拟南芥CARK11参与ABA介导的生理功能研究

为了探究CARK11在ABA介导的生理进程中的具体作用,本研究以拟南芥哥伦比亚生态型(WT)、CARK11功能缺失突变体cark11和回补激酶丧失CARK11^N203A(com-CARK11m)以及过表达转基因株系(OE-CARK11)为研究对象,探究CARK11在种子萌发、幼苗形态构建、主根生长与干旱响应中发挥的作用.结果显示：相比WT,ABA促进了cark11和com-CARK11m的种子萌发、子叶变绿和主根伸长,而OE-CARK11被抑制. qRT-PCR检测RAB18和RD29a的表达量,发现过表达CARK11促进RAB18和RD29a表达;ABA处理后,这种促进作用更强.干旱实验发现,OE-CARK11耐旱性增加,而cark11和com-CARK11m耐旱性弱于WT.这些结果表明：CARK11作为ABA信号通路的正调控因子抑制拟南芥的种子萌发、子叶变绿和主根生长;同时在干旱胁迫中具有积极作用;另外CARK11在ABA信号途径中的功能依赖激酶活性.     

抑制bri1表型的ABI突变体的筛选

油菜素甾醇(BR)是一类重要的调控植物生长发育的激素.脱落酸(ABA)是一类重要的帮助植物适应逆境的激素.之前的研究表明BR和ABA信号对萌发率的调控存在拮抗作用,BR合成或信号缺失的突变体在萌发率上对ABA更加敏感.为了进一步研究BR与ABA信号通路互作的分子机制,采用化学诱变剂甲基磺酸乙酯(EMS)诱变bri1-301(BR受体BRI1的一个弱突变体),0.75μmol/L ABA的培养基上筛选对ABA不敏感的突变体.经过多次连续世代筛选,获得了大约40个稳定的株系,发现其中8个株系与abi1、abi5 ABA不敏感突变体的萌发率相似.通过利用PCR扩增ABI1、ABI2、ABI3、ABI4和ABI5基因并测序,鉴定到bri1-301背景下的4个ABA不敏感突变体abi2-8、hab2-1、abi3-21和abi4-13,表明ABA与BR信号的互作可以不依赖BR受体调控种子的萌发,为ABA与BR信号互作提供了遗传的证据.     

拟南芥膜相关转录因子bZIP60活化后与转录因子MYB7互作共同调控种子萌发

膜相关转录因子在调控植物生长发育、逆境响应中发挥重要作用.拟南芥膜相关转录因子bZIP60在响应生物和非生物胁迫中发挥功能.本研究发现bZIP60的非常规剪接受ABA诱导,bZIP60的突变体zip60在ABA处理后绿色子叶率显著降低,说明bZIP60负调控ABA诱导的种子休眠,促进种子萌发.为了进一步研究bZIP60在其中的作用,以bZIP60Δ为诱饵蛋白对cDNA文库进行筛选,最后筛选到24个可能与bZIP60Δ相互作用的蛋白.体外pull-down实验证明bZIP60Δ蛋白与其中一个MYB7蛋白有直接相互作用,荧光素酶互补实验和双分子荧光互补实验(BiFC)证明bZIP60Δ蛋白与MYB7蛋白在烟草细胞中具有相互作用.MYB7之前报道在种子萌发过程中通过抑制ABI5的表达来解除ABA对种子萌发的抑制.这些实验表明bZIP60活化后可能通过与MYB7相互作用协同拮抗ABA信号,促进种子萌发.     

拟南芥锌指结构域ABRv1基因的功能研究

为了分析拟南芥脱落酸响应蛋白RING-V1基因(ABRv1)的功能,构建了高表达载体pBI121-ABRv1,经农杆菌转化,花序浸染后得到T0代转基因种子,并经卡那霉素板筛选得到转基因苗,再经过两代的筛选获取了纯合的高表达株系ABRv1.通过DNA及RNA水平鉴定了ABRv1基因的T-DNA插入突变体abrv1.对突变体、高表达转基因株系及野生型进行ABA诱导处理,突变体的萌发率不足10%,野生型萌发率为40%,而过表达株系萌发率为67%;突变体株系气孔几乎全部关闭,过表达株系气孔关闭不明显,大小是突变体株系的2~3倍.结果表明ABRv1基因在拟南芥的ABA信号应答响应中可能起负调控作用.     

