Welcome to Acta Agronomica Sinica,

Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (12): 1862-1869.doi: 10.3724/SP.J.1006.2020.04045


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 Online:2020-12-12 Published:2020-07-02
  • Contact: Hong ZHAI E-mail:757015572@qq.com;zhaihong@cau.edu.cn
  • 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)


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

Table 1

Primers used in this study"

Primer name
Primer sequence (5°-3°)
Primers for IbCAF1 amplification
Primers for identifying transformants
Primers for real-time quantitative PCR

Fig. 1

Sequence analysis of IbCAF1 A: sequence analysis of IbCAF1 protein. B: multiple sequence alignment of IbCAF1 and CAF1 proteins from other plants. C: phylogenetic analysis of IbCAF1 and CAF1 proteins from other plants. ItlCAF1: Ipomoea triloba (XP_031110715.1); InCAF1: Ipomoea nil (XP_019199562.1), NtCAF1: Nicotiana tabacum (XP_016511744.1); SpCAF1: Solanum pennellii (XP_015079674.1), CaCAF1: Capsicum annuum (NP_001312000.1), SlCAF1: Solanum lycopersicum (XP_004241342.1), StCAF1: Solanum tuberosum (XP_006361099.1)."

Fig. 2

Expression analysis of IbCAF1 in Lushu 3 A: expression analysis of IbCAF1 gene in different tissues of Lushu 3; B: expression analysis of the IbCAF1 gene in Lushu 3 after different times (h) in response to 200 mmol L-1 NaCl, 20% PEG-6000, 100 μmol L-1 ABA and 10 mmol L-1 H2O2, respectively. * and ** indicate significantly different at the 0.05 and 0.01 probability levels, respectively."

Fig. 3

Expression analysis of IbCAF1 gene in transgenic tobacco plants by qRT-PCR **: significantly different at the 0.01 probability level."

Fig. 4

IbCAF1 enhances salt and drought tolerance in transgenic tobacco plants A: responses of IbCAF1-transgenic and WT tobacco plants cultured for 4 weeks on half-MS medium supplemented without stress or with 200 mmol L-1 NaCl or 10% PEG-6000; B-H: DAB staining (B), NBT staining (C), H2O2 content (D), SOD activity (E), POD activity (F), proline content (G), and MDA content (H) in the leaves of IbCAF1 transgenic and WT tobacco plants cultured for 4w on half-MS medium supplemented with no stress, 200 mmol L-1 NaCl or 10% PEG-6000. * and ** indicate significantly different at the 0.05 and 0.01 probability levels, respectively."

Fig. 5

Relative expression of abiotic stress-responsive genes in the transgenic and WT plants * and ** indicate significantly different at the 0.05 and 0.01 probability levels, respectively."

