華中農(nóng)業(yè)大學(xué)在揭示miR159a-DUO1模塊調(diào)控柑橘花粉育性的機(jī)制上取得新進(jìn)展

　　近日，JIPB在線發(fā)表了華中農(nóng)業(yè)大學(xué)果蔬園藝作物種質(zhì)創(chuàng)新與利用全國(guó)重點(diǎn)實(shí)驗(yàn)室伊華林/吳巨勛課題組題為“The miR159a-DUO1 module regulates pollen development by modulating auxin biosynthesis and starch metabolism in citrus”的研究論文，揭示了miR159a-DUO1模塊整合生長(zhǎng)素合成和淀粉代謝途徑調(diào)控柑橘花粉發(fā)育的分子機(jī)制。

 

　　果實(shí)無核化是柑橘育種的重要目標(biāo)，是鮮食柑橘品種的核心需求之一，但是目前對(duì)于該性狀形成的分子機(jī)制的認(rèn)識(shí)仍較淺顯。柑橘童期長(zhǎng)且雜合度高，基于現(xiàn)有的育種手段選育無核柑橘新品種周期長(zhǎng)，利用分子設(shè)計(jì)育種進(jìn)行定向改良成為提高無核育種效率的可行且高效的育種手段。因此挖掘柑橘無核調(diào)控關(guān)鍵基因資源，深入研究柑橘無核性狀形成的分子調(diào)控機(jī)制，對(duì)加速柑橘分子育種具有重要意義。

 

　　在前期研究中，該課題組基于臍橙小RNA組和降解組測(cè)序鑒定出一個(gè)差異表達(dá)的csi-miR159a及其4個(gè)靶基因CsDUO1、 CsGAMYB、CsNOZZLE （SPOROCYTELESS）和CsPGIP1 （Wu et al., 2016）。該研究通過煙草瞬時(shí)表達(dá)實(shí)驗(yàn)和5'RACE驗(yàn)證DUO1、GAMYB、NZZ是柑橘中csi-miR159a真正的靶基因。在山金柑中超表達(dá)csi-miR159a導(dǎo)致小的無核果實(shí)產(chǎn)生和花粉發(fā)育缺陷。山金柑DUO1敲除株系花粉敗育和無核表型與csi-miR159a超表達(dá)株系一致。通過異花授粉實(shí)驗(yàn)，證明DUO1是csi-miR159a調(diào)控柑橘雄性不育的關(guān)鍵靶基因。透射電鏡和花粉DAPI染色進(jìn)一步表明csi-miR159a超表達(dá)株系和DUO1敲除系花粉第一次有絲分裂受阻和花粉中淀粉代謝異常。

 

　　為了解析其中的分子機(jī)制，通過轉(zhuǎn)錄組測(cè)序和DAP-Seq測(cè)序關(guān)聯(lián)分析，鑒定到DUO1下游關(guān)鍵靶基因YUC2/YUC6和SS4/STP8。隨后，通過酵母單雜交、LUC、EMSA等實(shí)驗(yàn)，驗(yàn)證DUO1可與YUC2/YUC6和SS4/STP8互作。經(jīng)研究發(fā)現(xiàn)，csi-miR159a通過靶向DUO1正調(diào)控生長(zhǎng)素合成基因YUC2/YUC6的表達(dá)，進(jìn)而參與生長(zhǎng)素合成及信號(hào)轉(zhuǎn)導(dǎo)的調(diào)控，從而參與調(diào)控柑橘花粉第一次有絲分裂；而csi-miR159a通過靶向DUO1直接調(diào)控淀粉合成基因SS4和糖轉(zhuǎn)運(yùn)基因STP8的表達(dá)，進(jìn)而調(diào)控柑橘花粉中淀粉代謝。該研究揭示了miR159a-DUO1模塊通過調(diào)節(jié)生長(zhǎng)素的生物合成和淀粉代謝來調(diào)控柑橘花粉發(fā)育的分子機(jī)制，為柑橘無核分子育種提供了理論依據(jù)及優(yōu)良基因資源。

 

　　華中農(nóng)業(yè)大學(xué)果樹系已畢業(yè)博士研究生徐彥輝為論文第一作者，華中農(nóng)業(yè)大學(xué)果樹系吳巨勛副教授為該論文通訊作者。華中農(nóng)業(yè)大學(xué)果樹系伊華林教授在實(shí)驗(yàn)設(shè)計(jì)、論文撰寫方面提供了指導(dǎo)和寶貴的意見，中南林業(yè)科技大學(xué)袁德義教授在論文撰寫方面提供了指導(dǎo)。博士研究生張力、尹民強(qiáng)和蔡珍梅以及碩士研究生田文秀參與了研究。本研究得到國(guó)家自然科學(xué)基金項(xiàng)目（32072541和31601729）、國(guó)家現(xiàn)代柑橘產(chǎn)業(yè)技術(shù)體系（CARS-26）等項(xiàng)目的資助。

 

　　【英文摘要】

 

　　Achieving seedlessness in citrus varieties is one of the important objectives of citrus breeding. Male sterility associated with abnormal pollen development is an important factor in seedlessness. However, our understanding of the regulatory mechanism underlying the seedlessness phenotype in citrus is still limited. Here, we determined that the miR159a-DUO1 module played an important role in regulating pollen development in citrus, which further indirectly modulated seed development and fruit size. Both the overexpression of csi-miR159a and the knocking out of DUO1 in Hong Kong kumquat （Fortunella hindsii） resulted in small and seedless fruit phenotypes. Moreover, pollen was severely aborted in both transgenic lines, with arrested pollen mitotic I and abnormal pollen starch metabolism. Through additional cross-pollination experiments, DUO1 was proven to be the key target gene for miR159a to regulate male sterility in citrus. based on DNA affinity purification sequencing （DAP-seq）， RNA-seq, and verified interaction assays, YUC2/YUC6, SS4 and STP8 were identified as downstream target genes of DUO1, those were all positively regulated by DUO1. In transgenic F. hindsii lines, the miR159a-DUO1 module down-regulated the expression of YUC2/YUC6, which decreased indoleacetic acid （IAA） levels and modulated auxin signaling to repress pollen mitotic I. The miR159a-DUO1 module reduced the expression of the starch synthesis gene SS4 and sugar transport gene STP8 to disrupt starch metabolism in pollen. Overall, this work reveals a new mechanism by which the miR159a-DUO1 module regulates pollen development and elucidates the molecular regulatory network underlying male sterility in citrus.