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作物学报 ›› 2022, Vol. 48 ›› Issue (6): 1558-1565.doi: 10.3724/SP.J.1006.2022.14093

• 研究简报 • 上一篇    

花生电压依赖性阴离子通道基因(AhVDAC)的克隆及在果针向地性反应中表达分析

李海芬(), 魏浩, 温世杰, 鲁清, 刘浩, 李少雄, 洪彦彬, 陈小平, 梁炫强*()   

  1. 广东省农业科学院作物研究所 / 广东省农作物遗传改良重点实验室, 广东广州 510640
  • 收稿日期:2021-05-18 接受日期:2021-09-10 出版日期:2022-06-12 网络出版日期:2021-10-12
  • 通讯作者: 梁炫强
  • 作者简介:E-mail: lihaifen@gdaas.cn
  • 基金资助:
    广东省重点领域研发计划项目(2020B020219003);财政部和农业农村部: 国家现代农业产业技术体系建设专项和广东省农业科学院学科团队建设项目(202104TD)

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 Published:2022-06-12 Published online:2021-10-12
  • Contact: LIANG Xuan-Qiang
  • 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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摘要：

为研究电压依赖性阴离子通道基因(AhVDAC)与花生果针向地性生长的相关性, 本研究克隆了花生AhVDAC基因全长cDNA序列, 并对其编码蛋白结构、亚细胞定位、原核表达蛋白及其在果针向地性反应过程的表达特性进行分析。结果表明, AhVDAC基因含有831 bp的开放阅读框, 编码一个含有276个氨基酸、分子量大小为29.7 kD、pI值为6.38的蛋白。亚细胞定位分析结果显示, AhVDAC基因主要定位在细胞质。构建了pPROEXHTa-AhVDAC原核表达载体, 诱导及分离纯化了37 kD的AhVDAC纯化蛋白。采用RT-PCR对花生果针入土前不同发育时期AhVDAC的表达进行分析发现, AhVDAC基因表达量在果针发育的第2天最高, 随后逐渐下降后维持在较低的表达水平。通过对离体培养的花生果针施加外源CaCl2和LaCl3发现, CaCl2明显促进花生果针向地性弯曲和AhVDAC的表达, 而LaCl3则减缓果针弯曲和AhVDAC的表达, 推测Ca2+的积累可能促进了AhVDAC的表达, 并通过生物膜上AhVDAC的运输对Ca2+进行不对称分布, 从而使得花生果针改变生长方向, 发生向地性弯曲。

关键词: 花生, 果针, 电压依赖性阴离子通道基因(AhVDAC), 克隆, 基因表达

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

表1

研究所用引物"

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

图1

AhVDAC的cDNA序列及氨基酸序列(A)、AhVDAC蛋白二级(B)和三级结构预测(C)"

图2

AhVDAC和其他植物VDAC系统进化树分析 图中小红星表示模式植物拟南芥中的VDAC蛋白, 小红点表示花生中与本次克隆基因相似性达100%的VDAC蛋白。"

图3

AhVDAC在洋葱表皮细胞的亚细胞定位 A: 空白对照; B: pCAMBIA1301-CaMV35S-GFP定位图; C: pCAMBIA1301-CaMV35S-AhVDAC-GFP定位图。"

图4

AhVDAC原核表达载体质粒PCR鉴定、菌液PCR鉴定和质粒双酶切鉴定 A: 重组质粒PCR鉴定; B: 菌液PCR鉴定; C: 双酶切鉴定。"

图5

AhVDAC原核表达SDS-PAGE分析及其蛋白纯化 1: 未诱导的pPROEXHTa-AhVDAC/BL21(DE3)pLysS的蛋白; 2~7: 诱导1~6 h的pPROEX HTa-AhVDAC/BL21(DE3)pLysS的蛋白; M: 蛋白marker。B: 诱导6 h的pPROEXHTa-AhVDAC/BL21(DE3)pLysS的蛋白。"

图6

花生果针不同发育时期AhVDAC的表达水平 不同小写字母表示不同时期间差异显著(P<0.05)。"

图7

CaCl2 和LaCl3 处理对花生离体果针弯曲度(A)和AhVDAC表达的影响(B)"

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