利用CRISPR/Cas9基因组编辑技术定向降低水稻落粒性

doi:10.3864/j.issn.0578-1752.2018.14.001

摘要/Abstract

摘要： 【目的】易落粒既不利于稻谷收获，也不适宜于水稻机械化生产。利用现阶段前沿的分子育种手段——CRISPR/Cas9技术对水稻落粒性主效基因qSH1进行定点编辑，并调查分析易落粒性状的改良效果，为创制稳产和适合机械化生产的水稻新种质奠定材料基础和探索新途径。【方法】以qSH1为靶标基因，根据CRISPR/Cas9技术原理设计靶标位点。将所设计的靶点序列在水稻参考基因组中比对分析以排除非特异性靶位点，最终筛选出qSH1-T1和qSH1-T5靶标位点。化学合成靶位点寡核苷酸序列，退火后分别与pYLgRNA-U3、pYLgRNA-U6a载体连接构建U3-qSH1T5-gRNA、U6a-qSH1T1-gRNA表达盒，最后将2个gRNA表达盒同时连接至pYLCRISPR/Cas9表达载体中，构建pYLCRISPR/Cas9-qSH1-T51表达载体。利用农杆菌介导转化易落粒的籼稻品种HR1128，以潮霉素抗性为筛选标记筛选获得T₀代转基因阳性植株。利用靶位点扩增测序法判断T₀代转基因植株在预期靶标位点是否发生突变，并进一步分析突变类型及基因型。将靶点序列在水稻参考基因组中比对，选择与靶点序列匹配度大于或等于15 bp且3′端具有NGG的位点作为潜在脱靶位点进行脱靶效应评估。利用潮霉素基因及靶点检测进一步筛选无T-DNA成分的qsh1突变植株并进一步构建qsh1突变系，并分析qsh1突变系的落粒性、qSH1的表达量及预测编码氨基酸序列。【结果】pYLCRISPR/Cas9-qSH1-T51载体成功地实现了对qSH1靶标位点的定点编辑。在T₀代转基因阳性植株中获得7个突变单株，其中qSH1-T1和qSH1-T5靶点的突变频率分别为54.55%和63.64%，突变基因型包括纯合突变、杂合突变、双等位突变和嵌合突变，突变类型包括碱基插入、碱基缺失及碱基突变。通过对T₁代植株进行潮霉素基因筛选、靶位点扩增测序，结果表明，在T₁代植株中Cas9载体骨架和qSH1突变位点都发生了分离，获得2种不含T-DNA成分的qsh1纯合突变株，并以此构建2个T₂代qsh1纯合突变系群体（17SZ01和17SZ02）。对46株转基因阳性植株进行脱靶效应分析，发现3个潜在脱靶位点均未发生突变，表明所设计的靶标位点具有较高的特异性。通过落粒性测定分析表明，与野生型对照相比，2个qsh1纯合突变系的落粒性显著降低。进一步分析发现，2个突变系氨基酸翻译均发生改变并提前终止，同时17SZ01突变系qSH1的表达量显著降低。【结论】利用CRISPR/Cas9技术对水稻基因组进行定点编辑定向改良水稻品种落粒性，是一条高效、安全的分子改良育种策略。

