Functional Analysis of Arabidopsis Thaliana Galactinol Synthase AtGolS2 in Response to Abiotic Stress

Shen Yang　Jia Bowei　Wang Jinyu　Cai Xiaoxi　Hu Bingshuang　Wang Yan　Chen Yue　Sun Mingzhe ^*　Sun Xiaoli ^*

Crop Stress Molecular Biology Laboratory, College of Agriculture, Heilongjiang Bayi Agriculture University, Daqing, 163000

* Co-corresponding authors, csmbl2016@126.com; hlj_mzsun@163.com

Abstract　Soil salt-alkalization is one of the adverse factors limiting crop yields. Identification of key salt-alkaline tolerant genes is of great significance for molecular breeding of stress-resistant crops. In this study, a T-DNA insertion Arabidopsis mutant atgols2 showing higher sensitivity to bicarbonate salt-alkaline stress was screened out against NaHCO₃ treatment. Further bioinformatic analysis revealed that the AtGolS2 gene encoded a galactinol synthase, which is a member of the glycosyltransferase family A superfamily. We predicted the protein interaction network of AtGolS2 via SMART online analysis, and found that these AtGolS2 interacting proteins were related to lipid metabolism, galactose biosynthesis and raffinose biosynthesis, and participated in abiotic stress responses. By using the online expression data, we showed that AtGolS2 expression responded to salt, osmotic, drought and ABA stresses. PCR amplication by using the three primers method verified the homozygous T-DNA insertion in atgols2. Phenotypic assays further uncovered that atgols2 mutant was more sensitive to high salt, osmotic and ABA stresses than the wild type Arabidopsis. Taken together, results in this study revealed the positive function of AtGolS2 in bicarbonate salt-alkaline, high salt, osmotic and ABA stresses, which will facilitate further research regarding the function and molecular mechanism of the GolS family genes in stress responses.

Keywords　Arabidopsis, Galactinol synthase, AtGolS2, Abiotic stress, Functional analysis

植物在生长发育过程中不可避免地受到干旱、低温和土壤盐碱化等非生物胁迫的影响。盐碱胁迫是影响作物生长发育并最终导致减产的主要因素之一(Ismail and Horie, 2017)。干旱、低温和盐碱胁迫的共同特点是造成细胞缺水，引起细胞水平衡失调、蛋白质大分子变性，破坏细胞膜结构等，影响植物的生长和发育(吴杨等, 2017)。当植物面对低温、干旱和高盐胁迫时，细胞内可溶性糖如葡萄糖、蔗糖、棉子糖系列寡糖(raffinose family oligosaccharides, RFOs)的含量增加，维持渗透平衡，增强植物对胁迫的耐受性(Salvi et al., 2018)。因此，鉴定关键的糖类代谢基因对于培育优良抗逆作物有重要意义。

肌醇半乳糖苷合酶(galactinol synthase, GolS)是从豌豆种子中发现，能够催化UDP-半乳糖和肌醇形成肌醇半乳糖苷，为RFOs提供活化的半乳糖基，调节植物体内RFOs的积累(Bachmann and Keller, 1995)。GolS只存在于开花植物中，拟南芥中有(Arabidopsis thaliana)7个(Selvaraj et al., 2017)，水稻(Oryza sativa L)中有2个(Shimosaka and Ozawa, 2015)，玉米(Zea mays L)中有10个(Zhou et al., 2012)。近年来研究发现在拟南芥中超表达玉米肌醇半乳糖苷合成酶(ZmGolS2)显著提高了植株叶片中的肌醇半乳糖苷和棉子糖含量，并且增强了植株的氧化胁迫耐性(Gu et al., 2019)。转AmGolS基因的红叶石楠(Photinia serratifolia)植株抗寒能力明显提高(Downie et al., 2003)。在番茄(Solanum lycopersicum)和匍匐筋骨草(Ajuga reptans)中，GolS的表达受低温诱导(Downie et al., 2003; Dos Santos et al., 2011)。同时GolS在植物重金属胁迫应答过程中也发挥了作用，TaGolS3表达受ZnCl₂和CuCl₂诱导，在拟南芥中超表达TaGolS3显著提高了转基因植株ROS清除能力、抗氧化酶活性和脯氨酸含量，而丙二醛(MDA)含量显著降低(Wang et al., 2016)。综上所述，GolS参与植物的氧化、低温和重金属胁迫等逆境响应，但在盐碱胁迫方面的报道较少。

