NMT是基因功能的活體檢測技術，已被103位諾貝爾獎得主所在單位，及北大、清華、中科院使用。

期刊：Plant Biotechnology Journal
主題：HvAKT2和HvHAK1通過增強葉肉H+穩態提升耐旱能力

標題：HvAKT2 and HvHAK1 Confer Drought Tolerance in Barley through Enhanced Leaf Mesophyll H+ Homeostasis

影響因子：6.840
檢測指標：K+、H+、Ca2+流速

檢測樣品：大麥葉肉細胞

K+、H+、Ca2+流實驗處理方法：

1）20% PEG瞬時處理大麥葉片
制備葉肉組織，測試液中平衡2h后，用20％ PEG瞬時處理
2）20%PEG預處理
制備葉肉組織，用20% PEG處理0、1、12、24h
K+、H+、Ca2+流實驗測試液成份：
0.1 mMCaCl2，0.5 mM KCl，pH 5.8

作者：浙江大學作物科學研究所張國平、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（谷歌機翻）

植物對鉀的吸收通常包括由搖床K+通道（AKT / KAT / KC）介導的低親和力機制和受HAK / KUP / KT轉運蛋白調控的高親和力機制，對此進行了廣泛的研究。但是，在適應旱地農業的農作物中，兩種K +吸收機制對干旱的耐受性的進化和遺傳作用尚未得到充分研究。

在這里，我們采用了進化生物信息學，生物技術和電生理方法來確定兩個重要的K+轉運蛋白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的基因沉默（BSMV-VIGS）克隆了HvAKT2和HvHAK1，并在大麥品種Golden Promise中進行了農桿菌介導的基因轉移。AKT2和HAK1的K+選擇性過濾器的標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化保守的。HvAKT2和HvHAK1都位于質膜上，并且對K+和Rb+的選擇性比其他測試陽離子高。

在這些轉基因品系中，HvAKT2和HvHAK1的過表達增強了K+吸收和H+穩態，從而導致了干旱耐受性。此外，與野生型和沉默株系相比，HvAKT2和HvHAK1過表達株系在整個質膜上表現出不同的K+，H+和Ca2+通量響應以及葉片中一氧化氮和*的產生。高親和力和低親和力的K+吸收機制及其與H+穩態的協調在野生大麥的干旱適應中起著重要作用。

這些發現有可能在未來氣候變化的情況下促進未來谷物抗逆性作物的育種計劃。

圖1植物和藻類中AKT2和HAK1進化分析

圖2 大麥葉片HvHA1表達量、H+-ATP酶活性和離子流變化