Improvement of Grain Shape and Fragrance by Using CRISPR/Cas9 System

doi:10.16819/j.1001-7216.2020.0104

Abstract

Abstract:

【Objective】 CRISPR/Cas9-mediated genome editing has become an important way for molecular breeding in rice. To promote rice breeding, the non-fragrant japonica rice variety Longjing11 was used as the test material. This study aims to edit GS3, GS9 and Badh2 genes for obtaining valuable and stable long-grain fragrant rice materials. 【Method】 The target genes GS3, GS9 and BADH2 were selected to construct the vector pYLCRISPR/ Cas9-GS3/ GS9/Badh2-gRNA by using CRISPR/Cas9 gene editing technology. The Agrobacterium-mediated transformation was used to transform Longjing11. Specific mutations were introduced into the GS3, GS9 and Badh2 genes in Longjing 11. 【Result】 The grain length of the transgenic free homozygote gs3/gs9/badh2 increased by 26.43% to 27.01%, yield per plant by 10.82% to 12.11%, 1000-grain weight by 18.34% to 41.36%, rice became fragrant as compared with wild type of Longjing 11. This study efficiently transformed round-grain rice into long-grain fragrant rice. 【Conclusion】The homozygous mutant lines featured by stable inheritance and long-grain fragrant quality were obtained by using CRISPR/Cas9 system. This study provides a convenient and effective way of combining multiple quality traits together, which could significantly accelerate breeding process from a breeding perspective.

Key words: rice, grain shape, quality breeding, fragrant rice, CRISPR/Cas9

摘要：

【目的】CRISPR/Cas9基因编辑技术已成为水稻分子育种的重要手段。为了促进水稻育种的发展,本研究以非香型粳稻品种龙粳11为试验材料,对GS3、GS9和Badh2基因进行编辑,以期获得能稳定遗传的长粒香水稻材料。【方法】利用CRISPR/Cas9技术,以GS3、GS9和Badh2为靶基因,构建敲除载体pYLCRISPR/Cas9-GS3/ GS9/Badh2-gRNA,通过农杆菌介导法,在龙粳11的GS3、GS9和Badh2基因中引入了特定的突变。【结果】T₂代无转基因的gs3/gs9/badh2纯合突变体与野生型龙粳11相比,粒长增加26.43%~27.01%,单株产量增加10.82%~12.11%,千粒重增加18.34%~41.36%,稻米变香,高效地将圆粒水稻变成长粒香型水稻。【结论】利用CRISPR/Cas9技术获得能够稳定遗传并具有长粒香品质的纯合突变株系,为组合多个品质性状提供了一种方便有效的方法,从育种角度加快了新品系创制过程。

关键词: 水稻, 粒型, 品质育种, 香米, CRISPR/Cas9

CLC Number:

Shanbin XU, Hongliang ZHENG, Lifeng LIU, Qingyun BU, Xiufeng Li, Detang ZOU. Improvement of Grain Shape and Fragrance by Using CRISPR/Cas9 System[J]. Chinese Journal OF Rice Science, 2020, 34(5): 406-412.

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.

