宁夏枸杞铁镉响应基因的筛选及其功能验证  

Isolation and Functional Verification of Genes Responding to Iron and Cadmium Stresses in Lycium barbarum

作　　者：余慧 王静 梁昕昕 辛亚平 周军 赵会君 YU Hui;WANG Jing;LIANG Xin-xin;XIN Ya-ping;ZHOU Jun;ZHAO Hui-jun(Key Laboratory of Ecological Protection of Agro-pastoral Ecotones in the Yellow River Basin,National Ethnic Affairs Commission of the People's Republic of China,College of Biological Science and Engineering,North Minzu University,Yinchuan 750021)

机构地区：[1]北方民族大学生物科学与工程学院国家民委黄河流域农牧交错区生态保护重点实验室,银川750021

出　　处：《生物技术通报》2023年第7期195-205,共11页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31960056);宁夏自然科学基金(2022AAC03278);经济林遗传改良创新团队(2022QCXTD04)。

摘　　要：为探讨铁(Fe)、镉(Cd)胁迫对宁夏枸杞LbFRO(ferric reduction oxidase)、LbZIP(zinc-regulated transporters and iron-regulated transporter-like protein)和LbNRAMP(natural resistance-associated macrophage protein)基因的表达模式的影响,为防治宁夏枸杞黄叶病、培育高效Fe利用枸杞品种和低镉累积品种提供理论基础及技术支持。以宁夏枸杞1号组培幼苗为试验材料,利用转录组测序技术分析筛选宁夏枸杞中与Fe和Cd吸收转运相关的基因;利用实时荧光定量PCR技术验证目的基因在Fe、Cd胁迫下的表达变化情况;利用常规PCR方法克隆获得全长序列并进行生物信息学分析;构建酵母表达载体转化Fe/Cd敏感型酵母菌株ΔCCC1/YK44进行转基因酵母功能验证。结果显示,LbIRT2、LbFRO以及LbNRAMP3受Fe和Cd胁迫后显著上调表达;系统进化树分析表明宁夏枸杞Fe、Cd响应基因与水稻和拟南芥亲缘关系较近;转基因酵母功能进一步验证了LbFRO、LbIRT2以及LbNRAMP3能增强酵母对Fe和Cd的敏感性。LbFRO、LbIRT2以及LbNRAMP3可能参与了枸杞对Fe和Cd的吸收和转运。In this paper,the effects of iron(Fe)and cadmium(Cd)stress on the expression patterns of genes of LbFRO(ferric reduction oxidase),LbZIP(zinc-regulated transporters and iron-regulated transporter-like protein)and LbNRAMP(natural resistanceassociated macrophage protein)in Lycium barbarum L.were investigated for providing theoretical basis and technical support in preventing and controlling yellow leaf disease of wolfberry in Ningxia,cultivation of high-efficiency Fe-utilizing wolfberry varieties and low-cadmium accumulation varieties.The seedlings of L.barbarum L.No.1 were used as experimental materials,and the genes related to iron(Fe)and cadmium(Cd)absorption and transport were isolated by transcriptome sequencing technology;the expression patterns of the target genes under Fe and Cd stress were verified by real-time fluorescence quantitative PCR;the full-length sequences of the target genes were cloned by conventional PCR method;and the yeast expression vector of those genes were constructed and transformed into Fe/Cd sensitive yeast strain ΔCCC1/YK44 for functional verification.The results showed that LbFRO,LbIRT1,LbIRT2 and LbNRAMP3 were significantly up-regulated under Fe and Cd stress,phylogenetic tree analysis showed that those genes of L.barbarum L.were closely related to rice and Arabidopsis thaliana,the functions of transgenic yeast further verified that LbFRO,LbIRT2 and LbNRAMP3 genes enhanced the sensitivity of yeast to iron(Fe)and cadmium(Cd),indicating that these genes may be involved in the absorption and transport of Fe and Cd in L.barbarum L.

关 键 词：宁夏枸杞 铁胁迫 镉胁迫 转基因酵母 实时荧光定量PCR 

分 类 号：X173[环境科学与工程—环境科学] S567.19[农业科学—中草药栽培]