本研究从一批拟南芥T-DNA插入突变体中筛选获得一个苏打盐碱胁迫十分敏感的突变体atgols2，进一步分析发现AtGolS2基因表达也响应高盐、高渗和ABA胁迫，且atgols2突变体对高盐、高渗和ABA胁迫更为敏感。本研究揭示了AtGolS2基因缺失对非生物胁迫耐性的影响，为后续解析GolS基因在逆境应答中的功能和作用机制奠定了基础。

1结果与分析

1.1苏打盐碱敏感的atgols2拟南芥突变体筛选

为鉴定耐苏打盐碱基因，本课题组前期从拟南芥生物资源保藏中心(Arabidopsis Biological Resource Center，ABRC)购买了一批拟南芥T-DNA插入突变体，从中筛选苏打盐碱敏感株系。图1所示为部分苏打盐碱敏感突变体(#6, #7, #9, #11, #15)在0 mmol/L或10 mmol/L NaHCO₃ 1/2MS培养基上的生长状态。正常条件下各株系正常生长，生长状态一致；而播种于含有10 mmol/L NaHCO₃培养基中的各株系种子萌发均受到抑制，尤其是#11突变体萌发的种子数最少(图1A)。萌发率统计结果也表明，10mM NaHCO₃处理下野生型(wild type, WT)、#6、#7、#9、#11、#15突变体种子萌发均变慢，7 d萌发率分别为52.9%、40.2%、42.7%、42.1%、35.8%、39.8% (图1B)。

图 1 苏打盐碱胁迫下拟南芥突变体表型及萌发率分析 注: A: 苏打盐碱胁迫下拟南芥突变体表型分析; B: 苏打盐碱胁迫下拟南芥突变体萌发率统计 Figure 1 Phenotype and germination rates analysis of Arabidopsis mutants under bicarbonate saline-alkali stress Note: A: Phenotype of Arabidopsis mutants under bicarbonate saline-alkali stress; B: Germination rates of Arabidopsis mutants under bicarbonate saline-alkali stress

本研究选取萌发率最低的#11突变体(SALK_101144)作为研究对象。根据拟南芥数据库TAIR获得#11突变体的T-DNA插入侧翼序列，发现T-DNA插入在AT1G56600基因。根据NCBI及TAIR注释，AT1G56600是一个编码肌醇半乳糖苷合酶的基因，命名为AtGolS2 (Nishizawa et al., 2008)。

1.2 AtGolS2蛋白保守结构域分析

利用SMART分析AtGolS2蛋白保守结构域，发现该蛋白具有一个糖基转移酶家族8结构域(Glyco_transf_8 domain)，属于糖基糖基转移酶家族A超家族中的一员(图2A)。在拟南芥中，该家族共有7个基因AtGolS1-7 (Nishizawa et al., 2008)。蛋白序列比对发现，拟南芥肌醇半乳糖苷合酶家族蛋白序列高度保守，均具有1个保守的Glyco_transf_8结构域(图2B)。该结构域具有将糖基从活化的核苷酸-糖供体转移到受体分子上合成低聚糖、多糖和糖缀合物的作用，可以增加植物体内多糖含量。已有文献表明AtGolS1通过增加棉子糖含量，提高氧化胁迫耐性(Song et al., 2016)；在白杨(Populus)中超量表达AtGolS3提高了杨树体内半乳糖醇和棉子糖的积累，增强了对活性氧的耐性(La Mantia et al., 2018)。由此推测具有相同结构域的AtGolS2可能通过参与脂多糖生物合成或糖原合成，提高半乳糖醇和棉子糖含量，进而参与非生物胁迫应答。

图2 AtGolS2蛋白保守结构域分析 注: A: AtGolS2蛋白保守结构域预测; B: 拟南芥AtGolS2同源蛋白多重序列比对, 红色横线标识Glyco_transf_8 domain保守结构域位置 Figure 2 Analysis of conserved domains in AtGolS2 protein Note: A: SMART prediction of the conserved domain in AtGolS2; B: Multiple alignment of AtGolS2 and homologous GolS proteins in Arabidopsis, the red line marked the position of Glyco_transf_8 domain

