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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (6): 1558-1565.doi: 10.3724/SP.J.1006.2022.14093

• RESEARCH NOTES • Previous Articles    

Cloning and expression analysis of voltage dependent anion channel (AhVDAC) gene in the geotropism response of the peanut gynophores

LI Hai-Fen(), WEI Hao, WEN Shi-Jie, LU Qing, LIU Hao, LI Shao-Xiong, HONG Yan-Bin, CHEN Xiao-Ping, LIANG Xuan-Qiang*()   

  1. Crops Research Institute, Guangdong Academy of Agricultural Science / Guangdong Provincial Key Laboratory of Crop Genetic Improvement, Guangzhou 510640, Guangdong, China
  • Received:2021-05-18 Accepted:2021-09-10 Online:2022-06-12 Published:2021-10-12
  • Contact: LIANG Xuan-Qiang E-mail:lihaifen@gdaas.cn;liangxuanqiang@gdaas.cn
  • Supported by:
    Key-Area Research and Development Program of Guangdong Province(2020B020219003);China Agriculture Research System of the Ministry of Finance and Ministry of Agriculture and Rural Affairs, and the Agricultural Competitive Industry Discipline Team Building Project of Guangdong Academy of Agricultural Science(202104TD)

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Abstract:

Peanut is a plant that blooms above ground and bears fruit underground. In our earlier studies, the voltage dependent anion channel (AhVDAC) gene was detected to be involved in gravitropism of peanut gynophores. In this study, the full-length cDNA of peanut AhVDAC gene was cloned, and the prokaryotic expression, subcellular localization, and the relative expression level of AhVDAC gene in gravitropism were analyzed. The results showed that the open reading frame of AhVDAC gene was 831 bp encoding a protein containing 276 amino acids, with a molecular weight of 29.7 kD and pI 6.38. Subcellular localization showed that AhVDAC gene was mainly located in cytoplasm. The prokaryotic expression vector pPROEXHTa-AhVDAC was constructed, and the 37 kD AhVDAC protein was induced, isolated, and purified. The relative expression level of AhVDAC in gynophores at different developmental days was analyzed by RT-PCR, and the results showed that the relative expression level of AhVDAC gene was the highest at the 2nd day, and then decreased gradually and maintained at a lower level. The application of exogenous CaCl2 and LaCl3 to peanut gynophores in vitro revealed that CaCl2 treatment could significantly promote the relative expression of AhVDAC gene and the geotropic bending of gynophores, whereas LaCl3 treatment could decelerate the relative expression of AhVDAC gene and geotropic bending of gynophores. In conclusion, we speculated that Ca2+ accumulation may promote the expression of AhVDAC and Ca2+was asymmetrically distributed through the transport of AhVDAC on biological membrane, so that the growth direction of peanut gynophore was changed and geotropic bending occurs.

Key words: peanut, gynophore, the voltage dependent anion channel gene (AhVDAC), clone, gene expression

Table 1

Primers used for this study"

引物用途
Primer function		 引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
基因克隆
Gene cloning		 AhVDAC-F ATGGTGAATGGTCCAGGTCTCTACT
AhVDAC-R CTAAGGCTTCAAGGCCACGG
亚细胞定位
Subcellular localization		 VDAC-M3 CGGGATCCATGGTGAATGGTCCAGGTCTC
VDAC-M4 AACTGCAGAGGCTTCAAGGCCACGG
原核表达载体构建
Construction of prokaryotic expression vector		 VDAC-M1 AACTGCAGTGGTGAATGGTCCAGGTCTCTACT
VDAC-M2 CCCAAGCTTCTA AGGCTTCAAGGCCACGG
荧光定量
Fluorescence quantitative PCR		 AhVDAC-qF GTAAGCCCGTTGACAAACACTG
AhVDAC-qR CACCAATGTTAGGGGGTCAAG
内参引物
Reference gene primer		 18S rRNA-F CAGCTCGCGTTGACTACG
18S rRNA-R CGAACACTTCACCGGACCAT

Fig. 1

cDNA sequence of AhVDAC gene and amino acids sequence (A), secondary structure (B), and three-dimensional structure (C) prediction of VDAC protein"

Fig. 2

Phylogenetic tree analysis of AhVDAC and VDAC from other plants The small red star in the figure represents the VDAC protein in the model plant Arabidopsis thaliana, and the small red dot represents the VDAC protein in peanut that is 100% similar to the gene cloned in this study."

Fig. 3

Subcelluar localization of the AhVDAC in nion epidermal cells A: blank control; B: the localization of pCAMBIA1301-CaMV35S-GFP; C: the localization of pCAMBIA1301-CaMV35S-AhVDAC-GFP."

Fig. 4

Identification of the pPROEX HTa-AhVDAC in BL21(DE3)pLysS by recombination plasmid PCR and Bacilli PCR and restriction digestion from recombination plasmid A: identification of recombination plasmid PCR; B: identification of Bacilli PCR; C: the restriction digestion by Pst I and Hind III. M: DNA marker DL2000; M1: DNA marker Hind IIIλ; M2: DNA marker DL2000."

Fig. 5

SDS-PAGE analysis of AhVDAC and protein purification 1: non-induced protein of BL21(DE3)pLysS with pPROEXHTa-AhVDAC; 2-7: induced protein of BL21(DE3)pLysS with pPROEX HTa-AhVDAC from 1 to 6 hours; M: protein molecular mass marker. B: purification the induced protein of BL21(DE3)pLysS with pPROEX HTa-AhVDAC at 6 h."

Fig. 6

Expression level of AhVDAC in different development stages of peanut gynophore Values with different lowercase letters show significant difference among different stages at the 0.05 probability level."

Fig. 7

Bending angle (A) and expression level (B) of AhVDAC in the peanut gynophores under the CaCl2 and LaCl3 treatments"

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