不同水稻<i>tms5</i>突变位点对雄性不育起点温度的影响

吴明基; 刘华清; 陈建民; 付艳萍; 杨绍华; 宋亚娜; 林艳; 王锋

doi:10.19303/j.issn.1008-0384.2023.07.001

不同水稻tms5突变位点对雄性不育起点温度的影响

doi: 10.19303/j.issn.1008-0384.2023.07.001

福建省农业科学院生物技术研究所/福建省农业遗传工程重点实验室，福建　福州　350003

基金项目: 福建省自然科学基金项目（2020J011359）；福建省农业高质量发展超越“5511”协同创新工程项目（XTCXGC2021002）；福建省科技重大专项（2020NZ08017）

详细信息

作者简介:
吴明基（1976 —），男，博士，助理研究员，主要从事水稻遗传育种研究，E-mail：839845032@qq.com

通讯作者:
王锋（1963 —），男，博士，研究员，主要从事水稻基因工程与遗传育种研究，E-mail：wf@fjage.org

中图分类号: S511
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出版历程
- 收稿日期: 2023-05-12
- 修回日期: 2023-05-22
- 网络出版日期: 2023-08-16
- 刊出日期: 2023-07-28

Critical Sterility-inducing Temperature of Rice Affected by tms5 Mutation Site

Institute of Biotechnology, Fujian Academy of Agricultural Sciences/Fujian Key Laboratory of Agricultural Genetic Engineering, Fuzhou, Fujian 350003, China

摘要

摘要: 目的研究不同水稻tms5突变位点对雄性不育起点温度的影响，探讨不育起点温度遗传调控途径。方法在水稻TMS5的6个外显子上设计11个CRISPR/Cas9基因编辑靶点，依次命名为T501 — T511，构建相应载体转化粳稻品种日本晴和籼稻品种明恢86，获得各靶点的tms5移码突变体。田间自然高温及人工气候箱（设日平均22 、24和28 ℃ 3种温度）条件下分析tms5突变体的花粉碘染及自交结实率，鉴定不育起点温度。结果粳稻日本晴tms5突变体的不育起点温度高于28 ℃，籼稻明恢86的不同tms5突变体不育起点温度为22～28 ℃。此外，同一遗传背景下，通过T501靶点编辑产生的tms5-1移码突变体不育起点温度均显著高于T502靶点的tm5-2突变体，其他位点上的tms5突变体育性特征与tm5-2突变体并无差异。基因表达量分析表明，tms5-1突变体幼穗Ub_L40基因表达量显著低于tm5-2突变体的表达量。结论水稻tms5突变体不育起点温度不仅受遗传背景的影响，tms5基因突变位点不同也会影响不育起点温度，特别是T501位点与其余位点突变体间不育起点温度差异显著，为研究水稻tms5两系不育起点温度的分子机理及遗传调控网络提供了新思路。
- 水稻 /
- CRISPR/Cas9 /
- TMS5 /
- 温敏核不育 /
- 不育起点温度
Abstract: Objective Effects of tms5 mutation site and related genetic regulatory factors in rice on the critical sterility-inducing temperature (CSIT) of the plant were investigated. Method Eleven CRISPR/Cas9 sequentially numbered T501 through T511 were designed to target 6 exons of tms5 to create mutants of Nipponbare (NIP, Oryza sativa ssp. Geng) and Minghui 86 (MH86, O. sativa ssp. Xian). Pollen male fertility and seed setting rate of the mutants were monitored under long day and high temperature field conditions in early August in Fuzhou (NHT) or at 22, 24, and 28 ℃ in phytotrons to identify the CSIT. Result The CSIT of the NIP mutants were higher than 28 ℃, while that of the MH86 mutants between 22 ℃ and 28°C. The tms5-1 T501 mutant had a significantly higher CSIT than the genetically identical tms5-2 counterparts T502, but the other 9 tm5 and the tm5-2 mutants did not differ on it. Expressions of the 3 Ub_L40 in young panicles were lower in the tms5-1 than the tm5-2 mutants of either rice varieties. Conclusion It appeared that the CSIT of tms5 mutants was affected by the genetic factors as well as the mutation site as the tms5-1 mutant T501 was shown significantly differed from the others.
- Rice /
- CRISPR/Cas9 /
- TMS5 /
- thermo-sensitive genic male sterility /
- critical sterility-inducing temperature