1.3 AtGolS2蛋白互作网络分析

利用SMART-Interactions分析AtGolS2可能参与的信号传导通路，模型预测结果表明RD20、USP、AT5G18200、UGE2、UGE5、DIN10、SIP2、STS、RFS1、RFS5可能与AtGolS2存在蛋白互作(图3)。通过NCBI蛋白数据库对互作蛋白进行功能注释分析(表1)，根据10个互作蛋白的性质可将它们分成以下三类：脂类代谢相关蛋白、半乳糖生物合成相关蛋白、棉子糖生物合成相关蛋白。

图3 AtGolS2蛋白互作网络示意图 Figure 3 Schematic diagram of AtGolS2 protein interaction network

表 1 AtGolS2互作蛋白功能注释 Table 1 Annotation of the function of AtGolS2 interacting protein

脂类代谢相关蛋白：RD20基因是Caleosin(油体钙)家族的一个成员，该家族蛋白促使种子在发芽过程中贮藏脂质，参与植物体内的脂类代谢，RD20参与响应盐、干旱和渗透胁迫(Aubert et al., 2011, Aubert et al., 2010; Sham et al., 2015; Park et al., 2018)。

半乳糖生物合成相关蛋白：UGE2、UGE5、USP、AT5G18200分别编码UDP-半乳糖差向异构酶、UDP-半乳糖焦磷酸化酶和UTP半乳糖-1-磷酸尿酸转移酶，在半乳糖的生物合成及分解过程中起到重要的作用。其中UGE2表达受低温，渗透胁迫诱导，UGE5表达受低温、渗透和盐胁迫诱导(Aznar et al., 2018)，USP表达受低温胁迫抑制(Decker and Kleczkowski, 2017)，AT5G18200受盐胁迫诱导(Kotake et al., 2007)。

棉子糖生物合成相关蛋白：DIN10、SIP2、STS、RFS1、RFS5分别编码棉子糖基水解酶和棉子糖特异性α-半乳糖苷酶棉子糖合酶，在棉子糖的生物合成及分解过程中起到重要的作用，同时也参与响应非生物胁迫。DIN10表达受低温，活性氧胁迫诱导(Maruyama et al., 2009; Lee et al., 2017)；SIP2表达受渗透胁迫诱导(Fujita et al., 2005)；STS表达受盐胁迫抑制；RFS5受低温、渗透、盐和干旱胁迫诱导(Nishizawa et al., 2008)。

通过对AtGolS2互作蛋白功能注释分析，发现互作蛋白与植物体内半乳糖和棉子糖生物合成相关，且大多响应非生物胁迫。推测AtGolS2可能与上述蛋白相互作用，影响体内糖类含量进而响应非生物胁迫。

1.4 AtGolS2基因非生物胁迫表达模式分析

基于Arabidopsis eFP Browser在线数据分析AtGolS2基因在非生物胁迫(冷, 渗透, 盐, 干旱)和激素处理(ABA, GA, ETH)下的表达模式。结果如图4所示，无论是地上(Shoot)还是地下(Root)部分，AtGolS2基因在低温、GA和ETH胁迫下，基因表达基本无变化。然而当遭受渗透、盐、干旱及ABA胁迫后AtGolS2基因表达量显著上升。尤其是在盐和渗透胁迫处理3h后，AtGolS2基因表达量上升倍数高达400和500倍，暗示AtGolS2基因可能通过ABA依赖途径参与盐、渗透和干旱胁迫应答。

图4 AtGolS2在非生物胁迫和激素处理下的表达模式 Figure 4 The expression pattern of AtGolS2 under abiotic stress and hormone treatment

