玉米高赖氨酸新突变opaque-16基因的图位克隆及功能分析

玉米opaque-16 (o16)是一个高赖氨酸新突变，具有良好的利用前景。对该突变的前期研究，虽然已经将其初步遗传定位，并筛选出10余个候选基因，但其基因尚未克隆。我们已用玉米野生型、o16突变型近等基因系（NILs），构建了o16突变杂交分离F2小群体、不透明和透明籽粒极端大群体。本项研究拟用NILs和这些群体，对前期研究鉴定出的候选基因、连锁位点进行PCR检测筛查，参考B73基因组进行染色体步行和精细定位，寻找共分离位点，图位克隆o16基因。然后，采用CRISPR-Cas9进行基因敲除测验，采用杂交技术进行等位测验，确证克隆基因。采用RT-qPCR分析该基因在NILs中不同组织和不同发育时期籽粒的时空表达，采用荧光素酶互补成像（LCI）技术分析O16蛋白、主要Zeins和O2蛋白间的互作关系。研究结果对优质资源的创新和品质育种具有重要意义。

Maize opaque-16 (o16) is a novel mutation of high lysine and has good utilization prospect. In the previous studies of the mutation, although it has been preliminarily genetically localized, and more than 10 of its candidate genes have been screened, but the gene has not been cloned. Using the wild type and o16 mutant near isogenic lines (NILs), we have constructed populations of small F2, large opaque and vitreous extreme bulk for o16 mutation. By using the NILs and these populations, this study intends to find co-separated locus and do map-based cloning for the o16 mutation through PCR detection and screening of candidate genes and linkage loci identified in previous studies, and chromosome walking and fine mapping via reference to the B73 genome. Then, to confirm the cloned O16 gene, gene knockout test by CRISPR-Cas9, and allelism test by hybridization will be carried out. To analyze the spatiotemporal expression of the O16 gene, RT-qPCR will be performed by use of different tissues and developmental kernels of Nils. To analyze the interactions between the O16 protein, and major Zeins and O2 protein, firefly luciferase complementation imaging (LCI) will be conducted. The research results are of great significance to the innovation of high quality resources and quality breeding.