WO2022247591A1 - 热激相关基因ZmHsf11及其在调控植物耐热性中的应用 - Google Patents

热激相关基因ZmHsf11及其在调控植物耐热性中的应用 Download PDF

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WO2022247591A1
WO2022247591A1 PCT/CN2022/090924 CN2022090924W WO2022247591A1 WO 2022247591 A1 WO2022247591 A1 WO 2022247591A1 CN 2022090924 W CN2022090924 W CN 2022090924W WO 2022247591 A1 WO2022247591 A1 WO 2022247591A1
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zmhsf11
plant
heat
related gene
gene
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French (fr)
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江海洋
秦倩倩
司伟娜
赵玉军
彭晓剑
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安徽农业大学
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    • C—CHEMISTRY; METALLURGY
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    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4636—Oryza sp. [rice]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4684—Zea mays [maize]
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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    • 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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    • 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
    • C12N15/8273—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 for drought, cold, salt resistance
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    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

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  • Corn is one of the important grain and feed crops in my country, and a slight change in the environment will reduce its yield and quality.
  • the impact of environmental factors on the growth and development of corn mainly includes biotic stress and abiotic stress.
  • the measures taken for biotic stress can be solved by using herbicides and pesticides; non-biotic stress It includes high temperature, drought, freezing damage, etc., and its effective methods cannot be used to solve such problems.
  • each stress can cause low or no yield (Liu et al., 2016).
  • the genes that have been reported to affect plant heat tolerance include plant hormones or signal transduction related genes, Ca 2+ signaling pathways, reactive oxygen species related genes, and heat shock transcription factors and heat shock protein genes that play a major role (Mittler et al. al., 2012).
  • Heat shock transcription factor (Heat Shock Transcription Factor, HSF) is the main conduction element in the heat stress signaling pathway. In the heat shock state, it activates the expression of related genes and plays a vital role in regulating the heat stress response process in plants ( Kotak et al., 2007).
  • the HSFs family is divided into three subfamilies, A, B, and C.
  • the protein structures produced by translation are similar, but these structures are different among the three subfamilies.
  • some B members may also contain a repressive region (RD) (Nover et al., 2001). Therefore, the functions of different HSF members may be involved in a variety of adversity stresses.
  • Current studies have shown that functional studies of HSFs mainly focus on stresses such as drought, salt, and high temperature.
  • the purpose of the present invention is to screen and identify a heat shock related gene ZmHsf11 capable of responding to high temperature and its application in regulating plant heat tolerance.
  • the present invention adopts the following technical solutions:
  • the present invention provides a heat shock related gene ZmHsf11, the nucleotide sequence of the heat shock related gene ZmHsf11 is shown in SEQ ID No.1, the heat shock related gene ZmHsf11 has the function of reducing plant heat resistance.
  • the present invention provides the protein encoded by the heat shock-related gene ZmHsf11, which is the protein described in (1) or (2):
  • a protein consisting of the amino acid sequence of SEQ ID No.2 in the sequence listing;
  • the invention provides a plant expression vector comprising the heat shock related gene ZmHsf11.
  • the plant expression vector includes inserting the heat shock-related gene ZmHsf11 nucleic acid molecule into the expression vector p1301a, which is a vector for overexpressing the ZmHsf11 gene, named p1301a-ZmHsf11.
  • the present invention also provides a plant cell comprising the heat shock related gene ZmHsf11, a plant comprising the plant cell, and a seed of the plant, and the plant is preferably rice or corn.
  • the present invention also provides a primer pair for cloning the heat shock related gene ZmHsf11, the primer pair includes an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown in SEQ ID No.3, The nucleotide sequence of the downstream primer is shown in SEQ ID No.4.
  • the present invention also provides the application of the heat shock related gene ZmHsf11 in regulating the heat resistance of plants, and the regulation of plant heat resistance is to reduce the heat resistance of plants or increase the heat resistance of plants.
