利用CRISPR/Cas9技术敲除<i>GS3</i>和<i>GS9</i>基因改良水稻粒型性状

doi:10.7668/hbnxb.20192762

摘要/Abstract

摘要：

为促进长粒型粳稻品种的选育,以粳稻品种东富139、龙粳31和东农427为试验材料,利用CRISPR/Cas9基因编辑技术构建了pYLCRISPR/Cas9-GS3-RNA和pYLCRISPR/Cas9-GS3-GS9-RNA 2个敲除载体,通过农杆菌转化法侵染受体材料的愈伤组织,对GS3和GS9基因进行定点编辑。最终,3个品种在T₂都获得了GS3单基因突变、GS9单基因突变和GS3、GS9双基因突变,且无T-DNA元件的纯合植株。在成熟期对T₂突变体及其野生型的农艺性状进行考察分析,结果表明,与野生型相比,3个品种的gs3突变植株的粒长、千粒质量均显著增加,粒宽、结实率和穗粒数无显著变化;gs9突变体粒长显著增加,粒宽显著减少,千粒质量、结实率和穗粒数无显著变化;gs3gs9突变体粒长增加,且增幅大于gs3和gs9,同时粒宽显著减少,千粒质量显著增加,而结实率和穗粒数无显著变化。综上,利用CRISPR/Cas9技术对东富139、龙粳31和东农427等3个粳稻品种的粒型进行改良,加快了长粒型粳稻新品种的选育进程。

关键词: 水稻, 粒型, GS3, GS9, CRISPR/Cas9

Abstract:

In order to promote the breeding of long-grain japonica rice varieties,two knock-out vectors,pYLCRISPR/Cas9-GS3-RNA and pYLCRISPR/Cas9-GS3-GS9-RNA,were constructed with japonica rice varieties Dongfu 139,Longjing 31 and Dongnong 427,the genes of GS3 and GS9 were edited by Agrobacterium tumefaciens in vitro.In the end,GS3,GS9 and GS3,GS9 double-gene mutations without T-DNA elements were obtained in the T₂.The results showed that the grain length and 1000-grain weight of gs3 mutant plants of three varieties were significantly increased compared with those of wild type,grain width,seed setting rate and grain number per panicle did not change significantly,grain length of gs9 mutant increased significantly,and grain width decreased significantly,while 1000-grain weight,seed setting rate and grain number per panicle did not change significantly,and grain length of gs3gs9 mutant increased more than gs3 and gs9,at the same time,grain width decreased significantly,1000-grain weight increased significantly,while seed setting rate and grain number per ear did not change significantly.To sum up,three japonica rice varieties,Dongfu 139,Longjing 31 and Dongnong 427 were improved by using CRISPR/Cas9 technique,which accelerated the breeding process of new long-grain japonica rice varieties.

Key words: Rice, Grain shape, GS3, GS9, CRISPR/Cas9

韩政宏, 段宇轩, 徐善斌, 王敬国, 刘化龙, 杨洛淼, 贾琰, 辛威, 郑洪亮, 邹德堂. 利用CRISPR/Cas9技术敲除GS3和GS9基因改良水稻粒型性状[J]. 华北农学报, 2022, 37(2): 9-17. doi: 10.7668/hbnxb.20192762.

HAN Zhenghong, DUAN Yuxuan, XU Shanbin, WANG Jingguo, LIU Hualong, YANG Luomiao, JIA Yan, XIN Wei, ZHENG Hongliang, ZOU Detang. Improvement of Grain Shape in Rice by Knocking GS3 and GS9 via CRISPR/Cas9 System[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(2): 9-17. doi: 10.7668/hbnxb.20192762.

