CN115044608A - 忽地笑LaOMT1及其编码基因在植物耐受盐胁迫中的应用 - Google Patents
忽地笑LaOMT1及其编码基因在植物耐受盐胁迫中的应用 Download PDFInfo
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- CN115044608A CN115044608A CN202210603452.3A CN202210603452A CN115044608A CN 115044608 A CN115044608 A CN 115044608A CN 202210603452 A CN202210603452 A CN 202210603452A CN 115044608 A CN115044608 A CN 115044608A
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Abstract
本发明公开了忽地笑LaOMT1蛋白编码基因在提高植物对耐受盐汞胁迫中的应用。将该基因在拟南芥的野生型中进行异源过量表达，发现获得的过表达的拟南芥植株在氯化钠处理下长势增加，主根长较野生型拟南芥WT显著增加，表明该基因可以作为目的基因导入植物来增强转基因植物对盐胁迫的耐受能力。本发明对培育耐盐植物品种提供新的基因靶点和资源。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及忽地笑LaOMT1蛋白及其编码基因在调控植物盐胁迫耐受中的应用。
背景技术
氧甲基化是不同生物体内普遍存在的一种反应。由氧甲基转移酶（O-methyltransferase, OMT）催化。在S-腺苷-L-甲硫氨酸（S-adenosyl-L-methionine，SAM）提供甲基的条件下，OMT能将底物的氧位甲基化，从而生成对应的甲基化产物，同时SAM转化成S-腺苷-L-高半胱氨酸（S-adenosyl-L-homocysteine, SAH）。研究表明，植物体内OMT参与许多化合物（色素、花香、木质素和防御素等）的合成。而且，不同的OMTs可以催化相同的底物，同一个OMT可以催化不同的底物，同一个OMT催化具有多羟基位点的底物时，其产物可能有多个。
植物OMT根据其编码的氨基酸数目，蛋白分子量大小和催化底物的不同分为Ⅰ类和Ⅱ类，Ⅰ类OMT分子量大小约27~30kDa,具有231–248个氨基酸，在木质素合成中起到关键作用。除Ⅰ类OMTs其余的OMT都归为Ⅱ类，分子量大小约38~42 kDa，具有344–383个氨基酸，主要是在类黄酮等植物次生代谢生物合成中起到重要作用。此外，研究表明，植物OMTs还可能在植物非生物胁迫响应中起到重要作用。目前，有关Ⅰ类OMT是否在植物耐受盐胁迫中起作用还未有关注。
发明内容
本发明的目的在于提供忽地笑LaOMT1蛋白及其编码基因在提高植物耐受盐胁迫中的应用。
本发明的目的可通过以下技术方案实现：
忽地笑LaOMT1蛋白编码基因，其核苷酸序列为：SEQ ID NO.1。
忽地笑LaOMT1蛋白，其氨基酸序列为：SEQ ID NO.2。
含有本发明所述的忽地笑LaOMT1蛋白编码基因的重组表达载体。
使用LaOMT1蛋白编码基因构建植物过表达载体时，可在其转录起始核苷酸前加入任何一种强启动子或诱导型启动子。为了便于对转基因植物进行筛选及鉴定，可对所用植物表达载体加工，如在载体上加入选择性标记基因（GUS基因、GFP基因等）。
忽地笑LaOMT1蛋白编码基因在通过基因工程提高植物盐胁迫耐受方面的应用；所述的忽地笑LaOMT1蛋白编码基因，其核苷酸序列为SEQ ID NO.1。
含有忽地笑LaOMT1蛋白编码基因的重组表达载体在通过基因工程提高植物盐胁迫耐受方面的应用；所述的忽地笑LaOMT1蛋白编码基因，其核苷酸序列为SEQ ID NO.1。
本发明的有益效果。
1、本发明通过研究，提供了忽地笑LaOMT1编码基因在提高拟南芥盐胁迫耐受中的生物功能。
2、构建了植物过表达载体pCHF1301-LaOMT1（图1A），并将其在拟南芥的野生型中进行异源表达（图1B）。对筛选出的T3代阳性苗进行表型鉴定，发现过表达LaOMT1蛋白编码基因的植株在盐胁迫处理下长势增强（图2），主要表现为主根长较野生型WT对照显著增加（图3），表明该基因可以作为目的基因导入植物，提高转基因植物的耐盐能力。
附图说明
图1A为LaOMT1编码基因过表达载体构建图。
图1B为转基因植株中LaOMT1蛋白编码基因表达量分析。其中WT为野生型拟南芥对照， OE为转LaOMT1蛋白编码基因的不同拟南芥转基因株系。
