大薯贮藏蛋白Dioscorin Da-dio5基因特异表达对块茎形态及发育的影响

Greater yam（Dioscorea alata L.）is an important food crop, its tuber yield and nutritional value is high. But it is difficult to dig the tuber, because it is often as long as one meter. How to get a new variety of high yield and short tuber is a challenging job in greater yam breeding. We have carried out the study on the main storage protein Dioscorin in the tuber of greater yam. We found that the protein appeared only in the early stage of tuber formation and expansion, and it was accumulated in a large amount. Overexpression of the Da-dio5 gene could cause the root of Arabidopsis thaliana to become shorter and thicker. We suspected that this gene might have a similar effect in the tuber of greater yam. Based on our established genetic transformation system of greater yam, the project will create the overexpression and directed knockout mutants by gene engineering technology. Using the transcriptomics and proteomics technology, our research will focus on the effect of Da-dio5 with the tuber growth, tuber yield, morphology, and the associated metabolic pathways for further excavating the key genes of tuber development. The results will provide a technical basis for the improvement of yield and morphological improvement of greater yam tuber, and supply a new resource for the molecular breeding.

大薯是重要的粮食作物，其块茎产量高，营养价值高，但往往长达一米，采挖困难。如何获得块茎粗短的高产新品种，是大薯育种中的一项有挑战性的工作。我们前期对大薯块茎中主要贮藏蛋白Dioscorin进行研究，发现该蛋白一般仅在块茎早期形成和膨大过程中特异性的出现并大量累积，过表达其家族中的Da-dio5基因，可引起拟南芥根明显变短变粗。我们猜测，该基因在大薯块茎中可能有类似作用。本研究拟在实验室建立的大薯遗传转化体系的基础上，通过基因工程的技术，获得Da-dio5过表达和定向敲除的突变体，进而通过转录组和蛋白质组技术，研究Da-dio5蛋白对块茎发育、块茎形态及产量的影响，以及与之相关代谢途径的共表达网络，发现块茎发育过程的关键基因。研究结果将为大薯产量提高及薯块形态改良提供一定技术基础，为大薯分子育种提供新的资源。

参薯（即大薯）是热区重要粮食作物，其块茎产量高，营养价值高，但其块茎形态多变，影响采挖和食用，解析块茎形成机制，获得粗短的高产新品种，是参薯育种中的重要工作。前期研究中，我们开展参薯块茎主要贮藏蛋白Dioscorin的研究，发现该类蛋白在块茎特异表达，且Da-dio5基因在拟南芥超表达，可引起拟南芥根明显变短变粗。基于此，本项目开展了对参薯Dioscorin的深入研究。根据现有不同参薯种质的转录谱数据，分析Ka/Ks值发现Da-dio5（Ka/Ks=0.6495）受中性选择。RT-qPCR实验表明Dioscorin基因在块茎和零余子高表达，在其他组织中表达较低，在块茎的萌发期和贮藏期，Da-dio1-5的表达下降，且植物激素和非生物胁迫处理均能诱导Dioscorin基因的表达。亚细胞定位实验表明Da-dio5定位于细胞质和液泡。原核表达和真核表达的Dioscorin重组蛋白基因均具有抗氧化活性。Da-dio5-promoter::GUS表达载体转化拟南芥表明，该启动子在拟南芥的顶端分生组织和下胚轴中表达。通过腋芽遗传转化体系，将Da-dio5超表达和CRISPR敲除载体转化参薯，使用PCR、RT-qPCR 和Western-blot进行阳性转化株进行鉴定。利用PCR检测的11株超表达抗性株中，Western-blot实验检测到1株阳性株。敲除表达植株的5 株抗性植株中，5株转基因植株的靶标序列附近有碱基的突变。通过参薯转录谱数据分析，发现Da-dio1-5基因属于氮代谢途径，WGCNA基因共表达网络分析发现，属于ERF、ALOG家族的转录因子与Dioscorin处于相同基因网络，这为后续工作开展提供的方向。