干旱胁迫下表观遗传机制对转C4型PEPC基因水稻种子萌发的影响

为探究干旱胁迫下表观遗传机制对高表达玉米(Zea mays) C₄型PEPC转基因水稻(Oryza sativa)种子萌发的影响, 以转C₄型PEPC水稻(PC)和野生型水稻Kitaake (WT)为试材, 采用10% (m/v)聚乙二醇6000 (PEG6000)模拟干旱条件, 通过单独和联合施用PEG6000、DNA甲基化抑制剂5-氮杂胞苷(5azaC)和可变剪接抑制剂大环内酯类(PB)进行种子发芽实验, 测定种子活力、萌发过程中可溶性糖和可溶性蛋白含量、α-淀粉酶活性以及PEPC、糖信号相关基因和部分剪接因子基因的表达。结果表明, 0.25 µmol·L^-1PB处理对2种供试水稻在干旱条件下种子萌发均表现出显著抑制作用, 使干旱条件下种子萌发过程中可溶性总糖、蔗糖、葡萄糖和果糖含量以及可溶性蛋白含量均有所下降, PB也抑制糖信号-蔗糖非发酵1 (SNF1)相关蛋白激酶(SnRKs)家族和剪接因子丝氨酸/精氨酸富集蛋白家族(SR proteins)相关基因的表达以及α-淀粉酶的活性, 但对PC的抑制作用小于WT。5 µmol·L^-15azaC处理对干旱条件下种子萌发的效果与可变剪接抑制剂相反。5 µmol·L ^-1 5azaC联合PEG6000干旱处理部分减缓了干旱对水稻种子发芽率的抑制作用, 使供试材料发芽率升高, 表明DNA甲基化和可变剪接机制参与了水稻芽期干旱耐性, 其中对PC的作用更大。

Effects of Epigenetic Mechanisms on C4 Phosphoenolpyruvate Carboxylase Transgenic Rice (Oryza sativa) Seed Germination Under Drought Stress

In order to reveal the effect of epigenetic mechanism under drought stress toward seed germination of transgenic rice within high maize C₄-type PEPC gene expressing, C₄-PEPC transgenic rice (PC) and wild type rice Kitaake (WT) were used in this study. By introducing DNA methylation inhibitor (5-azacytidine, 5azaC) and alternative splicing inhibitors (macrolides pladienolide B, PB), the drought simulation treatments with 10% (m/v) polyethylene glycol-6000 (PEG6000) alone or combining with the inhibitors were used for seed germination experiments. Seed vigor, soluble sugar and soluble protein content, α-amylase activity and the expression levels of related genes, PEPC-related genes, sugar signal-related genes, and some splicing factor genes during germination were measured. In the results, when treated with 0.25 µmol·L^-1, PB had showed a significant inhibitory effect on the seed germination of the two tested rice lines under drought conditions. The content of total soluble sugar, sucrose, glucose, fructose and soluble protein during seed germination after PB addition was reduced to a certain extent under drought conditions. PB treatment also inhibited the gene expression of sucrose nonfermenting-1 (SNF1)-related protein kinase SnRKs family and the splicing factor arginine/serine-rich proteins (SR proteins), and the activity of α-amylase as well, but the inhibitory effect on PC is less than those on WT. 5 µmol·L ^-1 5azaC treatment had an opposite effect with alternative splicing inhibitors. The combination treatment with 5azaC and PEG6000 partially alleviated the inhibitory effect of drought stress on rice seed germination, and the germination rate of the tested materials increased. It can be seen that the mechanism of DNA methylation and alternative splicing are involved in drought tolerance at the bud stage of rice lines, with a larger effect on PC.