基于PtIRT1基因被转录调控的机制解析枳易缺锌缺铁的原因

Zinc (Zn) and iron (Fe) deficiency are widespread in citrus production and difficult to correct as well, especially for those orchards with trifoliate orange (Poncirus trifoliata L. Raf.) as rootstock. The reason why Poncirus trifoliata is sensitive to Zn and Fe deficiency is still unclear. In our previous work, we isolated a Zn/Fe-regulated transporter (ZRT/IRT)-related protein, PtIRT1, from Poncirus trifoliata. And the function analysis suggested that PtIRT1 was able to uptake Zn and Fe in yeast very strongly, but its expression level in Poncirus trifoliata was significantly lower than that in Citrus junos (a kind of rootstock which is highly-tolerant to Zn and Fe deficiency) under conditions of Zn and Fe deficiency. The low transcription of PtIRT1 may limit uptake of Zn and Fe, which might be the reason why Poncirus trifoliata is sensitive to Zn and Fe deficiency. To verify our speculation, we plan to perform a comparative study using these two rootstocks as materials in the present project. Firstly, the promoter of PtIRT1 will be compared between Poncirus trifoliata and Citrus junos, in order to study whether the mutation of promoter affect its upstream transcription factors (TFs) to activate the transcriptional expression of PtIRT1. In addition, the upstream TFs of PtIRT1 will be identified and their different expression levels in Poncirus trifoliata and Citrus junos will be compared under Zn and Fe deficiency. The TF, that is lowly-expressed in Poncirus trifoliata but highly-expressed in Citrus junos, will be selected for overexpressing and RNAi interference in these two rootstocks via genetic transformation. The PTIRT1 expression and tolerance ability will be analyzed in transformed Poncirus trifoliata and Citrus junos, to determine if the low expression of key TF in Poncirus trifoliata results in weak transcriptional activation of PTIRT1. Our current project is the further extension of previous work, and it can not only enrich the theories of transcriptional controlling of nutrient uptake in citrus, but also lay the foundation for expanding the adaption area of Poncirus trifoliata by using PTIRT1 gene.

缺锌缺铁在柑橘生产中发生普遍且矫治困难，在枳砧上尤为严重，目前关于枳易缺锌缺铁的原因仍不清楚。对枳锌铁转运蛋白PtIRT1的研究发现，其在酵母中表现极强的锌铁吸收能力，但和耐缺锌缺铁的资阳香橙砧木相比，其在枳中表达水平较低。为研究枳PtIRT1表达水平低的原因以及明确这是否导致枳易缺锌缺铁，本项目以上述两种砧木为材料，首先比较PtIRT1启动子差异，研究是否因启动子差异影响上游转录因子对枳PtIRT1表达的激活；另外，比较PtIRT1上游转录因子在两种砧木中受缺锌缺铁诱导表达的差异，分析超表达或RNAi干涉转录因子后对枳和资阳香橙内源PtIRT1表达水平以及耐缺锌缺铁能力的影响，研究是否因为上游关键转录因子在枳中表达水平低导致激活PtIRT1表达减弱，最终导致枳易缺锌缺铁。本项目不仅可以丰富柑橘矿质营养吸收的转录调控理论，还将为高效利用PtIRT1基因扩大优良砧木枳的土壤适应范围奠定基础。

枳是柑橘生产中广泛使用的一种砧木，但在碱性土上极易缺铁黄化。在相同生长条件下，另外一种砧木资阳香橙表现耐缺铁黄化。在本实验室的前期研究基础上，本项目拟围绕铁转运基因IRT1的转录调控机制，解析枳易缺铁的原因。通过对枳和资阳香橙两种砧木的比较研究，首先证明两种砧木的IRT1基因及上游转录因子FIT基因均在增强植物耐缺铁中发挥功能，枳和资阳香橙耐缺铁的差异不是因为二者IRT1和FIT基因的功能差异所致。进一步研究发现，两种砧木的IRT基因启动子序列和在缺铁下的活性存在差异；酵母单杂、双荧光素酶和EMSA实验证明资阳香橙CjIRT1启动子与CjFIT转录因子能够结合，且鉴定出结合位点序列；和资阳香橙比较发现在该结合位点序列上，枳PtIRT1启动子中存在单个碱基差异，导致其不能与上游FIT结合，这可能是枳易缺铁的重要原因之一。另外，本项目利用CjIRT1启动子片段进行酵母文库筛选，鉴定出1个新的与其结合的转录因子。本项目从分子调控方面解析了枳易缺铁的原因，为开发耐缺铁的分子标记和进行基因编辑创制耐缺铁的柑橘砧木奠定了基础。