利用CRISPR/Cas9技术编辑粒长基因GS3改善粳稻花时

doi:10.16819/j.1001-7216.2018.7112

摘要/Abstract

摘要：

【目的】花时不遇是影响杂交水稻制种产量的直接因素之一,已有研究表明水稻粒型差异对花时有影响。本研究采用GS3功能缺失突变来研究粳稻花时差异,以期为粒型影响花时提供佐证。【方法】利用CRISPR/Cas9技术来定向编辑控制粒长基因GS3,获得13对粒型差异的近等基因系粳稻,小区种植,采用目测法来调查花时。【结果】获得转基因T₀植株并对其T₁植株测序分析,发现长白25、吉粳102、浙粳88、武运粳27和J42均发生单碱基插入移码突变,垦鉴稻6号、空育131、浙粳22、扬粳4227、南粳9108、J5933、J6167和J5938均发生部分碱基缺失突变。对T₁植株粒型考查表明,gs3突变体的粒长均显著长于野生型。对稳定的后代花时统计分析发现,粒型变长的突变体均比野生型花时有所提前,且吉粳102、空育131、浙粳88、武运粳27、扬粳4227这5个材料的gs3突变体花时均显著早于野生型,其余材料开花也提早,但不显著。【结论】长粒型粳稻GS3突变体的花时早于短粒粳稻野生型,这一研究可为粳稻粒型育种提供参考,加速长粒粳稻亲本选育,有望推动杂交粳稻发展。

关键词: 粳稻, 花时, CRISPR/Cas9, GS3

Abstract:

【Objective】Floweringtime asynchrony is one of the main factors limiting seed production of hybrid ricebetween male and female parents, and some studies have shown that flowering time is related to grainshape.Loss of function mutant ofGS3was used to study the difference of flowering timein this study so as to elucidate the impact of grain shape on flowering time.【Method】CRISPR/Cas9-mediated editing of GS3to achieve 13 pairs of near-isogenic line japonica varieties.Then the flowering time was investigated with visual methods. 【Result】T₁ individuals derived from T₀ generation were genotyped. The sequencing results show that some bases were mutatedwithsingle base insertion, includingChangbai25,Jijing102,Zhejing88,Wuyunjing27, J42 and otherswithbases deletion,including Kenjiandao6, Kongyu131, Zhejing22, Yangjing4227, Nanjing9108, J5933, J6167, J5938. For the grain shape of T₁, the gs3 mutants conferred longer grain length compared to the wild type, and the gs3 mutants of Jijing102, Kongyu131, Zhejing88, Wuyunjing27 and Yangjing4227 were significantly earlier in flowering time thanthose of the wild type. The others were also earlier, but not significant.【Conclusion】 Long-grain japonica rice has an earlier flowering time than short-grainjaponica rice, which could provide referencesto study the grain-type breeding of japonicarice, and it would accelerate the breeding process of long-grainjaponica parents and promote the development of hybrid japonicarice.

Key words: japonica rice, flowering time, CRISPR/Cas9, GS3

中图分类号:

孟帅, 徐鹏, 张迎信, 王宏, 曹立勇, 程式华, 沈希宏. 利用CRISPR/Cas9技术编辑粒长基因GS3改善粳稻花时[J]. 中国水稻科学, 2018, 1(1): 119-127.

MENGShuai, Peng XU, Yingxin ZHANG, Hong WANG, Liyong CAO, Shihua CHENG, SHENXihong. CRISPR/Cas9-mediated Editing of GS3to ImproveFloweringTime injaponicaRice[J]. Chinese Journal OF Rice Science, 2018, 1(1): 119-127.

图/表 7

参考文献 46

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引物名称 Primer name	引物序列 Sequence(5′-3′)
GS3-CAS9F	GGCAGTGACATGGCAATGGCGG
GS3-CAS9R	AAACCCGCCATTGCCATGTCAC
T3	ATTAACCCTCACTAAAGGGA
CAS9F	ACCAGACACGAGACGACTAA
CAS9R	ATCGGTGCGGGCCTCTTC
Hyg-F	GCTGTTATGCGGCCATTGTC
Hyg-R	GACGTCTGTCGAGAAGTTTC
GS3-F	CTATACATAGCTGCTGCACCGTCT
GS3-R	CAATCACGTACTCATCATGGCAGCA