http://www.hbnxb.net/CN/Y2022/V37/I2/9

图/表 11

表1 本研究中使用的引物

Tab.1 Primers used in this study

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
T1-F	GCCGCCGGCGCCGCCCGACCCATGCGG
T1-R	AAACCCGCATGGGTCGGGCGGCGCCGG
T2-F	GCCGACGCGCTCCACCGCGAGATCGG
T2-R	AAACCCGATCTCGCGGTGGAGCG
T3-F	GCCGTCCTGGCCTTTCCACGCGATGG
T3-R	AAACCCATCGCGTGGAAAGGCCA
Hyg-F	ACGGTGTCGTCCATCACAGTTTGCC
Hyg-R	TTCCGGAAGTGCTTGACATTGGGGA
NOS-F	GCGGTGTCATCTATGTTACTAG
GS3-seq-F	CATCTCACGGTTGTTATTT
GS3-seq-R	TATGCCATTGACTTCCTAT
GS9-seq-F	CGGCAACTCACTCTGCTCTG
GS9-seq-R	CGTAGGCCCTCACGAAAA
U-F	CTCCGTTTTACCTGTGGAATCG
gRNA-R	CGGAGGAAAATTCCATCCAC
B1'	TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG
B2	AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC
B2'	TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG
BL	AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC

图1 GS3、GS9基因结构及靶点位置

Fig.1 Gene structure and target site of GS3,GS9

图2 pYLCRISPR/Cas9-GS3-RNA载体和pYLCRISPR/Cas9-GS3-GS9-RNA载体

Fig.2 pYLCRISPR/Cas9-GS3-RNA vector and pYLCRISPR/Cas9-GS3-GS9-RNA vector

表2 转入pYLCRISPR/Cas9-GS3-GS9-RNA载体的T₀植株测序

Tab.2 Sequencing results of T₀ plants transformed into pYLCRISPR/Cas9-GS3-GS9-RNA vector

品种 Breed name	检测植株数 No.of plant examined	突变植株数 No.of plants with mutations	突变率/% Mutation rate	突变基因型 Mutant genosize
品种 Breed name	检测植株数 No.of plant examined	突变植株数 No.of plants with mutations	突变率/% Mutation rate	GS3单基因突变植株数 No.of plants with only GS3 mutations	GS9单基因突变植株数 No.of plants with only GS9 mutations	GS3、GS9均突变植株数 No.of plants with both GS3 and GS9 mutations
东富139	35	28	80.00	19	2	7
DF139
龙粳31	32	37	84.38	15	2	10
LJ31
东农427	33	30	90.91	23	1	6
DN427
总计Total	100	85	85.00	57	5	23

图3 3个粳稻品种野生型及T₁纯合突变植株测序
红色碱基为插入;红色虚线为碱基缺失;PAM序列用下划线及蓝色标注。

Fig.3 Sequencing results of wild type and T₁ homozygous mutant plants of three japonica rice varieties
Base insertion was marked with red letters;Base deletion was marked with red dotted line;PAM sequence was marked with underscore and blue.

图4 3个粳稻品种野生型及突变体粒型比较

Fig.4 Comparison of grain size of wild type and mutant in three japonica rice varieties

图5 3个粳稻品种野生型及突变体粒长比较
不同小写字母表示在0.05水平差异显著。图6—9同。

Fig.5 Comparison of grain length of wild type and mutant in three japonica rice varieties
Different small letters show significantly different at 0.05.The same as Fig.6—9.

图6 3个粳稻品种野生型及突变体粒宽比较

Fig.6 Comparison of grain width of wild type and mutant in three japonica rice varieties

图7 3个粳稻品种野生型及突变体千粒质量比较

Fig.7 Comparison of 1000-grain weight of wild type and mutant in three japonica rice varieties

图8 3个粳稻品种野生型及突变体结实率比较

Fig.8 Comparison of seed-setting rate of wild type and mutant in three japonica rice varieties

图9 3个粳稻品种野生型及突变体穗粒数比较

Fig.9 Comparison of grain number per panicle of wild type and mutant in three japonica rice varieties

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[50]	徐善斌,郑洪亮,刘利锋,卜庆云,李秀峰,邹德堂.利用CRISPR/Cas9技术高效创制长粒香型水稻[J].中国水稻科学,2020,34(5):406-412.doi:10.16819/j.1001-7216.2020.0104.
	Xu S B,Zheng H L,Liu L F,Bu Q Y,Li X F,Zou D T.Improvement of grain shape and fragrance by using CRISPR/Cas9 system[J].Chinese Journal of Rice Science,2020,34(5):406-412.

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利用CRISPR/Cas9技术敲除GS3和GS9基因改良水稻粒型性状

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