Figures/Tables 7

References 36

[1]	郭韬, 余泓, 邱杰, 李家洋, 韩斌, 林鸿宣. 中国水稻遗传学研究进展与分子设计育种[J]. 中国科学: 生命科学, 2019, 49(10): 1185-1212.
	Guo T, Yu H, Qiu J, Li J Y, Han B, Lin H X.Advances in rice genetics and breeding by molecular design in China[J]. Science in China: Life Sciences, 2019, 49(10): 1185-1212. (in Chinese with English abstract)
[2]	Wang M G, Mao Y F, Lu Y M, Wang Z D, Tao X P, Zhu J K.Multiplex gene editing in rice with simplified CRISPR-Cpf1 and CRISPR-Cas9 systems[J]. Journal of Integrative Plant Biology, 2018, 60(8): 626-631.
[3]	Cai Y P, Chen L, Liu X J, Guo C, Sun S, Wu C X, Jiang B J, Han T F, Hou W S.CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean[J]. Plant Biotechnology Journal, 2018, 16(1): 176-185.
[4]	Qi W W, Zhu T, Tian Z R, Li C B, Zhang W, Song R T.High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize[J/OL].BMC Biotechnology, 2016, 16(1): 58.
[5]	Zhang S J, Zhang R Z, Song G Q, Gao J, Li W, Han X D, Chen M L, Li Y L, Li G Y.Targeted mutagenesis using the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system in common wheat[J/OL].BMC Plant Biology, 2018, 18(1): 302.
[6]	Wang P C, Zhang J, Sun L, Ma Y Z, Xu J, Liang S J, Deng J W, Tan J F, Zhang Q H, Tu L L, Daniell H, Jin S X, Zhang X L.High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system[J]. Plant Biotechnology Journal, 2018, 16(1): 137-150.
[7]	Liu G Q, Li J Q, Godwin I D.Genome editing by CRISPR/Cas9 in sorghum through biolistic bombardment[J]. Methods in Molecular Biology, 2019, 1931: 169-183.
[8]	Samanta M K, Dey A, Gayen S.CRISPR/Cas9: An advanced tool for editing plant genomes[J]. Transgenic Research, 2016, 25(5): 561-573.
[9]	Ji X, Zhang H W, Zhang Y, Wang Y P, Gao C X.Establishing a CRISPR-Cas-like immune system conferring DNA virus resistance in plants[J/OL].Nature Plants, 2015, 1: 15144.
[10]	Hsu P D, Lander E S, Zhang F.Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014, 157(6): 1262-1278.
[11]	Smih F, Rouet P, Romanienko P J, Jasin M.Double strand breaks at the target locus stimulate gene targeting in embryonic stem cells[J]. Nucleic Acids Research, 1995, 23(24): 5012-5019.
[12]	Song X J, Huang W, Shi M, Zhu M Z, Lin H X.A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase[J]. Nature Genetics, 2007, 39(5): 623-630.
[13]	Fan C C, Xing Y Z, Mao H L, Lu T T, Han B, Xu C G, Li X H, Zhang Q F.GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein[J]. Theoretical and Applied Genetics, 2006, 112(6): 1164-1171.
[14]	Zhao D S, Li Q F, Zhang C Q, Zhang C, Yang Q Q, Pan L X, Ren X Y, Lu J, Gu M H, Liu Q Q. GS9 acts as a transcriptional activator to regulate rice grain shape and appearance quality[J/OL]. Nature Communications, 2018, 9(1): 1240.
[15]	Liu J F, Chen J, Zheng X M, Wu F Q, Lin Q B, Heng Y Q, Tian P, Cheng Z J, Yu X W, Zhou K N, Zhang X, Guo X P, Wang J L, Wang H Y, Wan J M.GW5 acts in the brassinosteroid signalling pathway to regulate grain width and weight in rice[J/OL].Nature Plants, 2017, 3: 17043.
[16]	Ishimaru K, Hirotsu N, Madoka Y, Murakami N, Hara N, Onodera H, Kashiwagi T, Ujiie K, Shimizu B I, Onishi A, Miyagawa H, Katoh E.Loss of function of the IAA-glucose hydrolase gene TGW6 enhances rice grain weight and increases yield[J]. Nature Genetics, 2013, 45(6): 707-711.
[17]	Che R H, Tong H N, Shi B H, Liu Y Q, Fang S R, Liu D P, Xiao Y H, Hu B, Liu L C, Wang H R, Zhao M F, Chu C C. Control of grain size and rice yield by GL2-mediated brassinosteroid responses[J/OL].Nature Plants, 2015, 2: 15195.
[18]	Qi P, Lin Y S, Song X J, Shen J B, Huang W, Shan J X, Zhu M Z, Jiang L, Gao J P, Lin H X.The novel quantitative trait locus GL3.1 controls rice grain size and yield by regulating Cyclin-T1;3[J]. Cell Research, 2012, 22(12): 1666-1680.
[19]	Li Y B, Fan C C, Xing Y Z, Jiang Y H, Luo L J, Sun L, Shao D, Xu C J, Li X H, Xiao J H, He Y Q, Zhang Q F.Natural variation in GS5 plays an important role in regulating grain size and yield in rice[J]. Nature Genetics, 2011, 43(12): 1266-1269.
[20]	Wang Y X, Xiong G S, Hu J, Jiang L, Yu H, Xu J, Fang Y X, Zeng L J, Xu E B, Xu J, Ye W J, Meng X B, Liu R F, Chen H Q, Jing Y H, Wang Y H, Zhu X D, Li J Y, Qian Q.Copy number variation at the GL7 locus contributes to grain size diversity in rice[J]. Nature Genetics, 2015, 47(8): 944-948.
[21]	Si L Z, Chen J Y, Huang X H, Gong H, Luo J H, Hou Q Q, Zhou T Y, Lu T T, Zhu J J, Shang G Y, Chen E W, Gong C X, Zhao Q, Jing Y F, Zhao Y, Li Y, Cui L L, Fan D L, Lu Y Q, Weng Q J, Wang Y C, Zhan Q L, Liu K Y, Wei X H, An K, An G, Han B.OsSPL13 controls grain size in cultivated rice[J]. Nature Genetics, 2016, 48(4): 447-456.
[22]	Wang S K, Wu K, Yuan Q B, Liu X Y, Liu Z B, Lin X Y, Zeng R Z, Zhu H T, Dong G J, Qian Q, Zhang G Q, Fu X D.Control of grain size, shape and quality by OsSPL16 in rice[J]. Nature Genetics, 2012, 44(8): 950-954.
[23]	Li M R, Li X X, Zhou Z J, Wu P Z, Fang M C, Pan X P, Lin Q P, Luo W B, Wu G J, Li H Q.Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system[J/OL].Frontiers in Plant Science, 2016, 7: 377.
[24]	Sakthivel K, Sundaram R M, Rani N S, Balachandran S M, Neeraja C N.Genetic and molecular basis of fragrance in rice[J]. Biotechnology Advances, 2009, 27(4): 468-473.
[25]	Chen S H, Yang Y, Shi W W, Ji Q, He F, Zhang Z D, Cheng Z K, Liu X N, Xu M L.Badh2, encoding betaine aldehyde dehydrogenase, inhibits the biosynthesis of 2-acetyl-1-pyrroline, a major component in rice fragrance[J]. Plant Cell, 2008, 20(7): 1850-1861.
[26]	Shan Q W, Zhang Y, Chen K L, Zhang K, Gao C X.Creation of fragrant rice by targeted knockout of the OsBadh2 gene using TALEN technology[J]. Plant Biotechnology Journal, 2015, 13(6): 791-800.
[27]	孙慧宇, 宋佳, 王敬国, 刘化龙, 孙健, 莫天宇, 徐善斌, 郑洪亮, 邹德堂. 利用CRISPR/Cas9技术编辑Badh2基因改良粳稻香味[J]. 华北农学报, 2019, 34(4): 1-8.
	Sun H Y, Song J, Wang J G, Liu H L, Sun J, Mo T Y, Xu S B, Zheng H L, Zou D T.Editing Badh2 gene to improve the fragrance of japonica rice by CRISPR/Cas9 technology[J]. Acta Agricultuae Boreali-Sinica, 2019, 34(4): 1-8. (in Chinese with English abstract)
[28]	曾栋昌, 马兴亮, 谢先荣, 祝钦泷, 刘耀光. 植物CRISPR/Cas9多基因编辑载体构建和突变分析的操作方法[J]. 中国科学: 生命科学, 2018, 48(7): 783-794.
	Zeng D C, Ma X L, Xie X R, Zhu Q L, Liu Y G.A protocol for CRISPR/Cas9-based multi-gene editing and sequence decoding of mutant sites in plants[J]. Science in China: Life Sciences, 2018, 48(7): 783-794. (in Chinese with English abstract)
[29]	Xie X R, Ma X, Zhu Q L, Zeng D C, Li G S, Liu Y G, CRISPR-GE: A convenient software toolkit for CRISPR- based genome editing[J]. Molecular Plant, 2017, 10(9): 1246-1249.
[30]	Ma X L, Zhang Q Y, Zhu Q L, Liu W, Chen Y, Qiu R, Wang B, Yang Z F, Li H Y, Lin Y R, Xie Y Y, Shen R X, Chen S F, Wang Z, Chen Y L, Guo J X, Chen L T, Zhao X C, Dong Z C, Liu Y G.A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants[J]. Molecular Plant, 2015, 8(8): 1274-1284.
[31]	Liu W Z, Xie X R, Ma X L, Li J, Chen J H, Liu Y G.DSDecode: A web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations[J]. Molecular Plant, 2015, 8(9): 1431-1433.
[32]	Zhang Y C, Li S M, Xue S J, Yang S H, Huang J, Wang L.Phylogenetic and CRISPR/Cas9 studies in deciphering the evolutionary trajectory and phenotypic impacts of rice ERECTA genes[J/OL]. Frontiers in Plant Science, 2018, 9: 473.
[33]	Wang M, Mao Y F, Lu Y M, Wang Z D, Tao X P, Zhu J K.Multiplex gene editing in rice with simplified CRISPR-Cpf1 and CRISPR-Cas9 systems[J]. Journal of Integrative Plant Biology, 2018, 60(8): 626-631.
[34]	Zhang J S, Zhang H, Botella J R, Zhu J K.Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties[J]. Journal of Integrative Plant Biology, 2018, 60(5): 369-375.
[35]	Wang Y, Geng L Z, Yuan M L, Wei J, Jin C, Li M, Yu K, Zhang Y, Jin H B, Wang E, Chai Z J, Fu X D, Li X G.Deletion of a target gene in indica rice via CRISPR/Cas9[J]. Plant Cell Reports, 2017, 36(8): 1333-1343.
[36]	Nan J Z, Feng X M, Wang C, Zhang X H, Wang R S, Liu J X, Yuan Q B, Jiang G Q, Lin S Y.Improving rice grain length through updating the GS3 locus of an elite variety Kongyu 131[J]. Rice, 2018, 11(1): 21.

