糯玉米中opaque-2基因近等基因系的创制与互作机理研究

Waxy gene and high lysine gene (Opaque-2 mutant gene, o2) are recessive genes in maize. Although waxy corn containing waxy gene has high economic value, its nutritional quality is not complete. Essential amino acids, such as lysine, methionine and tryptophan, are absent in waxy corn and lysine is the first limiting amino acids. Opaque-2 mutant gene can increase 80% lysine and tryptophan content of maize. So importing o2 in waxy corn can increase the lysine content of grain, which is important to improve the nutritional structure and food value of waxy corn seeds. In this study, o2 gene will be imported to 20 inbred lines of high grade waxy corn by SSR marker-assisted selection (MAS). o2w-NILs will be created to improve lysine content of waxy corn grain. Then o2w-NILs with the best quality will be selected by a series of quality analysis from all the o2w-NILs created and the corresponding waxy lines. The effect of o2 on grain quality of different waxy corns will be identified initially. Whether there are correlation between o2 and waxy gene will be judged preliminary by the change of their protein expression during different DAP. The changes of the key enzymes in the starch metabolic pathway and the interaction way between o2 and waxy gene will be studied by yeast hybrid, chromatin Immunoprecipitation and proteomics techniques, which will reveal the interaction mechanism. This study will help us lay the theoretical foundation of molecular marker-assisted breeding of waxy maize with o2.

糯性基因(waxy)和高赖氨酸基因(opaque-2)为玉米隐性基因，含waxy基因的糯玉米有很高经济价值，但营养不完全，尤其缺少赖氨酸、色氨酸等必需氨基酸，而赖氨酸是第一限制性氨基酸。导入o2可提高糯玉米籽粒赖氨酸含量，对完善其营养结构，提高食用价值具重要意义。本项目拟利用SSR分子标记和回交技术，将o2基因导入20份品质优良的糯玉米自交系，得到赖氨酸含量提高的双隐性近等基因系o2w-NILs；通过对o2w-NILs及其供受体籽粒的品质分析，找出最优o2w-NILs，初步判断o2对不同糯玉米品质的影响；研究O2与Waxy蛋白在授粉后不同时期表达变化规律，阐明二者是否具相关性；通过酵母杂交、CHIP-seq、蛋白质组学等方法，研究o2与waxy突变基因相互作用方式及淀粉代谢途径中关键酶的变化，从而揭示o2与waxy的互作机理，为建立在糯玉米中导入o2分子标记辅助育种方法奠定理论基础。

糯玉米源于中国，口感软糯，适口性好，有很高经济价值，但营养不完全，尤其缺少赖氨酸、色氨酸等必需氨基酸，而赖氨酸是第一限制性氨基酸。导入o2可提高糯玉米籽粒赖氨酸含量，对完善其营养结构，提高食用价值具重要意义。本项目利用SSR分子标记和回交技术，将o2基因导入25份品质优良的糯玉米自交系，成功构建了19份赖氨酸含量提高的双隐性近等基因系o2w2-NILs，赖氨酸最高可以提高60%以上；选出构建成功的5份o2w2-NILs连续繁育3代，发现 Bg-rbg转座子系统没有影响其稳定性；通过对o2w2-NILs及其供受体籽粒的品质分析，发现其在水分、粗脂肪、粗蛋白、粗淀粉、氨基酸总量和17种氨基酸组成等营养成分方面有不同差异，亚显微结构也发生了变化，其中赖氨酸含量、支链淀粉含量和非醇溶蛋白含量显著增加；利用酵母杂交技术证明了O2转录因子对WX和wx基因没有直接相互作用，O2蛋白既可以与AGPase大亚基Sh2基因部分片段结合又可以直接作用于SH2蛋白，还可以作用于蔗糖合酶（SuSy）Sh1基因等淀粉合成关键基因，推测O2和WX基因协同影响玉米淀粉合成。本研究为建立在糯玉米中导入o2分子标记辅助育种方法奠定了理论基础。