欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2020, Vol. 46 ›› Issue (12): 1862-1869.doi: 10.3724/SP.J.1006.2020.04045

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘薯IbCAF1基因的克隆及耐盐性、抗旱性鉴定

陈杉彬(), 孙思凡, 聂楠, 杜冰, 何绍贞, 刘庆昌, 翟红*()   

  1. 中国农业大学 / 农业农村部甘薯生物学与生物技术重点实验室 / 教育部作物杂种优势研究与利用重点实验室 / 北京市作物遗传改良重点实验室, 北京 100193
  • 收稿日期:2020-02-27 接受日期:2020-06-02 出版日期:2020-12-12 网络出版日期:2020-07-02
  • 通讯作者: 翟红
  • 基金资助:
    国家自然科学基金项目(31872878);国家重点研发计划项目(2018YFD1000700);国家重点研发计划项目(2018YFD1000704);国家现代农业产业技术体系建设专项(CARS-10)

Cloning of IbCAF1 and identification on tolerance to salt and drought stress in sweetpotato

Shan-Bin CHEN(), Si-Fan SUN, Nan NIE, Bing DU, Shao-Zhen HE, Qing-Chang LIU, Hong ZHAI*()   

  1. Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs / Laboratory of Crop Heterosis and Utilization, Ministry of Education / Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing 100193, China
  • Received:2020-02-27 Accepted:2020-06-02 Published:2020-12-12 Published online:2020-07-02
  • Contact: Hong ZHAI
  • Supported by:
    National Natural Science Foundation of China(31872878);National Key Research and Development Program of China(2018YFD1000700);National Key Research and Development Program of China(2018YFD1000704);China Agriculture Research System(CARS-10)

RichHTML

27

PDF

363

摘要：

CAF1 (CCR4-associated factor 1)基因在植物发育、抗病等方面发挥着重要的作用。本研究根据前期获得的差异表达EST序列, 克隆得到甘薯IbCAF1基因。IbCAF1基因的开放阅读框(open reading frame, ORF)长度为846 bp, 编码281个氨基酸, 分子量为32.13 kD, 等电点为4.83。氨基酸序列比对和系统进化树分析表明, IbCAF1与甘薯近缘野生种Ipomoea triloba (2x)同源蛋白ItlCAF1有较高的同源性, 序列一致性为96.8%。IbCAF1基因受到NaCl、PEG、ABA和H2O2的诱导表达。利用根癌农杆菌介导法将IbCAF1基因转入烟草, 过表达IbCAF1基因显著提高了转基因烟草植株的耐盐性和抗旱性。在200 mmol L -1 NaCl和10% PEG-6000的胁迫下, IbCAF1基因的过表达显著上调了转基因烟草植株中活性氧清除系统和脯氨酸合成相关基因的表达, 增加了SOD活性、POD活性、脯氨酸含量, 降低了H2O2含量和丙二醛含量。表明IbCAF1基因能够提高转基因烟草植株的耐盐性和抗旱性。本研究为后续甘薯IbCAF1基因耐盐抗旱基因工程研究奠定了基础。

关键词: 甘薯, IbCAF1, 转基因烟草, 耐盐, 抗旱

Abstract:

CAF1 (CCR4-associated factor 1) gene plays an important role in plant development and disease resistance. In this study, the IbCAF1 gene of sweetpotato was cloned according to the EST sequence. The ORF of IbCAF1 was 846 bp, encoding 281 amino acids, with a molecular weight of 32.13 kD and an isoelectric point of 4.83. The results of amino acid sequence alignment and phylogenetic tree analysis showed that IbCAF1 had higher homology with ItlCAF1, a homologous protein of Ipomoea triloba (2x), and the homology was 96.8%. IbCAF1 gene was induced and expressed by NaCl, PEG, ABA, and H2O2. The IbCAF1 gene was transferred into tobacco by Agrobacterium tumefaciens mediated transformation. The overexpression of IbCAF1 gene significantly improved the salt and drought tolerance of transgenic tobacco plants. After 200 mmol L -1NaCl and 10% PEG-6000 treatments, the transgenic tobacco plants showed significant upregulation of the genes involved in ROS scavenging system and proline biosynthesis related genes, significant increase of SOD activity, POD activity and proline content and significant decrease of H2O2 and malondialdehyde contents. These results demonstrate that the IbCAF1 gene could improve salt and drought tolerance in transgenic tobacco. This study will lay a foundation on salt and drought tolerance gene engineering of IbCAF1 gene in sweetpotato for the following research.

