利用CRISPR/CAS9技术定点编辑水稻花粉特异表达基因<i>OsIPA</i>

杨绍华; 陈惠妹; 周淑芬

doi:10.19303/j.issn.1008-0384.2017.011.001

利用CRISPR/CAS9技术定点编辑水稻花粉特异表达基因OsIPA

doi: 10.19303/j.issn.1008-0384.2017.011.001

福建省农业遗传工程重点实验室/福建省农业科学院生物技术研究所, 福建福州 350003

基金项目:

福建省科技计划项目——省属公益类科研院所基本科研专项 2014R1019-13

福建省科技计划重大专项 2015NZ0002-3

福建省农业科学院科技创新项目 2014QA-3

详细信息

作者简介:
杨绍华(1976-), 男, 副研究员, 研究方向:水稻遗传育种(E-mail:ysh@fjage.org)

中图分类号: S511
计量
- 文章访问数: 1279
- HTML全文浏览量: 238
- PDF下载量: 124
- 被引次数: 0
出版历程
- 收稿日期: 2017-10-11
- 修回日期: 2017-11-03
- 刊出日期: 2017-11-28

CRISPR/CAS9-mediated Editing for Pollen-specific OsIPA Genein Rice

Fujian Provincial Key Laboratory of Genetic Engineering for Agriculture/Institute of Biotechnology, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003, China

摘要

摘要: 水稻雄配子发育是水稻得以繁殖的关键发育过程，同时水稻的雄性不育在水稻杂种优势利用中也发挥着重要作用。花药/花粉特异表达基因可能在雄配子发育中发挥关键作用。本研究利用CRISPR/CAS9技术对水稻品种明恢86中的花药特异表达基因OsIPA定点编辑，获得OsIPA突变体，以期研究OsIPA基因在水稻花粉发育过程中的功能。试验最终成功的对OsIPA基因第1外显子上的2个不同的靶位点进行了定点突变，经检测发现有24株T₀代在相应的靶位点发生了突变，共有8种不同的类型，包括碱基缺失、替换以及单碱基的插入等不同类型。
- 水稻 /
- CRISPR/Cas9 /
- 基因编辑 /
- OsIPA
Abstract: Male gamete development is an essential step in rice reproduction process, and male sterility is crucial for the utilization of rice heterosis. Since the anther or pollen specific genes in rice might play a pivotal role in the male gamete development, the functions of OsIPA were of great interest to the scientists. This study applied CRISPR/CAS9 to generate an OsIPA mutant for an investigation. Two sites in the 1^st exon of OsIPA were selected as the targets. After sequencing, a total of 8 different types of mutant, including deletion, substitution and single nucleotide insertion, were found in 24T₀ transgenic plants.
- rice /
- CRISPR/Cas9 /
- gene editing /
- OsIPA

HTML全文

图 1 PCR鉴定转基因阳性植株

注：M为分子量标记，+为阳性对照，H₂O为以H₂O为模板的阴性对照。

Figure 1. PCR identification of positive transgenic plants

下载: 全尺寸图片幻灯片

图 2 野生型与T₀代突变体序列比对

注：下划线部分为PAM序列，碱基插入用大写字母表示，突变体序列中的缺失碱基用连字符表示。序列右侧的WT指野生型，-表示缺失，+表示插入。

Figure 2. DNA sequence alignment between wild type and mutants in T₀ generation

下载: 全尺寸图片幻灯片

图 3 T₁代部分纯合突变单株PCR产物测序峰图及花粉I₂-IK染色

注：A为明恢86，B为插入A纯合突变，C为插入T纯合突变，D为缺失GATGT纯合突变。箭头指编辑发生位点。测序峰图右侧为相应的花粉I₂-IK染色图，标尺为100 μm。

Figure 3. Sanger sequencing on PCR products of homozygous mutants

下载: 全尺寸图片幻灯片

参考文献(24)

