CN117305319A - 板栗非特异脂质转运蛋白CmnsLTP6.9基因及其应用 - Google Patents
板栗非特异脂质转运蛋白CmnsLTP6.9基因及其应用 Download PDFInfo
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Abstract
本发明公开了板栗非特异脂质转运蛋白CmnsLTP6.9基因及其应用。通过板栗不同组织定量PCR分析，发现CmnsLTP6.9基因在板栗雄花中特异表达，且受渗透/干旱强烈诱导；将CmnsLTP6.9基因通过农杆菌GV3101介导稳定转化拟南芥，通过生化分析，结果证实CmnsLTP6.9基因能显著缓解渗透/干旱胁迫下活性氧损伤，显著提高植株的渗透/干旱抗性。CmnsLTP6.9基因可用于构建板栗转基因植物，提高板栗等植物抗旱/渗透能力，在其他植物增强抗旱/渗透胁迫方面也具有很好的应用前景。
Description
技术领域
本发明属于植物基因工程技术领域，具体涉及板栗非特异脂质转运蛋白CmnsLTP6.9基因及其应用。
背景技术
板栗(Castanea mollissima Blume)原产于我国，是壳斗科栗属乔木，兼具经济和生态价值。板栗坚果营养丰富，被誉为“铁杆庄稼”，我国板栗产量占世界产量的80％以上(http://www.fao.org/home/en/)。板栗仁可用于食品加工，板栗壳、花和叶片还广泛用于医药、生物质和催化剂材料。板栗耐瘠薄，涵养水分，是荒山造林、绿化的重要生态树种。板栗大多生长在贫瘠山区，干旱和渗透胁迫是两种常见的非生物胁迫，也是限制板栗生长发育，影响果实品质和产量的关键环境因素。由于板栗童期长、遗传杂合度高，利用分子生物技术和基因工程改良是加速板栗育种的有效手段。挖掘调控板栗干旱和渗透胁迫的关键基因，解析其功能，对培育抗旱板栗新品种有重要意义。目前关于板栗抗旱、抗渗透胁迫的研究主要集中在生理水平，关于板栗干旱和渗透胁迫的关键基因的研究鲜有报道。
脂质作为重要的次生代谢产物，在植物应对环境胁迫的过程中发挥至关重要的作用，例如膜稳定、植物细胞外囊泡功能以及角质层和细胞壁完整性。在植物中，大量证据表明非特异性脂质转运蛋白(non-specific lipid transfer proteins，nsLTPs)参与一系列脂质相关的生物过程，包括油脂积累、角质蜡沉积、花粉壁形成和角质合成。nsLTPs还响应各种非生物/生物胁迫，如干旱、高盐、热、冷胁迫、病原体防御、渗透胁迫、IAA和ABA胁迫。如水稻OsLTPL159提高了水稻苗期早期的耐冷性；烟草NtLTPI.38通过调节脂质和类黄酮合成、抗氧化活性、离子稳态和ABA信号通路来增强烟草的耐盐性；棉花GhLTP4通过重塑脂质谱、调节脱落酸稳态和改善棉花的三羧酸循环来增强耐旱性；TaLTP40和TaLTP75通过将膜脂转移到生物膜来增强小麦的耐盐性。玉米ZmLTP3和拟南芥AtLTP3分别正向调节盐胁迫和干旱胁迫。然而，关于木本植物nsLTPs在非生物胁迫下的功能研究很少，板栗中抗旱/渗透胁迫相关的nsLTPs基因未有克隆和功能验证的报道。
发明内容
本发明的目的是提供板栗抗旱/渗透胁迫CmnsLTP6.9基因及其应用，为提高板栗等植物抗旱/渗透提供了可选择的候选基因，在增强植物抗旱/渗透胁迫方面具有很好的应用潜力。
本发明根据发明人前期通过高通量测序获得板栗转录组数据，结合板栗基因组信息，分离到一个板栗nsLTP家族基因，命名为CmnsLTP6.9，其核苷酸序列如SEQ ID NO.1所示，序列长度为351bp，编码的氨基酸序列如SEQ ID NO.2所示，长度为117个氨基酸。
