| 摘要: | 信使RNA的降解在基因表現調控中扮演重要角色，而CCR4相關因子1(CAF1)蛋白質是mRNA去腺苷化的重要酵素。OsCAF1B 是水稻CAF1基因家族的成員，研究顯示其表現在低溫和乾旱逆境下會被誘導表現，但其在水稻生殖發育與乾旱適應中的功能仍然未知。因此，我們利用CRISPR/Cas9基因編輯技術敲除OsCAF1B基因，並成功建立兩株未含有CAS9基因的OsCAF1B基因敲除株(KO lines)，這些突變株的位移突變OsCAF1B基因的蛋白質編碼序列轉譯成沒有功能OsCAF1B蛋白質。根據農藝性狀表現型的分析顯示，OsCAF1B基因剃除株與野生型水稻在芽長、根長、穀粒重量及發芽率方面無顯著差異。然而，OsCAF1B基因剃除株的結實率相較於野生型水稻有顯著下降。進一步分析發現，OsCAF1B基因剃除株的花粉活力和花粉發芽率皆有顯著降低於野生型水稻，顯示OsCAF1B在花粉發育及雄性生殖功能中具有關鍵作用。為了探討 OsCAF1B 在乾旱逆境中的生理功能，我們分析OsCAF1B基因剃除株和OsCAF1B過量表現株在20% PEG 6000 誘導的乾旱逆境下的存活率。結果顯示，OsCAF1B基因剃除株的存活率明顯較低，而OsCAF1B過量表現株則展現出較強的耐旱性。此外，基因表現分析顯示，在乾旱條件下，OsCAF1B基因剃除株中與乾旱反應的相關轉錄因子MYB108、DREB2B和OsHSFA3 的表達量相較於野生型水稻有顯著下降，顯示OsCAF1B參與了調控乾旱逆境適應的分子機制。這些研究結果證實OsCAF1B在水稻的生殖成功與乾旱耐受性中發揮關鍵作用，並且可能成為改良作物耐逆境能力的重要標靶基因。;Fine-tuning gene expression requires controlled decay of the messenger RNA (mRNA) and CCR4-associated factor 1 (CAF1) proteins are essential for mRNA deadenylation during mRNA degradation. In Oryza sativa, OsCAF1B, a member of the CAF1 gene family, is known to be activated by cold and drought stress. Compared to its homologs, OsCAF1B is substantially expressed in rice reproductive tissues, according to data from the Rice Annotation Project Database. Despite these discoveries, nothing is known about OsCAF1B′s function in reproduction and during drought stress. In order to examine OsCAF1B′s role during drought stress and the reproductive phase, we used a CRISPR/Cas9-mediated deletion technique. Agrobacterium-mediated transformation was used to introduce a CRISPR/Cas9 construct that targets OsCAF1B into rice calli generated from immature embryos. Two independents homozygous OsCAF1B knockout (KO) lines, KO-1 and KO+1, were generated and validated by genomic PCR and DNA sequencing. These lines carried -1 and +1 nucleotide insertions at the target location, respectively, most likely leading to non-functional OsCAF1B proteins. Agronomic parameters such as germination rate, shoot length, root length, and grain weight did not significantly change between the wild type (WT) and KO lines, according to phenotypic evaluations. In contrast to WT, which had a seed-setting percentage of 94.8%, KO lines had a much lower rate of 39.8% and 44.5% in KO+1 and KO-1, respectively. Pollen viability analysis showed a reduction from 60.4% in WT to 16% in KO+1 and 14.5% in KO-1. Similarly, pollen germination rates decreased drastically from 45.2% in WT to approximately 1.5% in both KO lines. Previously generated OsCAF1B overexpression (OE) lines were included for comparison under drought stress. Under 20% PEG-induced drought conditions, the survival rates of KO lines at the seedling stage were lower compared to OE lines. During germination, OE lines exhibited better growth than KO lines under similar drought stress conditions. These findings highlight the critical role of OsCAF1B in pollen development, as its disruption leads to substantial pollen sterility and reduced seed-setting rates. Additionally, OsCAF1B contributes to drought tolerance, underscoring its importance in both reproductive success and stress adaptation in rice. |