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图 1 CRISPR/Cas9系统介导的TMS5基因特异性突变

A：TMS5基因结构的示意图，黑色矩形代表TMS5的6个外显子，图中短竖线所指位置为各靶点在TMS5基因上的位置。B：CRISPR/Cas9介导的TMS5突变实例，每个靶点只列出了一种突变类型， T505、T508、T510靶点列出的是杂合型突变，T511靶点是双突变，其余位点列出的均为纯合型突变；图上三角形处为缺失或插入突变发生的位置。

Figure 1. CRISPR/Cas9-mediated genome editing of tms5

A.Schematic tms5 structure; black rectangles represent 6 exons of tms5; short vertical line refers to position of each target. B. CRISPR/Cas9-mediated tms5 mutants; only one mutation type is listed for each target; T505, T508, and T510 are listed as heterozygous mutations; T511 as biallelic mutation; and remainders as homozygous mutations; triangles point at deletion and insertion sites.

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图 2 T₁代tms5突变体在田间高温下的花粉育性

①A和B：明恢86（A）及日本晴（B）遗传背景的tms5突变体T₁代材料花粉育性；两种背景的tms5突变体（依次为T501至T511靶点突变）育性敏感期处于福州8月初的自然长日高温条件；MH86为明恢86，NIP为日本晴；M-#和N-#分别代表明恢86和日本晴遗传背景的突变体。C：明恢86和日本晴两种遗传背景的野生型、tms5-1突变体及tms5-2突变体穗部开花特征。D： M-T501-#1同一穗上出现可育与不育的颖花。②标尺长度：80 µm（A、B）， 0.5 cm（C、D）。

Figure 2. Pollen fertility of T₁ tms5 mutants under NHT

①Aand B: Pollen fertility of tms5 mutants in MH86 (A) and NIP (B) induced at 11 targets from T501 to T511; NHT was in early August in Fuzhou; M-# and N-# are mutant plants of MH86 and NIP, respectively. C: Flowering characteristics of WT, tms5-1, and tms5-2 mutants of NIP and MH86 backgrounds. D: Both fertile and sterile spikelets were set on same panicle of M-T501-#1. ②Scale bars represent 80 μm for A and B, 0.5 cm for C and D.

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图 3 T₂代tms5突变体在不同温度条件下的花粉育性

① 22℃ 、24 ℃、28 ℃、NHT分别表示明恢86及日本晴遗传背景的tms5-1及tms5-2突变体在人工气候箱22 ℃、24 ℃、28 ℃及福州8月初自然高温条件下(NHT)的花粉育性。MH86为明恢86，NIP为日本晴，M-#和N-#分别代表明恢86和日本晴遗传背景的突变体。②标尺长度：80 µm。

Figure 3. Pollen fertility of T₂ tms5 mutants under varied temperatures

① Pollen fertility of tms5-1 and tms5-2 mutants of MH86 and NIP backgrounds cultivated at 22 ℃, 24 ℃ or 28 ℃ in phytotrons, or under NHT, respectively; M-# and N-# represent mutant plants of MH86 and NIP backgrounds, respectively. ② Scale bars are 80 μm in length.

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图 4 Ub_L40基因的表达量差异分析

A、B和C分别表示明恢86及日本晴tms5突变体Ub_L401、Ub_L402和Ub_L404的表达特征，数据为平均数±标准差，n=3。

Figure 4. Differential expressions of Ub_L40

A, B and C: expressions of UbL401, UbL402, and UbL404 in tms5 mutants of MH86 and NIP, respectively. Data are mean ± standard deviation calculated from 3 replicates.