引物名称 Primer name	引物序列 Sequence(5′-3′)
GS3-CAS9F	GGCAGTGACATGGCAATGGCGG
GS3-CAS9R	AAACCCGCCATTGCCATGTCAC
T3	ATTAACCCTCACTAAAGGGA
CAS9F	ACCAGACACGAGACGACTAA
CAS9R	ATCGGTGCGGGCCTCTTC
Hyg-F	GCTGTTATGCGGCCATTGTC
Hyg-R	GACGTCTGTCGAGAAGTTTC
GS3-F	CTATACATAGCTGCTGCACCGTCT
GS3-R	CAATCACGTACTCATCATGGCAGCA

材料 Material	粒长 Grain length/mm	粒宽 Grain width/mm	长宽比Length/Width ratio	株高 Plant height/cm	花时 Flowering time/min	播始历期 Sowing to heading/d
WT-CB25	6.64±0.04	3.00±0.06	2.23±0.06	83.1±1.0	558.0±3.5	77.8±1.1
gs3-CB25	7.47±0.07**	2.92±0.02	2.58±0.03**	81.7±1.4*	553.0±4.3	77.2±0.8
WT-KJD6	6.40±0.02	3.07±0.02	2.10±0.01	61.9±1.9	541.0±3.4	68.0±0.7
gs3-KJD6	7.11±0.06**	3.09±0.04	2.33±0.03**	58.4±1.1*	538.0±4.8	68.2±0.2
WT-JJ102	6.36±0.02	2.74±0.02	2.25±0.02	67.8±1.3	555.7±6.3	69.0±0.7
gs3-JJ102	7.09±0.15**	2.75±0.03	2.65±0.06**	67.6±1.1*	544.5±6.0*	69.8±1.1
WT-KY131	6.42±0.16	3.06±0.02	2.13±0.03	68.5±1.7	545.0±5.9	69.3±0.5
gs3-KY131	7.36±0.06**	3.09±0.03	2.41±0.02**	66.8±0.9*	531.0±4.2**	69.5±0.5
WT-ZJ22	6.73±0.05	2.99±0.02	2.34±0.03	69.7±1.2	721.0±6.2	86.3±0.6
gs3-ZJ22	6.99±0.06*	2.97±0.02	2.41±0.04*	64.4±1.3*	718.0±6.5	85.3±0.8
WT-NJ9108	6.00±0.02	3.02±0.06	2.05±0.02	46.0±1.3	571.2±6.4	80.5±0.5
gs3-NJ9108	6.27±0.02*	3.06±0.03	2.13±0.02*	44.4±0.9	566.0±6.1	79.7±0.6
WT-ZJ88	6.10±0.05	2.89±0.08	2.24±0.07	62.3±0.9	629.0±6.0	76.7±0.6
gs3-ZJ88	6.33±0.05**	2.84±0.06	2.47±0.03**	54.3±1.5*	620.0±6.0*	77.3±0.5
WT-WYJ27	6.57±0.03	3.02±0.04	2.24±0.04	65.2±1.4	573.3±4.1	76.0±1.0
gs3-WYJ27	6.87±0.03**	3.04±0.07	2.35±0.02**	58.2±1.2*	566.5±2.4**	76.3±0.6
WT-YJ4227	6.13±0.02	3.04±0.03	2.13±0.03	58.1±1.3	560.0±6.5	76.7±0.6
gs3-YJ4227	6.39±0.09**	3.04±0.07	2.25±0.05**	57.6±1.3*	555.0±6.7	77.7±0.6
WT-J5933	8.68±0.02	2.48±0.02	3.67±0.37	80.8±0.4	567.7±5.5	83.3±0.5
gs3-J5933	8.88±0.03*	2.48±0.01	3.85±0.22*	80.5±0.7	561.0±6.1	82.7±0.6
WT-J6167	7.37±0.02	2.38±0.01	3.11±0.26	78.5±0.7	537.0±5.2	88.2±1.1
gs3-J6167	7.53±0.03**	2.36±0.02	3.23±0.30**	76.0±1.4*	535.0±6.2	88.7±0.9
WT-J5938	9.59±0.01	2.46±0.02	3.84±0.22	87.2±1.2	557.0±5.2	92.0±0.7
gs3-J5938	9.64±0.02*	2.48±0.02	3.96±0.24*	85.9±1.3*	551.8±6.4	91.3±0.5
WT-J42	7.46±0.02	2.66±0.04	2.89±0.37	88.4±1.1	518.0±3.5	83.3±0.5
gs3-J42	7.82±0.02**	2.60±0.05	2.91±0.36*	87.0±1.0*	518.3±3.7	82.7±0.6
和分别表示野生型与突变体在0.05,0.01水平上显著差异。 ,** Significant difference between WT and mutant at 0.05, 0.01 level, respectively.