Key words: sweetpotato, IbCAF1, transgenic tobacco, salt tolerance, drought tolerance

表1

本研究所用引物"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
IbCAF1扩增引物
Primers for IbCAF1 amplification		 IbCAF1-F: ATGGGTGTACAAGAAGATGTTTTG
IbCAF1-R: CTAAAAAACTTCTAGTCCGTACAATACT
鉴定转基因引物
Primers for identifying transformants		 IbCAF1-OPF: GAGGCTTACGCAGCAGGTC
IbCAF1-OPR: TTATAATTCATACTCGTGGAAGCGC
实时定量PCR引物
Primers for real-time quantitative PCR		 IbActin-F: AGCAGCATGAAGATTAAGGTTGTAGCAC
IbActin-R: TGGAAAATTAGAAGCACTTCCTGTGAAC
Actin-F: GAGGAATGCAGATCTTCGTG
Actin-R: TCCTTGTCCTGGATCTTAGC
IbCAF1RT-F: TCAGCTACCTCATCGACGAC
IbCAF1RT-R: AGTCAACCCGAGCTGAATCA
SOD-F: CTATTACCGACAAGCAGATTCCTC
SOD-R: TACCACAAGCAACCCTTCCAC
APX-F: GATGTTCCCTTTCACCCTGG
APX-R: CAGATAGACCCATTTGCTTCACA
POD-F: TCCGGGAGCCACACCATTGG
POD-R: TGGTCGGAATTCAACAG
P5CS-F: TTGTGACACGGACTGATGGAA
P5CS-R: TATCTAAGCCGCTGACGACCA

图1

IbCAF1基因序列分析 A: IbCAF1蛋白序列分析; B: 甘薯CAF1蛋白和其他植物中的CAF1蛋白序列比对; C: 甘薯CAF1蛋白和其他植物中的CAF1蛋白的同源进化树分析。ItlCAF1: 三裂叶野牵牛(XP_031110715.1); InCAF1: 牵牛(XP_019199562.1); NtCAF1: 烟草(XP_016511744.1); SpCAF1: 番茄近缘野生种(XP_015079674.1); CaCAF1: 辣椒(NP_001312000.1); SlCAF1: 番茄(XP_004241342.1); StCAF1: 马铃薯(XP_006361099.1)。"

图2

IbCAF1基因在鲁薯3号中的表达分析 A: IbCAF1基因在鲁薯3号不同组织中的表达; B: 200 mmol L-1 NaCl、20% PEG-6000、100 μmol L-1 ABA和10 mmol L-1 H2O2分别处理不同时间后, 鲁薯3号中IbCAF1基因的表达分析。*与**分别表示在0.05和0.01水平下差异显著。"

图3

转基因烟草植株的IbCAF1基因的qRT-PCR分析 **表示在0.01水平下差异显著。"

图4

IbCAF1增强了转基因烟草植株的耐盐性和抗旱性 A: 转IbCAF1基因烟草植株和WT烟草植株在无胁迫或添加200 mmol L-1 NaCl或10% PEG-6000的1/2 MS培养基上培养4周; B~H: 在无胁迫、200 mmol L-1 NaCl或10% PEG-6000的1/2 MS培养基上培养4周的IbCAF1转基因烟草和WT烟草叶片的DAB染色(B)、NBT染色(C)、H2O2含量(D)、SOD活性(E)、POD活性(F)、脯氨酸含量(G)、丙二醛含量(H)。*与**分别表示在0.05和0.01水平下差异显著。"

图5

转基因植株及WT植株的抗逆相关基因的表达分析 *与**分别表示在0.05和0.01水平下差异显著。"

