玉米胚乳蛋白质组平衡和优质蛋白玉米胚乳修饰机理研究

Corn seeds are mainly used as food for human and feed for livestock. However, essential amino acids like lysine and tryptophan are deficient in corn meal because of the abundance of its main storage protein zeins lack these amino acids. A natural mutant, opaque 2 (o2) causes reduction of zeins, an increase of non-zein proteins, and as a consequence, a doubling of lysine levels. The o2 mutant has several drawbacks, like reduced grain yield, soft chalky endosperm and greater susceptibility to pests and diseases, which precluded its direct commercialization. By selecting the o2 modifiers, breeders at CIMMYT first successfully converted the soft endosperm into hard endosperm and meanwhile maintained the high lysine trait. The modified o2 mutant is called quality protein maize (QPM). Because of rebalancing of endosperm proteome, seed still maintains wild-type levels of protein in o2 and QPM. The increase of non-zeins exhibited two patterns, i.e. global elevation of the majority and apparent increase of a few specific proteins. By taking advantage of RNA inference specifically against α-zein mRNAs, we can create transgenic lines with even higher-than-o2 levels of lysine. Still, endosperm proteome underwent rebalancing of zeins and non-zeins keeping total protein levels constant. Moreover, o2 modifiers are able to restore the soft endosperm caused by RNAi. Although QPM has been introduced to many countries, it still meets the challenge of its wide-spreading use. One of the reasons is the complicated mechanism of endosperm modification by o2 modifiers, which has precluded the fast breeding of new germplasms for hybrid QPM production. It was shown that in QPM the transcript and protein of the 27-kDa γ-zein accumulated 2- to 3-fold higher than the normal control and unmodified o2 mutant. Konckdown of the 27- and 16-kDa γ-zeins resulted in failure of modification, indicating that theγ-zeins act hypostatically to the o2 modifiers. However, none of the o2 modifiers has been cloned, because they only exist in QPM and it is impossible to isolate their mutants in the current reverse genetic mutant libraries, which were all generated in normal maize lines. In this project, we have two main goals: (1) Understanding how the proteomes in o2 and α-zeinRNAi mutants are rebalanced by using the transcriptomics and proteomics tools, and unraveling the identities and functions of the apparently increased proteins in proteome rebalancing and utilizing their regulatory mechanisms to improve exogenous protein expression in maize transgenic engineering; (2) Studying the transcriptome changes in QPM and the functions of the specifically up-regulated genes in endosperm modification; Generating mutations for o2 modifiers and their interactive factors in quality protein maize through EMS mutagenesis.

优质蛋白玉米是利用o2软质胚乳突变体为高赖氨酸供体，通过积累o2修饰因子恢复硬质胚乳表型的高营养玉米。o2突变体中，几乎不含赖氨酸的主要储存蛋白α-zein表达急剧下降，而赖氨酸含量平衡的非zein蛋白合成上升，这样既使胚乳总蛋白水平保持不变又使籽粒赖氨酸水平大幅度提高；现在通过RNAi干扰α-zein表达，赖氨酸水平甚至可以超过o2突变体，而且优质蛋白玉米修饰因子同样可以恢复RNAi引起的软质胚乳表型。优质蛋白玉米虽然已经产业化，但是大面积推广还面临挑战，主要原因是优质蛋白玉米胚乳修饰机理复杂，新品种育种缓慢。目前比较清楚的是γ-zein表达对修饰因子的修饰作用很关键。本项目重点研究两个问题：1、解析玉米胚乳蛋白质组平衡机制及在玉米籽粒基因工程中的意义；2、研究优质蛋白玉米胚乳转录组变化和特异表达上调基因在胚乳修饰中的功能；通过EMS诱导修饰因子和它们的互作因子突变体来推动修饰机理研究。

优质蛋白玉米是以o2突变体为基础材料选育的高赖氨酸硬质胚乳玉米，改善了非洲、亚洲等地的营养不良症。然而半个多世纪以来，优质蛋白玉米在选育过程中籽粒由软质变成硬质的遗传调控机制尚不清楚，大大限制了QPM玉米育种商业化进程。我们通过一系列遗传学实验证明了qγ27在遗传上通过顺式作用增加27-kD γ-zein表达，qγ27是一段包含了27-kD gamma-zein基因在内的15.26 kb的基因复制，因此提高了27-kD gamma-zein表达而促进优质蛋白玉米胚乳修饰。qγ27的基因复制发生在玉米驯化前，但结构不稳定，在优质蛋白玉米育种时受到人工选择。我们还解析了O2和PBF参与玉米品质和粒重形成的调控网络，发现O2和PBF通过影响淀粉合成对调控粒重，并证明了O2和PBF直接调控PPDKs和SSIII，提出将来培育优质蛋白玉米可用RNAi直接沉默醇溶蛋白基因，从而避免用o2突变体影响淀粉合成和粒重。