小米储藏过程中脂肪氧化酶介导的褪色机制研究

Millet colour is an important index to evaluate foxtail millet quality. Discoloration of millet during the storage reduces the value of the millet and also the quality stability of millet or millet products, which has been restricting the development of millet industry. In wheat, rice and foxtail millet, some evidences illustrated that lipoxygenase (LOX) was related to the degradation of carotenoids during storage or staleness. The millet colour of 400 varieties from the core germplasm of foxtail millet in China were analyzed during storage, which showed a wide variation. In this project, we propose to select 12 varieties from both the fastest and slowest discoloration groups (6 each) as research materials. At different storage stages, the LOX enzyme activities of different varieties will be analysed, and the composition and content of total caronenoids will be investigated. We plan to clone and analyze the sequence of all gene family members of LOX and compare their expression patterns in different millet varieties during grain maturing stages. Meanwhile, immunohistochemical technique and Western blot will be used to localize LOX protein in the millet and the expression profiles of LOX among different varieties at different storage stages to determine the LOX that plays a major role in the discoloration of millet. This project would add to our understanding of the molecular mechanism on millet discoloration during storage, which could lay a foundation for developing molecular markers and promoting the molecular breeding of high quality foxtail millet.

米色是评价小米品质的重要指标，然而小米储藏过程中普遍存在褪色问题,影响其外观与营养品质及其加工产品质量稳定性。在小麦、水稻以及小米中都有证据表明脂肪氧化酶与储藏或陈化中类胡萝卜素降解有关。我们对谷子核心种质资源中400份材料的小米分析发现，其储藏褪色特性存在很大变异。因此，本项目拟选择褪色快和慢的谷子品种各6个，分析其小米在储藏不同时期脂肪氧化酶（LOX）活性、总类胡萝卜素及其组成成分的变化；对这些品种中LOX基因家族成员进行全长序列克隆和比对，用定量RT-PCR比较LOX基因在不同品种籽粒成熟过程中的表达模式；利用免疫组化技术对小米形成过程中的LOX蛋白进行亚细胞定位，用Western-blot分析LOX在不同品种小米形成过程中以及不同储藏时期的表达水平，确定影响米色褪色的主效LOX蛋白。从而探索小米储藏过程中褪色的分子机制，为发掘小米褪色相关分子标记、促进谷子品质分子育种奠定基础。

谷子（Setaria italica (L.) Beauv.）是我国北方重要的粮食作物，其初加工产品-小米营养价值高，深受人们的喜爱。米色是评价小米品质的重要指标。然而，在小米储藏过程中由于类胡萝卜素降解导致的褪色问题，不但影响其食味品质，而且使其深加工产品的质量稳定性难以保证，这些都严重制约小米市场经济的发展。.本项目从400多份谷子品种中筛选出了褪色性状存在显著差异的品种作为试验材料，定期测定小米米色变化，同时测定了类胡萝卜素及其主要组分叶黄素和玉米黄质含量的变化，发现不同谷子品种小米储藏过程中类胡萝卜素的褪色差异较明显，褪色率介于10.46%-32.25%之间。 9806-1，豫谷1号，济谷11是褪色较快的品种，周里围，沁黄2号，苞米混子是褪色较慢的品种，其中9806-1褪色最快，苞米混子褪色最慢。对SiLOX蛋白籽粒发育不同时期及储藏前后的表达模式进行分析表明其表达模式存在显著的品种间差异，在褪色最慢品种苞米混子中各时期的表达都极低，而在褪色最快品种9806-1中的表达都很高。说明谷子脂氧合酶SiLOX是造成小米储藏期类胡萝卜素降解的关键因素。进一步通过定量qRT-PCR比较不同品种谷子籽粒不同成熟期SiLOXs基因家族成员的表达模式，结果显示 SiLOX4在9806-1中的表达均显著高于苞米混子，且其在苞米混子成熟末期完全不表达，因此推SiLOX4的测差异表达是造成小米褪色快慢的主要原因。.总之，本项目从生理及分子水平上共同探索小米储藏过程中的褪色机制，为进一步发掘控制小米褪色相关分子标记、提高谷子品质分子育种效率奠定基础。