材料 Material	粒长 Grain length/mm	粒宽 Grain width/mm	长宽比Length/Width ratio	株高 Plant height/cm	花时 Flowering time/min	播始历期 Sowing to heading/d
WT-CB25	6.64±0.04	3.00±0.06	2.23±0.06	83.1±1.0	558.0±3.5	77.8±1.1
gs3-CB25	7.47±0.07**	2.92±0.02	2.58±0.03**	81.7±1.4*	553.0±4.3	77.2±0.8
WT-KJD6	6.40±0.02	3.07±0.02	2.10±0.01	61.9±1.9	541.0±3.4	68.0±0.7
gs3-KJD6	7.11±0.06**	3.09±0.04	2.33±0.03**	58.4±1.1*	538.0±4.8	68.2±0.2
WT-JJ102	6.36±0.02	2.74±0.02	2.25±0.02	67.8±1.3	555.7±6.3	69.0±0.7
gs3-JJ102	7.09±0.15**	2.75±0.03	2.65±0.06**	67.6±1.1*	544.5±6.0*	69.8±1.1
WT-KY131	6.42±0.16	3.06±0.02	2.13±0.03	68.5±1.7	545.0±5.9	69.3±0.5
gs3-KY131	7.36±0.06**	3.09±0.03	2.41±0.02**	66.8±0.9*	531.0±4.2**	69.5±0.5
WT-ZJ22	6.73±0.05	2.99±0.02	2.34±0.03	69.7±1.2	721.0±6.2	86.3±0.6
gs3-ZJ22	6.99±0.06*	2.97±0.02	2.41±0.04*	64.4±1.3*	718.0±6.5	85.3±0.8
WT-NJ9108	6.00±0.02	3.02±0.06	2.05±0.02	46.0±1.3	571.2±6.4	80.5±0.5
gs3-NJ9108	6.27±0.02*	3.06±0.03	2.13±0.02*	44.4±0.9	566.0±6.1	79.7±0.6
WT-ZJ88	6.10±0.05	2.89±0.08	2.24±0.07	62.3±0.9	629.0±6.0	76.7±0.6
gs3-ZJ88	6.33±0.05**	2.84±0.06	2.47±0.03**	54.3±1.5*	620.0±6.0*	77.3±0.5
WT-WYJ27	6.57±0.03	3.02±0.04	2.24±0.04	65.2±1.4	573.3±4.1	76.0±1.0
gs3-WYJ27	6.87±0.03**	3.04±0.07	2.35±0.02**	58.2±1.2*	566.5±2.4**	76.3±0.6
WT-YJ4227	6.13±0.02	3.04±0.03	2.13±0.03	58.1±1.3	560.0±6.5	76.7±0.6
gs3-YJ4227	6.39±0.09**	3.04±0.07	2.25±0.05**	57.6±1.3*	555.0±6.7	77.7±0.6
WT-J5933	8.68±0.02	2.48±0.02	3.67±0.37	80.8±0.4	567.7±5.5	83.3±0.5
gs3-J5933	8.88±0.03*	2.48±0.01	3.85±0.22*	80.5±0.7	561.0±6.1	82.7±0.6
WT-J6167	7.37±0.02	2.38±0.01	3.11±0.26	78.5±0.7	537.0±5.2	88.2±1.1
gs3-J6167	7.53±0.03**	2.36±0.02	3.23±0.30**	76.0±1.4*	535.0±6.2	88.7±0.9
WT-J5938	9.59±0.01	2.46±0.02	3.84±0.22	87.2±1.2	557.0±5.2	92.0±0.7
gs3-J5938	9.64±0.02*	2.48±0.02	3.96±0.24*	85.9±1.3*	551.8±6.4	91.3±0.5
WT-J42	7.46±0.02	2.66±0.04	2.89±0.37	88.4±1.1	518.0±3.5	83.3±0.5
gs3-J42	7.82±0.02**	2.60±0.05	2.91±0.36*	87.0±1.0*	518.3±3.7	82.7±0.6
和分别表示野生型与突变体在0.05,0.01水平上显著差异。 ,** Significant difference between WT and mutant at 0.05, 0.01 level, respectively.