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表 1 本试验所用sgRNA序列

Table 1. sgRNA applied

靶位点 Target	序列（5′- 3′） Sequence	试验目的 Purpose
T501S	CAGGAACAGCGGCAAGTCATCGC	sgRNA构建/ sgRNA Construction
T501A	AACGCGATGACTTGCCGCTGTTC	sgRNA构建/ sgRNA Construction
T502S	CAGCCACCGCGCCGCCACCGGGT	sgRNA构建/ sgRNA Construction
T502A	AACACCCGGTGGCGGCGCGGTGG	sgRNA构建/ sgRNA Construction
T503S	CAGCACCGTCGAGGGCTACCCCG	sgRNA构建/ sgRNA Construction
T503A	AACCGGGGTAGCCCTCGACGGTG	sgRNA构建/ sgRNA Construction
T504S	CAGCCCATGTACGTCGCCACCCG	sgRNA构建/ sgRNA Construction
T504A	AACCGGGTGGCGACGTACATGGG	sgRNA构建/ sgRNA Construction
T505S	CAGAACCTCGTCCCCCTCGAGAT	sgRNA构建/ sgRNA Construction
T505A	AACATCTCGAGGGGGACGAGGTT	sgRNA构建/ sgRNA Construction
T506S	CAGGTAGGGGTATGTGATATACA	sgRNA构建/ sgRNA Construction
T506A	AACTGTATATCACATACCCCTAC	sgRNA构建/ sgRNA Construction
T507S	CAGATCAAGCAGCTGAAGCTGTC	sgRNA构建/ sgRNA Construction
T507A	AACGACAGCTTCAGCTGCTTGAT	sgRNA构建/ sgRNA Construction
T508S	CAGTGCTTTTACCGGAGATACGA	sgRNA构建/ sgRNA Construction
T508A	AACTCGTATCTCCGGTAAAAGCA	sgRNA构建/ sgRNA Construction
T509S	CAGTGGGCACACCCATCTGTTTG	sgRNA构建/ sgRNA Construction
T509A	AACCAAACAGATGGGTGTGCCCA	sgRNA构建/ sgRNA Construction
T510S	CAGTTCTGCTCGTTATACCGCAG	sgRNA构建/ sgRNA Construction
T510A	AACCTGCGGTATAACGAGCAGAA	sgRNA构建/ sgRNA Construction
T511S	CAGAAGTAGAGTTCATGCATTGA	sgRNA构建/ sgRNA Construction
T511A	AACTCAATGCATGAACTCTACTT	sgRNA构建/ sgRNA Construction

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表 2 供试引物

Table 2. Primers selected

引物名 Primer name	序列 Sequence (5′-3′)	试验目的 Purpose
HptF	AGGTCAGGCTCTCGCTAAAC	转基因阳性株检测/ Identify transgenic plants
Hpt-R	ACGTAAGGGATGACGCACAAT	转基因阳性株检测/ Identify transgenic plants
TMS5F	CCATCGTGCTTCGTGCCA	TMS5基因扩增、测序 PCR and Sanger sequence for TMS5
TMS5R	GAGTTCTTGGTACATGAGTGC	TMS5基因扩增、测序 PCR and Sanger sequence for TMS5
UBL401F	CTACCCCAAGGGGATCGAG	Real-Time PCR
UBL401R	GCAAGGCGGTCGATTGAACT	Real-Time PCR
UBL402F	TCGCTCAGGGTCCTCGCCTA	Real-Time PCR
UBL402R	CTAGATTCGGCATCCAGTAG	Real-Time PCR
UBL404F	TACACCATCCAGGAGCCCA	Real-Time PCR
UBL404R	GGTAGCTGGGCATACGAAG	Real-Time PCR
ActinF	CGTCTGCGATAATGGAACTG	Real-Time PCR
ActinR	TCTGGGTCATCTTCTCACGA	Real-Time PCR

