Morphological Alternation of Rice Plant by Site-Directed Mutagenesis on IPA1 Gene
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摘要:
目的 水稻株型改良是提高水稻产量的一个有效途径。水稻理想株型基因IPA1是一个调控水稻株型的关键基因,利用基因组编辑技术(TALENs技术)定点突变水稻IPA1基因,了解IPA1基因不同序列变异的株型效应,为进一步利用IPA1基因创制实用型水稻新株型材料奠定基础。 方法 利用TALENs技术定点突变优良恢复系明恢86的IPA1基因,通过测序鉴定突变体,种植于标准小区,调查分析其株型相关性状。 结果 利用TALENs技术获得了8种不同序列突变的水稻ipa1突变体,并通过转基因植株自交结合PCR分析筛选到去除了TALENs表达框,获得4种不含外源转基因成分的纯合突变体材料(IPA1基因表达区分别缺失2、4、16、23 bp)。表型分析发现,IPA1基因突变能够显著改变水稻的株高、有效穗数、穗长及穗粒数等性状。与野生型比较,缺失移码突变体株高降低7.9%~11.4%,有效穗数增加46.9%~68.4%,穗长短24.2%~29.3%,穗粒数减少31%~34%,结实率和千粒重差异不明显。 结论 利用TALENs技术定点突变水稻IPA1基因能够明显改变水稻株高、有效穗数、穗长及穗粒数等主要性状,产生水稻新株型。 Abstract:Objective The site-directed mutagenesis on grain-yield-related IPA1 to modifying the morphological characteristics of a rice plant for breeding purpose was studied. Method Minghui 86, a rice elite restorer, was used in the study. The primary gene that governs the gain yield of the rice plant, IPA1, was edited using the transcription activator-like effector nucleases (TALENs) to generate mutated plants for subsequent identification by gene sequencing and cultivation on standard plots to verify results of the mutagenesis. Eight transgenic rice plants that differed in ipa1 sequences had the TALENs boxes cleaved for self-crossing followed by screening with PCR analysis to arrive at 4 mutants with 2bp, 4bp, 16bp or 23bp segment deleted in their IPA1 genes. Result A phenotypic analysis on the 4 mutants free of exogenous transgenic elements confirmed that the site-directed mutagenesis on IPA1 had significantly altered the plant height, effective panicle number, panicle length, and grain number per panicle of the mutated rice plants. Compared with the wild-type Minghui 86, the plant height of the mutants was decreased by 7.9% to 11.4%, the effective panicle number increased by 46.9% to 68.4%, the panicle length shortened by 24.2% to 29.3%, the number of grains per panicle reduced by 31% to 34%, but the seed setting rate and 1000-grain weight were not changed significantly. Conclusion The site-directed mutagenesis applying TALENs on IPA1 could significantly modify the plant height, effective panicle number, panicle length, and grain number per panicle on the new rice plant. The technique could be applied for the breeding program on rice to increase grain yield. -
Key words:
- rice (Oryza sativa L.) /
- IPA1 gene /
- genomic editing /
- mutant /
- new plant architecture
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图 1 TALENs编辑水稻IPA1基因的靶位点
注:空心框为基因IPA1的非翻译区,实心框为IPA1的编码区,实线为内含子;箭头所示为miRNA156结合序列区;TIPA1-1、TIPA1-2为基于TALENs设计的2个靶位点。
Figure 1. Target sites in rice IPA1 gene for TALENs editing
Note: Empty frame encloses untranslated region; filled frame, coding region; and, solid line, intron. Arrow indicates binding sequence of microRNA156; TIPA1-1 and TIPA1-2 are two target sites designed based on TALENs.