[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] ZHOU Wen-Qi, QIANG Xiao-Xia, WANG Sen, JIANG Jing-Wen, WEI Wan-Rong. Mechanism of drought and salt tolerance of OsLPL2/PIR gene in rice [J]. Acta Agronomica Sinica, 2022, 48(6): 1401-1415.
[2] JIN Rong, JIANG Wei, LIU Ming, ZHAO Peng, ZHANG Qiang-Qiang, LI Tie-Xin, WANG Dan-Feng, FAN Wen-Jing, ZHANG Ai-Jun, TANG Zhong-Hou. Genome-wide characterization and expression analysis of Dof family genes in sweetpotato [J]. Acta Agronomica Sinica, 2022, 48(3): 608-623.
[3] HU Liang-Liang, WANG Su-Hua, WANG Li-Xia, CHENG Xu-Zhen, CHEN Hong-Lin. Identification of salt tolerance and screening of salt tolerant germplasm of mungbean (Vigna radiate L.) at seedling stage [J]. Acta Agronomica Sinica, 2022, 48(2): 367-379.
[4] ZHANG Hai-Yan, XIE Bei-Tao, JIANG Chang-Song, FENG Xiang-Yang, ZHANG Qiao, DONG Shun-Xu, WANG Bao-Qing, ZHANG Li-Ming, QIN Zhen, DUAN Wen-Xue. Screening of leaf physiological characteristics and drought-tolerant indexes of sweetpotato cultivars with drought resistance [J]. Acta Agronomica Sinica, 2022, 48(2): 518-528.
[5] ZHANG Si-Meng, NI Wen-Rong, LYU Zun-Fu, LIN Yan, LIN Li-Zhuo, ZHONG Zi-Yu, CUI Peng, LU Guo-Quan. Identification and index screening of soft rot resistance at harvest stage in sweetpotato [J]. Acta Agronomica Sinica, 2021, 47(8): 1450-1459.
[6] LI Hui, LI De-Fang, DENG Yong, PAN Gen, CHEN An-Guo, ZHAO Li-Ning, TANG Hui-Juan. Expression analysis of abiotic stress response gene HcWRKY71 in kenaf and transformation of Arabidopsis [J]. Acta Agronomica Sinica, 2021, 47(6): 1090-1099.
[7] MENG Jiang-Yu, LIANG Guang-Wei, HE Ya-Jun, QIAN Wei. QTL mapping of salt and drought tolerance related traits in Brassica napus L. [J]. Acta Agronomica Sinica, 2021, 47(3): 462-471.
[8] MA Meng, YAN Hui, GAO Run-Fei, KOU Meng, TANG Wei, WANG Xin, ZHANG Yun-Gang, LI Qiang. Construction linkage maps and identification of quantitative trait loci associated with important agronomic traits in purple-fleshed sweetpotato [J]. Acta Agronomica Sinica, 2021, 47(11): 2147-2162.
[9] LI Jian, WANG Yi-Ru, ZHANG Ling-Xiao, SUN Ming-Hao, QIN Yang, ZHENG Jun. Functional analysis of ZmCIPK24-2 gene from maize in response to salt stress [J]. Acta Agronomica Sinica, 2020, 46(9): 1351-1358.
[10] Li-Ge BAO,Ping LU,Meng-Sha SHI,Yue XU,Min-Xuan LIU. Screening and identification of Chinese sorghum landraces for salt tolerance at germination and seedling stages [J]. Acta Agronomica Sinica, 2020, 46(5): 734-744.
[11] HENG You-Qiang,YOU Xi-Long,WANG Yan. Pathogenesis-related protein gene SfPR1a from Salsola ferganica enhances the resistances to drought, salt and leaf spot disease in transgenic tobacco [J]. Acta Agronomica Sinica, 2020, 46(4): 503-512.
[12] ZHANG Huan, YANG Nai-Ke, SHANG Li-Li, GAO Xiao-Ru, LIU Qing-Chang, ZHAI Hong, GAO Shao-Pei, HE Shao-Zhen. Cloning and functional analysis of a drought tolerance-related gene IbNAC72 in sweet potato [J]. Acta Agronomica Sinica, 2020, 46(11): 1649-1658.
[13] LIU Xie-Xiang,CHANG Ru-Zhen,GUAN Rong-Xia,QIU Li-Juan. Establishment of screening method for salt tolerant soybean at emergence stage and screening of tolerant germplasm [J]. Acta Agronomica Sinica, 2020, 46(01): 1-8.
[14] Hai-Yan ZHANG,Bei-Tao XIE,Bao-Qing WANG,Shun-Xu DONG,Wen-Xue DUAN,Li-Ming ZHANG. Evaluation of drought tolerance and screening for drought-tolerant indicators in sweetpotato cultivars [J]. Acta Agronomica Sinica, 2019, 45(3): 419-430.
[15] SUN Xian-Jun,JIANG Qi-Yan,HU Zheng,ZHANG Hui-Yuan,XU Chang-Bing,DI Yi-Huan,HAN Long-Zhi,ZHANG Hui. Screening and identification of salt-tolerant rice germplasm in whole growth period [J]. Acta Agronomica Sinica, 2019, 45(11): 1656-1663.
Full text



No Suggested Reading articles found!