莲中介导矿质元素胁迫耐受性基因的筛选与功能验证

Isolation and Functional Verification of Genes Mediating Mineral Element Stress Tolerance in Lotus

【目的】莲是一种传统的药食同源作物,但其蕴藏的丰富基因资源仍缺乏功能鉴定。分离鉴定莲中参与微量矿质营养元素及重金属元素积累或耐受过程的功能基因,丰富莲中介导营养高效以及逆境耐受的基因资源,并为莲的遗传改良提供理论依据。【方法】以湘莲代表品种‘寸三莲’为试验材料。首先构建莲的酵母表达cDNA文库,并将文库转化酿酒酵母后分别在含有过量镉(Cd)、锰(Mn)、锌(Zn)、铜(Cu)、铁(Fe)、铝(Al)胁迫的平板上筛选,分离阳性酵母克隆中介导相关胁迫耐受性的目的基因。结合生物信息学分析和酵母互补试验对筛选出的耐受基因进行功能验证。【结果】构建了库容量超过106个单克隆、重组率100%、平均插入片段大于1 000 bp的酵母文库。通过筛选得到介导Cd耐受的基因13个、Mn耐受基因4个、Zn耐受基因4个、Cu耐受基因3个、Fe耐受基因7个、Al耐受基因1个,其中3个基因能够同时介导Fe和Mn的耐受性。这些基因在莲除6号染色外的其他染色体上都有分布。【结论】本研究构建了高质量的莲酵母表达cDNA文库,并筛选得到29个介导微量矿质营养元素或重金属元素胁迫抗性的基因,为莲的营养高效以及避免重金属污染的遗传改良积累了基因资源。

【Objective】Lotus(Nelumbo nucifera)is a traditional edible and medicinal homologous crop in China,yet its abundant genetic resources lack functional characterization.This research aimed to functionally identify genes involving in the accumulation/tolerance of trace mineral nutrients and heavy metal elements in lotus,thereby accumulated gene resources that facilitated increased nutrient efficiency and stress tolerance,so as to provide a theoretical basis for the genetic improvement of lotus.【Method】The representative Xianglian lotus variety,Cunsanlian was used as the experimental material.Initially,a yeast expression cDNA library of lotus was constructed and transformed into Saccharomyces cerevisiae.It was then screened on plates containing excessive cadmium(Cd),manganese(Mn),zinc(Zn),copper(Cu),iron(Fe),and aluminum(Al)stress to isolate target genes mediating stress tolerance in positive yeast clones.Finally,the functional verification of the selected tolerance genes was conducted through combining bioinformatics analysis and yeast complementation verification.【Result】A yeast cDNA library of lotus was constructed with a capacity exceeding 106 yeast monoclonal clones,a recombination rate of 100%,and an average insertion fragment size greater than 1000 bp.Following the screening on stress plates,13 genes for Cd tolerance,4 for Mn tolerance,4 for Zn tolerance,3 for Cu tolerance,7 for Fe tolerance,and 1 for Al tolerance were identified.Among these,3 genes were able to mediate tolerance to both Fe and Mn.These genes were distributed on all lotus chromosomes except chromosome 6.【Conclusion】A high-quality yeast expression cDNA library of lotus was constructed,and screened 29 genes mediating resistance to excess trace mineral nutrients or heavy metal elements,accumulating gene resources for lotus genetic improvement for enhanced nutrient efficiency and the prevention of heavy metal accumulation.