[1] Munns R, Tester M . Mechanisms of salinity tolerance. Annu Rev Plant Biol, 2008,59:651-681.
doi: 10.1146/annurev.arplant.59.032607.092911 pmid: 18444910
[2] 王佳丽, 黄贤金, 钟太洋, 陈志刚 . 盐碱地可持续利用研究综述. 地理学报, 2011,66:673-684.
doi: 10.11821/xb201105010
Wang J L, Huang X J, Zhong T Y, Chen Z G . Review on sustainable utilization of salt-affected land. Acta Geogr Sin, 2011,66:673-684 (in Chinese with English abstract).
[3] Yang S J, Vanderbeld B, Wan J X, Huang Y F . Narrowing down the targets: towards successful genetic engineering of drought tolerant crops. Mol Plant, 2010,3:469-490.
doi: 10.1093/mp/ssq016 pmid: 20507936
[4] Molin L, Puisieux A . C. elegans homologue of the Caf1 gene, which encodes a subunit of the CCR4-NOT complex, is essential for embryonic and larval development and for meiotic progression. Gene, 2005,358:73-81.
doi: 10.1016/j.gene.2005.05.023 pmid: 16039072
[5] Collart M A . The CCR4-NOT complex is a key regulator of eukaryotic gene expression. WIREs RNA, 2016,7:438-454.
doi: 10.1002/wrna.1332 pmid: 26821858
[6] Berthet C, Morera A M, Asensio M J, Chauvin M A, Morel A P, Dijoud F, Magaud J P, Durand P, Rouault J P . CCR4-associated factor CAF1 is an essential factor for spermatogenesis. Mol Cell Biol, 2004,24:5808-5820.
doi: 10.1128/MCB.24.13.5808-5820.2004 pmid: 15199137
[7] Cui Y J, Ramnarain D B, Chiang Y C, Ding L H, McMahon J S, Denis C L . Genome wide expression analysis of the CCR4-NOT complex indicates that it consists of three modules with the NOT module controlling SAGA-responsive genes. Mol Genet Genomics, 2008,279:323-337.
doi: 10.1007/s00438-007-0314-1 pmid: 18214544
[8] Feng L K, Yan Y B . The N-terminus modulates human Caf1 activity, structural stability and aggregation. Int J Biol Macromol, 2012,51:497-503.
doi: 10.1016/j.ijbiomac.2012.05.032 pmid: 22683897
[9] Sarowar S, Oh H W, Cho H S, Baek K H, Seong E S, Joung Y H, Choi G J, Lee S, Choi D . Capsicum annuum CCR4-associated factor CaCAF1 is necessary for plant development and defence response. Plant J, 2007,51:792-802.
doi: 10.1111/j.1365-313X.2007.03174.x pmid: 17587232
[10] Liang W X, Li C B, Liu F, Jiang H L, Li S Y, Sun J Q, Wu X Y, Li C Y . The Arabidopsis homologs of CCR4-associated factor 1 show mRNA deadenylation activity and play a role in plant defence responses. Cell Res, 2009,19:307-316.
doi: 10.1038/cr.2008.317 pmid: 19065152
[11] Kwon T, Yi Y B, Nam J . Overexpression of AtCAF1, CCR4-associated factor 1 homologue in Arabidopsis thaliana, negatively regulates wounding-mediated disease resistance. J Plant Biotechnol, 2011,38:278-284.
doi: 10.5010/JPB.2011.38.4.278
