铜胁迫下葡萄植株的生理响应和铜转运蛋白基因VvCTR1的克隆及分析

Physiological responses to copper stress in grape plants and cloning and analysis of copper transport VvCTR1 gene

[目的]本文旨在研究重金属铜对葡萄植物生长发育的影响,为揭示重金属铜对植物的伤害机制提供理论依据。[方法]采用过量的硫酸铜以及硫酸铜与其螯合剂混合液处理葡萄植株幼苗,以Hoagland溶液为对照,分析根和叶片的发育情况,叶绿素含量以及铜在植株中的分布情况;克隆了铜转运蛋白基因VvCTR1,分析其在果实发育过程中的表达量,并对其结构特征和物种间的同源性进行分析,同时分析铜胁迫下葡萄根、茎和叶中的VvCTR1表达情况。[结果]与对照相比,过量的硫酸铜、硫酸铜与柠檬酸或EDTA混合处理可以抑制葡萄植株新根的发育数量和长度,促进叶片叶绿素的降解和落叶速率,增加叶柄基部乙烯的产生以及多聚半乳糖醛酸酶和纤维素酶活性,尤其以硫酸铜和EDTA混合处理伤害更明显。Cu^(2+)在植株叶、茎、根中的含量从小到大分别依次为上部叶片、中部叶片、下部叶片,上部茎、中部茎、下部茎,老根、幼根;硫酸铜处理植株中的Cu^(2+)含量高于对照,并且随着处理时间的延长含量不断升高,尤其是硫酸铜与柠檬酸或者EDTA混合处理植株中Cu^(2+)含量更高。克隆的铜转运蛋白基因VvCTR1中无内含子,其编码的蛋白含有3个跨膜区TMD1~3,N端含有1个甲硫氨酸富含区。VvCTR1基因的表达量随着果实发育逐渐降低。无论在根中还是在不同部位的茎和叶中,VvCTR1都能被铜诱导。[结论]过量的铜会对葡萄植株造成伤害,尤其与Cu^(2+)的螯合剂柠檬酸或EDTA同时存在时,能使更多的Cu^(2+)进入植物体内,最终引起葡萄植株死亡。

[Objectives]This study aims to analyze the effects of heavy metal copper on the growth and development of grape plants,and provided theoretical basis for revealing the mechanism of damage by heavy metal copper to plants.[Methods]The excess CuSO4 and the mixture of CuSO4 and its chelating agent were applied on grape seedlings,the root and leaf development,leaf chlorophyll content,and the distribution of copper in plants were analyzed;copper transport VvCTR1 gene was isolated,and its expression levels were analyzed during fruit development.Its structure and homology among species were analyzed,and its expression levels were analyzed under the copper stress in roots,stems and leaves.[Results]Compared to the control,excess CuSO4,the mixture of CuSO4 and citric acid or EDTA inhibited the development of the number and length of new roots in grape,promoted the degradation of chlorophyll and leaf rate drop,and increased the production of ethylene and the polygalacturonase and cellulase activities in petiole base.Especially there were obvious damage in mixture of CuSO4 and EDTA treatment.The content of Cu^2＋in leaves,stems and roots from small to large was the morphologically up to down of the stems and leaves and old roots to new roots in turn.And the content of copper ion in CuSO4 treated plants was higher than control,and showed an increase with the extension of time,especially the mixture of CuSO4 and citric acid or EDTA treatment had more copper ions; there was no intron in copper transport VvCTR1 gene,and it contained three transmembrane regions of TMD1-3,which N contained a methionine rich region.Its expression decreased gradually with the fruit development,and the expression of VvCTR1 gene was enhanced under copper both in roots and in different positions of stems and leaves.[Conclusions]Excessive copper can cause damage to grape plants,especially when combined with Cu^2＋chelatecitric acid or EDTA,which can make more Cu^2＋ enter the plant and ultimately cause grape plant death.