【作者】 龙雨；

【导师】 武维华；

【作者基本信息】 中国农业大学 ， 植物学， 2014， 博士

【摘要】 钾是植物所必需的三大营养元素之一,在植物生长发育过程中起着非常重要的作用。植物通过根细胞质膜上的钾离子通道和钾离子转运体从环境中吸收钾离子。已有报道显示,Shake钾离子通道蛋白家族成员可能在植物钾离子吸收、转运过程中发挥重要作用。AKT1是模式植物拟南芥中重要的Shaker钾离子通道蛋白,它主要参与拟南芥根部钾离子吸收过程。然而在作物中,目前对于Shake钾通道蛋白参与钾吸收、转运的功能研究还比较少。水稻是最重要的粮食作物之一,OsAKT1是水稻中的一个Shaker钾通道蛋白,与拟南芥AKT1具有较高的同源性。推测,OsAKT1可能在水稻根部钾离子吸收过程中起重要作用。本论文工作主要通过电生理学的实验方法深入研究分析OsAKT1在水稻根部钾吸收过程中的生理功能及其分子调控机制。OsAKTl主要在水稻根中表皮和维管组织中表达较强。亚细胞定位分析显示,OsAKT1特异地定位于细胞质膜上。将OsAKT1转入拟南芥aktl突变体后,OsAKT1可以恢复aktl突变体根细胞原生质体中缺失的内向钾离子电流。表型检测发现,水稻(ysakt1突变体表现出明显的低钾敏感表型。膜片钳和非损伤离子流速测定结果显示,与Dongjin亲本材料相比,osakt1突变体根细胞的内向钾离子电流显著减小,并且突变体根部钾离子吸收速率也显著降低。在osakt1/OsAKT1恢复突变水稻材料中,植株的低钾敏感表型得以恢复,根细胞的内向钾离子电流也得以恢复。对膜片钳实验结果分析发现,水稻OsAKT1和拟南芥AKT1都具有内向钾通道活性,但两者在通道激活特性上具有显著差异。上述结果说明,OsAKT1具有内向钾离子通道活性,它在水稻根部钾吸收过程中发挥着重要作用。拟南芥中的研究结果显示,AKT1的活性受钙感受器CBL1/9和蛋白激酶CIPK23的调控。推测,OsAKT1的活性可能也受水稻OsCBLs和OsCIPKs蛋白的调控。互作结果显示,OsAKT1可以和多个OsCIPKs蛋白(OsCIPK3、9、19、23)互作,并且这一互作依赖于OsCBL1的存在。本论文进一步利用爪蟾卵母细胞和HEK293细胞作为异源表达系统,深入研究了这些OsCBLs和OsCIPKs蛋白对OsAKT1活性的调控作用。研究发现,在爪蟾卵母细胞中单独表达OsAKT1并未检测到OsAKT1的通道活性,共表达10OsCBLs与OsCIPK3/9/19/23的cRNA组合后,仍不能激活OsAKT1的活性。而在HEK293细胞中,单独表达的OsAKT1就具有内向钾离子通道活性,共表达OsCBL1-OsCIPK19/23后,可以显著增强OsAKT1的活性。一方面,OsCBL1-OsCIPK23复合体对OsAKT1的活性增强作用明显强于OsCBL1-OsCIPK19复合体；另一方面,OsCBL1-OsCIPK23也能在非洲蟾卵母细胞中激活AKT1的通道活性。说明,CBL1-CIPK23对调控AKT1这一分子机制在拟南芥和水稻中是保守的,OsCBL1-OsCIPK23可能在水稻体内调控OsAKT1介导的钾吸收。本论文研究结果表明,Shaker钾离子通道OsAKT1具有内向钾离子通道活性,它在水稻根部钾离子吸收过程中发挥着重要作用。并且,OsAKT1的钾离子吸收活性受上游OsCBLl-OsCIPK23的调控。该研究结果说明,AKT1和OsAKT1具有类似的生理功能和钾吸收调控通路,但它们在通道特性以及分子调控机制上仍存在部分差异。更多还原

【Abstract】 Potassium (K+) is one of the essential nutrient elements for plant growth and development. It is absorbed into the root cell through low and high affinity systems conferred by different K transporters and channels. Previous studied in our and other labs demonstrate that in model plant Arabidopsis, K+TRANSPORTER1(AKT1), belonging to Shaker K family, has both high and low affinity to K+, therefore has dominant contribution to K uptake of the root cells under both K deprivation and sufficient conditions. It is directly regulated by Arabidopsis CBL-INTERACTING PROTEIN KINASE23(CIPK23), activation of which in turn depends on interaction with Arabidopsis CALCINEURIN B-LIKE PROTEIN1(CBL1) and CBL9. This dissertation extends this key discovery found in model plant to rice, one of key staple crops in China in terms of contribution to food production.Shaker K+channels play important roles in K+uptake of plants Especially AKT1is one of the most important Shaker K+channels, and plays important role in K+uptake in the roots of Arabidopsis. Rice is one of the most important food resources. It has evolved machine to adapt low-K+environment. OsAKT1is homologous with AKT1and probobly important for K+uptake in rice root. The focus of this dissertation work is to study the function and molecular regulatory mechanism of OsAKT1in K+uptake process of rice roots by electrophysiological technology.In this dissertation work, the Shaker K+channel OsAKT1was characterized for its function in K+uptake in rice roots. Beta-glucuronidase based promoter activity analysis revealed that OsAK1primarily expresses in epidermal, cortex and xylem of the roots. The OsAKT1protein was localized in the plasma membrane. It displayed voltage-dependent inward-rectifying K-specific current while being exogenously expressed in mammalian cell HEK293but not Xenopus oocytes, as detected with patch clamp technique. The root cells of Arabidopsis AKT1knockout mutant (aktl) has no inward K current under patch clamp whole-cell configuration condition. Expressing OsAKTl in aktl rescued its inward K current. These data demonstrates that OsAKT1functions as an inward K channel like AKT1in the roots.To evaluate OsAKT1significance in K nutrient of rice, a rice mutant harboring a single T-DNA in OsAKT1was identified. The mutant osaktl had significant decreases of K uptake capacity and K-dependent growth as compared to those of wild type. Moreover, the inward K+current cross the plasma membrane of the root cell protoplasts was significant suppressed in the mutant. These data demonstrated that OsAKT1plays a crucial role in K nutrient of rice roots.In light of AKT1activity dependence on its interacting with CIPK/CBL complex, this dissertation work investigated which OsCIPK and OsCBL interact with OsAKTl with yeast two hybrid and BiFC assay. It was intriguing that OsAKTl interacts with OsCIPK3,9,19and23, all of which interact with OsCBL1. Exogenously expressing OsAKT1along with OsCIPKs and OsCBLl found that both OsCIPK23/OsCBLl and OsCIPK19/OsCBLl combination could significantly and comparably enhance OsAKT1-mediated inward K+current in HEK239Cells, which was much weaker for the OsCIPK19’s combination. These data implied that OsAKT1’s channel activity was under regulated by different OsCIPKs and OsCBL complex, which should be further investigated in vivo with genetic tools.In conclusion, this dissertation paves foundation on our understanding of OsAKT1-mediated K nutrient uptake mechanism in rice. Difference of AKT1operating machinery in Arabidopsis and rice was discussed.更多还原