背景:在有性生殖中,在胚珠发育过程中,核细胞分化成大孢子母细胞(MMC),通过减数分裂和有丝分裂,导致胚囊减少。在无囊无性生殖腕带中,在MMC旁边,其他核细胞分化成直接进入有丝分裂形成未减少的胚囊的先兆。IPT(异戊烯基转移酶)家族包含细胞分裂素(CK)途径中的关键基因,其在胚珠发育期间在拟南芥中表达。BbrizIPT9,B.brizantha(syn。Urochloabrizantha)IPT9基因,与其他禾本科植物的基因高度相似,还显示了与拟南芥IPT9,AtIPT9的相似性。在这项工作中,我们旨在研究BbrizIPT9与有性和无融合生殖植物胚珠发育的关系。
结果:RT-qPCR显示,性卵巢中的BbrizIPT9表达高于无融合生殖B.brizantha。原位杂交结果显示两种植物的MMC中BbrizIPT9的强信号,在大孢子发生的开始。通过分析AtIPT9敲低突变体,我们证实了增大的核细胞,在MMC旁边,百分比明显高于野生型,提示AtIPT9基因的敲除触发了额外的MMC样细胞的分化。
结论:我们的结果表明,在胚珠发育过程中,AtIPT9可能参与了单个MMC的适当分化。BbrizIPT9的表达,定位于雄性和雌性孢子细胞,和低于性交,IPT9基因敲除对拟南芥的影响,提示IPT9参与早期胚珠发育。
BACKGROUND: In Brachiaria sexual reproduction, during ovule development, a nucellar cell differentiates into a megaspore mother cell (MMC) that, through meiosis and mitosis, gives rise to a reduced embryo sac. In aposporic apomictic Brachiaria, next to the MMC, other nucellar cells differentiate into aposporic initials that enter mitosis directly forming an unreduced embryo sac. The IPT (isopentenyltransferase) family comprises key genes in the cytokinin (CK) pathway which are expressed in Arabidopsis during ovule development. BbrizIPT9, a B. brizantha (syn. Urochloa brizantha) IPT9 gene, highly similar to genes of other Poaceae plants, also shows similarity with Arabidopsis IPT9, AtIPT9. In this work, we aimed to investigate association of BbrizIPT9 with ovule development in sexual and apomictic plants.
RESULTS: RT-qPCR showed higher BbrizIPT9 expression in the ovaries of sexual than in the apomictic B. brizantha. Results of in-situ hybridization showed strong signal of BbrizIPT9 in the MMC of both plants, at the onset of megasporogenesis. By analyzing AtIPT9 knockdown mutants, we verified enlarged nucellar cell, next to the MMC, in a percentage significantly higher than in the wild type, suggesting that knockout of AtIPT9 gene triggered the differentiation of extra MMC-like cells.
CONCLUSIONS: Our results indicate that AtIPT9 might be involved in the proper differentiation of a single MMC during ovule development. The expression of a BbrizIPT9, localized in male and female sporocytes, and lower in apomicts than in sexuals, and effect of IPT9 knockout in Arabidopsis, suggest involvement of IPT9 in early ovule development.