  • the regulation of plant heat resistance is to increase the heat resistance of plants, and deleting or inhibiting the expression of ZmHsf11 gene in plants can improve the heat resistance of plants.
  • the present invention also provides a method for cultivating high-temperature-resistant plant varieties, the method comprising knocking out or inhibiting the expression of the heat-shock-related gene ZmHsf11, improving the survival rate of the plants after heat treatment, thereby enhancing the heat resistance of the plants.
  • the plant is rice or corn.
  • the beneficial effect of the present invention is that: a gene ZmHsf11 and its protein related to the negative regulation of heat tolerance are reported for the first time. Specifically, the present invention clones the ZmHsf11 gene from maize, and its expression level can be induced by high temperature and other adversity stresses. The ZmHsf11 gene was overexpressed in rice, and the functional identification of the ZmHsf11 gene was carried out. It was found that after overexpression, the survival rate of the overexpressed rice plants under heat treatment was significantly reduced.
  • the functional identification of the ZmHsf11 mutant obtained in the present invention shows that the heat resistance of the mutant is significantly improved after heat treatment, indicating that the ZmHsf11 gene negatively regulates the heat resistance of the plant, and the present invention provides a new technical basis for high temperature resistant maize breeding.
  • FIG. 1 is a graph showing tissue-specific expression analysis and induced expression pattern analysis of ZmHsf11 in Example 1 of the present invention
  • Fig. 2 is the subcellular localization map of the protein encoded by ZmHsf11 in Example 2 of the present invention
  • Fig. 3 is a GUS identification and semi-quantitative expression level identification diagram of rice overexpression-positive strains in Example 3 of the present invention.
  • Fig. 4 is the phenotype graph of the survival rate of rice overexpression plants under heat treatment conditions in Example 4 of the present invention.
  • Fig. 5 is a statistical diagram of the survival rate of rice overexpression plants under heat treatment conditions in Example 4 of the present invention.
  • Figure 6 is a graph showing the proline content of rice overexpression plants under heat treatment conditions in Example 4 of the present invention.
  • the protoplasts of maize B73 were obtained, and the p1305-GFP-ZmHsf11 recombinant plasmid and the nuclear localization signal plasmid NLS-RFP were co-transferred into the protoplasts, and then observed with a laser confocal microscope after culturing in the dark for 18 hours.
  • the seedlings grow out of the differentiation medium, they are transferred to the rooting medium and cultured in tissue culture bottles for 4 weeks.
  • RNA of transgenic rice was extracted and reverse transcribed into cDNA as a template for later use. Semi-quantitative experiments were used to observe the presence or absence of bands in transgenic rice, as well as the light and dark conditions of the bands.
  • Fig. 3A the leaf and stem cuts and the root system of the transgenic rice were stained blue, while the wild type was normal.
  • RT-PCR was used to detect the expression level of the ZmHsf11 gene in the overexpressed plants.
  • FIG. 3B there was no electrophoretic band in the wild type, but there were bands in the three strains of the overexpressed plants. Therefore, the above three strains were selected for follow-up research.
  • proline content the proline content of the wild type and Mu mutants treated at 45° C. for 0 h, 1 h, and 6 h were respectively measured with a kit.
  • the wild-type and Mu mutant plants were treated at 50°C for 4 hours, and the tip 5cm of the third leaf of the two groups of maize before and after treatment was cut off, and the leaves of the two groups were stained with DAB, and the staining results were observed.

