CN112501181A - 水稻抗逆相关基因OsTZF7及其编码蛋白与应用 - Google Patents
水稻抗逆相关基因OsTZF7及其编码蛋白与应用 Download PDFInfo
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Abstract
水稻抗逆相关基因OsTZF7及其编码蛋白与应用，涉及植物基因工程领域。OsTZF7基因是由序列表SEQ ID NO：1所示的核苷酸序列组成的RR‑TZF家族基因。利用该基因可培育抗旱、耐盐能力增强的转基因植物。还提供包含OsTZF7基因编码的蛋白，氨基酸序列如SEQ ID NO:2所示。还提供一种包含OsTZF7基因编码的重组载体。提供一种包含OsTZF7基因在提高植物抗逆性中的应用。OsTZF7基因受高盐，模拟干旱以及ABA的诱导表达，超量表达OsTZF7提高了水稻苗期耐盐性和抗旱性。对植物抗逆育种具有重要的意义，在农业领域具有广阔的应用空间和市场前景。
Description
技术领域
本发明涉及植物基因工程领域，尤其是涉及一种新的水稻抗逆相关基因OsTZF7及其编码蛋白与应用，利用该基因可以培育抗旱、耐盐能力增强的转基因植物。
背景技术
由于生活在自然环境中，植物一生中会面临例如干旱、高盐、高温、低温等一系列非生物逆境，而这些逆境严重影响了植物的生长发育。为了适应并在恶劣环境中生存，植物在感知环境胁迫后，激活一系列信号通路，通过逆境响应基因的表达，引起植物的生理生化反应和形态变化，最终适应各种逆境。高盐、干旱和低温等非生物胁迫是影响水稻生长发育的重要环境因素，严重时造成水稻的大幅减产。近年来，随着全球气候的变化，干旱气候、土壤盐渍化等问题的不断出现更将对水稻的种植提出更为严峻地考验；因此，寻找抗逆相关基因，并利用遗传工程培育抗逆水稻品种已成为作物新品种培育的方向。
CCCH型锌指蛋白是一类包含了一个或多个CCCH锌指结构域的蛋白家族，该结构域由三个半胱氨酸及一个组氨酸组成，并能与锌离子结合。串联CCCH锌指蛋白(Tandem CCCHzinc finger，TZF)属于CCCH型锌指蛋白的一个亚家族，在真核生物中广泛存在。近年来研究表明TZF蛋白在动植物的生长发育，逆境胁迫应答反应中发挥了重要的作用。拟南芥中多个TZF基因参与了逆境胁迫应答反应：AtTZF1(AtC3H23)影响ABA、GA以及糖介导的拟南芥生长发育和胁迫反应。AtTZF2(AtOZF1/AtC3H20)和AtTZF3(AtOZF2/AtC3H49)参与了ABA、JA以及氧化应激反应等。水稻中OsTZF1和OsTZF2(OsDOS)调控水稻叶片衰老，并且OsTZF1还参与了盐胁迫、干旱胁迫以及ABA、JA激素响应。本发明中OsTZF7基因受到盐胁迫、干旱胁迫以及应激激素ABA的诱导表达，超量表达该基因的转基因水稻对干旱胁迫及盐胁迫的抵抗力增强。本发明对于抗逆水稻新品种的培育具有重要意义。
发明内容
本发明的目的在于提供一种水稻抗逆相关基因OsTZF7及其编码蛋白，该基因响应逆境胁迫，超量表达OsTZF7可以提高转基因水稻对干旱胁迫及盐胁迫的抗性。
本发明的另一目的是提供水稻抗逆相关基因OsTZF7在提高水稻抗逆性中的应用。
所述水稻抗逆相关基因OsTZF7，来源于水稻，所述OsTZF7基因的序列包含SEQ IDNO:1所示的DNA序列、与SEQ ID NO:1至少90％同源的DNA序列、功能相当于SEQ ID NO:1所示序列的亚片段中的一种。
所述水稻抗逆相关基因OsTZF7的核苷酸序列如SEQ NO：1所述序列的1～768位碱基所示。
本发明还提供一种包含OsTZF7基因编码的蛋白，所述蛋白的氨基酸序列如SEQ IDNO:2所示，或者为所述SEQ ID NO:2序列的同源序列、或保守性变异体、或等位变异体、或天然突变体、或诱导突变体。
本发明还提供一种包含OsTZF7基因编码的重组载体，构建所述重组载体所选用的载体为Ti质粒或植物病毒载体，通过转基因，使OsTZF7基因在水稻中超量表达，提高水稻对干旱及盐胁迫耐受能力。
本发明还提供一种包含OsTZF7基因的一种转化体，所述转化体的宿主为水稻。
本发明另一优先方案是提供一种包含OsTZF7基因在提高植物抗逆性中的应用。