[12] Shimo H M, Terassi C, Lima Silva C C, de Lima Zanella J, Mercaldi G F, Rocco S A, Benedetti C E . Role of the Citrus sinensis RNA deadenylase CsCAF1 in citrus canker resistance. Mol Plant Pathol, 2019,20:1105-1118.
doi: 10.1111/mpp.12815 pmid: 31115151
[13] Walley J W, Kelley D R, Nestorova G, Hirschberg D L, Dehesh K . Arabidopsis deadenylases AtCAF1a and AtCAF1b play overlapping and distinct roles in mediating environmental stress responses. Plant Physiol, 2010,152:866-875.
doi: 10.1104/pp.109.149005 pmid: 19955262
[14] Liu Q C . Sweet potato omics and biotechnology in China. Plant OMICS: J Plant Mol Biol Omics, 2011,4:295.
[15] Park S C, Kim Y H, Jeong J C, Kim C Y, Lee H S, Bang J W, Kwak S S . Sweetpotato late embryogenesis abundant 14 ( IbLEA14) gene influences lignification and increases osmotic- and salt stress-tolerance of transgenic calli. Planta, 2011,233:621-634.
doi: 10.1007/s00425-010-1326-3
[16] Kim S H, Ahn Y O, Ahn M J, Lee H S, Kwak S S . Down-regulation of β-carotene hydroxylase increases β-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato. Phytochemistry, 2012,74:69-78.
doi: 10.1016/j.phytochem.2011.11.003
[17] Kim S H, Kim Y H, Ahn Y O, Ahn M J, Jeong J C, Lee H S, Kwak S S . Downregulation of the lycopene ε-cyclase gene increases carotenoid synthesis via the β-branch-specific pathway and enhances salt-stress tolerance in sweetpotato transgenic calli. Physiol Plant, 2013,147:432-442.
doi: 10.1111/j.1399-3054.2012.01688.x pmid: 22938023
[18] Kim S H, Jeong J C, Park S, Bae J Y, Ahn M J, Lee H S, Kwak S S . Down-regulation of sweetpotato lycopene β-cyclase gene enhances tolerance to abiotic stress in transgenic calli. Mol Biol Rep, 2014,41:8137-8148.
doi: 10.1007/s11033-014-3714-4
[19] Liu D G, He S Z, Zhai H, Wang L J, Zhao Y, Wang B, Li R J, Liu Q C . Overexpression of IbP5CR enhances salt tolerance in transgenic sweetpotato. Plant Cell Tiss Org Cult, 2014,117:1-16.
doi: 10.1007/s11240-013-0415-y
[20] Liu D G, Wang L J, Liu C L, Song X J, He S Z, Zhai H, Liu Q C . An Ipomoea batatas iron-sulfur cluster scafold protein gene, IbNFU1, is involved in salt tolerance. PLoS One, 2014,9:e93935.
doi: 10.1371/journal.pone.0093935 pmid: 24695556
[21] Liu D G, Wang L J, Zhai H, Song X J, He S Z, Liu Q C . A novel ɑ/β-hydrolase gene IbMas enhances salt tolerance in transgenic sweetpotato. PLoS One, 2014,9:e115128.
doi: 10.1371/journal.pone.0115128 pmid: 25501819
[22] Liu D G, He S Z, Song X J, Zhai H, Liu N, Zhang D D, Ren Z T, Liu Q C . IbSIMT1, a novel salt-induced methyltransferase gene from Ipomoea batatas, is involved in salt tolerance. Plant Cell Tissue Organ Cult, 2015,120:701-715.
doi: 10.1007/s11240-014-0638-6