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Abstract

本发明公开了热激相关基因ZmHsf11及其在调控植物耐热性中的应用，属于生物技术领域，本发明首次报道了一种耐热性负调控相关基因ZmHsf11及其蛋白，研究发现，ZmHsf11基因的表达可以被高温等逆境胁迫诱导。本发明通过对ZmHsf11基因进行功能鉴定，发现ZmHsf11基因在水稻中过量表达后，过表达水稻植株在热处理条件下的存活率显著降低。对本发明获得的ZmHsf11基因突变体进行功能鉴定，发现热处理后突变体耐热性显著提高，表明ZmHsf11基因对植株耐热能力起到负调控作用，从而为玉米耐高温育种提供了新的技术基础。

Description

热激相关基因ZmHsf11及其在调控植物耐热性中的应用 技术领域
本发明涉及分子生物学技术领域，具体涉及热激相关基因ZmHsf11、热激相关基因ZmHsf11编码的蛋白质的分离与鉴定，涉及热激相关基因ZmHsf11基因序列的获得方法，还涉及热激相关基因ZmHsf11在调控植物耐热性中的应用。
背景技术
玉米作为我国重要的粮饲作物之一，环境的稍微变化，都会降低其产量及品质。目前，环境的因素对玉米的生长与发育起到的影响，主要包括生物胁迫和非生物胁迫，其中对于生物胁迫采取的措施，可以通过利用除草剂和农药等来解决其问题；而非生物胁迫则包括高温、干旱、冻害等，无法利用其有效方法解决此类问题。特别是对农作物来说，每一种胁迫都会造成其低产或绝产(Liu et al.,2016)。
为此探究农作物在非生物胁迫下的响应分子机制，寻找其优良性状基因，提高作物产量和品质。另外，伴随着现代分子技术和分子遗传育种的迅速发展，将优良基因转化到优良株系中进行稳定遗传表达，从而提高作物在逆境胁迫下的耐受性。因此，研究抗逆基因和培育出优良性状的新品种，已成为植物抗逆遗传育种研究热点话题之一。
植物作为固着生物，高温胁迫对它们的生长发育繁殖有很大影响，对于无利趋避性，植物只能通过体内环境调节来达到稳态以抵抗外界环境。当受到短暂高温后，一些热激基因的表达在体内被激活，使机体恢复生长后产生获得耐热性，而基础耐热性则是植物体在受到热激时，体内的一些基因来调内环境的稳态，从而抵御外部环境。这就涉及到一些基因表达，如热激蛋白等。目前已经报道的影响植物耐热性的基因，包括植物激素或信号传导相关基因、Ca 2+信号通路、活性氧相关基因，以及起主要作用的热激转录因子和热激蛋白基因等(Mittler et al.,2012)。
热激转录因子(Heat Shock Transcription Factor,HSF)作为热胁迫信号途径中的主要传导元件，在热激状态下，激活相关基因的表达，调控植物体内的热胁迫响应过程发挥着至关重要作用(Kotak et al.,2007)。HSFs家族分成A、B、C三大亚族，其翻译产生的蛋白结构具有相似性，但这些结构在三大亚族中存在差异，例如某些B成员可能还含有抑制区(RD)(Nover et al.,2001)。所以说不同的HSF成员，其功能可能参与多种多样的逆境胁迫。目前的研究表明，HSF的功能研究主要集中在干旱、盐和高温等胁迫上。
发明内容
本发明的目的在于筛选鉴定出能够响应高温的热激相关基因ZmHsf11及其在调控植物耐热性中的应用。
为达到上述目的，本发明采用了下列技术方案：
本发明提供了一种热激相关基因ZmHsf11，所述的热激相关基因ZmHsf11的核苷酸序列如SEQ ID No.1所示，所述热激相关基因ZmHsf11具有降低植物耐热性的功能。
本发明提供了所述热激相关基因ZmHsf11编码的蛋白质，为如下(1)或(2)所述的蛋白质：