利用已克隆的OsTZF7基因为探针，从cDNA文库和基因组文库中筛选得到本发明的基因或同源基因。也可以采用PCR(polymerase chain reaction)技术，从基因组、mRNA和cDNA中扩增得到本发明的OsTZF7基因以及任何感兴趣的一段DNA或与其同源的一段DNA。采用以上技术，可以分离得到OsTZF7基因，将这一序列与任何一种可以引导外源基因在植物中表达的载体连接后转化植物，可获得对逆境响应增强的转基因植株。
本发明提供的载体携带OsTZF7基因的表达载体可通过使用Ti质粒、植物病毒载体，直接DNA转化、微注射、电穿孔等常规生物技术方法导入植物细胞。
本发明OsTZF7基因表达载体转化宿主是包括水稻在内的多种植物。
本发明的水稻基因对逆境产生明显响应，可应用于在植物抗逆育种。
本发明公开了一种水稻抗逆性相关基因OsTZF7及编码蛋白与应用。该基因是由序列表SEQ ID NO：1所示的核苷酸序列组成的RR-TZF家族基因。OsTZF7基因受高盐，模拟干旱以及ABA的诱导表达，超量表达OsTZF7提高了水稻苗期耐盐性和抗旱性。本发明的水稻基因及其编码蛋白对植物抗逆育种具有重要的意义，本发明在农业领域具有广阔的应用空间和市场前景。
附图说明
图1为OsTZF7逆境表达谱分析。
图2为OsTZF7过表达和CRISPR/Cas9敲除转基因水稻分析。
图3为转基因植株在盐胁迫中的表型鉴定。
图4为转基因植株在模拟干旱胁迫中的表型鉴定。
具体实施方式
下述实施例进一步描述本发明，但所述实施例仅用于说明本发明而不是限制本发明。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
实施例1水稻OsTZF7基因的克隆
1.RNA的提取：参照Promega公司的
Super总RNA提取试剂盒catalog#S104。取水稻材料放入研钵，迅速用液氮冷冻并研磨，直至组织完全研磨成粉末状。将粉末状样品迅速转移到无核酸酶的EP管中加入350μL裂解液体，反复吹打后加入等体积稀释液。室温放置5min后以最大速度离心5min。小心吸取上清液体到新的1.5mL无核酸酶EP管中，加入0.5倍上清体积的无水乙醇，用移液枪吸放3～4次以混匀。将混合液转移到离心柱内后12000g离心1min。弃滤液，将离心柱重新放回收集管中，向离心柱加入600μL RNA洗液，12000g离心1min，弃滤液。向离心柱中心加入50μL DNA酶I孵育液，室温静置30min。向离心柱加入600μLRNA洗液，12000g离心1min，弃滤液。重复一次后将离心柱重新安置于收集管上，12000g离心2min。将离心柱转移至洗脱管上，在离心柱膜中央加入50μL无核酸酶水，室温静置2min，12000g离心1min。将洗脱下来的洗脱液重新加回到离心柱中央，室温静置2min，12000g离心1min再次洗脱，在分光光度计上测定RNA含量。然后将RNA保存在-80℃。
2.反转录：参照vazyme公司的
II 1st Strand cDNASynthesis Kit反转录试剂盒catalog#R211，将RNA样品在65℃下孵育5min以打开高级结构，结束后立即将其置于冰上，配置以下反应体系：
(1)反应体系(20μL)
| 试剂名称 | 使用量(μL) |
| RNA | 6 |
| Oligo(dT)/Random hexamers | 2 |
| 2×RT Mix | |
| HiScript II Enzyme Mix | 2 |
| DEPC H<sub>2</sub>O | Up to 20μL |
(2)反应条件
| 温度 | 时间 |
| 25℃ | 5min |
| 50℃ | 60min |
| 85℃ | 2min |
将反应后样品放置4℃短期保存，-20℃长期保存。
3.cDNA的克隆:根据Rice Genome Annotation Project中水稻数据库信息中提供的OsTZF7的cDNA序列序列信息，设计引物，采用RT-PCR方法进行cDNA全长克隆。
引物序列如下(SEQ ID No:3～4)：
OsTZF7-F：TATGGCGAGCCGAGAGCACCT
OsTZF7-R：CTACATCACAAGCTCGGACAC
通过RT-PCR得到一个包含有完整开放读框的编码区，长度为768bp；回收后连接到pMD19-T载体上，并进行测序。测序结果在DNAMAN软件上进行分析，结果显示其与水稻OsTZF7基因完全匹配。
实施例2水稻基因OsTZF7在逆境胁迫下的表达分析
1.逆境胁迫处理
