利用CRISPR/Cas9技术创制广亲和水稻温敏雄性不育系

doi:10.16819/j.1001-7216.2023.221006

摘要/Abstract

摘要：

【目的】为突破传统杂交转育方法对水稻两系不育系选育的限制，利用CRISPR/Cas9技术对籼型恢复系台恢31的温敏雄性核不育基因TMS5进行编辑，以获得全新水稻温敏雄性不育系。【方法】选择水稻温敏不育基因TMS5第1外显子的2段序列作为靶点，构建双靶点pHUE411-TMS5-gRNA载体，通过农杆菌侵染获得116株T₀代转基因植株。【结果】经鉴定获得3种类型纯合突变体tms5-1、tms5-2和tms5-3，每一种突变均导致TMS5氨基酸序列的缺失。分期播种试验结果显示，幼穗分化敏感期温度为27℃时，tms5-1和tms5-3自交结实率分别为0.56%、0.03%，表现为高度不育，而tms5-2则完全不育。与野生型台恢31相比，不育系TB52S（即tms5-2）单株穗数增多，穗长变短，株高及每穗总粒数相较WT均下降25%，花药发白瘦小，无花粉粒充实，柱头、颖壳没有显著差异。TB52S幼穗分化4期时进行温度处理，确认TB52S的育性转换温度为26℃。TB52S分别与籼、粳型恢复系配组，F₁的结实率超过90%。【结论】利用CRISPR/Cas9技术对籼型恢复系台恢31的温敏雄性核不育基因TMS5进行编辑，获得能够稳定遗传的水稻广亲和温敏雄性不育系TB52S，为两系不育系选育提供新的技术支撑。

关键词: CRISPR/Cas9, 水稻, 幼穗分化敏感期, 自交结实, 育性转换温度

Abstract:

【Objective】In order to break the fetters in traditional hybridization in the breeding of genic male sterile rice lines, the thermo-sensitive genic male sterility gene TMS5 was edited by CRISPR/Cas9 technology to create new genic male sterile lines using indica restorer line Taihui 31 as material.【Method】Two sequences in the first exon of TMS5 were chosen as target sites for the construction of vector pHUE411-TMS5-gRNA and 116 T₀ generation transgenic plants were obtained by Agrobacterium infection method.【Result】Three homozygous mutants, tms5-1, tms5-2 and tms5-3 with amino acid deletion in TMS5 were verified by PCR and sequencing. The split sowing experiment showed that the self-fertilization rate of tms5-1 and tms5-3 were 0.56% and 0.03% at 27℃, respectively. Meanwhile, at the same temperature, the pollen of tms5-2 turned to be completely sterile. Compared with Taihui 31 (WT), TB52S (tms5-2) had more panicles, shorter panicle length. Its plant height and grain numbers per panicle reduced to nearly three quarters of WT. Furthermore, except for smaller anther with no pollens, the stigma, palea and lemma of TB52S didn’t change significantly as compared with WT. The fertility transfer temperature of TB52S was confirmed as 26℃ according to the temperature test during the IV phase of young panicle differentiation. Last but not least, the seed setting rate of hybrid F₁derived from the cross between TB52S and indica or japonica restorer lines was beyond 90%.【Conclusion】Using CRISPR/Cas9 technology, we obtained thermo-sensitive genic male sterile line TB52S which was genetically stable. It provides new technical support for the breeding of genic male sterile lines.

Key words: CRISPR/Cas9, rice, young panicle differentiation sensitive period, self-fertilization, critical temperature of fertility alternation

段敏, 谢留杰, 高秀莹, 唐海娟, 黄善军, 潘晓飚. 利用CRISPR/Cas9技术创制广亲和水稻温敏雄性不育系[J]. 中国水稻科学, 2023, 37(3): 233-243.

DUAN Min, XIE Liujie, GAO Xiuying, TANG Haijuan, HUANG Shanjun, PAN Xiaobiao. Creation of Thermo-sensitive Genic Male Sterile Rice Lines with Wide Compatibility Based on CRISPR/Cas9 Technology[J]. Chinese Journal OF Rice Science, 2023, 37(3): 233-243.

图/表 13

表1 本研究涉及的PCR扩增及测序引物

Table 1. Primers used for amplification and sequencing.

引物名称 Primer	寡核苷酸序列 (5’-3’) Oligonucleotide sequence (5’-3’)
T5T1-BsF	ATATATGGTCTCTGGCGAACAGCGGCAAGTCATCGC
T5T1-BsR	ATTATTGGTCTCTAAACTCACCTTGAGGTCCCTCCTC
T5T1-F0	GAACAGCGGCAAGTCATCGCGTTTTAGAGCTAGAAATAGC
T5T2-R0	AACTCACCTTGAGGTCCCTCCTCGCTTCTTGGTGCC
T5U3-F	GACAGGCGTCTTCTACTGGTGCTA
T5U3-R	TCACAAATTATCAGCACGCTAGTC
T5-SF1	GACAGGCGTCTTCTACTGGTGCTA
T5-SF2 HYG-F	GGCCAGCAATTACGAGTCCTTCTA GCGTCTGCTGCTCCATACAA
HYG-R	TGACATTGGGGAGTTTAGCG
T5-F	CCAACGCATAGCAGTAGTCG
T5-R	AATGAAATCTGCCATCGTATC

图1 gRNA靶点的位置(A)及终载体pHUE411-TMS5-Cas9组成(B)

Fig. 1. Targets site of gRNA (A) and structure of pHUE411-TMS5-Cas9 (B).