(1)由序列表中的SEQ ID No.2氨基酸序列组成的蛋白质；
(2)将序列表中的SEQ ID No.2氨基酸残基序列经过一至十个氨基酸残基的取代和/或添加且具有热激相关基因ZmHsf11基因功能由(1)衍生的蛋白质。
本发明提供了包含所述的热激相关基因ZmHsf11的植物表达载体。
进一步地，所述植物表达载体包括将热激相关基因ZmHsf11核酸分子插入表达载体p1301a，为过表达ZmHsf11基因载体，命名为p1301a-ZmHsf11。
本发明还提供了包含所述植物表达载体的宿主菌。
本发明还提供了包含所述热激相关基因ZmHsf11的植物细胞、包含所述植物细胞的植物、及所述植物的种子，所述植物优选为水稻或玉米。
本发明还提供了用于克隆所述热激相关基因ZmHsf11的引物对，所述的引物对包括上游引物和下游引物，所述的上游引物的核苷酸序列如SEQ ID No.3所示，所述的下游引物的核苷酸序列如SEQ ID No.4所示。
本发明还提供了所述热激相关基因ZmHsf11在调控植物耐热性中的应用，所述调控植物耐热性为降低植物的耐热性或增加植物的耐热性。
进一步地，所述的调控植物耐热性为增加植物的耐热性，在植株中缺失或抑制ZmHsf11基因表达能够提高植物的耐热性。
本发明还提供了一种耐高温植物品种的培育方法，所述方法包括敲除或抑制热激相关基因ZmHsf11表达，提高热处理后植株的存活率，从而增强植株的耐热性。
进一步地，所述的植物为水稻或玉米。
本发明的有益效果在于：首次报道了一种耐热性负调控相关基因ZmHsf11及其蛋白，具体的，本发明从玉米中克隆得到ZmHsf11基因，其表达水平可以被高温等逆境胁迫诱导，将所述ZmHsf11基因在水稻中进行过量表达，对ZmHsf11基因进行功能鉴定，发现过量表达后，热处理下的过表达水稻植株的存活率显著降低。对本发明获得的ZmHsf11突变体进行功能鉴 定，发现热处理后突变体耐热性显著提高，表明ZmHsf11基因对植株耐热能力起到负调控作用，本发明为玉米耐高温育种提供了新的技术基础。
附图说明
图1为本发明实施例1中ZmHsf11组织特异性表达分析及诱导表达模式分析图；
图2为本发明实施例2中ZmHsf11编码蛋白的亚细胞定位图；
图3为本发明实施例3中水稻过量表达阳性株系GUS鉴定和半定量表达水平鉴定图；
图4为本发明实施例4中水稻过量表达植株在热处理条件下的存活率表型图；
图5为本发明实施例4中水稻过量表达植株在热处理条件下的存活率统计图；
图6为本发明实施例4中水稻过量表达植株在热处理条件下的脯氨酸含量图；
图7为本发明实施例4中水稻过量表达植株在热处理条件下的DAB染色结果图；
图8为本发明实施例4中水稻过量表达植株在热处理条件下的台盼蓝染色结果图；
图9为本发明实施例5中玉米mu突变体PCR鉴定和RT-qPCR表达水平图；
图10为本发明实施例6中玉米mu突变体在热处理条件下的表型图；
图11为本发明实施例6中玉米mu突变体在热处理条件下的脯氨酸含量图；
图12为本发明实施例6中玉米mu突变体在热处理条件下的DAB染色结果图。
具体实施方式
以下通过实施例具体说明本发明的技术方案。然而，这些实施例仅用于举例说明的目的，并不意味着本发明的范围限于此。
应理解下述实施例中使用的实验方法如无特殊说明，均为常规方法。
还应当理解下述实施例中使用的实验材料、试剂等，如无特殊说明，均可从商业途径得到。
在植物基因组数据库网站(https://phytozome.jgi.doe.gov/pz/portal.html)，得到玉米ZmHsf11基因的CDS序列全长(SEQ ID No.1)以及蛋白序列(SEQ ID No.2)。