选取饱满的日本晴种子，蒸馏水清洗，3％NaClO消毒10min后清洗干净，30℃催芽，种子露白后转放到发芽盒内水培生长，三叶期后施加营养液(1/2MS大量)。在28℃，14h/10h的光照培养室中培养，至四叶期时进行各种逆境和激素处理：20％PEG6000、200mM NaCl、4℃和100μM脱落酸(ABA)。分别在处理前、处理后1h、3h、6h、12h、24h对处理样品分根部及地上部分取样。
2.RNA提取与第一链cDNA合成
同实施例1。
3.定量PCR分析
用基因特异引物Ubi5-qF/Ubi5-qR以及OsTZF7-qF/OsTZF7-qR分别对OsTZF7基因进行定量分析。采用2-ΔΔCT相对定量的方法(Livak,K.J.and T.D.Schmittgen(2001)."Analysis of relative gene expression data using real-time quantitative PCRand the 2(-Delta Delta C(T))Method."Methods 25(4):402-408.)对目的基因相对表达量进行分析。每个样品3个重复，每个实验重复2次以上。荧光定量PCR的反应体系为：
Premix Ex TaqTM II 5μL，Primer-F/R(10μmol/L)各0.4μL，ROX Reference Dye0.2μL，cDNA模板1μL，ddH2O 3μL；反应条件：95℃30s，95℃5s，60℃30s，40个循环，在每个循环60℃延伸最后5s进行荧光采集。
引物序列如下(SEQ ID No:5～8)：
OsTZF7-qF：GCGGAGATGGAAGAGTTGAT
OsTZF7-qR：TACATCACAAGCTCGGACAC
Ubi5-qF：CCTCTGTGAAGATTGTGATGCCCTAC
Ubi5-qR：CAAGACCACGCCAACGGATAAA
结果显示：当水稻幼苗受到PEG胁迫时，OsTZF7于叶片中的表达水平在受胁迫1h后上调至原来的5倍，而OsTZF7在根中会迅速做出反应导致表达水平上调至原来的4倍，并在24h后恢复到正常水平；当水稻幼苗受到盐胁迫时，OsTZF7表达水平在受胁迫1h后上调至正常生长状态下的4～10倍；当水稻受到冷胁迫时，OsTZF7基因表达水平明显下调，其中叶片中基因表达水平影响更大，并且在叶片受冷害24h后，OsTZF7的表达量会恢复到正常水平；当水稻受到外源ABA干扰时，在1h内OsTZF7的表达迅速下调至原来的0.3倍左右，之后随着外源ABA的持续影响，叶片中OsTZF7的表达水平在处理12h后上调至原来的3倍，最后在处理24h后恢复到正常水平，而根中OsTZF7的表达水平则会随着处理时间的增长而持续上调(图1)。以上结果说明，OsTZF7基因参与了水稻的盐胁迫和干旱逆境应答，可能在胁迫反应中发挥作用。
实施例3水稻OsTZF7基因在水稻中超量表达和CRISPR/Cas9及转基因植株的鉴定
1.水稻OsTZF7基因的超量表达及CRISPR/Cas9载体的构建
根据水稻OsTZF7基因的全长序列，设计扩增出完整编码开放阅读框的引物，并在上游引物上引入一个额外的碱基，以便构建过表达载体。以实施例1中获得的扩增产物为模板，经PCR扩增后，将水稻OsTZF7基因的cDNA克隆至过表达载体(如pCXUN)上，测序，在保证阅读框架正确的前提下再将该过表达载体转入农杆菌中，并转化模式植物水稻日本晴。
根据Rice Information GateWay(RIGW)水稻数据库信息中设计的PAM位点，设计CRISPR靶标位点(sgRNA)。该靶标位点引物为(SEQ ID No:9～10)：
OsTZF7-sgRNA-F：ggcatTGGGTGAGTACCTGTCCGCC
OsTZF7-sgRNA-R：aaacGGCGGACAGGTACTCACCCAt
合成引物二聚体后与纯化回收后的BsaI酶切CRISPR/Cas9入门载体pU3-sgRNA进行酶切连接，转化大肠杆菌DH5α，经菌液PCR鉴定和阳性单克隆测序，得到入门载体重组质粒pU3-OsTZF7-sgRNA。再通过Gateway***与目的载体pH-ubi-cas9进行LR反应。
LR反应体系(5μL)如下：
| 组分 | 体积 |
| 入门克隆(100ng/μL) | 3μL |
| 目标载体(150ng/μL) | 1μL |