水稻成花素基因Hd3a在ABA调控种子萌发中的功能研究

Hd3a(Heading date 3a)是拟南芥成花素基因FT(Flowering Locus T)的同源基因，作为水稻开花的强诱导因子，对短日照条件下水稻的开花具有重要的促进作用。Hd3a在水稻发育中是否具有其他功能，目前的认识仍非常有限。在我们的研究中，通过对Hd3a基因组序列进行生物信息学分析，发现Hd3a的启动子序列包含响应脱落酸(Abscisic Acid, ABA)信号的ABRE(ABA-Responsive Element)元件。荧光素酶发光反应和RT-qPCR分析都证实了Hd3a的转录表达随外源ABA增加而减少。通过不同浓度的ABA处理水稻野生型和hd3a缺失突变体种子，结果表明hd3a缺失突变体种子萌发明显被抑制，暗示了Hd3a缺失导致种子萌发中对ABA敏感性增强，同时hd3a突变体中ABA信号途径响应基因ABI5(ABA Insensitive 5)、ABL1(ABI5-Like1)的表达量升高，表明Hd3a可负调控ABA信号。综上所述，Hd3a可以响应ABA信号调控水稻种子萌发，这些研究结果对于深入理解水稻Hd3a基因的生物学功能具有重要意义。     

拟南芥RING finger结构域ABRv1基因的功能初步研究

拟南芥ABscisic acid responsive RING-v protein 1基因（ABRv1）编码一个含有RING finger结构域的E3连接酶。为了分析ABRv1的基因功能,我们首先构建了高表达载体pBI121- ABRv1,经农杆菌转化,花序浸染后得到T0代转基因种子,并经卡那霉素板筛选得到转基因苗,再经过两代的筛选获取了纯合的高表达株系ABRv1。通过DNA及RNA水平鉴定了该基因的T –DNA插入突变体abrv1。对突变体、高表达转基因株系及野生型进行ABA诱导处理,检测其萌发率、气孔关闭情况进行,发现ABA处理后突变体的萌发率降低,过表达株系萌发率升高;突变体株系气孔几乎未关闭,过表达株系气孔关闭非常明显。以上结果表明ABRv1基因在拟南芥的ABA信号应答响应中可能起负调控作用,为深入分析该基因的功能打下基础。     

拟南芥CARK5激酶响应脱落酸信号的研究

为了研究拟南芥脱落酸（Abscisic acid，ABA）受体的翻译后修饰，本研究以能够磷酸化ABA受体的激酶CARK5作为研究对象，通过构建了转基因株系来检测CARK5在ABA信号途径中的功能.结果显示： CARK5能够促进ABA介导的抑制种子萌发、幼苗的形态建成以及主根的生长，但是过表达激酶失活型CARK5m（CARK5N250A）则跟突变体cark5表型类似.拟南芥中过表达CARK5增强植株抗旱性；ABA处理后，ABA响应标记基因RAB18表达量增加.这些结果说明，CARK5通过可以磷酸化ABA受体，正调控ABA信号通路.     

AtSIAK在植物抗盐胁迫响应中的功能研究

AtSIAK基因在拟南芥中编码ABC1类蛋白激酶,RT-PCR检测AtSIAK基因在拟南芥的根、茎、叶、花、角果等组织中均有表达,与利用AtSIAK基因的启动子驱动GUS报告基因的研究结果一致.AtSIAK基因受不同胁迫(MV和NaCl)和激素ABA、SA处理均有明显的上调表达,其中在NaCl处理下最明显.不同浓度NaCl处理使35S::AtSIAK过表达植株比野生型(Columbia)和siak1突变体的种子萌发率和子叶变绿率高,表明AtSIAK基因参与抗盐胁迫的响应.     