本发明的一种热激相关基因ZmHsf11，所述的热激相关基因ZmHsf11的核苷酸序列如SEQ ID No.1所示。
本发明的一种热激相关基因ZmHsf11编码的蛋白质，为如下(1)或(2)所述的蛋白质：
(1)由序列表中的SEQI D No.2氨基酸序列组成的蛋白质；
(2)将序列表中的SEQI D No.2氨基酸残基序列经过一至十个氨基酸残基的取代和/或添加且具有热激相关基因ZmHsf11基因功能由(1)衍生的蛋白质。
本发明的所述的热激相关基因ZmHsf11的植物表达载体。包括将热激相关基因ZmHsf11核酸分子插入表达载体p1301a，为过表达ZmHsf11基因载体，命名为p1301a-ZmHsf11。
本发明的植物表达载体的宿主菌。
本发明的包含所述热激相关基因ZmHsf11的植物细胞、包含所述植物细胞的植物、及所述植物的种子，所述植物优选为水稻或玉米。
本发明的用于克隆所述的热激相关基因ZmHsf11的引物对，所述的引物对包括上游引物和下游引物，所述的上游引物的核苷酸序列如SEQ ID No.3所示，所述的下游引物的核苷酸序列如SEQ ID No.4所示。
本发明的热激相关基因ZmHsf11在降低植物的耐热性或增加植物的耐热性中的应用。
所述的调控植物耐热性为增加植物的耐热性；在植株中缺失或抑制ZmHsf11基因表达能够提高植物的耐热性。
本发明的一种耐高温植物品种的培育方法，所述方法包括敲除或抑制热激相关基因ZmHsf11表达，提高热处理后植株的存活率，从而增强植株的耐热性。
所述的植物为水稻或玉米。
实施例1
ZmHsf11基因的组织表达模式和诱导表达模式分析
1、玉米叶片处理
取玉米B73种植于直径40cm，高50cm的花盆中，每盆种3粒，共6盆，放于温棚中(28℃左右)，生长14d后，分别取根、茎、叶样品，液氮速冻后放置于-80℃保存，待生长至花期时又分别取花丝、苞叶、雄蕊样品，液氮速冻后放置于-80℃保存，全部样品RNA提取后反转录为cDNA样品保存于-20℃冰箱备用。
另取玉米B73种植于直径15cm，高10cm的花盆中，每盆4粒，共24盆，放于温棚中(28℃左右)，生长14d后，分别进行42℃、200Mm NaCl、20％PEG6000、100μM ABA处理，每种处理分别在0h、1h、3h、6h、12h、24h取样，每个样品三株混合取样，液氮速冻后放置于-80℃保存，全部样品RNA提取后反转录为cDNA样品保存于-20℃冰箱备用。
2、ZmHsf11引物设计
根据获得的ZmHsf11的cDNA序列全长，利用Primer5软件设计RT-qPCR定量引物：
qPCR-ZmHsf11-F：5’-CTGGGAGCGACCACGACG-3’；
qPCR-ZmHsf11-R：5’-AAACACTGGAGATTTTTACATAGG-3’。
以玉米内参基因GAPDH为对照，设计引物：
Zm-GAPDH-F:5'-CCTCTGGAAAA TTGTGGCGTG-3'；
Zm-GAPDH-R:5'-GCCCAAACGAACAGTCAAGTC-3'。
3、RT-qPCR反应体系及程序
反应体系：
cDNA模板浓度稀释10倍进行qPCR实验
Figure PCTCN2022090924-appb-000001
反应程序：
Figure PCTCN2022090924-appb-000002
扩增信号及数据的处理采用比较Ct法(ΔΔCt法)。相对表达值Cq＝2(-ΔΔCt)。每次荧光定量实验进行3次生物学重复和3次技术重复。