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表 3 各载体转化T₀代植株突变分析

Table 3. Mutation of T₀ transgenic plants

载体 Construct	靶点位置 Target site	品种 Variety	转基因植株数 Numbers of transgenic plants	含突变植株数 Numbers of mutation plants	突变率 Mutation rates/%
TMS501	TMS5第一外显子 First exon of TMS5	Nipponbare	21	15	71.4
TMS501		MH86	18	16	88.9
TMS502		Nipponbare	22	21	95.5
TMS502		MH86	11	11	100.0
TMS503		Nipponbare	29	11	37.9
TMS503		MH86	9	6	33.3
TMS504		Nipponbare	21	11	52.4
TMS504		MH86	15	7	46.7
TMS505		Nipponbare	21	6	28.6
TMS505		MH86	21	7	33.3
TMS506	TMS5第二外显子 2nd exon of TMS5	Nipponbare	19	13	68.4
TMS506		MH86	24	21	87.5
TMS507		Nipponbare	10	2	20.0
TMS507		MH86	11	1	9.1
TMS508	TMS5第三外显子 3rd exon of TMS5	Nipponbare	10	1	10.0
TMS508	TMS5第三外显子 3rd exon of TMS5	MH86	18	2	11.1
TMS509	TMS5第四外显子 4th exon of TMS5	Nipponbare	21	14	66.6
TMS509	TMS5第四外显子 4th exon of TMS5	MH86	18	13	72.2
TMS510	TMS5第五外显子 5th exon of TMS5	Nipponbare	10	1	10.0
TMS510	TMS5第五外显子 5th exon of TMS5	MH86	15	2	13.3
TMS511	TMS5第六外显子 6th exon of TMS5	Nipponbare	14	12	85.7
TMS511	TMS5第六外显子 6th exon of TMS5	MH86	11	9	81.8

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表 4 T₁代tms5材料结实率

Table 4. Seed setting rates of T₁ generation of tms5 mutants

背景 Background	材料 Lines	突变序列 § Mutation sequences	平均结实率 †Average seed setting rates/%
明恢86 MH86	MH86		87.5±4.3 a
	M-T501-#1	T501gaacagcggcaagtcatAcgccgg	6.9±9.6 b
	M-T501-#2	T501gaacagcggcaagtcatTcgccgg	4.0±5.6 b
	M-T501-#3	T501gaacagcggcaagtcatCcgccgg	5.4±6.9 b
	M-T502-#1	T502ccaccgcgcc-------ggtc	0 c
	M-T502-#2	T502ccaccgcgccgccaccgCggtcgg	0 c
	M-T502-#3	T502ccaccgcgccgccaccgTggtcgg	0 c
	M-T511-#1	T511gtagagttcatgcat-gaaggaa	0 c
	M-T511-#2	T511gtagagttcatgcatTtgaaggaa	0 c
	M-T511-#3	T511gtagagttcatgcatAtgaaggaa	0 c
	Nipponbare		89.5±2.0 a
日本晴 Nipponbare	N-T501-#1	T501gc-----------------gacc	48.9±22.1 c
	N-T501-#2	T501gaacagcggcaagtca--gccgg	54.2±15.8 b
	N-T501-#3	T501gaacagcggcaagtcatTcgccgg	44.1±12.2 c
	N-T502-#1	T502ccaccgcgccg------Cggtcggc	10.1±10.2 d
	N-T502-#2	T502ccaccgcgccgccaccgAggtcggc	8.8±10.1 d
	N-T502-#3	T502ccaccgcgccgccaccgTggtcgg	9.4±9.6 d
	N-T511-#1	T511gtagagttcatgca--gaaggaa	9.6±8.4 d
	N-T511-#2	T511gtagagttcatgcatCtgaaggaa	10.6±10.0 d
	N-T511-#3	T511gtagagttcatgcatAtgaaggaa	10.4±8.9 d
①§靶点位置的大写字母和短划线分别表示插入与缺失突变类型。 ②†结实率为10穗结实率的平均值±标准差。③同列数据后不同小写字母表示依据LSD多重比较，同一遗传背景材料间的差异达显著水平（P＜0.05），下同。 ①§ Capital letters and dashes at target sites indicate type of insertion and deletion mutations, respectively. ②† Average seed setting rates are mean ± standard deviation calculated from 10 panicles. ③ Data with different lowercase letters on same column indicate significant difference according to LSD multiple comparison (P＜0.05), Same for below.