本发明的第一个目的是提供一种表达载体，该表达载体含有上述的板栗CmnsLTP6.9基因。
本发明的第二个目的是提供一种宿主菌，该宿主菌含有上述的表达载体。优选，所述的宿主菌为大肠杆菌或根癌农杆菌单克隆细胞系。
本发明的第三个目的是提供CmnsLTP6.9基因在提高板栗等植物抗旱/渗透胁迫中的应用。
通过对离体板栗枝条分别经过干旱和250mM甘露醇处理1h，3h，6h和24h，发现CmnsLTP6.9受干旱和甘露醇显著诱导表达。采用根癌农杆菌GV3101介导，转化获得CmnsLTP6.9超量表达的转基因拟南芥株系，对转基因第三代(T3代)植株进行抗性能力评价，证实CmnsLTP6.9可显著增强拟南芥的抗旱/渗透胁迫能力。进一步，通过对干旱和渗透胁迫处理的转基因拟南芥进行3，3'-二氨基联苯胺(DAB)和硝基蓝四唑(NBT)染色分析，结果显示：CmnsLTP6.9可通过增强活性氧(ROS)清除能力，显著减少干旱和渗透胁迫导致的氧化损伤，提高抗性水平。以上结果表明，板栗CmnsLTP6.9基因可用于构建转基因植物，在基因工程改造提升板栗等植物抗旱/渗透胁迫能力等方面具有重要作用。
附图说明
图1是CmnsLTP6.9在板栗不同组织中的相对表达量分析。其中M1：4月13日，雄花序2cm左右；M2：4月29日；M3：5月13日；M4：5月25日，雄花盛花期。F1：雌花1；F2：雌花2；F1和F2分别对M3和M4采样时期。
图2是板栗经过250mM甘露醇和干旱处理后CmnsLTP6.9基因表达分析。图2A：CmnsLTP6.9响应渗透胁迫；图2B：CmnsLTP6.9响应干旱胁迫。
图3是超量表达CmnsLTP6.9后拟南芥在渗透和干旱处理下的表型(A)和相对含水量(B)、电解质渗漏率(C)、脯氨酸(D)和MDA(E)含量。对照是渗透和干旱未经处理，WT：未转基因拟南芥；nsLTP6.9L-OE：超量表达CmnsLTP6.9后拟南芥T3代植株；nsLTP6.9S-OE：超量表达CmnsLTP6.9S(无信号肽)后拟南芥T3代植株。
图4是超量表达CmnsLTP6.9的拟南芥在渗透和干旱条件下DAB和NBT染色情况(A)，H2O2浓度(B)、抗氧化酶SOD(C)和POD活性(D)检测结果。
具体实施方式
实施例1植物表达载体pCAMBIA2300-CmnsLTP6.9L-GUS-eGFP构建
通过板栗雌雄花差异转录组分析，筛选鉴定到一个雄花特异表达基因CmnsLTP6.9，采用qRT-PCR对CmnsLTP6.9在板栗不同组织中的表达特征进行分析，发现CmnsLTP6.9在板栗雄花中表达量显著高于茎、叶、雌花等组织(图1)。
将水培板栗幼嫩枝条，通过渗透(250mM甘露醇)和干旱(无水条件)处理1h、3h、6h、9h和24h后，采用实时荧光定量PCR分析CmnsLTP6.9基因在不同处理条件和时间后基因表达模式，采用CmnsLTP6.9特异引物qF：5'-CTGTGAGGTCAATGTGCTTGG-3'和qR：5'-CAACACTCCCATGATGGCTT AG-3'进行定量PCR扩增。以板栗看家基因β-actin为内参，引物为：aF：5'-TTGACTATGAGCAGGAACTT-3'和aR：5'-TTGTAGGTGGTCTCGTGAAT-3'。荧光定量采用Monad MonAmpTMGreen qPCR Mix(Vazyme)试剂盒，反应在ABI 7500实时定量PCR仪上进行(Applied Biosystems，USA)。基因的相对表达量通过2-ΔΔC(t)公式计算，即经内参β-actin校正后，以处理0h的样本表达水平为1计算相对表达量。结果表明CmnsLTP6.9L基因在500mM甘露醇处理1h、3h和6h后显著上调表达，且处理1h后即被显著诱导(图2A)；CmnsLTP6.9L基因在干旱处理6h后显著上调表达(图2B)。
