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作物学报 ›› 2023, Vol. 49 ›› Issue (2): 354-364.doi: 10.3724/SP.J.1006.2023.22002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

利用CRISPR/Cas9探究水稻OsPIN5c基因功能

杨晓祎1(), 王慧慧1, 张艳雯1, 侯典云1, 张红晓1, 康国章2, 胥华伟1,*()   

  1. 1河南科技大学农学院, 河南洛阳 471000
    2河南农业大学省部共建小麦玉米作物学国家重点实验室, 河南郑州 450046
  • 收稿日期:2022-01-13 接受日期:2022-06-07 出版日期:2022-07-08 网络出版日期:2022-07-08
  • 通讯作者: 胥华伟
  • 作者简介:E-mail: 1961827259@qq.com
  • 基金资助:
    河南省自然科学基金项目(182300410012);河南省自然科学基金项目(202300410151);河南省自然科学基金项目(202300410340);小麦玉米作物学国家重点实验室开放课题项目(SKL2021KF03)

Function analysis of OsPIN5c gene by CRISPR/Cas9

YANG Xiao-Yi1(), WANG Hui-Hui1, ZHANG Yan-Wen1, HOU Dian-Yun1, ZHANG Hong-Xiao1, KANG Guo-Zhang2, XU Hua-Wei1,*()   

  1. 1College of Agriculture, Henan University of Science and Technology, Luoyang 471000, Henan, China
    2National Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University, Zhengzhou 450046, Henan, China
  • Received:2022-01-13 Accepted:2022-06-07 Published:2022-07-08 Published online:2022-07-08
  • Contact: XU Hua-Wei
  • Supported by:
    Natural Science Foundation of Henan Province(182300410012);Natural Science Foundation of Henan Province(202300410151);Natural Science Foundation of Henan Province(202300410340);Open Research Fund of National Key Laboratory of Wheat and Maize Crop Science(SKL2021KF03)

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摘要：

生长素极性运输在植物生长发育中发挥着重要作用, 生长素输出载体蛋白(PIN-FORMED, PIN)是控制生长素极性运输的关键蛋白。虽然部分水稻OsPIN基因功能已有报道, 但水稻OsPIN5c的生物学功能仍有待研究。本研究在OsPIN5c第1外显子处设计靶点并构建CRISPR/Cas9载体, 通过转化粳稻品种日本晴获得24株转基因植株, PCR产物直接测序分析表明其中15株发生突变, 突变率为62.5%, 突变类型为双等位突变; 进一步从T1代突变体中筛选获得3种不同的纯合突变体, 将其命名为ospin5c-1、ospin5c-2和ospin5c-3。序列比对分析表明, 这3种类型的突变均造成移码突变和蛋白翻译提前终止, 由原来的398个氨基酸分别缩短为109、106和250个氨基酸; 跨膜螺旋结构域分析表明, 3种突变体中OsPIN5c蛋白的跨膜结构完全消失; 蛋白结构预测表明3种突变体中OsPIN5c蛋白螺旋结构明显减少。突变体的苗高、根长和不定根数均显著低于野生型植株。组织特异性表达表明OsPIN5c主要在根中表达。突变体根中OsPIN1a和OsPIN5b表达量提高; 生长素合成关键基因OsYUC1、OsYUC4、OsYUC6和OsYUC7的表达量也显著提高。ospin5c突变体根的向重性受到部分抑制。本研究利用CRISPR/Cas9基因编辑技术获得ospin5c纯合突变体并探讨了OsPIN5c基因功能, 为利用OsPIN5c基因进行作物遗传改良提供了潜在的基因资源。

关键词: OsPIN5c, CRISPR/Cas9, 基因功能, 水稻

Abstract:

Polar auxin transport (PAT) plays a key role in plant growth and development, and auxin efflux carriers PIN-FORMED (PIN) are the crucial proteins controlling PAT. Although the functions of some OsPIN genes have been reported, the function of OsPIN5c gene is still unclear. In this study, the target site was designed at the first exon of OsPIN5c and the recombinant CRISPR/Cas9 vector of OsPIN5c was constructed. Twenty-four independent transgenic rice lines were obtained by transformation using Nipponbare (Oryza sativa L. ssp. japonica) as the materials. PCR product sequencing indicated that 15 lines were identified as mutants and the corresponding mutation rate was 62.5%, the mutation type were biallelic heterozygous mutations. Three independent ospin5c homozygous mutants were further obtained in T1 generation lines and named as ospin5c-1, ospin5c-2, and ospin5c-3, respectively. Sequence alignment analysis showed that the three types of mutations resulted in frame-shift mutation and premature translation termination, which were shortened from 398 amino acid (aa) in wild-type (WT) plants to 109 aa, 106 aa and 250 aa, respectively. Transmembrane helices (TMH) indicated that the TMH of these three mutation proteins were disappeared totally. Protein structure demonstrated that the helix of three mutation proteins were obviously reduced than the native OsPIN5c protein. Phenotype structure indicated that, compared to the WT, the shoot height, root length and adventitious root number were significantly decreased in ospin5c mutants at seedling stage. Tissue-specific analysis showed that OsPIN5c was highly expressed in roots, and OsPIN genes (OsPIN1a and OsPIN5b), OsYUC genes (OsYUC1, OsYUC4, OsYUC6, and OsYUC7) were up-regulated significantly in ospin5c mutants. The gravitropism response of ospin5c mutants was partially inhibited. In conclusion, three ospin5c homozygous mutants were obtained via CRISPR/Cas9 technology and the function of OsPIN5c was investigated in this study, providing the potential gene resources for crop genetic improvement by using OsPIN5c gene.

Key words: OsPIN5c, CRISPR/cas9, gene function, rice

表1

引物及序列"

引物名称Primer name 正向引物Forward sequence (5'-3') 反向引物Reverse sequence (5'-3')
OsPIN5c-CRISPR TGTGTGGGTTCTCGTGGTGCATCAC AAACGTGATGCACCACGAGAACCCA
HPT CTGAACTCACCGCGACGTCTGTC TAGCGCGTCTGCTGCTCCATACA
OsPIN5c-Assay CCTCGTCGCCTGCTTCGCCG CCACACCGCTCTCACCAGCGG
OsPIN1a-qPCR CCTGAAATCCATCTCCATCCTC AACGTCGCCACCTTGTT
OsPIN5b-qPCR GCAAAGGAGTATGGGCTTCA GCAATCAGAATCGGCAGAGA
OsYUC1-qPCR AGGTGTTGGTCGTGGGATGCG GCGATGCCGAACGTGGATAGA
OsYUC4-qPCR CCTCGACCTCTGCAACCACAATG CGACAACAGGAGTACCAGCCAATC
OsYUC6-qPCR GGATACCAAAGCAACGTCCCC TGAAGCCAACAGAGTAGAGCCCTG
OsYUC7-qPCR ACCGGCTACCGCAGCAATGTG CGTACAGCCCCGACTCACCCT
OsACTIN1-qPCR CTTCATAGGAATGGAAGCTGCG CACCTTGATCTTCATGCTGCTA

图1

OsPIN5c gRNA靶位点示意图及阳性克隆筛选 A: OsPIN5c靶位点示意图; PAM: 前间隔序列邻近基序; 黑色下画线表示PAM序列。B: PCR筛选阳性克隆, M: DL2000 DNA marker; 1~12: PCR产物。"

图2

T0代突变植株的筛选及分析 A: PCR扩增OsPIN5c基因组片段; M: DL2000 DNA marker; 1~7: PCR产物。B: 突变类型分析, PAM: 前间隔序列邻近基序; 黑色下画线表示PAM序列。"

图3

T1代纯合ospin5c突变体的筛选 PAM: 前间隔序列邻近基序; 黑色下画线表示PAM序列; 红色箭头表示突变位点。"

