大麦HvNramp5转运子编码基因等位变异及其对镉吸收的调控机制研究

Understanding of the genetic mechanisms of cadmium (Cd) accumulation in crops and development of low Cd-contaminated cultivars are imperative for fundamentally solving the problem in agriculture caused by soil Cd pollution. Barley (Hordeum vulgare) is the fourth most important cereal crop globally after wheat, maize and rice, and it is huge potential risk of Cd contamination in barley. Therefore, revealing of the physiological mechanisms associated with Cd accumulation in barley is quite helpful for developing low Cd-contaminated cultivars. Based on our previous studies on the function of transporter HvNramp5, in the current project, three unknown aspects will be solved by further studies: 1. The sequence polymorphism of HvNramp5 and the relationship between the sequence polymorphism/gene expression and Cd uptakes in 100 barley cultivars; 2. The difference of Cd transport affinity of HvNramp5 haplotypes; 3. The mechanism of transcriptional regulation of HvNramp5 induced by Fe deficiency. Thus, the sequence polymorphism of HvNramp5 gene in 100 barley cultivars will be analyzed, and the gene haplotypes associated with low Cd accumulation are identified. The transport affinity of HvNramp5 haplotypes is compared to reveal its difference on Cd uptakes. Furthermore, the interaction factors of transcription factors with HvNramp5 are screened by Yeast-One-Hybrid system to explore the mechanism of transcriptional regulation of HvNramp5. It may be expected that the obtained results will reveal the genotypic variation and the mechanisms of transcriptional regulation of HvNramp5, and should innovate the theory of stress tolerance in plants and provide useful reference for breeding of barley cultivars with low Cd accumulation.

阐明作物镉（Cd）积累的遗传机制和培育低积累型新品种是根本上解决农业生产中土壤Cd污染问题的关键。大麦（Hordeum vulgare）是全球第四大禾谷类作物，揭示大麦Cd积累的生理机制对大麦遗传改良具有重要意义。在前期研究HvNramp5转运子的功能基础上，将深入解析待解决的三个科学问题：1. HvNramp5序列多态性及表达量差异与其Cd吸收能力的关系；2. HvNramp5不同单倍型转运蛋白对Cd转运的亲和性差异；3. HvNramp5表达受缺Fe诱导的转录调控机制。本项目拟对100份栽培大麦世界核心种质开展HvNramp5序列多态性分析，鉴定低Cd积累的基因单倍型；并利用酵母单杂交筛选参与调控HvNramp5转录的互作因子，揭示其表达受缺Fe诱导的转录调控机制。本研究结果将揭示大麦Cd积累基因型差异和HvNramp5的转录调控机制，不仅丰富作物抗逆理论，并为大麦遗传改良提供参考。

大麦（Hordeum vulgare）是全球第四大谷类作物，由于其环境适应性强且能在不同土壤条件下种植，故大麦受土壤镉（Cd）污染的潜在风险大。阐明作物Cd积累的遗传机制和培育低积累型新品种，是根本上解决农业生产中土壤Cd污染问题的关键，因此揭示大麦Cd积累的遗传机制对大麦遗传改良具有重要意义。本项目在前期研究HvNramp5转运子的功能基础上，分析了100份栽培大麦全球核心种质HvNramp5基因序列多态性及其基因型间的表达量差异，结果发现该基因在编码区外显子上保守，而在启动子序列上存在丰富的多态性，特别是在ATG上游373-583bp富含顺式作用元件的区间，并且发现高表达与低表达种质之间该片段上存在19个位点上的差异，而编码蛋白对Cd转运的亲和性无显著差异。为明确启动子片段对HvNramp5基因表达量的影响，选用该210bp片段作为诱饵片段进行酵母单杂筛库筛选HvNamp5的互作基因。酵母筛库实验共获得12个有完整的ORF的互作候选基因，结合烟草双荧光素酶实验、根系缺Fe诱导表达分析等实验，推测HORVU3Hr1G083250（BZIP转录因子）和HORVU4Hr1G089120（MYB转录因子）为关键的互作候选基因。为验证HORVU3Hr1G083250和HORVU4Hr1G089120是调控HvNramp5表达的关键互作基因，以大麦基因型Golden Promise幼胚为外植体诱导愈伤组织；构建了CRISPR/Cas9和RNAi大麦遗传转化材料，明确了以上两个基因通过结合HvNramp5基因启动子序列，正向调控HvNramp5基因的表达。另一方面，通过microRNA和m6A测序分析发现HvNramp5基因的表达也受到表观遗传的调控。基于以上研究结果，我们绘制了大麦根部镉吸收及HvNramp5基因转录调控分子网络。本研究结果明确了以下三个生物学问题：(1) HvNramp5序列多态性及表达量基因型差异与其Cd吸收能力的关系；(2) 大麦种质HvNramp5转运蛋白对Cd转运的亲和性差异；(3) HvNramp5表达的转录调控机制。本研究结果揭示了大麦Cd积累基因型差异和HvNramp5基因转录调控机制，不仅丰富作物镉积累分子调控理论，并为大麦低镉育种提供理论指导。