图 3 TALENs编辑基因IPA1的序列突变类型
注:下划线部分序列为TALENs识别序列,中间阴影部分为Spacer序列,缺失的碱基用“-”表示; 左边编号A起头的为未经过连续继代的抗性愈伤组织直接分化苗,编号B起头和C起头的分别为抗性愈伤组织连续继代2次和3次后的分化苗。右边数字为变化碱基数,缺失为“-”,插入为“+”;Homo为纯合突变,未标示者为杂合突变。
Figure 3. Mutation types of IPA1 produced by TALENs
Note: Underscored sequences are TALENs recognition sequences; shaded part, spacer sequence; and, "-", missing base. On the left, numbers begin with A are directly differentiated from non-successive resistant callus; numbers with B, successively sub-cultured twice; and, numbers with C, successively sub-cultured three times. On the right, data are counts of variable base; and, "-" represents deletion; "+", insertion; "Homo", homozygous mutation; and, unmarked heterozygous mutation.
图 4 不含转基因成分(TALENs载体成分)的T1植株筛选
注:泳道1~10为靶基因有突变的一个转基因T1代株系,其中无扩增条带的为不含转基因成分植株;泳道11:野生型明恢86;泳道12:清水;M:DNA Ladder。
Figure 4. Selection of T1 plants without transgenic (TALENs vector) components
Note: Lanes 1-10 are T1 transgenic plants mutated in target gene; and, those without amplified bands, non-transgenic. Lane 11 is wild-type Minghui 86; Lane 12, water; and, M, DNA ladder.
图 5 不含转基因成分的突变体筛选
注:A为测序检测野生型及缺失2 bp(-CT)纯合和杂合突变体靶序列; B为聚丙烯酰胺凝胶电泳结果,野生型(wt),缺失4 bp(-CTCT)的纯合(m)和杂合子(hm)。
Figure 5. Selection on mutants free of transgenic components
Note: A represents detection of target sequences in wild type and mutants with 2 bp-deletion (-CT) homozygous or heterozygous by sequencing; and, B, marker analysis of target site in wild type (wt) and mutants with 4 bp-deletion (-CTCT), homozygous (m) or heterozygote (hm) by polyacrylamide gel electrophoresis.
图 6 IPA1基因编辑突变体株型
注:ipa1-CK为杂合突变体分离得到的野生型;ipa1-2、ipa1-4、ipa1-16分别为T2代缺失2、4、16 bp的纯合突变体。
Figure 6. Plant architectures of wild-type and IPA1 gene edited mutants
Note:ipa1-CK is a wild-type from progeny of a heterozygous mutant; ipa1-2, ipa1-4, and ipa1-16 are T2 homozygous mutants with 2 bp, 4 bp, and 16 bp detection, respectively.
表 1 基因编辑的靶位点序列
Table 1. Target sequence for gene-editing
靶位点编号
Target Num.靶位点序列
Target sequencesTIPA1-1 左臂Left arm:GCCGCCACCGACTCGAG 右臂Right arm:TCCCATGGCTGGGTTGACA TIPA1-2 左臂Left arm:CGGTGCCGCCACCGACT 右臂Right arm:GGGTTGACAGAAGAG 表 2 基因编辑再生植株阳性率及靶位点突变率
Table 2. Positive transgenic and mutation rates of target gene editing on plants
载体名称
Vector name再生克隆数 阳性克隆数 阳性率/% 突变克隆数 阳性克隆突变率/% TIPA1-1 38 36 94.7 2 5.6 TIPA1-2 67 57 85.1 0 0.0 注:再生克隆数Num. of regenerated clones,阳性克隆数Num. of positive clones,阳性率Positive percentage,突变克隆数Num. of mutation clones,阳性克隆突变率Mutation percentage of positive clones。 表 3 ipa1纯合突变体主要农艺性状
Table 3. Main agronomic traits of IPA1 mutants
样品编号
Sample NO.株高
Plant height/cm有效穗数
Effective panicle number穗长Average of panicle length/cm 穗粒数
grains per panicle结实率
