【作者】 王璐；

【导师】 邱全胜；

【作者基本信息】 兰州大学 ， 生物学·植物学， 2022， 硕士

【摘要】 拟南芥KEA(Potassium efflux antiporter)基因家族编码K~+/H~+反向转运蛋白。KEAs蛋白是一种次级离子转运蛋白,其生化功能是将K+跨膜转运,以交换H+。KEA1和KEA2定位于叶绿体被膜的内膜,在离子和pH稳态平衡、渗透调节、光合作用、质体发育以及类囊体膜形成等方面具有重要作用。然而,关于KEA1和KEA2在干旱胁迫中的作用了解甚少。本研究采用遗传学、生理学和分子生物学等方法,在分析kea1 kea2突变体在干旱胁迫下生长表型的基础上,分析了 kea1 kea2突变体对ABA的响应以及气孔发育和气孔运动的情况。本研究探究了 KEA1和KEA2在干旱胁迫中的生物学功能,揭示植物耐旱胁迫的分子机制,并为离子转运蛋白在干旱胁迫中的研究奠定理论基础。本文取得的研究成果如下:(1)甘露醇胁迫分析。实验发现,随着甘露醇浓度增加,Col-0根生长抑制程度增加。kea1 kea2双突变体的根长在200 mM甘露醇处理下增加33%,在300 mM甘露醇处理下根长生长没有发生改变,在400 mM甘露醇处理下根长生长抑制32%。说明kea1 kea2突变体对甘露醇渗透胁迫具有耐受性;KEA1和KEA2参与植物对渗透胁迫的响应。(2)NaCl胁迫分析。在75、100、150 mM NaCl处理下,Col-0根长分别下降10%、21%、58%,说明随着NaCl浓度增加,Col-0根长生长抑制程度逐渐增加。然而,在75 mMNaCl处理下,kea1 kea2双突变体根长增加16%;在100 mM NaCl处理下,kea1 kea2双突变体根长没有发生变化;在150 mM NaCl处理下,kea1 kea2双突变体根长下降41%。说明kea1 kea2对NaCl胁迫有耐受性。(3)土壤干旱胁迫生长表型分析。在干旱处理前,kea1 kea2双突变体幼叶呈黄绿色,莲座叶小,生长发育迟缓。在干旱处理10天后,Col-0和kea1、kea2单突变体发生萎蔫,生长发育严重受损。然而,kea1 kea2突变体生长无明显变化,叶片绿色加深,含有较高叶绿素含量。同时发现,干旱处理下keal kea2双突变体具有较强的保水能力,较低MDA含量。(4)ABA处理分析。在0.5 μM ABA处理下,Col-0和kea1、kea2根长分别增加10%、10%、13%,而kea1 kea2根长增加47%,表明ABA促进kea1 kea2根系生长。在1.0 μMABA处理下,Col-0、kea1、kea2根长没有发生变化,kea1 kea2根长增加26%。在1.5 μM ABA处理下,Col-0和kea1、kea2根长分别下降20%、19%、25%;kea1 kea2根长仅下降15%。说明ABA对kea1 kea2根生长有促进作用。(5)ABA合成抑制剂FLU分析。在0.3 μM FLU处理下,Col-0的根长减少52%,kea1 kea2减少34%;在200 mM甘露醇处理下,Col-0的根长减少29%,kea1 kea2增加8%,说明kea1 kea2对甘露醇的渗透胁迫具有抗逆性。在甘露醇胁迫基础上叠加使用FLU发现,Col-0的根长减少27%,kea1 kea2减少30%,表明kea1 kea2在甘露醇胁迫下对FLU更敏感。暗示kea1 kea2对渗透胁迫的抗逆性可能是通过ABA起作用。(6)气孔发育和开度分析。与Col-0相比,kea1 kea2气孔密度增加17%,气孔面积减小18%,说明KEA1和KEA2具有调控气孔细胞发育的功能。KCl-光诱导气孔开放实验发现,与Col-0相比,kea1 kea2气孔开度减小6.3%,表明KEA1和KEA2具有调控气孔开度的功能。(7)干旱胁迫对气孔发育和开度影响分析。Col-0,kea1和kea2气孔密度分别增加23%、26%、27%;kea1 kea2增加39%。KCl-光诱导气孔开放实验发现,在正常条件下,相比Col-0,kea1 kea2气孔开度下降5%。在干旱胁迫下,Col-0气孔开度下降9%;kea1 kea2气孔开度下降11%。表明在干旱胁迫下,kea1 kea2的气孔开度更小;KEA1和KEA2参与调控干旱胁迫下的气孔运动。(8)胁迫应答基因表达分析。半定量PCR分析发现,在甘露醇处理下,kea1 kea2的RAB18、SnRK2.2、SnRK2.3、SnRK2.6及CAT2基因表达增加,表明kea1 kea2可能通过增强ABA信号应答和调节细胞内H2O2稳态平衡而耐受渗透胁迫。(9)75 mMNaCl处理分析。75 mMNaCl处理下,kea1 kea2株高增加15%,叶绿素含量增加33%,MDA含量无变化,POD酶活性升高,表明75 mM NaCl处理可以促进kea1 kea2生长,提高叶绿素含量,促进清除过氧化氢,减缓氧化损伤。(10)75 mMNaCl处理气孔观察。在正常条件下,与Col-0相比,kea1 kea2气孔密度增加91%。75 mM NaCl处理下,Col-0气孔密度降低26%,kea1 kea2下降46%,恢复至野生型水平。表明75 mMNaCl处理可以恢复kea1 kea2对气孔发育的影响。在正常条件下,相比Col-0,kea1 kea2气孔开度下降6%;在75 mMNaCl处理下,相比Col-0,kea1 kea2双突变体气孔开度下降13%。在75 mM NaCl处理下,Col-0气孔开度下降6%;kea1 kea2气孔开度下降13%。表明,kea1 kea2对75 mM NaCl处理更敏感。总之,我们的研究表明,kea1 kea2在干旱胁迫下具有较强耐逆性;KEA1和KEA2可能通过调节ABA信号转导途径以及气孔发育和运动调控植物对干旱胁迫的应答。更多还原

【Abstract】 Arabidopsis KEA(Potassium efflux antiporters)gene family encodes K+/H+antiporters whose biochemical function is to transfer the K+across a membrane in exchange for H+.KEA1 and KEA2,which are targeted to the inner envelope membrane of chloroplasts,are required for a variety of biological processes,such as ion and pH homeostasis,the regulation of osmotic,the formation,and development of plastid,photosynthesis,and the formation of the thylakoid membrane.However,it remains unclear about the function of KEA1 and KEA2 in drought stress.Using genetics,physiological and molecular biology methods,This study analyzed the growth phenotypes of keal kea2 double