is the key for the development of two-line hybrid rice. The allele of tms5 is the major TGMS genetic resource for two-line hybrid rice breeding in China. Targeted editing on TMS5 of fertile rice using CRISPR/Cas-9 technology could effectively create thermo-sensitive genic male sterile rice. This study used the approach to edit the TMS5 gene successfully. The CRISPR/Cas-9 vector of TMS502 was constructed, then the T-DNA was transformed into GH89. Ten transgenic plants were thus regenerated, with 6 of them containing either the insertion or deletion mutations. The T0 plant of tms5-l was a homozygous mutant, and tms 5-4 a bi-allelic mutant. Both of them displayed thermo-sensitive male sterile phenotype. There were 4 homozygous mutant genotypes in the T1 generation of the non-transgenic plants. The mutants showed the same characteristics as shown in the T0 generation without any new mutations. Male fertility of T2 generation of tms5 mutants was tested in natural high temperature environment and in phytotrons at 3 temperature levels. The results indicated that the critical sterility-inducing temperature of the tms5 sterile lines was approximately 24 C, which satisfied the requirements for two-line male sterile line breeding. The successful conversion of GH89 line into a two-line male sterile rice using the gene editing technology suggested that CRISPR/Cas-9 could be applied to facilitate the development of two line hybrid rice breeding in the future.-
Key words:
- hybrid rice /
- two-line male sterile line /
- gene /
- CRISPR/Cas-9 /
- tms5
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图 1 PCR鉴定T0代转基因阳性植株
注：M为分子量标记，H2O是以无菌水为模板的阴性对照，+为潮霉素阳性对照。
Figure 1. Positive PCR identification of T0 transgene plants
图 2 T1代tms5纯合突变体测序
注：A为GH89野生型；B为tms5-1-1(插入A)；C为tms5-2-1(插入T)；D为tms5-5-5(缺失G)；E为tms5-9-1(缺失CCACCGG)。
Figure 2. Representative sequences of T1 homozygous tms5 mutants
图 3 日均温22℃和28℃的温度条件下tms5-1突变体与野生型的花药形态及花粉育性
注：标尺长度上四图为1 mm，下四图为60 μm。
Figure 3. Anther morphology and pollen fertility of tms5-1-wt and tms5-1-1 under day average temperature of 22℃ or 28℃
表 1 试验所用引物
Table 1. Primers used for the study
引物名 序列(5′-3′) 试验目的 TMS502-S CAGCCACCGCGCCGCCACCGGGT sgRNA构建 TMS502-A AACACCCGGTGGCGGCGCGGTGG sgRNA构建 TMS5F1 CCATCGTGCTTCGTGCCA TMS5基因扩增、测序 TMS5R1 CTGCTTGATCTCGCTCCC TMS5基因扩增、测序 HptF AGGTCAGGCTCTCGCTAAAC 转基因阳性株检测 HptR ACGTAAGGGATGACGCACAAT 转基因阳性株检测 
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表 2 人工气候箱处理的光温条件
Table 2. Light and temperature conditions in phytotron
时间/
(时：分)日加权平均温度/℃ 光照/
(μmol·m-2·s-1)28 24 22 2:00 25 20 18 0 5:00 25 23 21 0 5:30 25 23 21 150 8:00 27 25 23 300 11:00 33 27 25 480 16:00 29 25 23 300 20:00 27 23 21 150 2:00 25 20 18 0
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表 3 tms5突变体花粉育性鉴定
Table 3. Pollen fertility of tms5 mutants
材料 花粉黑染率/% 自交结实率/% 22℃ 24℃ 28℃ 自然条件 22℃ 24℃ 28℃ 自然条件 tms5-1-1 75.1 1.2 0 0 36.2 0 0 0 tms5-5-5 78.5 0.8 0 0 38.1 0 0 0 tms5-9-1 83.1 0.5 0 0 35.2 0 0 0 tms5-1-wt 90.6 91.2 91.8 89.6 85.1 83.5 82.3 81.4 注：花粉黑染率及自交结实率均为10株的平均值，自然条件指福州7月1~20日的长日高温条件。
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