CN114250246B - 一种抗极端高温生长条件的猕猴桃种质材料及培育方法 - Google Patents
一种抗极端高温生长条件的猕猴桃种质材料及培育方法 Download PDFInfo
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- CN114250246B CN114250246B CN202111570913.3A CN202111570913A CN114250246B CN 114250246 B CN114250246 B CN 114250246B CN 202111570913 A CN202111570913 A CN 202111570913A CN 114250246 B CN114250246 B CN 114250246B
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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Abstract
本发明提供一种对极端高温生长条件有强抗性的猕猴桃种质材料及培育方法，所述猕猴桃种质材料通过过量表达热激转录因子HsfA2‑1获得。所述猕猴桃种质材料在抗高温胁迫方面具有极强的抗性，对50℃极端高温胁迫至少能持续抵抗2小时，并且在经受胁迫后能够继续正常生长。本发明以‘东红’猕猴桃中热激转录因子AcHsfA2‑1为例，详细阐述该种质材料的培育方法。其他与AcHsfA2‑1的核苷酸序列或者氨基酸序列同源性高于90％的基因在此方面的应用，均在本发明的保护范围内。本发明培育方法设计合理、操作方便，易于推广应用。
Description
技术领域
本发明属于植物分子生物技术和基因工程领域，涉及一种抗极端高温生长条件的猕猴桃种质材料及培育方法。
背景技术
植物定植于土壤中，无法通过移动躲避环境胁迫，因此抵抗环境胁迫的能力对植物的生长发育至关重要。随着全球变暖的加剧，高温胁迫对植物生长发育的危害日益严重，增强高温抗性对植物的生存和繁衍越来越重要。高温胁迫导致植物细胞内活性氧增多、抗氧化酶活性降低和花粉萌发率下降，进而严重影响作物产量和品质。来自中国和印度的统计数据显示，年均温度上升0.5～1％将导致小麦产量减少5～10％，而基于此数据的预测显示，相较于15℃，当全球年均温度上升至30℃时，作物的产量将减少50％以上。
猕猴桃是起源于中国的多年生藤本果树，果实富含维生素C和多种微量元素，营养价值高，被誉为“水果之王”，然而在猕猴桃的栽培过程中，高温胁迫始终影响着果实的产量和品质。例如，当温度高于27℃时，毛花猕猴桃(Actinidia eriantha)的花粉萌发率显著降低，不利于果实的形成；高温胁迫减少美味猕猴桃‘海沃德’果实中维生素C的含量，降低果实品质；此外，高温抑制美味猕猴桃植株中抗氧化酶(如超氧化物歧化酶SOD)的活性，进而抑制植株的抗逆性。因此，培育抗高温胁迫的种质材料具有重要的意义。
根据已有的研究进展，当遭遇高温胁迫时，植物会迅速产生多种生理变化以响应胁迫，减少高温胁迫对生长的危害。热激转录因子(Heat shock transcription factors，Hsfs)和热激蛋白(Heat-shock proteins，Hsps)是植物中与高温胁迫关系最为密切的两类基因，其基因和蛋白的表达受高温胁迫显著诱导。在高温胁迫下，大量热激转录因子和热激蛋白基因的表达量显著上升，进而响应高温胁迫。
在植物响应高温胁迫的基因调控网络中，热激转录因子可调控热激蛋白的基因表达水平，其表达量越高，对热激蛋白表达的正调控效应往往越显著，例如，在模式植物拟南芥中，Hsf基因的过表达能够引起大量热激转录蛋白基因表达上调。而热激蛋白的高表达有利于调控下游与高温抗性相关基因的表达，增强植物的耐热性，例如，苹果和柑橘中Hsp基因的表达水平均与耐热性呈正相关。因此，基于热激转录因子的功能，结合猕猴桃遗传转化技术培育抵抗高温胁迫的种质材料，具有较大的可行性和重大的产业意义。
