College of Life Sciences, Inner Mongolia Agricultural University, Inner Mongolia Key Laboratory of Plant Stress Physiology and Molecular Biology, Erdos Road, Hohhot, 010018, Inner Mongolia, China.
Wheat Research Institute, Henan Academy of Agricultural Sciences, Henan Key Laboratory of Wheat Biology, National Engineering Laboratory for Wheat, Key Laboratory of Wheat Biology and Genetic Breeding in Central Huang-Huai Region, Ministry of Agriculture, Zhengzhou, 450002, China.
BMC Plant Biol. 2019 Aug 9;19(1):349. doi: 10.1186/s12870-019-1950-4.
AFP is a negative regulator of ABA signaling that promotes ABI5 protein degradation and weakens regulation of ABA signaling by targeting upstream genes of ABI5, and TaABI5 gene was seed-specific, and accumulated during wheat grain maturation and dormancy acquisition, which played an important role in seed dormancy; TaAFP has a conserved domain with AFP, so TaAFP may also play an important role in seed dormancy in wheat.
Two allelic variants of TaAFP were identified on chromosome 2BS in common wheat, and designated as TaAFP-B1a and TaAFP-B1b. Sequence analysis showed a 4-bp deletion in the 5'UTR region of TaAFP-B1b compared with TaAFP-B1a. Based on the 4-bp deletion, a co-dominant functional marker of TaAFP-B was developed and designated as AFPB. The genotype generating a 203-bp fragment (TaAFP-B1b) was more resistant to pre-harvest sprouting than the genotype producing a 207-bp fragment (TaAFP-B1a) in a test of 91 white-grained Chinese wheat cultivars and advanced lines. The average germination index(GI) values of TaAFP-B1a and that of TaAFP-B1b were 45.18 and 30.72%, respectively, indicating a significant difference (P < 0.001). Moreover, the 4-bp deletion located in the 5'UTR not only affected the transcription level of TaAFP-B but also affected the mRNA decay, reduced the translation level of GUS and tdTomatoER and GUS activity in wheat leaves of transient expression. The transcript expression and the mRNA half-life value of TaAFP-B1a in developing seeds and mature seeds were much higher than those of TaAFP-B1b.
We identified a 4-bp InDel in the 5'UTR of TaAFP-B, which affected the mRNA transcription level, mRNA decay, translation levels of GUS and tdTomatoER, GUS activity, and was significantly associated with seed dormancy in common wheat. A functional marker was developed and validated based on this InDel.
AFP 是 ABA 信号的负调控因子，通过靶向 ABI5 的上游基因促进 ABI5 蛋白降解，削弱 ABA 信号的调控，TaABI5 基因是种子特异性的，在小麦籽粒成熟和休眠获得过程中积累，在种子休眠中发挥重要作用；TaAFP 与 AFP 具有保守结构域，因此 TaAFP 可能在小麦种子休眠中也发挥重要作用。
在普通小麦 2BS 染色体上鉴定到 TaAFP 的两个等位变异体，分别命名为 TaAFP-B1a 和 TaAFP-B1b。序列分析显示 TaAFP-B1b 与 TaAFP-B1a 相比，在 5'UTR 区有 4 个碱基缺失。基于这 4 个碱基缺失，开发了 TaAFP-B 的共显性功能标记，命名为 AFPB。在 91 个白粒中国小麦品种和品系的预收获发芽试验中，产生 203bp 片段（TaAFP-B1b）的基因型比产生 207bp 片段（TaAFP-B1a）的基因型具有更高的抗预收获发芽能力。TaAFP-B1a 和 TaAFP-B1b 的平均发芽指数（GI）值分别为 45.18%和 30.72%，差异显著（P<0.001）。此外，位于 5'UTR 中的 4 个碱基缺失不仅影响 TaAFP-B 的转录水平，还影响 mRNA 衰减，降低小麦叶片瞬时表达中 GUS 和 tdTomatoER 的翻译水平和 GUS 活性。发育种子和成熟种子中 TaAFP-B1a 的转录表达和 mRNA 半衰期值明显高于 TaAFP-B1b。
我们在 TaAFP-B 的 5'UTR 中鉴定到一个 4 个碱基缺失，该缺失影响了 mRNA 转录水平、mRNA 衰减、GUS 和 tdTomatoER 的翻译水平、GUS 活性，并与普通小麦种子休眠显著相关。在此插入缺失的基础上，开发并验证了一个功能标记。