图4

无转基因成分植株筛选 A: PCR筛选结果; M: DL2000 DNA marker; 1~25: HPT PCR扩增结果; P: 阳性对照(质粒); N: 阴性对照(WT)。B: 叶片潮霉素抗性筛选结果, 1~25: ospin5c离体叶片潮霉素筛选。"

图5

OsPIN5c突变蛋白的氨基酸变化 A: 靶位点氨基酸变化; PAM: 前间隔序列邻近基序; 下画线对应的氨基酸表示突变氨基酸; 星号表示翻译的终止。B: 突变蛋白比对分析。"

图6

OsPIN5c突变蛋白的跨膜螺旋结构域及三级结构分析 A: 跨膜螺旋结构域分析; B: 三级结构分析。"

图7

幼苗期ospin5c突变体表型 A: 7 d ospin5c突变体表型分析。B: 14 d ospin5c突变体表型分析(n ≥ 15)。**表示P < 0.01水平差异。"

图8

基因表达分析 A: OsPIN5c组织特异性表达分析。B: OsPIN和OsYUC表达分析; *表示P < 0.05水平差异, **表示P < 0.01水平差异, ***表示P < 0.001水平差异。"

图9

野生型(WT)和ospin5c突变体根向重性分析 A: 重力刺激后野生型(WT)和ospin5c突变体表型, 0~24 h表示重力刺激处理时间。B: 重力刺激后野生型(WT)和ospin5c突变体根弯曲角度统计分析(n ≥ 12); *表示P < 0.05水平差异, **表示P < 0.01水平差异。"