关键词: CRISPR/Cas9, 基因编辑, 水稻, qSH1, 落粒性

Abstract: 【Objective】Seed shattering is not good for seeds harvest，nor suitable for mechanized production of rice. In this study, using the latest molecular breeding method-CRISPR/Cas9 system to site-directed edit of the rice seed shattering gene qSH1 and evaluated the improvement effect, in order to lay materials foundation and explore new method for creating stable-yielding rice germplasm suitable for mechanized seed production. 【Method】The sgRNA was designed based on the principle of CRISPR/Cas9 technology. After excluding non-specific target sites by analysis of sequence alignment in the rice genome database, the target sites of qSH1-T1 and qSH1-T5 were selected. The oligonucleotides of sgRNA were chemically synthesized, and then inserted into plasmid pYLgRNA-U3, pYLgRNA-U6a for constructing the U3-qSH1T5-gRNA and U6a-qSH1T1-gRNA expression-boxes respectively. And the pYLCRISPR/cas9-qsh1-t51 expression vector was constructed by linking expression-boxes to plasmid pYLCRISPR/Cas9. The vector of pYLCRISPR/cas9-qsh1-t51 was introduced into the callus induced from mature seeds of an indica rice variety HR1128 by the Agrobacterium-mediated transformation. At T₀ generation, the positive transgenic plants were selected by hygromycin-resistant, and the target site for each plant were detected via a test of PCR combined Sanger-sequencing for confirming whether it was mutated. Besides blasting sgRNA sequence with rice genome in NCBI (https://blast.ncbi.nlm.nih.gov), Gramene (http://ensembl.gramene.org), highly identical sites with more 15 matching bases and NGG site at 3′region were selected to assess off-target efficiency and specificity of sgRNA. By using hpt gene and target site PCR amplification, mark-free qsh1 mutants were selected and qsh1 mutation lines were constructed by qsh1 mutants self-cross, and the level of rice seed shattering, the expression level of qSH1 and amino acid sequence of gene production of qsh1 lines were analyzed.【Result】The pYLCRISPR/cas9-qsh1-t51 expression vector successfully actualized gene-specific editing of qSH1. We obtained 7 mutants in T₀ transgenic generation and the mutation type ratios of qSH1-T1, qSH1-T5 were 54.55%, 63.64% respectively. Their genotypes included homozygous, heterozygous, biallelic and chimeric mutations. The mutation types were mainly insertions, deletions and substitution. After analysis of T₁ transgenic plants by hpt gene and target site PCR amplification, we obtained 2 mutant types which were homozygous and mark-free, and 2 T₂ generation qsh1 mutation lines (namely as 17SZ01, 17SZ02) were constructed further. We examined three putative off-target sites in the rice genome that showed the highly similarity with our target sites. And any off-target mutations at these sites in 46 plants of T₀ and T₁ generations were not observed, which indicated the sgRNA was highly specificity. Compared with the wild type, the 2 lines showed a significant decreased level of seed shattering (P<0.05). Furthermore, the amino acid sequence were changed of mutation lines, and the qSH1 expression level also decreased significantly (P<0.05) of the 17SZ01 mutation line. 【Conclusion】The strategy of site-directed mutagenesis qSH1 by CRISPR/Cas9 works effective for reducing the seed shattering in rice.

Key words: CRISPR/Cas9, gene editing, rice, qSH1, seed shattering

盛夏冰，谭炎宁，孙志忠，余东，汪雪峰，袁贵龙，袁定阳，段美娟. 利用CRISPR/Cas9基因组编辑技术定向降低水稻落粒性[J]. 中国农业科学, 2018, 51(14): 2631-2641.

SHENG XiaBing, TAN YanNing, SUN ZhiZhong, YU Dong, WANG XueFeng, YUAN GuiLong, YUAN DingYang, DUAN MeiJuan. Using CRISPR/Cas9-Mediated Targeted Mutagenesis of qSH1 Reduces the Seed Shattering in Rice[J]. Scientia Agricultura Sinica, 2018, 51(14): 2631-2641.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2018.14.001

https://www.chinaagrisci.com/CN/Y2018/V51/I14/2631

参考文献

[1] 叶兴锋, 徐林峰, 施聪, 童川, 沈圣泉. 中等落粒性的改良型超级稻‘协青早A/M9308’应用价值评价. 中国农学通报, 2012, 28(21): 131-134.

YE X F, XU L F, SHI C, TONG C, SHENG S Q. Application evaluation on improved type of the super rice ‘Xieqingzao A/M9308’ with medium shattering habit. Chinese agricultural science bulletin, 2012, 28(21): 131-134. (in Chinese)

[2] HISAMTSU S, SAKAUE M, TAKIZAWA A, KATO T, KAMOSHITA M, ITO J, KASHIWAZAKI N. Knockout of targeted gene in porcine somatic cells using zinc-finger nuclease. Animal Science Journal, 2015, 86(2): 132-137.

[3] SUN N, ZHAO H M. Transcription activator-like effector nucleases (TALENs): A highly efficient and versatile tool for genome editing. Biotechnology and Bioengineering, 2013, 110(7): 1811-1821.

[4] CHEN K, GAO C. Targeted genome modification technologies and their applications in crop improvements. Plant Cell Reports, 2014, 33(4): 575-583.

[5] WIEDENHEFT B, STERNBERG S H, DOUDNA J A. RNA-guided genetic silencing systems in bacteria and archaea. Nature, 2012, 482: 331-338.

[6] CONG L, RAN F A, COX D, LIN S, BARRETTO R, HAIBB N, HSU P D, WU X, JIANG W, MARRAFFINI L A, ZHANG F. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339(6121): 819-823.