图2 T0代转基因植株（部分）阳性检测结果 M―标记；1~24―T0单株。A―潮霉素标记, 条带大小506 bp；B―载体+基因片段, 条带大小831 bp。扩增出相应片段表示植株为阳性。

Fig. 2. Positive identification of T0 transgenic plants (partial). M, Marker; 1-24, T0 plants. A, Hygromycin gene, 506 bp in length; B, The band with 831 bp length was amplified by primer T5U3-F/R. The 863 bp fragment means a positive test result.

图3 T0代转基因植株及野生型(台恢31) TMS5第一靶点测序结果 A, T0代杂合植株第一靶点测序结果, 箭头表示双峰开始位置；B, 野生型（台恢31）第一靶点测序结果。

Fig. 3. Sequencing of the first target site of TMS5 in T0 transgenic plants and WT (Taihui 31). A, Sequencing of T0 transgenic plants at the first target site. The arrow indicates the starting point of double peaks; B, Sequencing results of WT.

图4 tms5突变株与野生型靶位点突变序列比对分析

Fig. 4. Comparison of mutation sequences at target sites between tms5 mutants and WT.

图5 tms5突变株与野生型氨基酸序列比对分析

Fig. 5. Comparison of amino acid sequence between tms5 mutants and WT.

表2 tms5突变体分期播种的不育性鉴定

Table 2. Sterility identification of tms5 mutants seeded at different dates.

播种期 Sowing date	突变体类型 Mutant type	播始历期 Days from sowing to heading/d	幼穗分化敏感期 Young panicle differentiation period	敏感期温度均值 Mean temperature of period/℃	自交结实率 Self-fertilization rate/%
05-14（I）	tms5-1	90	07-23 -7月29日	27.0	0.56±1.26
	tms5-2	91	07-24 -7月30日	27.4	0
	tms5-3	90	07-23 -7月29日	27.0	0.03±0.13
05-24（II）	tms5-1	87	07-30 -8月5日	28.1	0
	tms5-2	87	07-30 -8月5日	28.1	0
	tms5-3	86	07-29 -8月4日	28.3	0
06-04（III）	tms5-1	79	08-02 -8月8日	27.8	0
	tms5-2	80	08-03 -8月9日	27.8	0
	tms5-3	80	08-03 -8月9日	27.8	0
06-14（IV）	tms5-1	76	08-09 -8月15日	27.3	0
	tms5-2	77	08-10 -8月16日	27.0	0
	tms5-3	76	08-09 -8月15日	27.3	0

图6 tms5突变体在I期和IV期播种时的花粉育性

Fig. 6. Pollen fertility of tms5 mutants seeded at sowing dates I and IV.

图7 不育系TB52S及野生型成熟期表型对比

Fig. 7. Comparison of phenotype between TB52S mutant and WT.

表3 水稻温敏核不育系TB52S农艺性状

Table 3. Agronomic traits of thermo-sensitive genic male sterile mutant TB52S.

材料 Material	株高 Plant height/cm	单株穗数 Panicles per plant	每穗总粒数 Grains per panicle	穗长 Panicle length/cm	柱头外露率 Chapiter exposure percentage/%
TB52S	86.1±3.0	13.5±2.0	243.2±63.4	23.0±1.9	53.3±3.7
台恢31 Taihui 31(WT)	113.3±3.1	8.3±0.6	316.3±71.7	26.8±2.0	-

图8 突变体TB52S在不同温度处理下的花粉育性、花药形态及自交结实率（比例尺为5 mm，F表示可育，S表示不育）

Fig. 8. Pollen fertility and self-fertilization rate of TB52S mutant under different temperature treatments during the young panicle differentiation. (Bar=5 mm; F, Fertile; S, Sterile)

图9 突变体TB52S在不同光照长度处理下的花粉育性、花药形态及自交结实率（比例尺为5 mm，S表示不育）

Fig. 9. Pollen fertility and self-fertilization rate of TB52S mutant under different photoperiod period treatments during the young panicle differentiation. (Bar=5 mm; S showed sterile)

表4 水稻温敏核不育系突变体TB52S与不同恢复系配组F1农艺性状

Table 4. Agronomic traits of F1 hybrids derived from the cross between TB52S and restorer lines.

组合 Combination	株高 Plant height/cm	有效穗数 Effective panicle number	穗长 Panicle length /cm	每穗粒数 Grains per panicle	结实率 Seed-setting rate/%	千粒重1000-grain weight/g	单株产量 Yield per plant/g
TB52S/台恢1050 TB52S/Taihui 1050	122.3	10.3	24.7	209.4	93.3	28.8	60.2
TB52S/台恢468 TB52S/Taihui 468	129.3	13.7	24.8	246.0	90.9	26.8	79.2
TB52S/台恢1628 TB52S/Taihui 1628	139.3	11.0	27.3	288.9	88.9	26.4	75.1
TB52S/TP39	126.3	8.3	21.5	258.2	96.1	24.6	49.9
TB52S/HR11	125.7	7.0	25.0	316.7	90.8	25.2	50.8
TB52S/HR4	123.7	8.3	24.8	307.0	94.6	25.4	55.6

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