1.5 atgols2 T-DNA插入突变体鉴定

根据atgols2突变体T-DNA插入侧翼序列，绘制T-DNA插入结构图。如图5A所示，T-DNA插入在AtGolS2基因启动子区中。根据插入位点设计基因特异引物(P1, P2)，采用三引物法PCR鉴定atgols2是否为T-DNA插入纯合突变体。当以LB (T-DNA序列特异引物)+P2 (基因特异反向引物)进行PCR扩增时，WT中未扩增出条带，而突变体植株可见500 bp的目的条带，说明突变体中有T-DNA插入(图5B)。以P1＋P2引物组合进行PCR扩增，WT中扩增出626 bp大小的目的条带，而突变体植株没有条带，说明突变体是纯合的(图5C)。上述结果表明鉴定的6株突变体植株均为拟南芥AtGolS2基因的T-DNA纯合插入突变体。

图5 New ICT based fertility management model in private dairy farm India as well as abroad

1.6 atgols2突变体盐、渗透胁迫和ABA处理下表型分析

为验证AtGolS2基因在盐、渗透胁迫和ABA处理下的功能，对比WT与突变体atgols2种子在含125 mmol/L NaCl、250 mmol/L Mannitol、0.6 μm ABA以及正常1/2 MS培养基上的生长状况。正常条件下，WT和atgols2生长状态一致且萌发速率一致；在含有125 mmol/L NaCl、250 mmol/L Mannitol和0.6 μmol/L ABA培养基中WT和atgols2均生长迟缓，萌发速率受到影响。萌发率统计结果表明：在含有125 mmol/L NaCl、250 mmol/L Mannitol和0.6 μmol/L ABA培养基中，培养2天时，atgols2种子萌发率均显著低于WT(图6B)，培养7天后，atgols2和WT均能全部萌发，说明AtGolS2基因的缺失可能抑制种子萌发速率，但不影响种子最终萌发。在NaCl和Mannitol处理下培养7天后，atgols2突变体展叶率低于WT，幼苗长势明显不如WT (图6A)。上述结果表明AtGolS2基因缺失降低了拟南芥对高盐、高渗和ABA胁迫的耐性，抑制拟南芥的正常生长。

图6 盐, 渗透胁迫和ABA处理下atgols2突变体表型及萌发率统计 注: A: 盐, 渗透胁迫和ABA处理下的atgols2突变体表型; B: 盐, 渗透胁迫和ABA处理2天的atgols2突变体萌发率; *在p<0.05水平上差异显著(n=30), **在p<0.01水平上差异显著(n=30) Figure 6 Phenotype and germination rates analysis of atgols2 mutant under salt, osmotic and ABA treatment Note: A: Phenotype of atgols2 mutant under salt, osmotic and ABA treatment; B: Germination rates of atgols2 mutant under salt, osmotic and ABA treatment for 2 day; *: Significantly different at p<0.05 level (n=30); **: Significantly different at p< 0.01 level (n=30)

2讨论

土地盐碱化是世界上普遍存在的问题，严重影响植物的生长发育，限制作物的质量和产量。世界上有8.31亿hm²的土壤因盐分过高而无法有效利用(Jin et al., 2008)。盐胁迫主要由中性盐如NaCl、Na₂SO₄引起，而盐碱胁迫主要由碱性盐如碳酸氢盐(HCO₃^－)和碳酸盐(CO₃^2－)引起。盐胁迫对植物的危害主要包括离子胁迫、渗透胁迫和氧化胁迫，而盐碱胁迫在此基础上增加了HCO₃^－或CO₃^2－引起的离子胁迫及高pH胁迫，比盐胁迫危害更大(刘奕媺等, 2018)。因此，鉴定耐盐碱关键基因对于提高作物耐盐碱能力和开发利用盐碱地具有重要意义。