SettingPercentage/%千粒重1000-grain weight/g ipa1-CK 102.2Aa 9.8A 25.6 A 178.2Aa 81.4a 28.3 a ipa1-2 90.6Bb 15.8C 19.3B 122.7Bb 79.20 a 28.1 a ipa1-4 90.7Bb 16.5C 19.4B 121.2Bb 83.70 a 28.6 a ipa1-16 94.1Bc 14.4B 18.1C 113.2Bc 82.50 a 28.8 a 注:ipa1-CK为杂合突变体分离得到的野生型;ipa1-2、ipa1-4、ipa1-16分别为T2代缺失2、4、16 bp的纯合突变体。表中同列数据后无相同大、小写字母者分别表示同一性状不同样品材料之间差异达极显著水平(P < 0.01)和显著水平(P < 0.05)。
Note:ipa1-CK is a wild-type from progeny of a heterozygous mutant; ipa1-2, ipa1-4, and ipa1-16 are T2 homozygous mutants with 2 bp, 4 bp, and 16 bp detection, respectively. Data in a same column with different capital letters indicate extremely significant differences (P < 0.01) and with different lowercase letters, significant differences (P < 0.05). -
[1] 袁隆平.杂交水稻超高产育种[J].杂交水稻, 1997, 12(6):1-3. http://d.old.wanfangdata.com.cn/Periodical/nmzfzy201520059YUAN L P.Hybrid rice breeding for super high yield[J].Hybrid Rice, 1997, 12(6):1-3.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/nmzfzy201520059 [2] 杨守仁.水稻超高产育种问题探讨[J].中国农学通报, 1990, 6(3):5-8. http://d.old.wanfangdata.com.cn/Periodical/shandnykx200801004YANG S R. Discussion on rice breeding for super high yield[J].Chinese Agricultural Science Bulletin, 1990, 6(3):5-8.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/shandnykx200801004 [3] GAJ T, GERSBACH C A, BARBAS Ⅲ C F. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering[J]. Trends Biotechnol, 2013, 31: 397-405. doi: 10.1016/j.tibtech.2013.04.004 [4] 王福军, 赵开军.基因组编辑技术应用于作物遗传改良的进展与挑战[J].中国农业科学, 2018, 51(1):1-16. http://d.old.wanfangdata.com.cn/Periodical/zgnykx201801001WANG F J, ZHAO K J.Progress and challenge of crop genetic improvement via genome editing[J].Scientia Agricultura Sinica, 2018, 51(1):1-16.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zgnykx201801001 [5] JIAO Y, WANG Y, XUE D, et al. Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice[J]. Nat Genet, 2010, 42(6): 541-544. doi: 10.1038/ng.591 [6] MIURA K, IKEDA M, MATSUBARA A, et al.OsSPL14 promotes panicle branching and higher grain productivity in rice[J]. Nat Genet, 2010, 42:545-549. doi: 10.1038/ng.592 [7] LU Z, YU H, XIONG G, et al. Genome-wide binding analysis of the transcription activator ideal plant architecture1 reveals a complex network regulating rice plant architecture[J]. Plant Cell, 2013, 25(10): 3743-3759. doi: 10.1105/tpc.113.113639 [8] 张建新, 郑家团, 谢华安, 等.水稻新株型明恢86及其系列组合的选育研究[J].江西农业大学学报, 2000, 22 (4):485-490. doi: 10.3969/j.issn.1000-2286.2000.04.004ZHANG J X, ZHENG J T, XIE H A, et al. Breeding of the rice ideal plant type minghui86 and its combinations[J]. Acta Agriculturae Universitis Jiangxiensis, 2000, 22 (4):485-490.(in Chinese) doi: 10.3969/j.issn.1000-2286.2000.04.004 [9] HEIGWER F, KERR G, WALTHER N, et al. E-TALEN: a web tool to design TALENs for genome engineering[J].Nucleic Acids Res, 2013, 41: e190. doi: 10.1093/nar/gkt789 [10] 苏军, 胡昌泉, 翟红利, 等.