[7] SHAN Q, WANG Y, LI J, ZHANG Y, CHEN K, LIANG Z, ZHANG K, LIU J, XI J, QIU J L, GAO C. Targeted genome modification of crop plants using a CRISPR-Cas system. Nature Biotechnology, 2013, 31: 686-688.

[8] JINEK M, CHYLINSKI K, FONFARA I, HAUER M, DOUDNA J A, CHARPENTIER E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012, 337: 816-821.

[9] MALI P, YANG L, ESVELT K M, AACH J, GUELL M, DICARLO J E, NORVILLE J E, CHURCH G M. RNA-guided human genome engineering via Cas9. Science, 2013, 339(6121): 823-826.

[10] CHANG N, SUN C, GAO L, ZHU D, XU X, ZHU X, XIONG J W, XI J J. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Research, 2013, 23(4): 465-472.

[11] WANG F J, WANG C L, LIU P Q, LEI C L, HAO W, GAO Y, LIU Y G, ZHAO K J. Enhanced rice blast resistance by CRISPR/Cas9- targeted mutagenesis of the ERF transcription factor gene OsERF922. Plos One, 2016, 11(4): e0154027.

[12] ZHOU H, HE M, LI J, CHEN L, HUANG Z F, ZHENG S Y, ZHU L Y, NI E, JIANG D G, ZHAO B R, ZHUANG C X. Development of commercial Thermo-sensitive genic male sterile rice accelerates hybrid rice breeding using the CRISPR/Cas9-mediated TMS5 editing system. Scientific Reports, 2016, 6: 37395-37406.

[13] ZONG Y, WANG Y P, LI C, ZHANG R, CHEN K L, RAN Y D, QIU J L, WANG D W, GAO C X. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nature Biotechnology, 2017, 35(5): 438-441.

[14] JAKOCIUNAS T, BONDE I, HERRGARD M, HARRISON S J, KRISTENSEN M, PEDERSEN L E, JENSEN M K, KEASLING J D. Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae. Metabolic Engineering, 2015, 28: 213-222.

[15] XU R F, LI H, QIN R Y, WANG L, LI L, WEI P C, YANG J B. Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice. Rice, 2014, 7(1): 5-8.

[16] NAGAO S, TAKAHASI M Z. Trial construction of 12 linkage groups in Japanese rice. Faculty Agriculture Hokkaido University, 1963, 53: 72-130.

[17] OBA S, SUMI N, FUJIMOTO F, TASUKE Y. Association between grain shattering habit and formation of abscission layer controlled by grain shattering gene sh-2 in rice (Oryza sativa L.). Japanese Journal of Crop Science, 1995, 64: 607-615.

[18] ZHU Y, ELLSTRAND N C, LU B R. Sequence polymorphisms in wild, weedy, and cultivated rice suggest seed-shattering locus sh4 played a minor role in Asian rice domestication. Ecology & Evolution, 2012, 2(9): 2106-2113.

[19] CAI H W, MORISHIMA H. Genomic regions affecting seed shattering and seed dormancy in rice. Theoretical & Applied Genetics, 2000, 100(6): 840-846.

[20] FUKUTA Y, YAGI Y. Mapping of a shattering resistance gene in a mutant line SR-5 induced from an indica rice (Oryza sativa L.) variety, Nan-jing11. Breeding Science, 1998, 48: 345-348.

[21] ZHOU Y, LU D F, LI C Y, LUO J H, ZHU B F, ZHU J J, SHANGGUAN Y Y, WANG Z X, SANG T, ZHOU B, HAN B. Genetic control of seed-shattering in rice by the APETALA2 transcription factor SHATTERING ABORTION1. The Plant Cell, 2012, 24(3): 1034-1048.

[22] KONISHI S, IZAWA T, LIN S Y, EBANA K, FUKUTA Y, SASAKI T, YANO M. An SNP caused loss of seed shattering during rice domestication. Science, 2006, 312(5778): 1392-1396.

[23] 王军, 徐详, 杨杰, 王芳权, 范方军, 朱金燕, 李文奇, 仲维功. 江苏省和东北地区杂草稻落粒性基因的序列分析. 分子植物育种, 2014, 12(6): 1097-1102.

WANG J, XU X, YANG J, WANG F Q, FAN F J, ZHU J Y, LI W Q, ZHONG W G. Sequence analysis of genes associated with seed shattering of weedy rice in region of jiangsu and Northeast of China. Molecular Plant Breeding, 2014, 12(6): 1097-1102. (in Chinese)

[24] 王黎明, 陈勇, 王林. 广东湛江杂草稻qSH1基因片段序列分析. 中国农学通报, 2010, 26(19): 31-33.