| LR Clonase<sup>TM</sup> II Enzyme Mix | 1μL |
25℃孵育2h。经过大肠杆菌转化和鉴定，最终测序正确后说明CRISPR/Cas9-Os39基因编辑载体构建成功。经热激转化至农杆菌EHA105后通过农杆菌介导的转化方法，将重组载体转化日本晴水稻。
2.农杆菌介导法进行水稻转基因。
将成熟水稻种子去壳后，用15％次氯酸钠溶液表面消毒30min，无菌水清洗4～6遍；无菌滤纸上晾干后，在诱导培养基NBD上26℃暗培养14～20d诱导愈伤组织；将掐芽后愈伤组织转移至继代培养基NBD上继代培养10～14d，挑选鲜黄而结实的愈伤组织在农杆菌的AAM培养基中培养1h，吹干后转入共培养基NBD-AS中黑暗条件下23℃共培养3d，无菌水清洗愈伤组织数遍(直至清洗液透明无浑浊)后转移至含有抗生素的筛选培养基NBS上进行筛选培养；2～3轮筛选(每次15d左右)后死亡愈伤上长出淡黄色抗性愈伤；将抗性愈伤转移至分化培养基RGH上23℃光照条件下进行分化；当不定芽长至5～8cm时，将其转移至生根培养基MSR中诱导生根，根系长好的转基因幼苗经过水培炼苗后移栽至田间培养。其中所述AAM培养液、NBD诱导培养基、NBD-AS共培养培养基、NBS筛选培养基、RGH分化培养基、MSR生根培养基配方参照Hiei等(Hiei,Y.,et al.(1994)."Efficient transformation of rice(Oryzasativa L.)mediated by Agrobacterium and sequence analysis of the boundariesof the T-DNA."Plant J 6(2):271-282.)和Lin等(Lin,Y.J.and Q.Zhang(2005)."Optimising the tissue culture conditions for high efficiency transformationof indica rice."Plant Cell Rep 23(8):540-547.)的文献。
生根结束后，除去生根培养基后，将苗泡于水中数日进行炼苗，然后移栽入土中生长。
3.超量表达阳性植株中目的基因的表达水平检测
RNA提取及定量PCR方法见实施例1及实施例2。
提取转基因T0代植物叶片RNA，采用定量PCR法检测了OsTZF7超量表达植株中目的基因的表达水平(图2A)，在检测的3株转基因植株中，表达量均超过40倍。
4.基因敲除转基因水稻的敲除位点检测
CTAB法提取提取转基因植株和非转基因植株的基因组DNA,以此为模板,使用基因两端的引物OsTZF7-F/OsTZF7-R进行PCR扩增,凝胶电泳检测扩增产物,并进行测序,根据测序结果进行序列比对,检测敲除位点的突变情况。结果显示，OsTZF7基因敲除的两个株系均为纯合突变，其中4号株系为碱基缺失，两条染色体均缺失一个碱基；6号株系为碱基***，两条染色体均***一个碱基(图2B)。
实施例4转基因水稻的苗期盐胁迫及PEG处理。
将OsTZF7过表达和基因敲除的转基因家系种子去壳消毒(75％酒精处理1min，1.5％NaClO处理20min，无菌水清洗5次)，在含有50mg/L潮霉素的1/2MS培养基上发芽，野生型对照晚一天播于不含潮霉素的1/2MS培养基上。待发芽2～3天后挑选发芽好且长势一致的种子，转移到96板上，放入水稻营养液中进行培养,待苗长至4叶期时,将苗转移至含120mM NaCl进行盐胁迫处理，处理3天后将苗转移至正常营养液中进行复水,在光照培养室生长4天后观察表型，统计植株的存活率。；将苗转移至含20％PEG的水稻营养液中进行模拟干旱胁迫处理,处理15天后将苗转移至正常营养液中进行复水,在光照培养室生长7天后观察表型，统计植株的存活率。处理过程中观察表型并拍照。
结果显示，正常生长条件下，OsTZF7转基因植株与野生型植株没有明显的差异；用120mM NaCl胁迫处理3天后，相比野生型，OsTZF7过表达转基因植株水稻叶片翠绿，茎秆直立，表现出明显的耐盐性，而OsTZF7基因敲除植株则更早出现叶片枯黄现象，表现出盐敏感性(图3A)。复水4天后，OsTZF7过表达植株存活率为27.08％，WT存活率为10.415％，而OsTZF7基因敲除植株的存活率为0，即各株系存活率OsTZF7OE>WT>OsTZF7Cas9(图3B)。PEG模拟干旱处理15天后，OsTZF7过表达转基因植株的叶片更为挺拔，卷曲程度低于野生型；相反，基因敲除转基因植株萎蔫程度要高于野生型；即过表达转基因植株存活率高于野生型，而基因敲除转基因植株存活率低于野生型(图4)。该结果表明过表达OsTZF7提高了水稻苗期对盐胁迫以及模拟干旱胁迫的抗性。