植物ABI3转录因子研究进展

植物脱落酸不敏感蛋白3(abscisic acid-insensitive 3,ABI3)属于植物特有的B3蛋白家族,是脱落酸(ABA)信号转导过程中重要的调控因子之一,可与ABI5(abscisic acid-insensitive 5)共同作用,来诱导ABA响应基因的表达,从而调节种子休眠和萌发.其功能缺失突变体在种子萌发、幼苗生长以及气孔运动过程中都对ABA不敏感.除此之外,ABI3还在非生物胁迫方面发挥重要作用.文章综述了植物ABI3转录因子的结构特点、生物学功能等方面的最新研究进展,以期对ABI3转录因子的分子调控网络有一定了解,对作物遗传改良有新的思考.     

ABC1类蛋白激酶AtSIAK在植物抗盐胁迫响应中的功能研究

在拟南芥中克隆了一个编码ABC1 类蛋白激酶的基因SIAK (Salt Induction ABC1-like Kinase), RT-PCR检测SIAK基因在拟南芥的根、茎、叶、花、角果等组织中均有表达,与利用SIAK基因的启动子驱动GUS报告基因的研究结果一致. SIAK基因受不同胁迫（ABA、SA、MV和NaCl）处理均有明显的上调表达,其中在NaCl处理下最明显.在不同浓度NaCl处理下,35S::SIAK 表达植株比WT 和siak1 突变体的种子萌发率和子叶变绿率,表明AtSIAK基因参与抗盐胁迫的响应.     

Functional analysis of the ABA-responsive protein family in ABA and stress signal transduction in Arabidopsis

The phytohormone abscisic acid (ABA) plays an important role in plant growth and development, for example in seed dormancy and germination, as well as in plant responses to environmental stresses, such as drought and high salinity. Previous studies have shown that ABA regulates the expression of genes with an ABA-responsive element (ABRE) and their corresponding physiological responses. Bioinformatics analysis identified a GRAM domain-containing gene family that has a multiple ABRE cis-element, which was termed the ABA-responsive protein (ABR) family. To analyze the function of the ABR family, we identified homozygous T-DNA insertion mutants and constructed abr1, 2, 3 double mutants and triple mutant. The abr1, abr2 and abr3 single mutants showed a normal phenotype; however, the germination of seeds of the double mutants and triple mutant were insensitive to ABA, NaCl, mannitol and glucose. ABR1-GFP was distributed as a punctate structure in the cytosol and may be localized in the endomembrane system. The ABR2-GFP and ABR3-GFP proteins localized in the cytoplasm. In addition, ABR1, ABR2 and ABR3 were expressed in various tissues, and could be induced by several abiotic stresses, especially by ABA. The expressions of these genes were significantly suppressed in aba2, abi1 and abi2 null mutants. These results suggested that the ABR family may act downstream of ABI1 and ABI2 in the ABA signal transduction process in plants.     

Characterization of Arabidopsis calreticulin mutants in response to calcium and salinity stresses

As an important calcium-binding protein,calreticulin plays an important role in regulating calcium homeostasis in endoplasmic reticulum (ER) of plants.Here,we identified three loss-of-function mutants ofcalreticulin genes in Arabidopsis to demonstrate the function of calreticulin in response to calcium and salinity stresses.There are three genes encoding calreticulin in Arabidopsis,and they are named AtCRT1,2,and 3,respectively.We found that both single mutant of crt3 and double mutant of crtl crt2 were more sensitive to low calcium environment than wild-type Arabidopsis.Moreover,crt3 mutant showed more sensitivity to salt treatment at germination stage,but tolerance to salt stress at later stage compared with wild-type plant.However,there was no obvious growth difference in the mutant crt1 and crt2 compared with wild-type Arabidopsis under calcium and salt stresses.These results suggest that calreticulin functions in plant responses to calcium and salt stresses.     

