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

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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)

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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

Table 1

Primers used in this study"

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

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."

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