mutants under drought stress.We further analyzed the response of keal kea2 mutants to ABA signals and the roles of KEA1 and KEA2 in the regulation of stomatal development and movement.This study aims at understanding the biological functions of KEA1 and KEA2,which help us understand the molecular mechanisms of plant tolerance to drought stress.The major results of this paper are as follows:(1)Mannitol stress analysis.With the increasing concentration of mannitol,the inhibition growth of root length was increased in Col-0.At 200 mM mannitol,the root length of keal double mutants was increased by 33%.At 300 mM mannitol,no significant changes in root length were observed in keal kea2.At 400 mM mannitol,the root length of kea1 kea2 was decreased by 32%.These results indicate that keal kea2 double mutants are tolerant to osmotic stress.KEA1 and KEA2 play an important role in response to osmotic stress.(2)NaCl stress analysis.Under 75,100,and 150 mM NaCl treatment,the root length of Col-0 was decreased by 10%,21%,and 58%,respectively,indicating that with the increase in NaCl concentrations,the inhibition growth of root length was gradually increased in Col-0.However,at 75 mM NaCl,the root length of keal kea2 double mutants was increased by 16%;at 100 mM NaCl,no significant changes in root length were observed in keal kea2:at 150 mM NaCl,the root length of keal kea2 double mutants was reduced by 32%.These results indicate that the keal kea2 double mutants is tolerant to NaCl stress.(3)Analysis of seedling growth in soil under drought stress.Before drought treatment,keal kea2 double mutants showed pale green leaves and severely stunted growth.After 10 days of drought treatment,Col-0,keal,and kea2 single mutant were wilted,and their growth and development were severely impaired;no significant changes in growth phenotype were observed in keal kea2,and the leaves became greener and had higher chlorophyll content.Meanwhile,keal kea2 double mutants had stronger water retention ability and lower MDA content under drought stress.(4)Analysis of ABA treatment.At 0.5 μM ABA,the root length of Col-0,keal,and kea2 was increased by 10%,10%,and 13%,respectively,while the root length of keal kea2 double mutants was increased by 47%,suggesting that ABA promotes root growth of keal kea2.At 1.0μM ABA,the root length were no significant changes in Col-0,kea1,and kea2 single mutant,but increased by 26%in keal kea2.At 1.5 μM ABA,the root length of Col-0,kea1,and kea2 was decreased by 20%,19%,and 25%,respectively.The root length of keal kea2 was decreased by 15%.These results suggest that ABA has a stronger effect on root growth of keal kea2 double mutants.This may indicate that keal kea2 double mutants have a low level of ABA contents.(5)Analysis of ABA synthesis inhibitor FLU.At 0.3 μM FLU,the root length of Col-0 and keal kea2 double mutants were decreased by 52%and 34%,respectively.At 200 mM mannitol,the root length was decreased by 29%in Col-0 and increased by 8%in keal kea2,indicating that keal kea2 double mutants are resistant to osmotic stress.We did FLU treatment assay at mannitol stress.When treated with mannitol and FLU,compared with that treated with mannitol,The root length of Col-0 and keal kea2 double mutants was decreased by 27%and 30%,respectively,indicating that keal kea2 double mutants are more sensitive to FLU under mannitol stress.These results suggest that the resistance of keal kea2 to osmotic stress may be mediated by ABA.