大豆GmALMT33基因在镉胁迫应答中的功能分析

Functional Analysis of Soybean GmALMT33 Gene in Response to Cadmium Stress

由于工业发展以及生活废弃物污染的不断加剧,作物中的重金属浓度超标,严重威胁人体的健康。铝激活苹果酸转运体编码一类阴离子通道蛋白,在植物有机酸的跨膜转运中发挥重要的作用。为研究GmALMT33基因在大豆应对镉胁迫中的功能,本研究以大豆黑农48的叶片cDNA为模板,利用RT-PCR克隆得到GmALMT33基因。该基因CDS区全长1622 bp,编码553个氨基酸,含有1个ALMT结构域。qRT-PCR结果表明,GmALMT33在大豆根部的表达水平最高;镉胁迫后,该基因表达量呈现先升高后降低的趋势。构建植物表达载体pCPB-GmALMT33并对烟草、大豆毛状根进行遗传转化,转基因植株抗逆表型与生理指标分析表明,镉(66μmol/L CdCl_(2))胁迫下,转基因烟草叶片黄化、褪绿,边缘褐化程度明显低于野生型烟草。转基因大豆毛状根复合体植株茎秆和叶脉呈现的红褐色毒害症状程度明显弱于转空载体植株。在镉胁迫处理7 d后,转基因烟草叶片的超氧化物歧化酶、抗坏血酸过氧化物酶活性及可溶性糖含量均高于野生型对照,丙二醛含量均低于对照。在镉胁迫处理0 d、1 d、3 d后,转基因大豆毛状根复合体根和叶的超氧化物歧化酶、抗坏血酸过氧化物酶活性、可溶性糖含量均高于转空载体对照,丙二醛含量均低于对照,表明GmALMT33基因提高了植株的耐镉能力。本研究为进一步探讨GmALMT33基因的作用机制提供了依据,并为大豆抗逆育种提供了新的基因。

Due to industrial development and increasing pollution from domestic wastes,heavy metal concentrations in crops are exceeding standards,posing a serious threat to human health.Aluminum-activated malate transporters(ALMT)encode a class of anion channel proteins that play important roles in the transmembrane transport of plant organic acids.In order to investigate the function of GmALMT33 gene in response to Cd stress in soybean,the GmALMT33 gene was cloned from soybean using RT-PCR in this study.The CDS region of the gene is 1622 bp in lengh,encodes 553 amino acids,and contains the ALMT structural domain.qRT-PCR results showed that the expression level of GmALMT33 was the highest in the roots of soybeans,and the expression of this gene showed a tendency of first increasing and then decreasing after cadmium stress.We constructed the plant expression vector pCPB-GmALMT33 and genetically transformed tobacco and soybean hairy roots.Phenotypic analysis of the transgenic plants showed that under cadmium stress(66μmol/L CdCl_(2)),the leaf blades of the transgenic tobacco were yellowed and greenish,and the degree of browning at the edges was significantly lower than that of wild-type tobacco.The transgenic soybean hairy root complex plants showed significantly weaker degree of reddish-brown toxicity symptoms in stalks and leaf veins than the trans-space vector plants,which indicated that the GmALMT33 gene improved the toxicity symptoms of transgenic soybean hairy root complex.After 7 d of cadmium stress treatment,the SOD,APX activity and soluble sugar content of transgenic tobacco leaves were higher than those of the wild-type control,and the MDA content was lower than that of the control.After 0 d,1 d and 3 d of cadmium stress treatment,the SOD,APX activity and soluble sugar content of roots and leaves of transgenic soybean hairy root complex were higher than those of the trans-space vector control,and the MDA content was lower than that of the control.GmALMT33 gene improved the cadmium tolerance ability of plants.This study provides a basis for further exploring the mechanism of action of GmALMT33 gene and provides a new gene for soybean stress tolerance breeding.