[1]	NONOMURA K I, MIYOSHI K, EIGUCHI M, et al. The MSP1 gene is necessary to restrict the number of cells entering into male and female sporogenesis and to initiate anther wall formation in rice[J]. Plant Cell, 2003, 15(8):1728-1739. doi: 10.1105/tpc.012401
[2]	ZHAO X, PALMA J D, OANE R, et al. OsTDL1A binds to the LRR domain of rice receptor kinase MSP1, and is required to limit sporocyte numbers[J]. Plant Journal, 2008, 54(3):375-387. doi: 10.1111/j.1365-313X.2008.03426.x
[3]	GUO J X, LIU Y G.Molecular control of male reproductive development and pollen fertility in rice[J].J Integr Plant Biol, 2012, 54(12):967-978. doi: 10.1111/jipb.2012.54.issue-12
[4]	JUNG K H, HAN M J, LEE Y S, et al. Rice Undeveloped Tapetum1 is a major regulator of early tapetum development[J]. Plant cell, 2005, 17(10):2705-2722. doi: 10.1105/tpc.105.034090
[5]	LI N, ZHANG D S, LIU H S, et al. The rice tapetum degeneration retardation gene is required for tapetum degradation and anther development[J]. Plant Cell, 2006, 18(11):2999-3014. doi: 10.1105/tpc.106.044107
[6]	TAN H, LIANG W, HU J, et al.MTR1 Encodes a Secretory Fasciclin Glycoprotein Required for Male Reproductive Development in Rice[J].Developmental Cell, 2012, 22(6):1127-1137. doi: 10.1016/j.devcel.2012.04.011
[7]	LI H, YUAN Z, VIZCAY-BARRENA, et al. PERSITENT TAPETAL CELL1 encodes a PHD-finger protein that is required for tapetal cell death and pollen development in rice[J].Plant physiology, 2011, 156(2):615-630. doi: 10.1104/pp.111.175760
[8]	YI J, MOON S, LEE S, et al. Defective Tapetum Cell Death 1(DTC1) Regulates ROS Levels by Binding to Metallothionein during Tapetum Degeneration[J]. Plant physiology, 2016, 170(3):1611-1623. https://www.researchgate.net/publication/26261371_Improvement_in_sperm_DNA_quality_using_an_oral_antioxidant_therapy
[9]	JUNG K H, HAN M J, LEE D, et al. Wax-deficient anther1 Is Involved in Cuticle and Wax Production in Rice Anther Walls and Is Required for Pollen Development[J]. Plant Cell, 2006, 18(11):3015-3032. doi: 10.1105/tpc.106.042044
[10]	HAN M J, JUNG K H, YI G, et al. Rice Immature Pollen1(RIP1) is a regulator of late pollen development[J]. Plant cell physiology, 2006, 47(11):1457-1472. doi: 10.1093/pcp/pcl013
[11]	YI J, MOON S, HWANG I, et al.. The rice gene DEFECTIVE TAPETUM and MEIOCYTES 1(DTM1) is required for early tapetum development and meiosis[J]. Plant Journal, 2012, 70(2):256-270. doi: 10.1111/tpj.2012.70.issue-2
[12]	BORG M, BROWNFIELD L, TWELL D.Male gametophyte development:a molecular perspective[J].J Exp Bot, 2009, 60(5):1465-1478. doi: 10.1093/jxb/ern355
[13]	HOBO T, SUWABE K, AYA K, et al. Various spatiotemporal expression profiles of anther-expressed genes in rice[J]. Plant Cell Physiol, 2008, 49(10):1417-1428. doi: 10.1093/pcp/pcn128
[14]	SUWABE K, SUZUKI G, TAKAHASHI H, et al. Separated Transcriptomes of male gametophyte and tapetum in rice:balidity of a laser microdissection(LM) microarray[J]. Plant Cell Physiol, 2008, 49(10):1407-1416. doi: 10.1093/pcp/pcn124
[15]	TANG X, ZHANG Z Y, ZHANG W J, et al. Global gene profiling of laser-captured pollen mother cells indicates molecular pathways and gene subfamilies involved in rice meiosis[J].Plant Physiology, 2010, 154(4):1855-1870. doi: 10.1104/pp.110.161661
[16]	GUPTA V, KHURANA R, TYAGI A K.Promoters of two anther-specific genes confer organ-specific gene expression in a stage-specific manner in transgenic systems[J]. Plant Cell Reports, 2007, 26:1919-1931. doi: 10.1007/s00299-007-0414-8
[17]	SWAPNA L, KHURANA R, VIJAYA KUMAR S, et al.Pollen-Specific Expression of Oryza sativa Indica Pollen Allergen Gene (OsIPA) Promoter in Rice and Arabidopsis Transgenic Systems[J]. Mol Biotechnol, 2011, 48(1):49-59. doi: 10.1007/s12033-010-9347-5
[18]	MA X, CHEN L, ZHU Q, et al. Rapid decoding of sequence-specific nuclease-induced heterozygous and biallelic mutations by direct sequencing of PCR products[J]. Mol Plant, 2015, 8(8):1285-1287. doi: 10.1016/j.molp.2015.02.012
[19]	CHEN R, ZHAO X, SHAO Z, et al.Rice UDP-Glucose Pyrophosphorylase1 Is Essential for Pollen Callose Deposition and Its Cosuppression Results in a New Type of Thermosensitive Genic Male Sterility[J]. Plant Cell, 2007, 19(3):847-861. doi: 10.1105/tpc.106.044123
[20]	MU H, KE J, LIU W, et al. UDP-glucose pyropho-sphorylase2(OsUgp2), a pollen-preferential gene in rice, plays a critical role in starch accumulation during pollen maturation[J]. Chinese Science Bulletin, 2009, 54(2):234-243.
[21]	WANG A, XIA Q, XIE W, et al.The classical Ubisch bodies carry a sporophytically produced structural protein (RAFTIN) that is essential for pollen development[J]. PNAS, 2003, 100(24):14487-14492. doi: 10.1073/pnas.2231254100
[22]	邵高能, 谢黎虹, 焦桂爱, 等.利用CRISPR/CAS9技术编辑水稻香味基因Badh2[J].中国水稻科学, 2017, 31(2):216-222. http://ricesci.cn/CN/volumn/volumn_1283.shtml
[23]	王加峰, 郑才敏, 刘维, 等.基于CRISPR/Cas9技术的水稻千粒重基因tgw6突变体的创建[J].作物学报, 2016, 42(8):1160-1167. http://www.doc88.com/p-3733129922134.html
[24]	ZHOU H, HE M, LI J, et al. Development of Commercial Thermo-sensitive Genic Male SterileRice Accelerates Hybrid Rice Breeding Using the CRISPR/Cas9-mediated TMS5 Editing System[J]. Scientific Reports, 2016, 6:37395. doi: 10.1038/srep37395