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Ca2+流指示的微藻氮脅迫信號(hào)轉(zhuǎn)導(dǎo)研究
NMT是基因功能的活體檢測(cè)技術(shù)，已被103位諾貝爾獎(jiǎng)得主所在單位，及北大、清華、中科院使用。
期刊：Plant Biotechnology Journal
主題：HvAKT2和HvHAK1通過(guò)增強(qiáng)葉肉H+穩(wěn)態(tài)提升耐旱能力
標(biāo)題：HvAKT2 and HvHAK1 Confer Drought Tolerance in Barley through Enhanced Leaf Mesophyll H+ Homeostasis
影響因子：6.840
檢測(cè)指標(biāo)：K+、H+、Ca2+流速
檢測(cè)樣品：大麥葉肉細(xì)胞
K+、H+、Ca2+流實(shí)驗(yàn)處理方法：
1）20% PEG瞬時(shí)處理大麥葉片
制備葉肉組織，測(cè)試液中平衡2h后，用20％ PEG瞬時(shí)處理
2）20%PEG預(yù)處理
制備葉肉組織，用20% PEG處理0、1、12、24h
K+、H+、Ca2+流實(shí)驗(yàn)測(cè)試液成份：
0.1 mMCaCl2，0.5 mM KCl，pH 5.8
作者：浙江大學(xué)作物科學(xué)研究所張國(guó)平、鄔飛波、陳仲華、馮雪
英文摘要
Plant K+ uptake typically consists low‐affinity mechanisms mediated by Shaker K+ channels (AKT/KAT/KC) and high‐affinity mechanisms regulated by HAK/KUP/KT transporters, which are extensively studied. However, the evolutionary and genetic roles of both K+ uptake mechanisms for drought tolerance are not fully explored in crops adapted to dryland agriculture.
Here, we employed evolutionary bioinformatics, biotechnological and electrophysiological approaches to determine the role of two important K+ transporters HvAKT2 and HvHAK1 in drought tolerance in barley. HvAKT2 and HvHAK1 were cloned and functionally characterized using barley stripe mosaic virus‐induced gene silencing (BSMV‐VIGS) in drought‐tolerant wild barley XZ5 and agrobacterium‐mediated gene transfer in the barley c*r Golden Promise. The hallmarks of the K+ selective filters of AKT2 and HAK1 are both found in homologues from strepotophyte algae, and they are evolutionarily conserved in strepotophyte algae and land plants. HvAKT2 and HvHAK1 are both localized to the plasma membrane and have high selectivity to K+ and Rb+ over other tested cations.
Overexpression of HvAKT2 and HvHAK1 enhanced K+ uptake and H+ homeostasis leading to drought tolerance in these transgenic lines. Moreover, HvAKT2‐ and HvHAK1‐overexpressing lines showed distinct response of K+, H+, and Ca2+ fluxes across plasma membrane and production of nitric oxide and hydrogen peroxide in leaves as compared to the wild type and silenced lines. High‐ and low‐affinity K+ uptake mechanisms and their coordination with H+ homeostasis play essential roles in drought adaptation of wild barley.
These findings can potentially facilitate future breeding programs for resilient cereal crops in a changing global climate.
中文摘要（谷歌機(jī)翻）
植物對(duì)鉀的吸收通常包括由搖床K+通道（AKT / KAT / KC）介導(dǎo)的低親和力機(jī)制和受HAK / KUP / KT轉(zhuǎn)運(yùn)蛋白調(diào)控的高親和力機(jī)制，對(duì)此進(jìn)行了廣泛的研究。但是，在適應(yīng)旱地農(nóng)業(yè)的農(nóng)作物中，兩種K +吸收機(jī)制對(duì)干旱的耐受性的進(jìn)化和遺傳作用尚未得到充分研究。
在這里，我們采用了進(jìn)化生物信息學(xué)，生物技術(shù)和電生理方法來(lái)確定兩個(gè)重要的K+轉(zhuǎn)運(yùn)蛋白HvAKT2和HvHAK1在大麥的耐旱性中的作用。使用耐旱的野生大麥XZ5中的大麥條紋花葉病毒誘導(dǎo)的基因沉默（BSMV-VIGS）克隆了HvAKT2和HvHAK1，并在大麥品種Golden Promise中進(jìn)行了農(nóng)桿菌介導(dǎo)的基因轉(zhuǎn)移。AKT2和HAK1的K+選擇性過(guò)濾器的標(biāo)志都存在于鏈藻科藻類的同系物中，并且在鏈藻科藻類和陸地植物中是進(jìn)化保守的。HvAKT2和HvHAK1都位于質(zhì)膜上，并且對(duì)K+和Rb+的選擇性比其他測(cè)試陽(yáng)離子高。
在這些轉(zhuǎn)基因品系中，HvAKT2和HvHAK1的過(guò)表達(dá)增強(qiáng)了K+吸收和H+穩(wěn)態(tài)，從而導(dǎo)致了干旱耐受性。此外，與野生型和沉默株系相比，HvAKT2和HvHAK1過(guò)表達(dá)株系在整個(gè)質(zhì)膜上表現(xiàn)出不同的K+，H+和Ca2+通量響應(yīng)以及葉片中一氧化氮和過(guò)氧化氫的產(chǎn)生。高親和力和低親和力的K+吸收機(jī)制及其與H+穩(wěn)態(tài)的協(xié)調(diào)在野生大麥的干旱適應(yīng)中起著重要作用。
這些發(fā)現(xiàn)有可能在未來(lái)氣候變化的情況下促進(jìn)未來(lái)谷物抗逆性作物的育種計(jì)劃。
圖1植物和藻類中AKT2和HAK1進(jìn)化分析
圖2 大麥葉片HvHA1表達(dá)量、H+-ATP酶活性和離子流變化