玉米醇溶蛋白基因染色体重排和拷贝数变化的遗传解析

The Quality Protein Maize (QPM) was successfully selected from the o2 mutant lines, which have the advantage of high lysine content that greatly improved the malnutrition in Africa, America, and other places. The genome structure of 27-kD γ-zein gene and (22, 19-kD) α-zein are unstable under the pressure of artificial selection, which leads to the gene rearrangement and recombination events occurring and a complex gene clusters with linkage and conserved haplotype adapting in breeding selection. However, the molecular mechanism of the genome evolution is still unknown. In this project, we constructed the genetic model and created the plants that occurred rearrangement and recombination events in zein genes. Based on these materials, the linkage marker and PacBio BAC sequencing were utilized to detect the 27-kD γ-zein gene rearrangement frequency and the (22, 19-kD) α-zein recombination frequency in different genetic background. The aim of this project is to decipher the molecular mechanism of rearrangement and recombination under artificial selection in breeding system by using the genomic sequencing data. The results of this project will provide a basic genetic knowledge for understanding the genome conservation and variation feature, thus benefit to breed the non-o2-regulated lines to create high yield and quality variety in maize.

优质蛋白玉米以o2突变体为基础选育而成，改善了非洲、美洲等地的营养不良症。玉米醇溶蛋白基因27-kD γ-zein和(22, 19-kD) α-zein在人工选择的压力下基因组结构不稳定而发生重排或重组，形成连锁且保守的单体型，以基因簇的形式形成适应性的表型，然而其发生的分子机制还不清楚。申请人在前期的基础中创建了用于分析基因重排和重组的遗传材料和检测模型，初步获得了发生重排和重组的单株。在此基础上拟利用开发的连锁标记检测不同遗传背景下27-kD γ-zein基因发生重排的频率和(22, 19-kD) α-zein发生基因重组的频率，同时将筛选的单株构建BAC库结合三代测序的方法，深入解析其发生重排和重组的分子机制，验证基因结构中序列的保守性和变异性，解析不同遗传背景下醇溶蛋白基因单体型和基因簇的演变机制，为创制新型的非o2调控的具有高配合力的优质蛋白玉米自交系奠定基础。

拷贝数变异(Copy number variations, CNVs)与基因表达水平的改变有关，是玉米遗传变异的主要来源之一。前期研究发现优质蛋白玉米γ27醇溶蛋白高表达是因为含有两个γ27拷贝的S等位基因，该位点的结构不稳定，容易发生基因重排。为了得到重排发生频率以及重排模型，我们利用qγ27紧密连锁的功能标记0707-1和K0326Y-deletion材料与不同自交系的正反交组合，检测到基因重排的平均频率是1.27 x 10-3，并对得到的单拷贝重排单株测序，得到γ27位点的重排模型。基因发生重排后，一条染色体失去基因拷贝的同时，另一条染色体会得到相应的拷贝数。我们利用qRT-PCR来筛选Uniform Mu群体，得到了三拷贝γ27基因的材料，并通过测序确定三拷贝重排单株重排类型为Rabb型。对三拷贝材料胚乳细胞观察发现，蛋白体的密度增加。将三拷贝材料回交导入到αRNAi材料中，获得的αRNAi-M材料不仅含高赖氨酸品质，而且胚乳表现硬质。为了更进一步了解αRNAi-M的胚乳修饰机制，我们对授粉后18天的胚乳进行了透射电镜观察，发现蛋白体的数量和大小都是有所增加的，与优质蛋白玉米的胚乳修饰机制一致。说明三拷贝γ27等位基因为优质蛋白玉米育种提供了一个优良等位修饰位点。

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