申请人利用板栗转录组数据库和板栗基因组(https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_000763605.2/)，克隆到CmnsLTP6.9基因，其功能未知。提取板栗品种的雄花RNA，反转录为单链cDNA。以此cDNA为模板，以CmnsLTP6.9-BamHⅠ-F(SEQ ID NO.3)为正向引物，CmnsLTP6.9-KpnⅠ-R(SEQ ID NO.4)为反向引物，采用TaKaRa ExDNAPolymerase试剂盒，根据其说明书进行PCR反应，扩增CmnsLTP6.9基因全长序列，反应体系如下：
扩增程序为：94℃变性30s，54℃退火30s，72℃延伸1min，进行32个循环，终止反应。反应产物通过1％琼脂糖凝胶电泳，并采用QiAGel Extraction kit(QIAGEN,Hilden,Germany)试剂盒回收目的条带DNA，目的条带DNA通过/>-T1Simple CloningKit进行T-A克隆，载体连接反应体系如下：
目的条带DNA约30ng
-T1Simple Cloning Vctor 1μL
ddH2O补至5μL
连接产物热激转化至大肠杆菌DH5α。挑取白色单克隆，用克隆引物进行PCR阳性鉴定，阳性克隆菌液送至武汉天一华煜基因科技有限公司测序，测序结果用SnapGene软件进行比对。测序序列(SEQ ID NO.1)与板栗基因组参考序列比对，选择测序正确质粒。质粒和PCAMBIA 2300-GUS-GFP双元载体采用Bam HI和KpnⅠ内切酶进行双酶切，质粒酶切产物经过1.0％琼脂糖凝胶电泳，取约350bp大小的目的条带回收。酶切后载体(约12kb)回收片段(约350bp)通过T4连接酶连接，反应体系如下：
使用PCR仪控温，16℃过夜(12-16h)连接。取连接产物5μL热激转化至大肠杆菌DH5α，挑取饱满的单克隆菌落，使用CmnsLTP6.9-BamHⅠ-F和CmnsLTP6.9-KpnⅠ-R引物进行阳性鉴定，选取含有目的基因条带的菌液扩大培养，并提取质粒，使用限制性内切酶KpnⅠ和BamHⅠ进行酶切验证pCAMBIA2300-CmnsLTP6.9-GUS-eGFP质粒载体构建成功，最后将阳性克隆菌液添加30％甘油后置于-80℃保存，将pCAMBIA2300-CmnsLTP6.9-GUS-eGFP质粒置于-20℃保存。
实施例2农杆菌介导拟南芥蘸花法遗传转化
取1μg左右的pCAMBIA2300-CmnsLTP6.9-GUS-eGFP载体质粒，加入到200μL农杆菌GV3101感受态细胞中，热激法转化，挑取单克隆细菌扩繁，利用CmnsLTP6.9-BamHⅠ-F和CmnsLTP6.9-KpnⅠ-R进行PCR筛选阳性克隆。验证后的农杆菌GV3101加30％甘油保存于-80℃备用。将野生型拟南芥种子均匀撒播于营养土表面，于4℃避光春化2d后在22℃的光照16h/暗8h培养箱中培养10d左右单株移栽于土钵中生长。待拟南芥开始抽薹时，及时掐去主花序，促进更多的侧花序生长，当拟南芥大多数花序刚开始露白但未开放时进行以下步骤：
(1)GV3101农杆菌菌液培养
挑取阳性单克隆于5mL LB培养基(Rif＝50mg/L，Kan＝50mg/L)，28℃、200rpm振荡培养至OD600值为0.4～0.6；取2mL该菌液于200mL LB培养基(利福平＝50mg/L，卡那霉素＝50mg/L)，28℃、200rpm振荡培养至OD600值为1.8左右，4500rpm、常温离心20min，收集所有菌体。配置新鲜浸染液(1/2MS，5.0％蔗糖，0.05％ Silwet L-77，pH＝5.7)。用新鲜浸染液重悬菌体，将OD600值调至0.8备用。