[1] Benkova E, Michniewicz M, Sauer M, Teichmann T, Seifertova D, Jurgens G, Friml J. Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell, 2003, 115: 591-602.
pmid: 14651850
[2] Friml J. Auxin transport—shaping the plant. Curr Opin Plant Biol, 2003, 6: 7-12.
pmid: 12495745
[3] Petrasek J, Mravec J, Bouchard R, Blakeslee J J, Abas M, Seifertova D, Wisniewska J, Tadile Z, Kubes M, Covanova M, Dhonukshe P, Skupa P, Benkova E, Perry L, Krecek P, Lee O R, Fink G R, Geisler M, Murphy A S, Luschnig C, Zazimalova E, Friml J. PIN proteins perform a rate-limiting function in cellular auxin efflux. Science, 2006, 312: 914-918.
pmid: 16601150
[4] Karki S, Rizal G, Quick W P. Improvement of photosynthesis in rice (Oryza sativa L.) by inserting the C4 pathway. Rice (New York), 2013, 6: 1-8.
[5] Woodward A W. Auxin: regulation, action, and interaction. Ann Bot, 2005, 95: 707-735.
doi: 10.1093/aob/mci083
[6] Zhao Y. Auxin biosynthesis and its role in plant development. Annu Rev Plant Biol, 2010, 61: 49-64.
doi: 10.1146/annurev-arplant-042809-112308 pmid: 20192736
[7] Michiewicz M, Brewer P B, Friml J. Polar auxin transport and asymmetric auxin distribution. Arabidopsis Book, 2007, 5: e0108.
[8] Krecek P, Skupa P, Libus J, Naramoto S, Tejos R, Friml J, Zazimalova E. The PIN-FORMED (PIN) protein family of auxin transporters. Genome Biol, 2009, 10: 249.
doi: 10.1186/gb-2009-10-12-249 pmid: 20053306
[9] Okada K, Ueda J, Komaki M K, Bell C J, Shimura Y. Requirement of the auxin polar transport system in early stages of Arabidopsis floral bud formation. Plant Cell, 1991, 3: 677-684.
doi: 10.2307/3869249
[10] Luschnig C, Gaxiola R A, Grisafi P, Fink G R. EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana. Genes Dev, 1998, 12: 2175-2187.
doi: 10.1101/gad.12.14.2175
[11] Muller A, Guan C, Galweiler L, Tanzler P, Huijser P, Marchant A, Parry G, Bennett M, Wisman E, Palme K. AtPIN2 defines a locus of Arabidopsis for root gravitropism control. EMBO J, 1998, 17: 6903-6911.
doi: 10.1093/emboj/17.23.6903
[12] Friml J, Wisniewska J, Benkova E, Mendgen K, Palme K. Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis. Nature, 2002, 415: 806-809.
doi: 10.1038/415806a
[13] Wang J, Hu H, Wang G, Li J, Chen J, Wu P. Expression of PIN genes in rice (Oryza sativa L.): tissue specificity and regulation by hormones. Mol Plant, 2009, 2: 823-831.
doi: 10.1093/mp/ssp023
[14] Miyashita Y, Takasugi T, Ito Y. Identification and expression analysis of PIN genes in rice. Plant Sci, 2010, 178: 424-428.
doi: 10.1016/j.plantsci.2010.02.018
[15] Li Y, Zhu J, Wu L, Shao Y, Wu Y, Mao C. Functional divergence of PIN1 paralogous genes in rice. Plant Cell Physiol, 2019, 60: 2720-2732.
doi: 10.1093/pcp/pcz159
[16] Chen Y, Fan X, Song W, Zhang Y, Xu G. Over-expression of OsPIN2 leads to increased tiller numbers, angle and shorter plant height through suppression of OsLAZY1. Plant Biotechnol J, 2012, 10: 139-149.
doi: 10.1111/j.1467-7652.2011.00637.x
[17] Inahashi H, Shelley I J, Yamauchi T, Nishiuchi S, Takahashi-nosaka M, Matsunami M, Ogawa A, Noda Y, Inukai Y. OsPIN2, which encodes a member of the auxin efflux carrier proteins, is involved in root elongation growth and lateral root formation patterns via the regulation of auxin distribution in rice. Physiol Plant, 2018, 164: 216-225.