发明内容
本发明的目的是公布一种猕猴桃种质材料，其体内与抗高温胁迫相关的热激转录因子HsfA2-1的表达量增强2000～4000倍，其中HsfA2-1的核苷酸序列为SEQ：NO.1，编码氨基酸序列为SEQ：NO.2，在无高温胁迫的正常环境下可正常生长；在高温胁迫下，可抗至少持续2小时的50℃极端高温的生长条件，叶片依然呈现绿色，未出现边缘卷曲萎蔫干枯的现象；在高温胁迫消除后，该种质材料可继续正常生长。
本发明的另一目的是公布该种质材料的培育方法：首先利用普通PCR技术，以中华猕猴桃‘东红’叶片的cDNA为模板，应用序列为SEQ NO.3和SEQ NO.4的引物，进行热激转录因子AcHsfA2-1(SEQ：NO.1)的克隆；然后将AcHsfA2-1(SEQ：NO.1)的PCR产物通过琼脂糖凝胶电泳、分离、纯化后，搭载到可用于植物遗传转化的表达载体中；再将含有AcHsfA2-1序列(SEQ：NO.1)的重组表达载体通过电击或液氮冻融等方法转化到农杆菌EHA105菌株中；最后通过农杆菌介导的猕猴桃叶盘法，将AcHsfA2-1转化到‘东红’植株中，筛选转化成功的种质材料，即可获得可抗至少持续2小时50℃极端高温生长条件的猕猴桃种质材料。
本发明以‘东红’猕猴桃中热激转录因子AcHsfA2-1为例，详细阐述该种质材料的培育方法。其他与AcHsfA2-1的核苷酸序列或者氨基酸序列同源性高于90％的基因在此方面的应用，均在本发明的保护范围内。
本发明公布的猕猴桃种质材料，在抗高温胁迫方面具有极强的抗性。普通的猕猴桃植株，经过2小时50℃极端高温胁迫后，叶片萎蔫干枯，部分叶片凋落；高温胁迫消除后的9天内，植株仍呈现萎蔫干枯、生长滞缓的状态，基本失去生命力。但是，本发明公布的猕猴桃种质材料，经过2小时50℃极端高温胁迫后，叶片依然呈现健康的绿色，未出现萎蔫干枯的现象；高温处理消除后的9天内，本发明种质材料未出现生长迟缓的现象，并且在经受胁迫后仍能继续正常生长。本发明培育方法设计合理、操作方便，易于推广应用。
附图说明
图1为种质材料的鉴定。图中(a)普通PCR技术鉴定成功转化AcHsfA2-1的种质材料；(b)实时定量PCR技术鉴定成功转化AcHsfA2-1的种质材料；(c)俯视图：以野生型为对照，检测种质材料的极端高温抗性；(d)正视图：以野生型为对照，检测种质材料的极端高温抗性。图中“WT”为“野生型”的英语缩写。
具体实施方式
本发明结合附图和实施例作进一步的说明。
实施例1
(一)研究方法
1.高温胁迫处理普通猕猴桃植株：选取生长于1/2MS生根培养基中、长势健壮的普通猕猴桃组培苗，每瓶接种有3棵作为三个生物学重复。将植株放置于50℃的恒温烘箱内，模拟自然高温胁迫条件，时间持续2小时。然后将植株重新放置于20℃的组培室内，正常培养，观察表型变化。
(二)研究结果
经过2小时50℃极端高温处理后，普通猕猴桃植株叶片萎蔫干枯，部分叶片凋落；高温处理消除后的9天内，植物仍呈现萎蔫干枯、生长滞缓的状态，基本失去生命力(附图1c和1d)。
实施例2
(一)研究方法
1.热激转录因子AcHsfA2-1的克隆
利用普通PCR技术，以中华猕猴桃‘东红’叶片的cDNA为模板，应用序列为SEQ NO.3和SEQ NO.4的引物，进行热激转录因子AcHsfA2-1的克隆。PCR体系包括15μLMaxBuffer、1.2μL SEQ NO.3引物、1.2μL SEQ NO.4引物、0.6μL dNTP Mix(10mM each)、0.6μLMax Super-Fidelity DNA Polymerase、1μL cDNA模板、10.4μL无核酸酶水，总体积为30μL。PCR程序为：5min 95℃，38个热循环(95℃15s、58℃15s、72℃90s)，5min 72℃，终止。