农杆菌介导籼稻明恢86高效稳定转化体系的建立[J]; 福建农业学报, 2003, 18(4):209-213. doi: 10.3969/j.issn.1008-0384.2003.04.003SU J, HU C Q, ZHAI H L, et al.Establishment of a highly efficient and stable tranf orming system mediated by Agrobacterium tumefacien in indica rice[J].Fujian Journal of Agricultural Sciences, 2003, 18(4):209-213.(in Chinese) doi: 10.3969/j.issn.1008-0384.2003.04.003 [11] YOSHIDA S.Physiological aspects of grain yield[J]. Annu Rev Plant Physiol, 1972, 23:437-464. doi: 10.1146/annurev.pp.23.060172.002253 [12] DONALD C M.The breeding of crop ideotypes[J]. Euphytica, 1968, 17:385-403. doi: 10.1007/BF00056241 [13] HAMBLIN J. The ideotype concept: Useful or outdated[M]//BUXTON D R, SHIBLES R, FORXBERG R A, et al.International Crop Science I. Madison: Crop Science Society of America, Inc Press, 1993: 589-597. [14] REYNOLDS M P, ACEVEDO E, SAYRE K D, et al. Yield potential in modern wheat varieties: its association with a less competitive ideotype[J]. Field Crops Res, 1994, 37:149-160. doi: 10.1016/0378-4290(94)90094-9 [15] DONALD C M. A barley breeding program based on an ideotype[J]. J Agric Sci, 1979, 93:261-269. doi: 10.1017/S0021859600037941 [16] RASMUSSON D C. A plant breeder's experience with ideotype breeding[J]. Field Crops Res, 1991, 26: 191-200. doi: 10.1016/0378-4290(91)90035-T [17] KOKUBUN M. Design and examination of soybean ideotypes[J]. Japan Agric Res Quart, 1988, 21:237-243. [18] PENG S, KHUSH G S, CASSMAN K G. Evolution of the new plant ideotype for increased yield potential[M]//CASSMAN K G. Breaking the Yield Barrier. Los Ba os: International Rice Research Institute Press, 1994: 5-20. [19] KHUSH G S. Breaking the yield frontier of rice[J]. GeoJournal, 1995, 35:29-332. doi: 10.1007/BF00812620 [20] PENG S. KHUSH G S, VIRK P, et al.Progress in ideotype breeding to increase rice yield potential[J]. Field Crops Res, 2008, 108: 32-38. doi: 10.1016/j.fcr.2008.04.001 [21] MARSHALL D R. Alternative approaches and perspectives in breeding for higher yields[J]. Field Crops Res, 1991, 26: 171-190. doi: 10.1016/0378-4290(91)90034-S [22] ZHANG L, YU H, MA B, et al. A natural tandem array alleviates epigenetic repression of IPA1 and leads to superior yielding rice[J]. Nat Commun, 2017, 8:14789. doi: 10.1038/ncomms14789 [23] WANG J, YU H, XIONG G, et al. Tissue-specific ubiquitination by IPA1 INTERACTING PROTEIN1 modulates IPA1 protein levels to regulate plant architecture in rice[J]. Plant Cell, 2017, 29:697-707. doi: 10.1105/tpc.16.00879 [24] SONG X, LU Z, YU H.et al. IPA1 functions as a downstream transcription factor repressed by D53 in strigolactone signaling in rice[J]. Cell Res, 2017, 27:1128-1141. doi: 10.1038/cr.2017.102 [25] LI M, LI X, ZHOU Z, et al. Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system[J]. Front Plant Sci, 2016, 7:377. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=Doaj000004616239 [26] ORDONIO R L, MATSUOKA M.New path towards a better rice architecture[J].Cell Res, 2017, 27:1189-1190. doi: 10.1038/cr.2017.115