doi: 10.1111/ppl.12707 pmid: 29446441
[18] Wu D, Shen H, Yokawa K, Baluska F. Overexpressing OsPIN2 enhances aluminium internalization by elevating vesicular trafficking in rice root apex. J Exp Bot, 2015, 66: 6791-6801.
doi: 10.1093/jxb/erv385
[19] Wu D, Shen H, Yokawa K, Baluska F. Alleviation of aluminium-induced cell rigidity by overexpression of OsPIN2 in rice roots. J Exp Bot, 2014, 65: 5305-5315.
doi: 10.1093/jxb/eru292
[20] Zhang Q, Li J, Zhang W, Yan S, Wang R, Zhao J, Li Y, Qi Z, Sun Z, Zhu Z. The putative auxin efflux carrier OsPIN3t is involved in the drought stress response and drought tolerance. Plant J, 2012, 72: 805-816.
doi: 10.1111/j.1365-313X.2012.05121.x
[21] Lu G, Coneva V, Casaretto J A, Ying S, Mahmood K, Liu F, Nambara E, Bi Y M, Rothstein S J. OsPIN5b modulates rice (Oryza sativa) plant architecture and yield by changing auxin homeostasis, transport and distribution. Plant J, 2015, 83: 913-925.
doi: 10.1111/tpj.12939
[22] Hou M, Luo F, Wu D, Zhang X, Lou M, Shen D, Yan M, Mao C, Fan X, Xu G, Zhang Y. OsPIN9, an auxin efflux carrier, is required for the regulation of rice tiller bud outgrowth by ammonium. New Phytol, 2021, 229: 935-949.
doi: 10.1111/nph.16901
[23] 单奇伟, 高彩霞. 植物基因组编辑及衍生技术最新研究进展. 遗传, 2015, 37: 953-973.
Dan Q W, Gao C X. Research progress of genome editing and derivative technologies in plants. Hereditas (Beijing), 2015, 37: 953-973. (in Chinese with English abstract)
[24] Liu W, Xie X, Ma X, Li J, Chen J, Liu Y G. DSDecode: a web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations. Mol Plant, 2015, 8: 1431-1433.
doi: 10.1016/j.molp.2015.05.009 pmid: 26032088
[25] 吴世洋, 杨晓祎, 张艳雯, 侯典云, 胥华伟. 利用CRISPR/Cas9基因编辑技术构建水稻ospin9突变体. 中国农业科学, 2021, 54: 3805-3817.
Wu S Y, Yang X Y, Zhang Y W, Hou D Y, Xu H W. Generation of ospin9 mutants in rice by CRISPR/Cas9 genome editing technology. Sci Agric Sin, 2021, 54: 3805-3817. (in Chinese with English abstract)
[26] Xin Z, Chen J. A high throughput DNA extraction method with high yield and quality. Plant Methods, 2012, 8: 26.
doi: 10.1186/1746-4811-8-26 pmid: 22839646
[27] Ma X, Chen L, Zhu Q, Chen Y, Liu Y. Rapid decoding of sequence-specific nuclease-induced heterozygous and biallelic mutations by direct sequencing of PCR products. Mol Plant, 2015, 8: 1285-1287.
doi: 10.1016/j.molp.2015.02.012 pmid: 25747846
[28] 刘巧泉, 陈秀花, 王兴稳, 彭凌涛, 顾铭洪. 一种快速检测转基因水稻中潮霉素抗性的简易方法. 农业生物技术学报, 2001, 9: 264.
Liu Q Q, Chen X H, Wang X W, Peng L T, Gu M H. A rapid simple method of assaying hygromycin resistance in transgenic rice plants. J Agric Biotechnol, 2001, 9: 264. (in Chinese with English abstract)
[29] 张洪, 马骏, 胡国成, 斯华敏, 付亚萍, 戴良英, 孙宗修. 用叶片检测转基因水稻对潮霉素反应的可靠性研究. 浙江农业学报, 2005, 17: 341-345.
Zhang H, Ma J, Hu G C, Si H M, Fu Y P, Dai L Y, Sun Z X. Reliability study on the response of HPT gene in transgenic rice to hygromycin by leaf assay. Acta Agric Zhejiangensis, 2005, 17: 341-345. (in Chinese with English abstract)
[30] Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol, 2016, 33: 1870-1874.
doi: 10.1093/molbev/msw054 pmid: 27004904
[31] Waterhouse A M, Procter J B, Martin D M A, Clamp M, Barton G J. Jalview Version 2: a multiple sequence alignment editor and analysis workbench. Bioinformatics, 2009, 25: 1189-1191.
doi: 10.1093/bioinformatics/btp033 pmid: 19151095