本研究以发掘植物盐碱胁迫响应基因为出发点，利用课题组前期购买的拟南芥T-DNA插入突变体筛选出对苏打盐碱胁迫最敏感的#11号突变体——atgols2 (图1)。现有研究已证实GolS参与干旱、氧化、低温胁迫响应，但在苏打盐碱胁迫方面的报道鲜有。本研究发现atgols2突变体对苏打盐碱敏感性增加，为GolS参与苏打盐碱应答提供了证据。此外，本研究还发现AtGolS2基因表达显著受盐、渗透、干旱和ABA处理诱导(图4)，且atgols2突变体种子萌发对高盐、高渗和ABA胁迫更加敏感(图6B)，说明AtGolS2基因正调控拟南芥对非生物胁迫的耐性。与本研究结论一致，杨树中过表达AtGolS3/AtGolS2导致体内抗氧化酶合成基因表达增强，抗氧化能力增强，气孔开度减小，耐旱能力增强(Yu et al., 2017; La Mantia et al., 2018)。在二穗短柄草(Brachypodium distachyon)中转入AtGolS2基因，植物体内叶绿素含量增多，耐旱能力增强(Himuro et al., 2014)。拟南芥中过表达CaGolS1/CaGolS2通过减少ROS积累，增强了高温耐受性(Salvi et al., 2018)。红叶石楠中转入AmGolS基因，转基因植株抗寒能力明显提高(Downie et al., 2003; Dos Santos et al., 2011)。本研究发现了AtGolS2基因参与苏打盐碱和ABA应答，未来将进一步评价GolS基因在作物苏打盐碱性状改良中的应用价值。AtGolS2编码蛋白属于糖基转移酶家族A超家族，该家族负责编码合成肌醇半乳糖苷合酶，该酶催化RFOs生物合成的第一步。RFOs利用肌醇半乳糖苷提供的半乳糖基合成一系列不同的寡糖(Saravitz et al., 1987)。已有研究发现在拟南芥中超量表达AtGolS2增加了半乳糖醇和棉子糖的含量，减小叶片气孔开度，降低蒸腾速率，提高了耐旱性(Nishizawa et al., 2008)。在大豆(Glycine max)和水稻中转入AtGolS2，与WT相比半乳糖醇水平含量增多，耐旱能力增强，产量得到提升(Honna et al., 2016; Selvaraj et al., 2017)。TsGolS2是从盐芥(Thellungiella salsuginea)克隆得到的，与AtGolS2具有高度同源性，在拟南芥中过表达TsGolS2，转基因植物中半乳糖醇、棉子糖和α-酮戊二酸的含量显着增加，提高了对高盐和高渗胁迫的耐受性(Sun et al., 2013)。此外，AtGolS3、CaGolS1、CaGolS2表达均促进了棉子糖的积累(Downie et al., 2003; Dos Santos et al., 2011)。然而，GolS基因是否通过促进半乳糖醇和棉子糖等寡糖积累，参与ABA应答过程尚需进一步验证。

植物中的各种生理活动主要是通过细胞中的蛋白质进行调控和调节，蛋白质功能的发挥不是凭借单个蛋白质独立执行，而是依靠蛋白质与蛋白质相关作用执行其功能。本研究利用SMART-Interactions预测了AtGolS2蛋白互作网络，发现网络中的互作蛋白可分成三类：脂类代谢相关蛋白、半乳糖生物合成相关蛋白、棉子糖生物合成相关蛋白(图3; 表1)，在调控脂类代谢，半乳糖、棉子糖的生物合成及分解过程中起到重要的作用。此外，这些互作蛋白编码基因的表达均响应非生物胁迫(表1)。其中RD20基因编码Caleosin(油体钙)蛋白，该基因表达受干旱、盐和ABA诱导，与WT相比，rd20敲除拟南芥突变体气孔开度增大，表现出更高的蒸腾速率。在缺水条件下RD20通过控制植物气孔开度，提高植物耐旱能力，同时，rd20敲除拟南芥突变体也表现出盐敏感表型(Aubert et al., 2010; Aubert et al., 2011; Sham et al., 2015; Park et al., 2018)。DIN10基因编码糖基水解酶，其表达响应冷胁迫及活性氧胁迫，在拟南芥中超量表达DIN10可提高拟南芥耐冷性(Maruyama et al., 2009; Lee et al., 2017)。SIP2编码棉子糖特异性α-半乳糖苷酶，拟南芥中超量表达SIP2可提高耐旱性(Fujita et al., 2005)。STS和RFSS编码棉子糖合酶，sts和rfss拟南芥突变体体内棉子糖含量均降低，表现出冷敏感表型(Nishizawa et al., 2008)。后续可通过酵母双杂交、pull down、免疫共沉淀等方法验证AtGolS2与上述蛋白间的相互作用，为深入揭示AtGolS2耐苏打盐碱信号传导通路提供理论依据。