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表 5 T₂代tms5 突变体花粉育性及自交结实率

Table 5. Pollen fertility and seed setting rates of T₂ tms5 mutants

植株编号 Plant ID	花粉黑染率 Stained pollens rate/%				自交结实率 Seed setting rate/%
植株编号 Plant ID	22 ℃	24 ℃	28 ℃	自然条件 Natural condition	22 ℃	24 ℃	28 ℃	自然条件 Natural condition
MH86	86.7±7.8 a	87.1±6.9 a	87.2±5.8 a	88.3±7.1 a	83.4±8.7 a	83.1±6.4 a	85.2±7.2 a	85.9±6.5 a
M-T501-#1	87.5±10.3 a	61.6±13.5 c	0 b	20.5±11.5 c	67.8±20.3 b	30.7±12.3 c	0 b	8.5±10.6 c
M-T501-#2	80.1±12.0 b	62.7±10.4 c	0 b	28.2±16.3 b	65.3±16.5 bc	40.2±20.5 b	0 b	21.3±16.3 b
M-T501-#3	83.3±8.7 ab	71.2±20.2 b	0 b	25.6±15.6 bc	62.2±8.5 c	41.4±17.4 b	0 b	18.7±21.5 b
M-T502-#1	12.1±10.6cd	0 d	0 b	0 d	1.5±0.3 d	0 d	0 b	0 d
M-T502-#2	9.3±5.8d	0 d	0 b	0 d	1.8±0.6 d	0 d	0 b	0 d
M-T502-#3	13.9±10.1 c	0 d	0 b	0 d	1.9±1.3 d	0 d	0 b	0 d
Nipponbare	92.5±5.9a	90.6±7.3 a	91.1±6.3 a	92.5±6.5 a	87.6±8.2 a	88.1±9.3 a	87.8±6.6 a	90.5±2.3 a
N-T501-#1	84.2±5.7 b	78.2±20.4 b	35.1±11.3 c	67.4±6.9 c	84.1±8.3 a	66.4±10.6 b	13.1±7.2 c	45.2±33.1 c
N-T501-#2	86.2±19.5 b	80.2±12.9 b	37.1±8.8 bc	75.7±15.2 b	83.2±7.8 a	68.5±10.3 b	14.8±5.1 bc	52.1±35.8b
N-T501-#3	82.3±21.2 b	80.1±18.1 b	40.1±10.5 b	69.1±9.6 c	83.1±10.9 a	50.5±12.2 c	18.6±9.5 b	50.7±28.3 bc
N-T502-#1	82.2±6.8 b	70.0±20.2 c	6.1±2.5 d	25.2±19.0 e	72.3±9.2 bc	35.3±28.1 d	3.5±1.6 c	14.1±11.2 d
N-T502-#2	85.5±6.3 b	72.2±18.5 c	7.6±6.6 d	31.1±16.9d	75.8±12.3 b	33.4±16.9 d	7.5±8.1 c	18.6±10.3 d
N-T502-#3	84.1±9.2 b	70.5±16.7 c	9.2±8.1 d	26.3±27.7 e	69.6±9.2 c	25.4±20.3 e	4.2±2.5 c	17.6±16.3 d
花粉黑染率及自交结实率均为5株的平均值，自然条件指福州8月份的长日高温条件。 Data are means on 5 plants; NHT was defined as long day and high temperature in August in Fuzhou.

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