CN117700508B - 基因PagDR1在提高林木耐旱性上的应用 - Google Patents
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Abstract
本发明公开了基因PagDR1在提高林木耐旱性上的应用，所述基因PagDR1为编码SEQ ID No.2所示氨基酸序列的基因。本发明首先从84K杨基因组中克隆获得PagDR1基因并构建表达载体35S::PagDR1‑Venus，利用遗传转化技术获得35S::PagDR1‑Venus 84K杨过表达植株。将野生型与该过表达株系进行干旱处理，并检测其离子渗透率，发现过表达植株相较于野生型表现出更高的耐旱性和较低的离子渗透率，研究结果表明过表达PagDR1基因可以显著提高林木的耐旱性。
Description
技术领域
本发明属于基因工程领域，涉及一种林木抗旱相关基因PagDR1及其应用。
背景技术
植物在自然环境下容易受到干旱、盐、高温、低温和重金属胁迫。非生物胁迫影响植物的分子、生理和生化形态进一步影响植物的多种生命活动。为了应对胁迫，植物进化出一系列复杂的防御机制帮助其生存和保持生态平衡。高温、降雨量减少的逐步加剧导致干旱胁迫的发生，成为了制约林木生长的重要因素。林木可以调节气候、保持生态平衡，同时对于提供木质、纤维素等材料也具有很大的价值。但是，干旱胁迫对于林木的生长威胁很大，甚至影响生态平衡。因此，研究林木应答非生物胁迫的机制对于培育林木耐旱新物种具有重要的意义。
蛋白质的翻译后修饰是真核生物中广泛存在的一种重要调节机制，对生物的生长发育起着重要的作用。研究表明很多转录因子通过翻译后修饰，调节其活性或稳定性来影响植物对逆境胁迫的响应。
干旱胁迫对于木本植物会造成严重的损害，一方面会使土壤中的可利用水分减少，引起植物脱水；另一方面使植物蒸腾作用增强，植物组织含水量降低，从而损伤植物细胞膜系统、引发细胞机械性损伤、抑制植物光合作用等正常生理活动。
发明内容
本发明所要解决的技术问题为：提供一种基因PagDR1新的用途。
本发明的技术方案为：基因PagDR1在提高林木耐旱性上的应用，所述基因PagDR1为编码SEQ ID No.2所示氨基酸序列的基因。
进一步地，由于密码子的简并性，编码SEQ ID No.2所示氨基酸序列的基因可以有很多不同的组合。优选地，所述基因PagDR1的核苷酸序列如SEQ ID No.1所示。
进一步地，所述应用为在林木中过表达基因PagDR1，从而提高林木耐旱性。
进一步地，所述林木为杨树。
与现有技术相比，本发明具有以下有益效果：
本发明首先从84K杨基因组中克隆获得PagDR1基因并构建表达载体35S::PagDR1-Ven us，利用遗传转化技术获得35S::PagDR1-Venus 84K杨过表达植株。将野生型与该过表达株系进行干旱处理，并检测其离子渗透率，发现过表达植株相较于野生型表现出更高的耐旱性和较低的离子渗透率，研究结果表明过表达PagDR1基因可以显著提高林木的耐旱性。
附图说明
图1基因PagDR1的CDS片段电泳图。
图2干旱处理后PagDR1的表达水平。
图3 35S::PagDR1-Venus 84K杨过表达株系的鉴定及表达量检测。
图4干旱胁迫下35S::PagDR1-Venus 84K杨过表达植株的表型观察及分析。
具体实施方式
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试验材料，如无特殊说明，均为从商业渠道购买得到的。
1.基因克隆
设计PagDR1扩增引物，利用设计的引物从84K杨cDNA中扩增得到CDS序列并将其连接Venus构建表达载体，测序结果显示基因编码区全长共有2640bp(SEQ ID No.1，编码的蛋白序列如SEQ ID No.2所示)。利用NCBI网站对PagDR1进行了结构域预测，发现其含有常见的SAP，PHD，SP-RING，PINIT和SXS结构域。表1为扩增所设计的引物序列。CDS扩增体系如下(共50μL)
表1基因PagDR1的CDS引物序列
| Primer name | Primer sequences(5`-3`) |
| PagDR1 CDS-F | ATGGATTTAGTTGCTAGT |
| PagDR1 CDS-R | TTCAGAGTCCGAGTCAAT |
实验结果：
经过1.0％的琼脂糖凝胶电泳检测扩增产物为2640bp的片段，如图1所示，将扩增片段通过酶切连接到含有Venus报告基因的表达载体中，提取阳性质粒进行测序。测序结果与SEQ ID No.1所示核苷酸序列一致，说明35S::PagDR1-Venus表达载体构建完成。
2.过表达84K杨植株
将构建成功的含35S::PagDR1-Venus的表达载体转入农杆菌中，通过叶盘转化法转入84K杨材料。通过筛选培养基诱导生芽生根获得过表达84K杨植株，选择长势良好的抗性幼苗移栽至土里继续栽培，并进一步进行转基因植株鉴定。(图3)。
3.干旱处理
将长势良好的1月龄84K杨从土壤里完整取出，用ddH2O洗掉根部土壤并用吸水纸除去残留水分后，放在干燥洁净的培养瓶中进行干旱处理，在0、0.5、1、2h收样提取RNA反转录cDNA用于qRT-PCR检测PagDR1基因在不同处理时间段的表达变化(图2)。
将同一时期的野生型和35S::PagDR1-Venus过表达84K杨株系栽到土里正常培养1个月之后进行干旱处理，期间进行表型观察并对其进行离子渗透率检测。结果如图4所示，干旱处理4天后，野生型叶片出现卷曲，顶端弯曲；过表达株系叶片平展，顶端直立，表现出更强的耐干旱表型。此外对几种株系的相对离子渗透率结果也显示，干旱处理后过表达株系的相对离子渗透率明显低于84K杨。这些结果均表明PagDR1是一个重要的正向调节因子，可以增强林木对于干旱胁迫的耐受性。
Claims (2)
1.基因PagDR1在提高杨树耐旱性上的应用，所述基因PagDR1为编码SEQ ID No.2所示氨基酸序列的基因，所述应用为在杨树中过表达基因PagDR1，从而提高杨树耐旱性。
2. 根据权利要求1所述的应用，其特征在于，所述基因PagDR1的核苷酸序列如SEQ IDNo.1所示。
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| CN105504032A (zh) * | 2014-09-26 | 2016-04-20 | 中国科学院植物研究所 | 一种与植物抗逆性相关的GmSIZ1a/b蛋白及其编码基因与应用 |
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