(6)Analysis of stomatal development and stomatal aperture.Compared with Col-0,the stomatal density of keal kea2 was increased by 17%;the stomatal area of keal kea2 was decreased by 18%.These results suggest that KEA1 and KEA2 may function in regulating stomatal development.Under the KCl-light-induced stomatal opening,compared with Col-0,the stomatal aperture of keal kea2 was decreased by 6.3%,suggesting that KEA1 and KEA2 may regulate stomatal aperture.(7)Effects of drought stress on stomatal development and stomatal aperture.The stomatal density of Col-0,keal,and kea2 were increased by 23%,26%,and 29%,respectively;the stomatal density of keal kea2 was increased by 39%.These results indicate that KEA1 and KEA2 regulate stomatal development under drought stress.Under normal conditions,stomatal aperture at KCl-light-induced stomatal opening was decreased by 5%in keal kea2 compared to Col-0.Under drought stress,compared with the control,the stomatal aperture was decreased by 9%in Col-0 and 11%in keal kea2.These results indicate that keal kea2 has a smaller stomatal aperture,and KEA1 and KEA2 regulate stomatal aperture under drought stress.(8)Analysis of stress response genes.Semi-quantitative PCR analysis showed that the expression of RAB18,SnRK2.2,SnRK2.3,SnRK2.6,and CAT2 genes in kea1kea2 was significantly increased under mannitol treatment,suggesting that the keal kea2 is tolerated to osmotic stress by enhancing response to ABA signaling and by regulating cellular H2O2 homeostasis.(9)Analysis of kea1 kea2 treated with 75 mM NaCl.At 75 mM NaCl,the plant height was increased by 15%,chlorophyll content was increased by 33%,and MDA content did not change in keal kea2 double mutants,indicating that 75 mM NaCl treatment promotes the growth,increases chlorophyll content,promotes the removal of hydrogen peroxide and reduces oxidative damage in keal kea2 double mutants.(10)Stomatal observation under 75 mM NaCl treatment.Under normal conditions,the stomatal density of keal kea2 was increased by 91%compared with Col-0.At 75 mM NaCl,stomatal density was decreased by 26%in Col-0 and decreased by 46%in keal kea2.These results indicate that 75 mM NaCl treatment could restore stomatal development of keal kea2 double mutants.Under normal conditions,the stomatal aperture of keal kea2 was 6%less than that of Col-0.Under 75 mM NaCl treatment,the stomatal aperture was decreased by 13%in keal kea2 and 6%in Col-0.These results indicate that keal kea2 double mutants are more sensitive to 75 mM NaCl treatment.In conclusion,our study shows that the keal kea2 double mutants is resistant to drought and salt stress.KEA1 and KEA2 may regulate plants response to drought and salt stress by regulating ABA signal transduction pathways as well as stomatal development and movement.更多还原