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

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

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CaCl_(2)和LaCl_(3)发现,CaCl_(2)明显促进花生果针向地性弯曲和AhVDAC的表达,而LaCl_(3)则减缓果针弯曲和AhVDAC的表达,推测Ca^(2+)的积累可能促进了AhVDAC的表达,并通过生物膜上AhVDAC的运输对Ca^(2+)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。

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 c DNA 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 831bp 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 p PROEXHTa-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 CaCl_(2)and LaCl_(3)to peanut gynophores in vitro revealed that CaCl_(2)treatment could significantly promote the relative expression of AhVDAC gene and the geotropic bending of gynophores,whereas LaCl_(3)treatment could decelerate the relative expression of AhVDAC gene and geotropic bending of gynophores.In conclusion,we speculated that Ca^(2+) accumulation may promote the expression of AhVDAC and Ca^(2+) 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.