Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (19): 3985-3999.doi: 10.3864/j.issn.0578-1752.2025.19.014

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Functional Study of AkNAC2 from Actinidia kolomikta

ZOU PeiYi(), LIU MeiYan, WANG Ying, LI RanHong()   

  1. College of Life Sciences and Technology, Mudanjiang Normal University, Mudanjiang 157011, Heilongjiang
  • Received:2025-04-18 Accepted:2025-07-14 Online:2025-10-01 Published:2025-10-10
  • Contact: LI RanHong

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Abstract:

【Objective】The NAC transcription factors constitute the largest family of transcription factors in plants, and play an important role in plant resistance to abiotic stresses such as cold. Actinidia kolomikta is one of the most cold-resistant kiwifruits. Based on the transcriptome data of Actinidia kolomikta, a NAC transcription factor family member responding to cold-AkNAC2 was obtained. The function of AkNAC2 in cold response was further studied by constructing overexpression and inhibition expression lines, which provided available gene resources for cultivating cold-resistant kiwifruit varieties.【Method】The leaves cDNA of Actinidia kolomikta were used as experimental materials to clone AkNAC2, and its sequence information and protein structure were analyzed by bioinformatics. The subcellular localization of AkNAC2 was analyzed by GFP labeling. The tissue-specific expression information of AkNAC2 was obtained by transcriptome data, and the response of AkNAC2 to cold, salt and waterlogging stress was detected by qRT-PCR. The overexpression lines of AkNAC2 Nicotiana benthamiana and the inhibitory expression lines of Actinidia kolomikta were constructed. The phenotypic, physiological and biochemical indexes and the expression of CBF pathway related genes were compared between the transgenic lines and the control after cold treatment.【Result】The CDS length of AkNAC2 is 897 bp. and has a domain. AkNAC2 encoding 298 amino acids, was a hydrophilic acidic protein, its promoter contained multiple cis-elements related to abiotic stress. Phylogenetic analysis indicated that AkNAC2 was closely related to NAC2 of Actinidia eriantha. Subcellular localization showed that AkNAC2 is localized in the nucleus. The expression level of AkNAC2 was the highest in leaves, followed by pistillate flowers, staminate flowers, fruits and stems, and the lowest in roots. The expression of AkNAC2 could be induced by cold, salt and waterlogging stress, and the response of AkNAC2 to cold stress was the most obvious. Six Nicotiana benthamiana transgenic lines were obtained by Agrobacterium-mediated, named OE1-OE6, Among them, the gene expression levels of OE2, OE4 and OE6 were higher, which were used as follow-up research materials. Under 4 ℃, compared with wild-type Nicotiana benthamiana, the relative conductivity and malondialdehyde content of the three transgenic Nicotiana benthamiana lines were lower, the content of protective soluble sugar and soluble protein significantly increased, the activity of peroxidase and superoxide dismutase increased, and the antioxidant capacity significantly increased. The expression levels of CBF pathway-related genes (NbtICE1, NbtCOR47, NbtCBF1, NbtCBF2, NbtCBF3) were significantly up-regulated. The AkNAC2 inhibitory expression lines of Actinidia kolomikta were obtained by transient transformation. qRT-PCR showed that the expression of AkNAC2 in the inhibitory expression lines was significantly lower than that in the wild type and the empty vector group. On the 4th day of treatment, the expression level reached the lowest, which was 0.40 times that of the wild type and 0.34 times that of the empty vector group. After 4 °C low temperature treatment, the relative conductivity and malondialdehyde content of the inhibited expression lines were significantly increased, the contents of protective substances such as soluble sugar and soluble protein were significantly decreased, the activities of peroxidase and superoxide dismutase were significantly decreased, and the expression levels of CBF pathway-related genes (AkCBF1.1 and AkCBF3) were significantly down-regulated.【Conclusion】A low temperature response gene AkNAC2 of Actinidia kolomikta was cloned, it may positively regulate the cold resistance of plants by affecting the expression of key genes in the CBF pathway and reducing oxidative stress.

