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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (11): 1649-1658.doi: 10.3724/SP.J.1006.2020.04051

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

Cloning and functional analysis of a drought tolerance-related gene IbNAC72 in sweet potato

ZHANG Huan1(), YANG Nai-Ke1, SHANG Li-Li2, GAO Xiao-Ru1, LIU Qing-Chang1, ZHAI Hong1, GAO Shao-Pei1, HE Shao-Zhen1,*()   

  1. 1 Key Laboratory of Sweet Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs / Laboratory of Crop Heterosis & Utilization and Joint Laboratory for International Cooperation in Crop Molecular Breeding, Ministry of Education, China Agricultural University, Beijing 100193, China
    2 Yantai Academy of Agricultural Sciences, Yantai 265500, Shandong, China
  • Received:2020-02-29 Accepted:2020-07-02 Online:2020-11-12 Published:2020-07-13
  • Contact: Shao-Zhen HE E-mail:zhanghuan1111@cau.edu.cn;sunnynba@cau.edu.cn
  • Supported by:
    This study was supported by the National Key Research and Development Program of China(2018YFD1000700);This study was supported by the National Key Research and Development Program of China(2018YFD1000704);the Beijing Food Crops Innovation Consortium Program(BAIC09-2020);the China Agriculture Research System(CARS-10,甘薯)

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Abstract:

NAC (NAM/ATAF/CUC) is a plant-specific transcription factor family, which plays an important role in plant growth, development and stress responses. In this study, we cloned IbNAC72, a drought tolerance-related gene from sweet potato [Ipomoea batatas (L.) Lam.] variety Lizixiang by RACE method. The IbNAC72 cDNA of 1319 bp in length, had an open reading frame (ORF) of 1008 bp, and encoded a 335 amino acids polypeptide, with a molecular weight of 37.4 kD and an isoelectric point (pI) of 8.76. The genomic DNA of IbNAC72 gene was 1199 bp and was deduced to contain 3 exons and 2 introns. Sequence alignment and phylogenetic analysis revealed that IbNAC72 had a close relationship with the predicted protein products of Ipomoea nil. RT-qPCR analysis showed that IbNAC72 was expressed at the highest level in the leaves of sweet potato, and it was strongly induced by PEG-6000 and NaCl, respectively. IbNAC72 was transformed into tobacco via Agrobacterium-mediated transformation. Its overexpression significantly enhanced drought tolerance in the transgenic tobacco plants. Under drought stress, transgenic plants developed stronger root system; the SOD activity was significantly increased whereas the MDA content was significantly decreased in transgenic plants compared to those of wild type plants. This study showed that IbNAC72 gene was closely related to drought tolerance, providing a basis for in-depth study on the drought tolerance molecular mechanism of IbNAC72 in sweet potato.

Key words: sweet potato, NAC transcription factor, IbNAC72, overexpression, drought tolerance

Table 1

Primers used for IbNAC72 gene cloning and functional analysis"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 用途
Function
NAC-1 CGGAAAGTTGGGATCAAGAA 3′-RACE扩增
NAC-2 CAGGAAGACCGGAAGTTCAA 3′-RACE amplification
NAC-3 TTGAACTTCCGGTCTTCCTG 5′-RACE扩增
NAC-4 ATTCGCCCTGCTAGGAAGAT 5′-RACE amplification
NAC-F ATGGGTGTGAAAGATATGGACC ORF、DNA序列扩增
NAC-R TTACTGCCTGAACGCTAAGCTAC ORF and DNA sequence amplification
NAC-QF TCTTCAACAAGACGACAAGTCAAG RT-qPCR检测
NAC-QR AGGAAAACTCAATGCCATTG RT-qPCR detection
Actin-F AGCAGCATGAAGATTAAGGTTGTAGCAC 甘薯内参基因检测
Actin-R TGGAAAATTAGAAGCACTTCCTGTGAAC Sweet potato internal control
NAC-OE-F (Xba I) GCTCTAGAATGGGTGTGAAAGATATGGACC 过表达载体构建
NAC-OE-R (Sac I) CGAGCTCTTACTGCCTGAACGCTAAGCTAC Construction of overexpression vector

Fig. 1

Sequence analysis of IbNAC72 A: multiple protein sequence alignment of IbNAC72 with other plant NAC72 proteins; the red lines indicate the five subdomains a to e. B: phylogenetic analysis of NAC72 proteins. C: comparison of the genomic structures of IbNAC72 and AtNAC72; boxes indicate exons, and lines indicate introns."

Fig. 2

Expression analysis of IbNAC72 A: transcriptional levels of IbNAC72 in different tissues of 4-week-old in vitro-grown Lizixiang plants; B: transcriptional levels of IbNAC72 in different tissues of 3-month-old field-grown Lizixiang plants; C: expression level of IbNAC72 in the Lizixiang plants under 100 mmol L-1 NaCl treatment; D: expression level of IbNAC72 in the Lizixiang plants under 20% PEG-6000 treatment. The error bars indicate ± SD (n = 3). Different lowercase letters indicate significant difference at the 0.05 probability level based on one-way ANOVA."

Fig. 3

Construction and identification of IbNAC72 overexpression vector A: PCR amplification of IbNAC72 gene; B: double enzyme digestion detection of pC3301-121-IbNAC72."

Fig. 4

Production and identification of IbNAC72 transgenic tobacco plants A-D: transformation, screening and regeneration of IbNAC72 transgenic tobacco plants; E-I: the leaves, stems, and roots of the overexpression plants show positive reactions in the GUS assay, whereas no GUS expression is detected in WT; H: PCR analysis of GUS-positive plants; I: transcript levels of IbNAC72 in transgenic and WT plants. M: BL2000 DNA markers; W: water as a negative control; P: pCAMBIA3301-121-IbNAC72 as a positive control; C: WT as a negative control; WT: wild-type tobacco plants; T1-T3: IbNAC72-overexpressing tobacco lines. The error bars indicate ± SD (n = 3). ** indicates significant difference at the 0.01 probability level based on Student’s t-test."

Fig. 5

Overexpression of IbNAC72 enhances drought tolerance of transgenic tobacco plants A: phenotypes of transgenic plants and WT grown for four weeks under drought stress; B: plant height of transgenic plants and WT grown for four weeks under drought stress; C: root phenotype of transgenic plants and WT grown for four weeks under drought stress; D: fresh weight of the root of transgenic plants and WT grown for four weeks under drought stress. The error bars indicate ± SD (n = 3). ** indicates significant difference at the 0.01 probability level based on Student’s t-test. Abbreviations are the same as those given in Fig. 4."

Fig. 6

Effects of drought stress on SOD activity and MDA accumulation in transgenic plants A: the superoxide dismutase (SOD) activity in transgenic plants and WT grown for four weeks under drought stress; B: the malondialdehyde (MDA) content in transgenic plants and WT grown for four weeks under drought stress. The error bars indicate ± SD (n = 3). ** indicate significant difference at the 0.01 probability level based on Student’s t-test. Abbreviations are the same as those given in Fig. 4."

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