WANG L M, CHEN Y, WANG L. Sequence analysis of qSH1 gene fragments of weedy rice from Zhanjiang of Guangdong province. Chinese Agricultural Science Bulletin, 2010, 26(19): 31-33. (in Chinese)

[25] ONISHI K, TAKAGI K T, KONTANI M, TANAKA T, SANO Y. Different patterns of genealogical relationships found in the two major QTLs causing reduction of seed shattering during rice domestication. Genome, 2007, 50(8): 757-766.

[26] MA X L, ZHANG Q, ZHU Q, LIU W, CHEN Y, QIU R, WANG B, YANG Z, LI H, LIN Y, XIE Y, SHEN R, CHEN S, WANG Z, CHEN Y, GUO J, CHEN L, ZHAO X, DONG Z, LIU Y G. A Robust CRISPR/Cas9 system for convenient, high-Efficiency multiplex genome editing in monocot and dicotplants. Molecular Plant, 2015, 8(8): 1274-1284.

[27] 王慧娜, 初志战, 马兴亮, 李日清, 刘耀光. 高通量PCR模板植物基因组DNA制备方法. 作物学报, 2013, 39(7): 1200-1205.

WANG H N, CHU Z Z, MA X L, LI R Q, LIU Y G. A high through-Put protocol of plant genomic DNA preparation for PCR. Acta Agronomica Sinica, 2013, 39: 1200-1205. (in Chinese)

[28] LI M, LI X, ZHOU Z, WU P, FANG M, PAN X, LIN Q, LOU W, WU G, LI H. Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system. Frontiers in Plant Science, 2016, 7(12217): 377.

[29] FENG C, YUAN J, WANG R, LIU Y, BIRCHLER J A, HAN F. Efficient target genome modification in maize using CRISPR/Cas9 system. Journal of Genetics and Genomics, 2016, 43(1): 37-43.

[30] WANG Y P, CHENG X, SHAN Q W, ZHANG Y, LIU J X, GAO C X, QIU J L. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology, 2014, 32(9): 947.

[31] 唐丽, 李曜魁, 张丹, 毛毕刚, 吕启明, 胡远艺, 韶也, 彭彦, 赵炳然, 夏石头. 基于基因组编辑技术的水稻靶向突变特征及遗传分析. 遗传, 2016, 38(8): 746-755.

TANG L, LI Y K, ZHANG D, MAO BG, LüQ M, HU Y Y, SHAO Y, PENG Y, ZHAO B R, XIAO S T. Characteristic and inheritance analysis of targeted mutagenesis mediated by genome editing in rice. Hereditas, 2016, 38(8): 746-755. (in Chinese)

[32] ZHANG H, ZHANG J S, WEI P L, ZHANG B T, GOU F, FENG Z Y, MAO Y F, YANG L, ZHANG H, XU N F, ZHU J K. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnology Journal, 2014, 12(6): 797.

[33] HUANG X, ZHAO Y, WEI X H, LI C Y, WANG A H, ZHAO Q, LI W J, GUO Y L, DENG L W, ZHU C R, FAN D L, LU Y Q, WENG Q J, LIU K Y, ZHOU T Y, JING Y F, SI L Z, DONG G J, HUAN T, LU T T, FENG Q, QIAN Q, LI J Y, HAN B. Genome-wide association study of flowering time and grain yield traits in a worldwide collection of rice germplasm. Nature Genetics 44: 32-39.

[34] RODRIGUEZ-LEAL D, LEMMON Z H, MAN J, BARTLETT M E, LIPPMAN Z B. Engineering quantitative trait variation for crop improvement by genome editing. Cell, 2017, 171(2): 470-480.

[35] LI X T, XIE Y Y, ZHU Q L, LIU Y G. Targeted genome editing in genes and cis-regulatory regions improves qualitative and quantitative traits in crops. Molecular Plant, 2017, 10(11): 1368-1370.

[36] SHOEMAKER C J, GREEN R. Translation drives mRNA quality control. Nature Structural & Molecular Biology, 2012, 19(19): 594-601.

[37] YIN L, TAO Y, Zhao K, SHAO J M, LI X B, LIU G Z, LIU S Q, ZHU L H. Proteomic and transcriptomic analysis of rice mature seed-derived callus differentiation. Proteomics, 2007, 7(5): 755-768.