[23] Wang B, Zhai H, He S Z, Zhang H, Ren Z T, Zhang D D, Liu Q C . A vacuolar Na+/H+ antiporter gene, IbNHX2, enhances salt and drought tolerance in transgenic sweetpotato. Sci Hortic, 2016,201:153-166.
doi: 10.1016/j.scienta.2016.01.027
[24] Wang F B, Tong W J, Zhu H, Kong W L, Peng R H, Liu Q C, Yao Q H . A novel Cys2/His2 zinc fnger protein gene from sweetpotato, IbZFP1, is involved in salt and drought tolerance in transgenic Arabidopsis. Planta, 2016,243:783-797.
doi: 10.1007/s00425-015-2443-9 pmid: 26691387
[25] Wang F B, Zhai H, An Y Y, Si Z Z, He S Z, Liu Q C . Overexpression of IbMIPS1 gene enhances salt tolerance in transgenic sweetpotato. J Integr Agric, 2016,15:271-281.
doi: 10.1016/S2095-3119(14)60973-4
[26] Zhai H, Wang F B, Si Z Z, Huo J X, Xing L, An Y Y, He S Z, Liu Q C . A myo-inositol-1-phosphate synthase gene, IbMIPS1, enhances salt and drought tolerance and stem nematode resistance in transgenic sweet potato. Plant Biotechnol J, 2016,14:592-602.
doi: 10.1111/pbi.2016.14.issue-2
[27] Li R J, Kang C, Song X J, Yu L, Liu D G, He S Z, Zhai H, Liu Q C . A ζ-carotene desaturase gene, IbZDS, increases β-carotene and lutein contents and enhances salt tolerance in transgenic sweetpotato. Plant Sci, 2017,262:39-51.
doi: 10.1016/j.plantsci.2017.05.014 pmid: 28716419
[28] Kang C, Zhai H, Xue L Y, Zhao N, He S Z, Liu Q C . A lycopene β-cyclase gene, IbLCYB2, enhances carotenoid contents and abiotic stress tolerance in transgenic sweetpotato. Plant Sci, 2018,272:243-254.
doi: 10.1016/j.plantsci.2018.05.005 pmid: 29807598
[29] Zhang H, Gao X R, Zhi Y H, Li X, Zhang Q, Niu J B, Wang J, Zhai H, Zhao N, Li J G, Liu Q C, He S Z . A non-tandem CCCH-type zinc-finger protein, IbC3H18, functions as a nuclear transcriptional activator and enhances abiotic stress tolerance in sweet potato. New Phytol, 2019,223:1918-1936.
doi: 10.1111/nph.15925 pmid: 31091337
[30] 杨元军, 王玉萍, 翟红, 刘庆昌 . 甘薯块根总RNA的高效快速提取方法. 分子植物育种, 2008,6:193-196.
Yang Y J, Wang Y P, Zhai H, Liu Q C . A simple and rapid procedure for RNA isolation from storage roots of sweetpotato ( Ipomoea batatas). Mol Plant Breed, 2008,6:193-196 (in Chinese with English abstract).
[31] Wang L J, He S Z, Zhai H, Liu D G, Wang Y N, Liu Q C . Molecular cloning and functional characterization of a salt tolerance-associated gene IbNFU1 from sweetpotato. J Integr Agric, 2013,12:27-35.
doi: 10.1016/S2095-3119(13)60202-6
[32] Jiang T, Zhai H, Wang F B, Zhou H N, Si Z Z, He S Z, Liu Q C . Cloning and characterization of a salt tolerance-associated gene encoding trehalose-6-phosphate synthase in sweetpotato. J Integr Agric, 2014,13:1651-1661.
doi: 10.1016/S2095-3119(13)60534-1
[33] 喻娜, 郭新勇, 焦天奇, 祝建波 . 转小拟南芥ApHRD基因烟草获得及其抗旱性鉴定. 西北植物学报, 2010,30:2385-2393.