引物名称 Primer name	引物序列（5'-3'） Primer sequence（5'-3'）
B1-F	GCCGAGTGACATGGCAATGGCGG
B1-R	AAACCCGCCATTGCCATGTCACT
B2-F	GCCGCGATTGCTTCCTGCTCGGTT
B2-R	AAACAACCGAGCAGGAAGCAATCG
B3-F	GCCGCAAGTACCTCCGCGCAATCG
B3-R	AAACCGATTGCGCGGAGGTACTTG
U-F	CTCCGTTTTACCTGTGGAATCG
gRNA-R	CGGAGGAAAATTCCATCCAC
B1'	TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG
B2	AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC
B2'	TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG
B3	AGCGTGGGTCTCGTCTTGGTCCATCCACTCCAAGCTC
B3'	TTCAGAGGTCTCTAAGACACTGGAATCGGCAGCAAAGG
BL	AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC
Seq1-F	TTCAAAGCAAAGCACCAAGC
Seq1-R	GCTGGGGAAAACTTACAATG
Seq2-F	TCCACTCGACTCCTCACTCA
Seq2-R	GATTAGCGTAGGCCCTCACG
Seq3-F	ATCCATCTCCGTATCTCT
Seq3-R	GGTAGTCACCACCCTA
Hyg-F	ACGGTGTCGTCCATCACAGTTTGCC
Hyg-R	TTCCGGAAGTGCTTGACATTGGGGA
NOFS	GCGGTGTCATCTATGTTACTAG
M13F-47	CGCCAGGGTTTTCCCAGTCACGAC