结果如图1所示，ZmHsf11基因在玉米B73的各个组织中均有表达，但在玉米的茎、雌蕊和雄蕊中表达水平明显高于其他组织。在热处理下，1h表达水平明显升高，随着热激时间增加表达水平又逐渐降低；在高盐、渗透和ABA处理下，表达水平均低于对照0h的表达水平。以上结果表明了ZmHsf11可能参与热激、盐、渗透和ABA等非生物胁迫途径。
实施例2
ZmHsf11基因的克隆及亚细胞定位
1、引物设计：
通过对ZmHsf11的CDS序列及亚细胞定位载体p1305-GFP序列分析，设计了如下引物：
ZmHsf11-F(XbaI):5'-gctctagaATGGCCGCCGAGCATGCCA-3'；
ZmHsf11-R(SmaI):5'-cccccgggCCTCGAGTCGTTGGACCC-3'。
2、载体构建：
(1)以实施例1中获得的cDNA为模板进行PCR克隆，反应体系如下：
Figure PCTCN2022090924-appb-000003
Figure PCTCN2022090924-appb-000004
PCR反应程序：
Figure PCTCN2022090924-appb-000005
PCR反应结束后，加入3μL的10×loading buffer，然后跑琼脂糖凝胶电泳，用胶回收试剂盒回收目的片段。
(2)将(1)中的PCR回收产物与p1305-GFP空载体质粒一起进行酶切，酶切体系如下：
Figure PCTCN2022090924-appb-000006
在金属浴37℃反应3h，酶切结束后加入3μL loading buffer终止反应，然后进行琼脂糖凝胶电泳，回收酶切产物；
(3)连接反应，连接体系如下：
Figure PCTCN2022090924-appb-000007
混匀后，PCR仪16℃1h；
(4)将连接后的产物转入大肠杆菌中，进行扩繁，获得重组质粒。
3、原生质体转化：
获得玉米B73的原生质体，将p1305-GFP-ZmHsf11重组质粒与核定位信号质粒NLS-RFP 一起共转到原生质体中，黑暗培养18h后用激光共聚焦显微镜观察。
结果如图2所示，p1305-GFP空载体在细胞核核和细胞膜上均有表达，而p1305-ZmHsf11:GFP只在细胞核上出现绿色荧光，核定位信号为红色荧光，重叠后为黄色荧光。结果表明，ZmHsf11蛋白是一个定位在细胞核上的蛋白质。
实施例3
水稻过量表达ZmHsf11植株的获得
1、同实施例2中方法类似，用下述引物获得过表达重组质粒，并将重组质粒转入到农杆菌中：
ZmHsf11-F(KpnI):5'-gggtaccA TGGCCGCCGAGCA TGCCA-3'；
ZmHsf11-R(PstI):5'-gctctagaTCACCTCGAGTCGTTGGACCC-3'。
2、农杆菌介导的水稻愈伤组织的遗传转化过程
(1)愈伤组织诱导
将消毒过的种子接种到倒有诱导培养基的组培瓶中，每瓶至多接种8粒，置于组培室培养14-20d。
(2)愈伤组织的侵染
将含有目的基因的农杆菌菌液划线于含抗性的YEP固体培养基上，放于28℃培养箱中，培养2d；挑取单菌落于含有5mL培养基的15mL摇菌管中，培养36h；在150mL悬浮培养基中加入3mL葡萄糖和800-900μL的菌液以及150μL的AS，进行扩大培养。
将愈伤组织放于组培瓶中，加入50mL菌液，轻摇10min，重复一次；将侵染过的愈伤组织置于含有K+和Rif的固体YEP固体培养基上，24℃，培养3d，观察培养皿的生菌量；利用灭菌的ddH2O，每次洗涤愈伤组织5min，重复5-7次，然后转入干净的瓶子中，用羧苄水浸泡30min(分2次进行)，在滤纸上晾干，接种到选择培养基中，放于组培室，培养2周。
(3)愈伤组织的分化