Yu N, Guo X Y, Jiao T Q, Zhu J B . Transformation of ApHRD gene and drought-tolerance identification of transgenic plants in tobacco. Acta Bot Boreali-Occident Sin, 2010,30:2385-2393 (in Chinese with English abstract).
[34] Huo J X, Du B, Sun S F, He S Z, Zhao N, Liu Q C, Zhai H . A novel aldo-keto reductase gene, IbAKR, from sweet potato confers higher tolerance to cadmium stress in tobacco. Front Agric Sci Eng, 2018,5:206-213.
[35] Gill S S, Tuteja N . Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem, 2010,48:909-930.
doi: 10.1016/j.plaphy.2010.08.016 pmid: 20870416
[36] Smirnoff N, Cumbes Q J . Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry, 1989,28:1057-1060.
doi: 10.1016/0031-9422(89)80182-7
[37] Bao A K, Wang S M, Wu G Q, Xi J J, Zhang J L, Wang C M . Overexpression of the Arabidopsis H+-PPase enhanced resistance to salt and drought stress in transgenic alfalfa (Medicago sativa L.). Plant Sci, 2009,176:232-240.
doi: 10.1016/j.plantsci.2008.10.009
[38] Kumar V, Shriram V, Kishor P B K, Jawali N, Shitole M G . Enhanced proline accumulation and salt stress tolerance of transgenic indica rice by over-expressing P5CSF129A gene. Plant Biotechnol Rep, 2010,4:37-48.
doi: 10.1007/s11816-009-0118-3
[1] 周文期, 强晓霞, 王森, 江静雯, 卫万荣. 水稻OsLPL2/PIR基因抗旱耐盐机制研究[J]. 作物学报, 2022, 48(6): 1401-1415.
[2] 王兴荣, 李玥, 张彦军, 李永生, 汪军成, 徐银萍, 祁旭升. 青稞种质资源成株期抗旱性鉴定及抗旱指标筛选[J]. 作物学报, 2022, 48(5): 1279-1287.
[3] 靳容, 蒋薇, 刘明, 赵鹏, 张强强, 李铁鑫, 王丹凤, 范文静, 张爱君, 唐忠厚. 甘薯Dof基因家族挖掘及表达分析[J]. 作物学报, 2022, 48(3): 608-623.
[4] 胡亮亮, 王素华, 王丽侠, 程须珍, 陈红霖. 绿豆种质资源苗期耐盐性鉴定及耐盐种质筛选[J]. 作物学报, 2022, 48(2): 367-379.
[5] 张海燕, 解备涛, 姜常松, 冯向阳, 张巧, 董顺旭, 汪宝卿, 张立明, 秦桢, 段文学. 不同抗旱性甘薯品种叶片生理性状差异及抗旱指标筛选[J]. 作物学报, 2022, 48(2): 518-528.
[6] 张思梦, 倪文荣, 吕尊富, 林燕, 林力卓, 钟子毓, 崔鹏, 陆国权. 影响甘薯收获期软腐病发生的指标筛选[J]. 作物学报, 2021, 47(8): 1450-1459.
[7] 宋天晓, 刘意, 饶莉萍, Soviguidi Deka Reine Judesse, 朱国鹏, 杨新笋. 甘薯细胞壁蔗糖转化酶基因IbCWIN家族成员鉴定及表达分析[J]. 作物学报, 2021, 47(7): 1297-1308.
[8] 李辉, 李德芳, 邓勇, 潘根, 陈安国, 赵立宁, 唐慧娟. 红麻非生物逆境胁迫响应基因HCWRKY71表达分析及转化拟南芥[J]. 作物学报, 2021, 47(6): 1090-1099.
[9] 赵佳佳, 乔玲, 武棒棒, 葛川, 乔麟轶, 张树伟, 闫素仙, 郑兴卫, 郑军. 山西省小麦苗期根系性状及抗旱特性分析[J]. 作物学报, 2021, 47(4): 714-727.
[10] 韩贝, 王旭文, 李保奇, 余渝, 田琴, 杨细燕. 陆地棉种质资源抗旱性状的关联分析[J]. 作物学报, 2021, 47(3): 438-450.
[11] 蒙姜宇, 梁光伟, 贺亚军, 钱伟. 甘蓝型油菜耐盐和耐旱相关性状的QTL分析[J]. 作物学报, 2021, 47(3): 462-471.
[12] 闫彩霞, 王娟, 赵小波, 宋秀霞, 姜常松, 孙全喜, 苑翠玲, 张浩, 单世华. 全生育期鉴定筛选耐盐碱花生品种[J]. 作物学报, 2021, 47(3): 556-565.
[13] 王翠娟, 柴沙沙, 史春余, 朱红, 谭中鹏, 季杰, 任国博. 铵态氮素促进甘薯块根形成的解剖特征及其IbEXP1基因的表达[J]. 作物学报, 2021, 47(2): 305-319.
[14] 马猛, 闫会, 高闰飞, 后猛, 唐维, 王欣, 张允刚, 李强. 紫甘薯SSR标记遗传图谱构建与重要农艺性状QTL定位[J]. 作物学报, 2021, 47(11): 2147-2162.
[15] 李健, 王逸茹, 张凌霄, 孙明昊, 秦阳, 郑军. 玉米ZmCIPK24-2基因在盐胁迫应答中的功能研究[J]. 作物学报, 2020, 46(9): 1351-1358.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2