引物名称 Primer name	引物序列（5'-3'） Primer sequence（5'-3'）
B1-F	GCCGAGTGACATGGCAATGGCGG
B1-R	AAACCCGCCATTGCCATGTCACT
B2-F	GCCGCGATTGCTTCCTGCTCGGTT
B2-R	AAACAACCGAGCAGGAAGCAATCG
B3-F	GCCGCAAGTACCTCCGCGCAATCG
B3-R	AAACCGATTGCGCGGAGGTACTTG
U-F	CTCCGTTTTACCTGTGGAATCG
gRNA-R	CGGAGGAAAATTCCATCCAC
B1'	TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG
B2	AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC
B2'	TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG
B3	AGCGTGGGTCTCGTCTTGGTCCATCCACTCCAAGCTC
B3'	TTCAGAGGTCTCTAAGACACTGGAATCGGCAGCAAAGG
BL	AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC
Seq1-F	TTCAAAGCAAAGCACCAAGC
Seq1-R	GCTGGGGAAAACTTACAATG
Seq2-F	TCCACTCGACTCCTCACTCA
Seq2-R	GATTAGCGTAGGCCCTCACG
Seq3-F	ATCCATCTCCGTATCTCT
Seq3-R	GGTAGTCACCACCCTA
Hyg-F	ACGGTGTCGTCCATCACAGTTTGCC
Hyg-R	TTCCGGAAGTGCTTGACATTGGGGA
NOFS	GCGGTGTCATCTATGTTACTAG
M13F-47	CGCCAGGGTTTTCCCAGTCACGAC