综上所述，尽管已引证一些具体实例对本发明作了详细的说明，但对本领域技术人员来说，只要不离开本发明的精神和范围，作各种变化或修正是显然的。
序列表
<110> 福建省亚热带植物研究所；厦门大学
<120> 水稻抗逆相关基因OsTZF7及其编码蛋白与应用
<130> OsTZF7
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 768
<212> DNA
<213> Oryza sativa
<400> 1
atggcgagcc gagagcacct cctgctcgac ccggcggcgc tggccgtctc ttgggctgac 60
cccgctgcgg tggagatccc gcccgagctc ctcgccgcgc tgggtgagta cctgtccgcc 120
aggcgtagcg acggggaggc cgaggccgac gccgaggcgg aggctgatga tgagttcatg 180
atgtacgagt tcaaggtgcg gcggtgcgcg cgggcgcgga gccacgactg gacggcgtgc 240
ccgtacgcgc accctggcga ggccgcgagg cggcgtgacc cgaggcgcgt ggcgtacacg 300
ggcgagccgt gcccggactt ccgccgccgg ccgggcgccg cgtgcccgag gggcagcacg 360
tgcccgttcg cgcacggcac gttcgagctc tggctccacc cgtcgcgcta ccgcacgcgg 420
ccgtgccgcg cgggcgtcgc gtgccgccgc cgcgtctgct tcttcgcgca caccgccggc 480
gagctccgcg ccgggtccaa ggaagactcg ccgctgtcgc tctcccccaa gtcgaccctg 540
gcctccctct gggagtcgcc gccggtgtcg ccggtggagg ggcggaggtg ggtggacggc 600
atcgatgaat gcgacgcgga cgcggagatg gaagagttga tgttcgcaat gcgggagctc 660
ggcctccgga aggtgaggcc gtcggcatcg tccgtaacgc cggtgctacc gccggtgacg 720
gacgaggacg ggccggattt cgggtgggtg tccgagcttg tgatgtag 768
<210> 2
<211> 255
<212> PRT
<213> Oryza sativa
<400> 2
Met Ala Ser Arg Glu His Leu Leu Leu Asp Pro Ala Ala Leu Ala Val
1 5 10 15
Ser Trp Ala Asp Pro Ala Ala Val Glu Ile Pro Pro Glu Leu Leu Ala
20 25 30
Ala Leu Gly Glu Tyr Leu Ser Ala Arg Arg Ser Asp Gly Glu Ala Glu
35 40 45
Ala Asp Ala Glu Ala Glu Ala Asp Asp Glu Phe Met Met Tyr Glu Phe
50 55 60
Lys Val Arg Arg Cys Ala Arg Ala Arg Ser His Asp Trp Thr Ala Cys
65 70 75 80
Pro Tyr Ala His Pro Gly Glu Ala Ala Arg Arg Arg Asp Pro Arg Arg
85 90 95
Val Ala Tyr Thr Gly Glu Pro Cys Pro Asp Phe Arg Arg Arg Pro Gly
100 105 110
Ala Ala Cys Pro Arg Gly Ser Thr Cys Pro Phe Ala His Gly Thr Phe