从选择培养基中，挑选出新的愈伤组织，接至分化培养基中，放于组培室，培养出分化苗。
(4)生根培养
待分化培养基长出苗，将其移到生根培养基中，在组培瓶中，培养4周。
(5)炼苗期
在生根瓶中加入自来水，等5d后移入田中。
3、验证水稻阳性植株
(1)GUS染色
剪取水稻叶片，置于PCR管中，加入100μL的GUS染液，放在37℃培养箱避光染色过夜，观察叶片侵染情况(图3)。
(2)RT-PCR法
提取转基因水稻的RNA，并反转录为cDNA作为模板备用，利用半定量实验，观察转基因水稻有无条带，以及条带亮暗情况。
结果如图3A所示，转基因水稻的叶片和茎切口处以及根系被染成蓝色，而野生型正常。同时用RT-PCR检测过表达植株的ZmHsf11基因的表达量，如图3B所示，野生型中无电泳条带，而过表达植株中三个株系均有条带。因此，选用以上三个株系进行后续研究。
实施例4
ZmHsf11水稻过量表达植株耐热性鉴定
1、转基因水稻表型实验
收获T02种子后，将其种子放于移液枪的枪盒中，加入适量水，放置于28℃温室中，生长14d左右，移栽到小圆盆(D＝14.8cm，H＝12cm)中，放于28℃温室生长15d，再放于45℃温室处理22h，恢复室温后重新在温室28℃培养20d，观察水稻热处理前后的表型，并分别统计野生型与过表达水稻的存活率。
结果如图4和图5所示，在经过高温处理及恢复生长后，过表达植株的存活率明显低于野生型的存活率，表明在水稻中过表达ZmHsf11基因降低了植株的耐热能力。
2、生理生化指标测定
(1)脯氨酸含量测定：用试剂盒测定分别水稻45℃处理0h、1h、6h的脯氨酸含量。
结果如图6所示，在0h时，过表达植株的脯氨酸含量低于野生型，而随着热处理1h时，过表达植株的脯氨酸含量又高于野生型，当热处理6h时，可以看到过表达株系OE1和OE2又低于野生型WT。结果表明在水稻中ZmHsf11基因可能在热处理1h后开始起作用。
(2)DAB染色和台盼蓝染色：对水稻植株进行50℃处理2.5h，然后对处理前后的植株分别进行DAB染色和台盼蓝染色，观察染色结果。
结果如图7和图8所示，在DAB染色后，过表达植株的叶片颜色深度和面积明显高于野生型。同时在台盼蓝染色后，过表达植株叶片的死亡细胞区域也明显高于野生型。以上这些结果均表明在水稻植株中，过表达ZmHsf11基因降低过表达植株对高温胁迫的应答能力，表明ZmHsf11基因对植株耐热性调控可能起到负调控的作用。
实施例5
ZmHsf11玉米Mu突变体植株鉴定
利用购买获得的以玉米W22品种为背景的Mu突变体材料(来源于玉米遗传合作股份中心 Maize Genetics Cooperation Stock Center)，为T0代，通过PCR技术，验证T0代植株，筛选出含有Mu转座子的玉米，将筛选的含有Mu转座子的玉米进行自交，得到T01代，继续使用上述方法，获得T02代，确定纯合株系的种子，利用RT-qPCR实验验证突变体中ZmHsf11基因表达水平。
结果如图9所示，在Mu突变体(图中编号为Mu11)中ZmHsf11基因表达量低于野生型中的表达量，表明Mu突变体中ZmHsf11基因表达受到抑制或降低，该Mu突变体为ZmHsf11基因功能缺失的突变体。
实施例6
ZmHsf11玉米突变体植株耐热性鉴定
1、突变体表型实验
将野生型W22品种和Mu突变体种子播种在小圆盆中，放于温室生长至三叶一心期，控制盆中水分含量一致，将植株放于光照培养箱，培养过夜，然后放于45℃温室处理12h，再放置室温后，将其放于温室恢复培养2d，观察野生型与Mu突变体叶片萎蔫情况(图10)。
结果如图10所示，热处理后野生型叶片出现明显萎蔫，而Mu突变体植株叶片未出现萎蔫，表明Mu突变体植株的耐热性高于野生型植株。
