郑58 /opaque2近等基因系中opaque2基因突变位点分析与高效分子标记开发
优质蛋白鲜食玉米分子标记辅助聚合育种过程中,opaque2突变体材料是最常用的高赖氨酸玉米供体。因此我们对已获得的郑58/o2近等基因系突变表型、突变位点以及连锁标记开发进行了详细研究。结果表明郑58/o2表现出典型的o2突变体表型,其子粒赖氨酸含量显著增加,SDS-PAGE显示胚乳发育不同时期22-kD α-醇溶蛋白的积累均明显低于郑58。荧光定量PCR结果表明α-醇溶蛋白家族基因Z1A、Z1B、Z1C和Z1D的表达均显著低于郑58,但是荧光定量PCR实验发现郑58/o2胚乳发育不同时期Opaque2基因均正常表达。测序分析发现郑58/o2中o2基因ATG后713bp处缺失10个碱基,ORF预测OPAQUE2蛋白翻译提前终止。针对突变缺失位点开发基因内分子标记o2-indel-1,利用该标记进行回交转育,结果表明o2-indel-1与o2突变表型完全连锁,错选率为0。因此opaque2基因突变位点的解析有助于高效分子标记的开发,有效降低错选率,提高优质蛋白鲜食玉米多基因聚合育种的选择效率。
英文摘要:
Opaque2 mutant gene enhances lysine content in the endosperm as compared with the wild genotype and o2 mutant is the main donor of quality protein fresh maize breeding. Opaque2 gene mutation site, kernel phenotype and development of functional molecular marker of Zheng58/opaque-2 near-isogenic line were investigated in this study. The research results show that zheng58/o2 had an evident similarity to the o2 mutant phenotype, which had increase in lysine over the zheng58, and reduction of the accumulation of 22-kDa α-zeins by SDS-PAGE. However, expression level of the o2 gene was normal in the developing endosperm of the o2 mutant from 10 DAP to 42 DAP at all times by qRT-PCR. The expression of α-zein genes (Z1A, Z1B, Z1C and Z1D) in the o2 mutant were shown to be significantly reduced. Sequence analysis of o2 mutant gene showed that 10-nucleotide deletion mutant were found at 713 bp downstream of ATG in the mutant sequence which resulted in frame shift and early termination of OPAQUE2 protein translation. Functional molecular marker o2-indel-1 had been developed based on the deletion mutation, which was completely associated with o2 mutant phenotype during marker-assisted backcross breeding. Analysis of o2 mutation site contribute to the development of functional molecular marker and increase the efficiency of quality protein fresh maize breeding through marker assisted selection.
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