野蔷薇内源多胺的HPLC测定方法及其组织变化规律  

HPLC method for determination of endogenous polyamines in Rosa multiflora Thunb.and its changes in different tissues

作　　者：陈锐 陈金萍 杨雯翔 邓致运 程文翰 CHEN Rui;CHEN Jinping;YANG Wenxiang;DENG Zhiyun;CHENG Wenhan(School of Bioengineering, Jingchu University of Technology, Jingmen 448000, China;Hubei Engineering Research Center for Specialty Flowers Biological Breeding, Jingmen 448000, China;College of Agricultural, Xinjiang Agricultural University, Urumq 830052, China)

机构地区：[1]荆楚理工学院生物工程学院,湖北荆门448000 [2]特色花卉生物育种湖北省工程研究中心,湖北荆门448000 [3]新疆农业大学农学院,新疆维吾尔自治区乌鲁木齐830052

出　　处：《安徽大学学报（自然科学版）》2022年第2期101-108,共8页Journal of Anhui University(Natural Science Edition)

基　　金：湖北省荆门市科技局重点项目(2018YFZD027);荆楚理工学院大学生创新创业项目(201811336019,S201811336062);2021年中央财政支持地方高校发展专项资金项目(zycztd001)。

摘　　要：以野蔷薇为材料,提取内源多胺,用高效液相色谱法HPLC进行腐胺、亚精胺、精胺含量测定.结果表明,多胺提取过程中,样品用量0.2 g、冰浴浸提液300μL、苯甲酰化反应37℃,提取效率最高.HPLC检测参数:流动相为V(甲醇)∶V(水)(60∶40),进样量15μL,流速1 mL·min^(-1),柱温30℃,检测波长254 nm,保留时间15 min时,3种内源多胺可完全分离并定量测定,相关系数均大于0.99,测定加标回收率为87.17%~103.64%,表明该方法可靠.在野蔷薇体细胞胚胎发生过程中,叶片培养3 h,内源多胺无明显变化,培养3 d后,多胺总量和腐胺含量显著增加,5 d后,亚精胺和精胺含量显著增加,亚精胺含量最高,精胺其次,腐胺含量极显著下降,同时多胺总量下降,说明在野蔷薇体细胞胚胎发生的启动阶段,腐胺向下游产物亚精胺和精胺发生了转化;在培养25 d的愈伤组织中,多胺总量显著下降,腐胺和精胺含量很低,说明在野蔷薇愈伤组织增殖阶段,可能精胺作用不大.该试验建立了内源多胺的2 mL提取体系,该方法降低了样品材料的用量,多胺提取过程更简便高效,并初步探索了野蔷薇体细胞胚胎发生过程中内源多胺的变化规律.In this paper,wild rose was used to extract endogenous polyamines,and the content of endogenous putrescine,spermidine,and spermine was determined by HPLC.The results showed that in the process of polyamine extraction,the extraction efficiency was highest when the sample amount was 0.2 g,the ice bath leaching solution was 300μL and the benzoylation was 37℃.HPLC detection parameters were the following:V(methanol)∶V(water)was 60∶40 and the injection volume was 15μL,flow rate was 1 mL·min^(-1),the column temperature was 30℃,the detection wavelength was 254 nm,and the retention time was 15 min,the three endogenous polyamines could be completely separated and quantitatively determined.The correlation coefficients were all greater than 0.99,and the standard recovery rate was 87.17%~103.64%,indicating that the method was reliable.In the process of wild rose somatic embryogenesis,the leaves were cultured for 3 h,and the endogenous polyamines did not change significantly.After 3 d,the total polyamines and putrescine content increased significantly.After 5 d,the contents of spermidine and spermine increased significantly.The content of spermidine was the highest,followed by spermine.The content of putrescine decreased significantly.At the same time,the total amount of polyamines decreased,indicating that during the initiation stage of wild rose somatic embryogenesis,putrescine was transformed into spermidine and spermine.In the callus of 25 d,the total amount of polyamines decreased significantly,and the levels of putrescine and spermine were very low,indicating that the effect of spermine might not be significant at the proliferation stage of wild rose callus.In this experiment,It had been established that the 2 mL extraction system of endogenous polyamines,which reduced the amount of sample materials,thus the extraction process of polyamines was more simple and efficient.And the authors had preliminarily explored the change rule of endogenous polyamines in the process of somatic embryogenesis in wild rose.

关 键 词：野蔷薇 高效液相色谱法HPLC 多胺 体细胞胚胎发生 

分 类 号：S601[农业科学—园艺学]