[32] Krogh A, Larsson B, von Heijne G, Sonnhammer E L L. Predicting transmembrane protein topology with a hidden markov model: application to complete genomes. J Mol Biol, 2001, 305: 567-580.
doi: 10.1006/jmbi.2000.4315 pmid: 11152613
[33] Madeira F, Park Y M, Lee J, Buso N, Gur T, Madhusoodanan N, Basutkar P, Tivey A R N, Potter S C, Finn R D, Lopez R. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res, 2019, 47: W636-W641.
doi: 10.1093/nar/gkz268
[34] Iino M, Tarui Y, Uematsu C. Gravitropism of maize and rice coleoptiles: dependence on the stimulation angle. Plant Cell Environ, 1996, 19: 1160-1168.
pmid: 11539324
[35] Bennett T, Brochington S F, Rothfels C, Graham S W, Stevenson D, Kutchan T, Rolf M, Thomas P, Wong G K, Leyser O, Glover B J, Harrison C J. Paralogous radiations of PIN proteins with multiple origins of noncanonical PIN structure. Mol Biol Evol, 2014, 31: 2042-2060.
doi: 10.1093/molbev/msu147 pmid: 24758777
[36] Rahman A, Takahashi M, Shibasakei K, Wu S, Inaba T, Tsurumi S, Baskin T I. Gravitropism of Arabidopsis thaliana roots requires the polarization of PIN2 toward the root tip in meristematic cortical cells. Plant Cell, 2010, 22: 1762-1776.
doi: 10.1105/tpc.110.075317
[37] Zhang J, Zhang H, Li S, Li J, Yan L, Xia L. Increasing yield potential through manipulating of an ARE1 ortholog related to nitrogen use efficiency in wheat by CRISPR/Cas9. J Integr Plant Biol, 2021, 63: 1649-1663.
doi: 10.1111/jipb.13151
[38] Zhang J, 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 Integr Plant Biol, 2018, 60: 369-375.
doi: 10.1111/jipb.12620
[39] Wang Y, Liu X, Zheng X, Wang W, Yin X, Liu H, Ma C, Niu X, Zhu J K, Wang F. Creation of aromatic maize by CRISPR/Cas. J Integr Plant Biol, 2021, 63: 1664-1670.
doi: 10.1111/jipb.13105
[40] Zhang L, Wang T, Wang G, Bi A, Wassie M, Xie Y, Cao L, Xu H, Fu J, Chen L, Zhao Y, Hu T. Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses. J Integr Plant Biol, 2021, 63: 1410-1415.
doi: 10.1111/jipb.13101
[41] 侯智红, 吴艳, 程群, 董利东, 芦思佳, 南海洋, 甘卓然, 刘宝辉. 利用CRISPR/Cas9技术创制大豆高油酸突变系. 作物学报, 2019, 45: 839-847.
doi: 10.3724/SP.J.1006.2019.84157
Hou Z H, Wu Y, Cheng Q, Dong L D, Lu S J, Nan H Y, Gan Z R, Liu B H. Creation of high oleic acid soybean mutation plants by CRISPR/Cas9. Acta Agron Sin, 2019, 45: 839-847. (in Chinese with English abstract)
[42] 张旺, 冼俊霖, 孙超, 王春明, 石丽, 于为常. CRISPR/Cas9编辑花生FAD2基因研究. 作物学报, 2021, 47: 1481-1490.
doi: 10.3724/SP.J.1006.2021.04214
Zhang W, Xian J L, Sun C, Wang C M, Shi L, Yu W C. Preliminary study of genome editing of peanut FAD2 genes by CRISPR/Cas9. Acta Agron Sin, 2021, 47: 1481-1490. (in Chinese with English abstract)
[43] 石育钦, 孙梦丹, 陈帆, 成洪涛, 胡学志, 付丽, 胡琼, 梅德圣, 李超. 通过CRISPR/Cas9技术突变BnMLO6基因提高甘蓝型油菜的抗病性. 作物学报, 2022, 48: 801-811.
doi: 10.3724/SP.J.1006.2022.14077
Shi Y Q, Sun M D, Chen F, Cheng H T, Hu X Z, Fu L, Hu Q, Mei D S, Li C. Genome editing of BnMLO6 gene by CRISPR/Cas9 for the improvement of disease resistance in Brassica napus L. Acta Agron Sin, 2022, 48: 801-811. (in Chinese with English abstract)
doi: 10.3724/SP.J.1006.2022.14077
[44] Ma X, 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 dicot plants. Mol Plant, 2015, 8: 1274-1284.
doi: 10.1016/j.molp.2015.04.007 pmid: 25917172
