低温胁迫下褪黑素对葡萄悬浮细胞生长及生化特性的影响

Effects of melatonin on growth and biochemical properties of Vitis vinifera suspension cells under low temperature stress

【目的】分析低温胁迫下褪黑素(MT)对葡萄悬浮细胞生长、生化特性及冷响应相关基因表达的影响,筛选适宜MT浓度,探究MT增强葡萄耐冷性的作用机制。【方法】以‘黑比诺’葡萄悬浮细胞为试材,在低温(4℃)胁迫下,考察不同浓度MT[0(CK),50(T_(1)),100(T_(2)),200(T_(3)),300μmol/L(T_(4))]处理对悬浮细胞生长、丙二醛(MDA)含量、脯氨酸(Pro)含量、抗氧化酶活性的影响及T_(2)处理冷响应相关的基因表达变化趋势。【结果】(1)在低温胁迫下,各MT处理悬浮细胞干重在培养周期内均呈先增加再减少最后趋于平稳或降低的趋势,在处理末期均明显高于CK。(2)各处理悬浮细胞MDA和Pro含量均随处理时间延长表现为先升后降,在24 h或48 h时达到峰值;各MT处理MDA含量均显著低于CK,Pro含量则显著高于CK,并均以T_(2)处理最低或最高;(3)各MT处理细胞抗氧化酶活性均显著高于同期CK,并均以T_(2)处理最高;随着时间延长,各处理的POD和CAT活性先升后降,SOD和APX活性则先降后升,且分别在24 h或6 h时达最高。(4)T_(2)处理悬浮细胞冷响应相关基因(VvCBFs、VvICE1、VvRD29B和VvKIN-2)相对表达量在不同时间均较相应CK极显著上调。【结论】在低温胁迫下,适宜浓度(100μmol/L)MT可通过提高葡萄悬浮细胞抗氧化酶活性和冷响应相关基因的相对表达量,增加Pro含量,降低MDA含量,最终增强葡萄悬浮细胞低温耐受性。

[Objective]The study aims to analyze the effects of melatonin(MT)on the growth,biochemical characteristics,and expression of cold-responsive genes in grapevine(Vitis vinifera L.)suspension cells under low temperature stress,to screen the appropriate MT concentrations and to investigate the mechanism of MT in enhancing cold tolerance of grapevine.[Methods]Using‘Pinot Noir’grapevine suspension cells as test materials,we investigated the effects of different concentrations of MT[0(CK),50(T_(1)),100(T_(2)),200(T_(3)),300μmol/L(T_(4))]on the growth of the suspension cells,the content of malondialdehyde(MDA),the content of proline(Pro),and the antioxidant enzyme activities,and changes in gene expression related to cold response to T_(2) treatment,under low temperature(4℃)stress,[Results](1)Under low temperature stress,the dry weight of cells in each MT treatment showed a trend of first in crease then decrease,which was finally stable or decreased,and was higher than CK at the end of treat-ment.(2)MDA and Pro contents of each treatment were firstly increased and then decreased with the time of treatment,which reached the peak at 24 h or 48 h.MDA content of each MT treatment was lower than that of CK,while the Pro content was higher than that of CK,with T_(2) treatment being the lowest or the highest.(3)The antioxidant enzyme activities of each MT treatment were higher than that of CK,with T_(2) treatment being the highest.With the prolongation of time,the activities of POD and CAT of each treatment firstly rose and then fell,while the activities of SOD and APX firstly fell and then rose,which reached the highest at 24 h or 6 h,respectively.(4)The relative expression of cold-responsive genes(VvCBFs,VvICE1,VvRD29B,and VvKIN-2)in T_(2)-treated suspension cells were up-regulated com-pared with CK.[Conclusion]Under low temperature stress,the appropriate concentration(100μmol/L)of MT increases the expression of antioxidant enzymes and cold-response genes,increases Pro content,de-creases MDA content,and enhances low temperature tolerance of grapevine suspension cells.