Involvement of DEG5 and DEG8 proteases in the turnover of the photosystem II reaction center D1 protein under heat stress in Arabidopsis thaliana

Deg5,deg8 and the double mutant,deg5deg8 of Arabidopsis thaliana were used to study the physiological role of the DEG proteases in the repair cycle of photosystem II (PSII) under heat stress. PSII activity in deg mutants showed increased sensitivity to heat stress, and the extent of this effect was greater in the double mutant, deg5deg8, than in the single mutants, deg5 and deg8. Degradation of the D1 protein was slower in the mutants than in the WT plants. Furthermore, the levels of other PSII reaction center proteins tested remained relatively stable in the mutant and WT plants following high-temperature treatment. Thus, our results indicate that DEG5 and DEG8 may have synergistic function in degradation of D1 protein under heat stress.     

Characterization of Arabidopsis calreticulin mutants in response to calcium and salinity stresses

As an important calcium-binding protein, calreticulin plays an important role in regulating calcium homeostasis in endoplasmic reticulum （ER） of plants. Here, we identified three loss-of-function mutants of calreticulin genes in Arabidopsis to demonstrate the function of calreticulin in response to calcium and salinity stresses. There are three genes encoding calreticulin in Arabidopsis, and they are named AtCRT1, 2, and 3, respectively. We found that both single mutant of crt3 and double mutant of crtl crt2 were more sensitive to low calcium environment than wild-type Arabidopsis. Moreover, crt3 mutant showed more sensitivity to salt treatment at germination stage, but tolerance to salt stress at later stage compared with wild-type plant. However, there was no obvious growth difference in the mutant crtl and crt2 compared with wild-type Arabidopsis under calcium and salt stresses. These results suggest that calreticulin functions in plant responses to calcium and salt stresses.     

拟南芥SnRK2.2/2.3激酶参与调节镉胁迫响应的机制探究

为探究拟南芥SnRK2.2和SnRK2.3基因对Cd胁迫响应的分子机制. 以野生型（WT）、双突变体SnRK2.2/2.3、过表达SnRK2.2和过表达SnRK2.3的转基因植物为材料,研究SnRK2.2和SnRK2.3基因与Cd胁迫响应的关系.发现过表达两个基因可以提高拟南芥对Cd的耐受性,表现为可以减少Cd、丙二醛（MDA）及活性氧（ROS）的累积量,增加抗氧化酶CAT、POD和SOD的活性. qRT PCR结果显示在Cd胁迫下,两种过表达植株中铁转运蛋白IRT1和转录因子FIT、bHLH038和bHLH039表达水平受到明显抑制,ABA合成相关基因AAO3和NCED3的表达量显著上调.在Cd胁迫下,两种过表达植株中ABA含量显著高于WT和双突变体. 以上结果表明：拟南芥遭受Cd胁迫时,SnRK2.2和SnRK2.3基因通过下调IRT1基因表达从而减少植物对Cd的吸收,同时通过增加内源ABA含量来缓解Cd对植物的毒害.     

UV-B预处理对拟南芥uvr8-2突变体响应干旱胁迫的影响

以拟南芥uvr8-2突变体植株为材料,研究UV-B预处理对植株响应干旱胁迫的影响以及植物激素在此过程中的作用.实验结果表明:从形态观察到生理指标,UV-B预处理显著提高了拟南芥uvr8-2突变体的干旱适应性,与野生型Ler的结果一致;UV-B预处理显著提高了拟南芥uvr8-2突变体抗氧化酶SOD、POD、CAT活性,抗氧化酶基因POD和CAT的表达量,以及植物激素ABA、JA、SA的质量分数,降低了细胞膜受损程度.由此推测:UV-B预处理诱导uvr8-2植株的干旱适应性中存在一条不依赖于UVR8的信号转导途径,即通过增加植物逆境响应激素ABA、JA和SA的质量分数,调控抗氧化酶基因CAT和POD表达和增强抗氧化酶活性,从而缓解干旱胁迫下拟南芥的叶片萎焉、相对含水量下降等现象.