3材料与方法

3.1植物材料

atgols2拟南芥(Arabidopsis thaliana) T-DNA插入突变体(SALK_101144)购买于拟南芥种子库ABRC，WT拟南芥(哥伦比亚生态型)由作物逆境分子生物学实验室保存。

3.2 atgols2拟南芥突变体非生物胁迫耐性分析

WT和atgols2突变体种子经5% NaClO消毒10 min，再用ddH₂O清洗6~10次，4℃避光处理3 d。将灭菌后的WT和突变体拟南芥种子播种在正常、或含有10 mmol/L NaHCO₃ (苏打盐碱胁迫)、或含有125 mmol/L NaCl (高盐胁迫)、或含有250 mmol/L Mannitol (高渗胁迫)、或含有0.6 μmol/L ABA的1/2MS培养基中，置于培养箱培养7 d，统计萌发率并拍照。每次实验每个株系30粒种子，实验包括3次生物学重复和3组技术重复。培养箱设定温度18~22℃，湿度50%~70%，光周期16 h光照/8 h黑暗。

3.3拟南芥atgols2 T-DNA插入突变体PCR鉴定

根据拟南芥数据库TAIR获得atgols2突变体T-DNA插入侧翼序列信息，在T-DNA插入序列的前后位置分别设计基因特异引物，标记为P1 (5'-CGTGTCCACATAATAACCAATCAGA-3')和P2 (5'-CCCCTTTCACGTAGTCTCCAGTT-3')。利用SALK系列突变体T-DNA上的固定引物LB (5'-ATTTTGCCGATTTCGGAAC-3')，采用三引物法PCR鉴定拟南芥atgols2突变体是否为纯合T-DNA插入突变体。采用全式金公司EasyPure基因组DNA提取试剂盒提取拟南芥基因组DNA，以此为模板，分别进行插入鉴定(P1+LB-anti)和纯和鉴定(P1＋P2)的PCR反应。反应条件如下：95℃ 5 min；95℃ 30 s，55℃ 30 s，72℃ 40 s，共30个循环，72℃ 10 min。PCR反应体系：gDNA模板2 μL、2×EasyTaq®PCR SuperMix 7.5 μL、Sense primer 0.3 μL、Anti-sense primer 0.3μL、ddH₂O 4.9 μL。PCR产物进行琼脂糖凝胶电泳分析。

3.4 AtGolS2基因生物信息学分析

通过Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html)在线获得AtGolS2的基因组序列、CDS序列、氨基酸序列以及蛋白功能注释等基本信息，利用SMART (http://smart.embl.de/)在线预测软件分析蛋白保守结构域。通过NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi)在线Protein Blast，筛选出拟南芥AtGolS2同源基因，下载其氨基酸序列，利用Clustal X1.83将AtGolS2与其同源基因进行蛋白氨基酸序列比对。利用STRING (https://version11.string-db.org/cgi/network)分析AtGolS2蛋白可能参与的互作网络,并通过Uniprot (https://www.uniprot.org/)蛋白数据库获得互作蛋白功能注释，进行聚类分析。

3.5基于在线数据库AtGolS2基因非生物胁迫表达模式分析

基于在线数据库Arabidopsis eFP Browser (http://bar.utoronto.ca/eplant/)获得AtGolS2基因在冷、渗透、盐、干旱、ABA、GA和ETH处理下的表达数据(Kilian et al., 2007)。下载原始数据，统计于Excel，按胁迫类型进行分类，取胁迫处理0 h、0.5 h、1 h、3 h、6 h表达数据，利用Excel绘制热图。

作者贡献

沈阳和贾博为是本研究的执行人；王金玉、才晓溪和孙明哲负责完成atgols2表型观察和数据处理；胡冰霜、陈悦和王研负责AtGolS2生物信息学分析；孙晓丽是本研究的构思者和负责人，指导实验设计，数据分析，论文撰写与修改。全体作者都阅读并同意最终的文本。

致谢

本研究由黑龙江八一农垦大学研究生创新科研项目YJSCX2019-Y06和国家自然科学基金项目31971826共同资助。

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