2、生理生化指标测定
(1)脯氨酸含量测定：用试剂盒分别测定野生型与Mu突变体45℃处理0h、1h、6h的脯氨酸含量。
结果如图11所示，随着热激时间增加，脯氨酸含量急剧上升，同时不同时间处理下，Mu突变体的脯氨酸含量均略高于或者高于野生型植株的脯氨酸含量。
(2)DAB染色
对野生型与Mu突变体植株进行50℃处理4h，剪取处理前后两组玉米的第三片叶的尖端5cm，对两组叶片进行DAB染色，观察染色结果。
结果如图12所示，在DAB染色后，Mu突变体植株的叶片颜色深度明显低于野生型植株。以上这些结果表明了ZmHsf11基因功能的缺失提高了植株的耐热能力，进而表明ZmHsf11基因对植株耐热能力起到负调控作用。
最后应当理解的是，以上所述仅显示了本发明的优选实施例而已，并不用于限制本发明，本领域的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理和方法，在不背离本发明精神和范围的前提下，本发明还会有各种变化和改进，本发明要求保护范围由所附的权利要求书、说明书及其等效物界定。

Claims (15)

  1. 一种热激相关基因ZmHsf11，其特征在于：所述热激相关基因ZmHsf11的核苷酸序列如SEQ ID No.1所示。
  2. 根据权利要求1所述的热激相关基因ZmHsf11，其特征在于：所述热激相关基因ZmHsf11具有降低植物耐热性的功能。
  3. 一种热激相关基因ZmHsf11编码的蛋白质，其特征在于：为如下(1)或(2)所述的蛋白质：
    (1)由序列表中的SEQ ID No.2氨基酸序列组成的蛋白质；
    (2)将序列表中的SEQ ID No.2氨基酸残基序列经过一至十个氨基酸残基的取代和/或添加，且具有热激相关基因ZmHsf11功能的由(1)衍生的蛋白质。
  4. 一种包含权利要求1或2所述的热激相关基因ZmHsf11的植物表达载体。
  5. 根据权利要求4所述的植物表达载体，其特征在于：包括将热激相关基因ZmHsf11核酸分子插入表达载体p1301a，为过表达ZmHsf11基因载体，命名为p1301a-ZmHsf11。
  6. 一种包含权利要求4或5所述的植物表达载体的宿主菌。
  7. 一种包含权利要求1或2所述的热激相关基因ZmHsf11的植物细胞。
  8. 一种包含权利要求7的植物细胞的植物。
  9. 根据权利要求8所述的植物，其特征在于，所述植物为水稻或玉米。
  10. 一种从权利要求8或9的植物中获得的种子。
  11. 一种用于克隆权利要求1或2所述的热激相关基因ZmHsf11的引物对，其特征在于：所述的引物对包括上游引物和下游引物，所述的上游引物的核苷酸序列如SEQ ID No.3所示，所述的下游引物的核苷酸序列如SEQ ID No.4所示。
  12. 一种权利要求1或2所述的热激相关基因ZmHsf11在调控植物耐热性中的应用，所述调控植物耐热性为降低植物的耐热性或增加植物的耐热性。
  13. 根据权利要求12所述的应用，其特征在于：所述的调控植物耐热性为增加植物的耐热性，在植株中缺失或抑制ZmHsf11基因表达能够提高植物的耐热性。
  14. 一种耐高温植物品种的培育方法，其特征在于：所述方法包括敲除或抑制权利要求1或2所述的热激相关基因ZmHsf11表达，提高热处理后植株的存活率，从而增强植株的耐热性。
  15. 根据权利要求14所述的耐高温植物品种的培育方法，其特征在于：所述的植物为水稻或玉米。
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