图2为不同浓度氯化钠处理下各植株的表型。其中WT为野生型拟南芥，OE为转LaCOMT蛋白编码基因的不同拟南芥转基因株系。
图3为1不同浓度氯化钠处理下各植株的主根长统计结果。其中WT为野生型拟南芥， OE为转LaOMT1蛋白编码基因的不同拟南芥转基因株系。
具体实施方式：
以下结合具体实施方式对本发明做进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。下列实施例中，如无特殊说明，序列表中各核苷酸序列的第1位均为相应DNA的5’末端核苷酸，末位均为相应DNA的3’末端核苷酸。
实例1、LaOMT1蛋白编码基因在拟南芥中的遗传转化。
一、重组载体的构建
提取忽地笑幼苗的总RNA，将RNA用逆转录酶合成cDNA。
以双酶切法构LaOMT1蛋白编码基因的过表达载体，设计包含LaOMT1蛋白编码基因完整开放阅读框（ORF）及Kpn I和Xba I两个酶切位点的引物（不包含终止密码子），引物序列如SEQ ID NO.3和SEQ ID NO.4 所示。PCR扩增得到含有LaOMT1蛋白编码基因的PCR产物。
上述PCR扩增产物和pCAMBIA1301-GFP载体质粒分别用Kpn I和Xba I双酶切。将酶切后的PCR产物与骨架载体采用T4连接酶在25ºC连接2 h，得到重组载体，将序列正确的重组载体命名为pCHF1301-LaOMT1。pCHF1301-LaOMT1的部分结构如图1A所示，pCHF1301-LaOMT1能表达SEQ ID NO.2所示的蛋白质。
将上述得到的重组表达载体pCHF1301-LaOMT1转化到根癌农杆菌EHA105，得到含有重组表达载体的农杆菌。
二、拟南芥的遗传转化及筛选
将获得的含pCHF1301-LaOMT1的根癌农杆菌EHA105通过浸花侵染法转化拟南芥Columbia-0生态型，并收获T1代种子。将T1代拟南芥种子播种于含有20 mg/L的1/2 MS固体培养基上，进行潮霉素抗性筛选，筛选得到的抗潮霉素的转基因株系幼苗分别移栽至装有蛭石的盆中，用1/2 MS液体培养基适时浇灌，22 ºC长日照条件下生长。观察并记录T2及T3代转基因拟南芥的生长发育过程及表型性状。
提取筛选获得的T3代纯合转基因拟南芥的总RNA，反转录得到cDNA，利用设计的用于LaOMT1蛋白编码基因扩增引物（引物序列如SEQ ID NO.5和SEQ ID NO.6 所示）进行表达量检测。同时采用拟南芥Actin2作为内参，设计引物（引物序列如SEQ ID NO.7和SEQ IDNO.8 所示）。结果如图1B所示，在野生型拟南芥WT拟南芥植株中，未能检测到LaOMT1基因，而筛选到的3个过表达LaOMT1蛋白编码基因的拟南芥株系则能检测到LaOMT1基因的表达。
实例2、忽地笑LaOMT1蛋白编码基因的转基因拟南芥在与野生型植株在氯化钠胁迫下的耐受性比较。
将野生型拟南芥WT、和过表达LaOMT1编码蛋白基因的拟南芥不同株系（OE-4、OE-15和OE-22）同时播种于含有0 mM（对照）、50 mM、100 mM和150 mM氯化钠的1/2 MS固体培养基中，处理7天后观察拟南芥根系生长状态的变化。结果表明在正常的1/2 MS培养基上，过表达LaOMT1编码蛋白基因的拟南芥与野生型拟南芥WT拟南芥植株形态差异略有差异。而在含有不同浓度氯化钠的1/2 MS固体培养基上，转基因植株形态更大、长势更好（图2）。具体表现为地下部根系更发达，其中转基因植株主根长显著大于野生型拟南芥WT（图3）。表明该基因可以作为目的基因导入植物，提高转基因植物耐受盐胁迫的能力。
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Claims (3)
1.忽地笑LaOMT1蛋白编码基因在通过基因工程增强植物耐受盐胁迫方面的应用；所述的LaOMT1蛋白编码基因所编码的蛋白氨基酸序列如SEQ ID NO.2所示；所述植物为拟南芥。
2.含有忽地笑LaOMT1蛋白编码基因的重组表达载体在通过基因工程增强植物耐受盐胁迫方面的应用；所述的忽地笑LaOMT1蛋白编码基因所编码的蛋白氨基酸序列如SEQ IDNO.2所示；所述植物为拟南芥。
3.根据权利要求2所述的应用，其特征在于将含有忽地笑LaOMT1蛋白编码基因的过表达载体导入拟南芥并得到过表达LaOMT1蛋白编码基因的拟南芥，从而增强拟南芥的盐胁迫耐受性。
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