2.表达载体构建
将上述PCR产物通过琼脂糖凝胶电泳、分离、纯化后，搭载到可用于植物遗传转化的表达载体中，可以是任意商用表达载体。PCR产物和表达载体的连接体系为：2μL 5×CEII Buffer，1μLII，3μL线性化表达载体，4μL回收的PCR产物，总体积为10μL。连接反应条件为37℃30min。
通过42℃45s的热激处理，将连接产物转化到5α大肠杆菌感受态中，筛选成功转化包含正确PCR产物的重组表达载体的菌株，用于后续的猕猴桃遗传转化。
3.猕猴桃遗传转化
将含有正确AcHsfA2-1序列的重组表达载体通过电击或液氮冻融等方法转化到农杆菌EHA105菌株中。通过农杆菌介导的猕猴桃叶盘法，将AcHsfA2-1转化到‘东红’植株中，筛选转化成功的种质材料。
4.猕猴桃种质材料高温抗性检测
将成功转化AcHsfA2-1的种质材料置于50℃极端高温的生长条件下，持续2小时。以生长状态接近、没有转化AcHsfA2-1的‘东红’猕猴桃(以下称为“野生型”)植株为对照，同时置于相同的生长条件下处理相同时间。高温处理后，将成功转化AcHsfA2-1和野生型两类植株，继续置于25℃的正常生长条件下培养。
(二)研究结果
获得成功转化热激转录因子AcHsfA2-1的种质材料
利用普通PCR技术检测所有转化农杆菌的猕猴桃植株，以野生型为参照，发现其中两棵成功过量表达AcHsfA2-1，分别命名为Line2和Line7。与野生型植株相比，Line7中AcHsfA2-1的表达量上升了1900倍，Line2中则上升了4000倍(附图1a和1b)。同时，Line2和Line7中自身的热激蛋白基因AcHsp20-1，AcHsp20-2和AcHsp20-3的表达量也显著提高(附图1b)。
种质材料对50℃极端高温胁迫能至少持续抵抗2小时
成功转化AcHsfA2-1的种质材料，经过2小时50℃极端高温处理后，叶片依然呈现处理前健康的绿色，未出现萎蔫干枯的现象；高温处理消除后的9天内，该种质材料仍继续正常生长，未出现生长迟缓的现象(附图1c和1d)。
序列表
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Claims (1)
1.一种抗极端高温生长条件的猕猴桃种质材料的培养方法，特征在于：通过以下步骤实现：首先利用普通PCR技术，以中华猕猴桃‘东红’叶片的cDNA为模板，应用序列为SEQNO.3和SEQ NO.4的引物，进行热激转录因子AcHsfA2-1的克隆；然后将AcHsfA2-1的PCR产物通过琼脂糖凝胶电泳、分离、纯化后，搭载到可用于植物遗传转化的表达载体中；再将含有序列如SEQ：NO.1所示的AcHsfA2-1的重组表达载体通过电击或液氮冻融方法转化到农杆菌EHA105菌株中；最后通过农杆菌介导的猕猴桃叶盘法，将AcHsfA2-1转化到‘东红’植株中，筛选转化成功的种质材料，即获得能抗至少持续2小时50℃极端高温生长条件的猕猴桃种质材料。
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| WO2008064341A1 (en) * | 2006-11-21 | 2008-05-29 | Ceres, Inc. | Nucleotide sequences and corresponding polypepetides conferring enhanced heat tolerance in plants |
| CN103275990A (zh) * | 2013-02-06 | 2013-09-04 | 中国农业科学院作物科学研究所 | 拟南芥热激转录因子基因hsfa2及其编码蛋白与应用 |
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