Improvement of Grain Shape in Rice by Knocking GS3 and GS9 via CRISPR/Cas9 System

doi:10.7668/hbnxb.20192762

Abstract

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

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.

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References 50

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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

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

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

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

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