(2)浸染花序
将拟南芥莲座叶片和土固定好，倒置于浸染液中，使植物上部分花序浸入浸染液10min左右；蘸好花序的拟南芥卧倒平放于托盘，避光处理24h(注意保湿)。第二天移入正常生长的培养箱中培养，第三天重复浸染一次。生长一个月左右收集T0代种子。抗性培养基(1/2MS，含卡那霉素100mg/L)播种，生长2周左右的苗子，长出真叶并且很绿的苗子初步视为阳性苗，黄化苗子为非阳性苗，进一步用激发光照射阳性植株，阳性株带有绿色荧光，而非阳性植株无绿色荧光。将阳性苗子移栽至营养土中生长并收取T1代种子；如上自交纯化、筛选，获得转基因T3代植株。通过实时荧光定量PCR分析转基因拟南芥植株中的CmnsLTP6.9基因表达量，采用CmnsLTP6.9特异引物qF：5'-CTGTGAGGTCAATGTGCTTGG-3'和qR：5'-CAACACTCCCATGATGGCTT AG-3'进行定量PCR扩增。具体过程同实施例2中定量分析方法。选取表达量最高的株系进行后续试验研究。
实施例3CmnsLTP6.9转基因拟南芥抗性鉴定
为了深入探究CmnsLTP6.9的基因功能，同时获得CmnsLTP6.9无信号肽的转基因拟南芥株系CmnsLTP6.9S-OE(CmnsLTP6.9S基因序列如SEQ ID NO.5所示)，用于研究信号肽在CmnsLTP6.9中的作用。
将T3转基因纯系种子和野生型种子经酒精消毒后播种于1/2MS固体培养基(含卡那霉素100mg/L)中(方法同上)，于4℃冰箱中春化2d，移至22℃、16h光照/8h黑暗的人工气候箱中培养。
选取生长一致1个月龄拟南芥用于渗透和干旱处理。停止给拟南芥植物浇水两周以模拟干旱胁迫；用250mM甘露醇溶液灌溉幼苗以诱导渗透胁迫。将所有植物转移至23±2℃、16小时/8小时光/暗循环的人工气候箱中。处理和未处理的表型拍照观察，结果显示：与未转基因拟南芥(WT)和转基因植株(nsLTP6.9S-OE)相比，转基因植株(nsLTP6.9L-OE)在500mM甘露醇和干旱处理下生长正常，表现明显的耐受性(图3)。同时分析了渗透和干旱处理后叶片相对含水量和相对电解质渗漏量、叶片脯氨酸和丙二醛(MDA)含量，采用脯氨酸测定试剂盒和MDA测定试剂盒(南京建成生物工程研究所，中国南京)测定。所有检测进行了三次重复。进一步，使用DAB和NBT染色来检测渗透和干旱处理下叶片中H2O2和O2-的积累，使用过氧化氢试剂盒(南京建成生物工程研究所，中国南京)来检测H2O2浓度。抗氧化酶SOD和POD活性的测定采用南京建成研究所的试剂盒分析。每个测定重复三次。DAB和NBT染色结果显示：转基因植株(nsLTP6.9L-OE)中H2O2和O2-含量显著降低，抗氧化酶SOD和POD活性显著增强(图4)。
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.板栗非特异脂质转运蛋白CmnsLTP6.9基因，其特征在于，所述CmnsLTP6.9基因的核苷酸序列如SEQ ID NO.1所示。
2.含有权利要求1所述CmnsLTP6.9基因的表达载体或宿主菌。
3.用于扩增权利要求1所述CmnsLTP6.9基因的引物，其特征在于，所述引物的序列如SEQ ID NO.3和4所示。
4.权利要求1所述的基因或权利要求2所述的载体或宿主菌在增强植物抗渗透胁迫或干旱胁迫中的应用。
5.根据权利要求4所述的应用，其特征在于，所述的植物为板栗或拟南芥。
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