Key words: Actinidia kolomikta, AkNAC2, transcription factor, gene cloning, Agrobacterium-mediated, CBF pathway

Table 1

Primers used in this study"

引物 Primer 正向引物 Forward primer (5′-3′) 反向引物 Reverse primer (5′-3′)
AkNAC2 CAGTGGTCTCACAACATGACGAGTCAGTTGGAGTTGC CAGTGGTCTCATACACTAGAATGGCTTGTCCATGAACATGA
TRV2-AkNAC2 AAGGTTACCGAATTCTCTAGCCAACGTGGACAGGTCCG CGTGAGCTCGGTACCGGATCTCACGTGCTCGGACCCAC
GFP-AkNAC2 GCTCTAGAATGACGAGTCAGTTGGAGTTGC GCGTCGACCTAGAATGGCTTGTCCATGAACATGA
qAkNAC2 TCACGGTCAGTCCAAAACTCG TTGCTTGGTGGTACGATGGC
qAkGAPDH GTCGGTGGAGACCACATCAT CACTGTCGATGTCTCAGTGGT
qNbtCBF1 CCAAGTCATACAAGGGGTGGT ATTAGCCCCTCGGCCATATTC
qNbtCBF2 TCATCGGAGGCCGAAAACAT ATCCCGGCATGCAAAATAGC
qNbtCBF3 TAGACGAGGAGGCGCTTTTC GAATGCTCAGCTTCCATGCC
qNbtICE1 GGCAACCCCAATTTGAGTTCTT CCATCACTAAACCCCAGTGGA
qNbtCOR47 CCGATGACACTGCCATGATTG GCGGTGAAGTTTGTCCATGAG
qNbtActin ACTGAAGCGCCTCTTAACCC AGACGGAGAATGGCATGTGG
qAkICBF1.1 TCCGAGGATGCCAAGAGAGA TCCCAACTAAAATCCCGCCC
qAkCBF3 AAGTTCAGGGAGACGAGGCA TCGGCGGTTGGGTAGGT

Fig. 1

Cloning of AkNAC2 M: Marker (DL2000); 1: Target gene"

Fig. 2

Bioinformatic analysis of the AkNAC2 A: Tertiary structure of AkNAC2; B: Protein interaction network of AkNAC2; C: Phylogenetic tree"

Table 2

Prediction of the cis-elements in the promoter of AkNAC2"

可能的功能 Possible function 顺式元件 Cis-element 核心序列 Core sequence 数目 Number
低温响应元件Low temperature responsive element ABRE ACGTG 6
MYC CATTTG 3
热诱导元件Heat shock element STRE AGGGG 2
盐胁迫响应元件Salt stress response element W-box TTGACC 2
G-box CACGTT 2
生长素响应元件Auxin response element P-box CCTTTTG 2
光响应元件Light responsive element G-box CACGTC 4
水杨酸响应元件Salicylic acid response element TGACG-motif TGACG 6

Fig. 3

Subcellular localization of AkNAC2"

Fig. 4

Expression patterns of AkNAC2 in Actinidia kolomikta Different letters represent significant levels of difference at P<0.05. The same as below"

Fig. 5

PCR verification of the constructed vectors A: PCR of the overexpressed vector pHK-35S-AkNAC2; B: PCR of repression expression vector pTRV2-AkNAC2. M: Marker (DL2000); 1-2: Recombinant bacterial solution"

Fig. 6

PCR results of transgenic Nicotiana benthamiana DNA M: Marker (DL2000); 1: Positive control; 2: Nicotiana benthamiana WT; 3: Water; 4-9: Transgenic Nicotiana benthamiana OE1-OE6"

Fig. 7

qRT-PCR results of transgenic Nicotiana benthamiana"

Fig. 8

Phenotypes of Nicotiana benthamiana under 4 ℃"

Fig. 9

Physiological changes of Nicotiana benthamiana under cold stress"

Fig. 10

Analysis of the expression level of CBF pathway genes in Nicotiana benthamiana"

Table 3

Prediction of promoter predicted sites"

AtNAC002的下游结合序列
The downstream binding sequence of AtNAC002		 基因
Gene		 结合序列
Predicted sequence
NbtICE1 ACGTAATT
NbtCBF1 ACTCAACT
NbtCBF2 AAGTAACT
NbtCBF3 AAGCAACT
NbtCOR47 ACGGAACC

Fig. 11

Expression levels of AkNAC2 suppresses lines A: The inhibition efficiency of AkNAC2; B: Expression level of AkNAC2 in different treatment time"

Fig. 12

Phenotypes of Actinidia kolomikta under 0 ℃"

Fig. 13

Physiological changes of Actinidia kolomikta under cold stress"

Fig. 14

Analysis of gene expression level of CBF pathway genes in Actinidia kolomikta"

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