[45] Lu Y, Tian Y, Shen R, Yao Q, Wang M, Chen M, Dong J, Zhang T, Li F, Lei M, Zhu J. Targeted, efficient sequence insertion and replacement in rice. Nat Biotechnol, 2020, 38: 1402-1407.
doi: 10.1038/s41587-020-0581-5
[46] Wei C, Wang C, Jia M, Guo H X, Luo P Y, Wang M G, Zhu J K, Zhang H. Efficient generation of homozygous substitutions in rice in one generation utilizing an rABE8e base editor. J Integr Plant Biol, 2021, 63: 1595-1599.
doi: 10.1111/jipb.13089
[47] Niu Q, Wu S, Xie H, Wu Q, Liu P, Xu Y, Lang Z. Efficient A. T to G. C base conversions in dicots using adenine base editors expressed under the tomato EF1alpha promoter. Plant Biotechnol J, 2021, doi: 10.1111/pbi.13736.
doi: 10.1111/pbi.13736
[48] Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi J J, Qiu J, Gao C. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol, 2013, 31: 686-688.
doi: 10.1038/nbt.2650 pmid: 23929338
[49] Li J, Norville J E, Aach J, McCormack M, Zhang D, Bush J, Church G M, Sheen J. Multiplex and homologous recombination-mediated genome editing via guide RNA/Cas9. Nat Biotechnol, 2013, 31: 688-691.
doi: 10.1038/nbt.2654
[50] Xie K, Yang Y. RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plants, 2013, 6: 1975-1983.
[51] 马兴亮, 刘耀光. 植物CRISPR/Cas9基因组编辑系统与突变分析. 遗传, 2016, 38: 118-125.
Ma X L, Liu Y G. CRISPR/Cas9-based genome editing systems and the analysis of targeted genome mutations in plants. Hereditas (Beijing), 2016, 38: 118-125 (in Chinese with English abstract).
[52] Mravec J, Skupa P, Baill Y A, Hoyerova K, Krecek P, Bielach A, Petrasek J, Zhang J, Gaykova V, Stierhof Y D, Dobrev P I, Schwarzerova K, Rolcik J, Seifertova D, Luschnig C, Benkova E, Zazimalova E, Geisler M, Friml J. Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter. Nature, 2009, 459: 1136-1140.
doi: 10.1038/nature08066
[53] Fukaki H, Fujisawa H, Tasaka M. How do plant shoots bend up? The initial step to elucidate the molecular mechanisms of shoot gravitropism using Arabidopsis thaliana. J Plant Res, 1996, 109: 129-137.
pmid: 11539858
[54] Chen R, Hilson P, Sedbrook J, Rosen E, Caspar T, Masson P H. The Arabidopsis thaliana AGRAVITROPIC 1gene encodes a component of the polar-auxin-transport efflux carrier. Proc Natl Acad Sci USA, 1998, 95: 15112-15117.
doi: 10.1073/pnas.95.25.15112
[55] Grunewald W, Friml J. The march of the PINs: developmental plasticity by dynamic polar targeting in plant cells. EMBO J, 2010, 29: 2700-2714.
doi: 10.1038/emboj.2010.181 pmid: 20717140
[56] Harrison B R, Masson P H. ARL2, ARG1 and PIN3 define a gravity signal transduction pathway in root statocytes. Plant J, 2008, 53: 380-392.
pmid: 18047472
[57] Keuskamp D H, Pollmann S, Voesenek L A, Peeters A J, Pierik R. Auxin transport through PIN-FORMED 3 (PIN3) controls shade avoidance and fitness during competition. Proc Natl Acad Sci USA, 2010, 107: 22740-22744.
doi: 10.1073/pnas.1013457108
[58] Wang L, Guo M, Li Y, Ruan W, Mo X, Wu Z, Sturrock C J, Yu H, Lu C, Peng J, Mao C. LARGE ROOT ANGLE1, encoding OsPIN2, is involved in root system architecture in rice. J Exp Bot, 2018, 69: 385-397.
doi: 10.1093/jxb/erx427 pmid: 29294052
[59] Farooq M, Jan R, Kim K. Gravistimulation effects on Oryza sativa amino acid profile, growth pattern and expression of OsPIN genes. Sci Rep, 2020, 10: 17303.
doi: 10.1038/s41598-020-74531-w
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