115 120 125
Glu Leu Trp Leu His Pro Ser Arg Tyr Arg Thr Arg Pro Cys Arg Ala
130 135 140
Gly Val Ala Cys Arg Arg Arg Val Cys Phe Phe Ala His Thr Ala Gly
145 150 155 160
Glu Leu Arg Ala Gly Ser Lys Glu Asp Ser Pro Leu Ser Leu Ser Pro
165 170 175
Lys Ser Thr Leu Ala Ser Leu Trp Glu Ser Pro Pro Val Ser Pro Val
180 185 190
Glu Gly Arg Arg Trp Val Asp Gly Ile Asp Glu Cys Asp Ala Asp Ala
195 200 205
Glu Met Glu Glu Leu Met Phe Ala Met Arg Glu Leu Gly Leu Arg Lys
210 215 220
Val Arg Pro Ser Ala Ser Ser Val Thr Pro Val Leu Pro Pro Val Thr
225 230 235 240
Asp Glu Asp Gly Pro Asp Phe Gly Trp Val Ser Glu Leu Val Met
245 250 255
<210> 3
<211> 21
<212> DNA
<213> Oryza sativa
<400> 3
tatggcgagc cgagagcacc t 21
<210> 4
<211> 21
<212> DNA
<213> Oryza sativa
<400> 4
ctacatcaca agctcggaca c 21
<210> 5
<211> 20
<212> DNA
<213> Oryza sativa
<400> 5
gcggagatgg aagagttgat 20
<210> 6
<211> 20
<212> DNA
<213> Oryza sativa
<400> 6
tacatcacaa gctcggacac 20
<210> 7
<211> 26
<212> DNA
<213> Oryza sativa
<400> 7
cctctgtgaa gattgtgatg ccctac 26
<210> 8
<211> 22
<212> DNA
<213> Oryza sativa
<400> 8
caagaccacg ccaacggata aa 22
<210> 9
<211> 25
<212> DNA
<213> Oryza sativa
<400> 9
ggcattgggt gagtacctgt ccgcc 25
<210> 10
<211> 25
<212> DNA
<213> Oryza sativa
<400> 10
aaacggcgga caggtactca cccat 25
Claims (7)
1.水稻抗逆相关基因OsTZF7，其特征在于包括：(1)SEQ ID NO:1所示的DNA序列；或(2)与SEQ ID NO:1至少90％同源的DNA序列；或(3)功能相当于SEQ ID NO:1所示序列的亚片段。
2.如权利要求1所述水稻抗逆相关基因OsTZF7，其特征在于其核苷酸序列如SEQ NO：1所述序列的1～768位碱基所示。
3.水稻抗逆相关基因OsTZF7编码蛋白，其特征在于所述蛋白的氨基酸序列如SEQ IDNO:2所示，或为所述SEQ ID NO:2序列的同源序列、或保守性变异体、或等位变异体、或天然突变体、或诱导突变体。
4.包含如权利要求1所述水稻抗逆相关基因OsTZF7基因编码的重组载体。
5.如权利要求4所述重组载体，其特征在于构建所述重组载体所选用的载体为Ti质粒或植物病毒载体，通过转基因，使OsTZF7基因在水稻中超量表达，用于提高水稻对干旱及盐胁迫耐受能力。
6.将权利要求1所述水稻抗逆相关基因OsTZF7或权利要求4所述重组载体导入宿主细胞而成的转化体，所述宿主为水稻。
7.如权利要求1所述水稻抗逆相关基因OsTZF7在提高植物抗逆性中的应用。
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