CN116947987A - GhMACPF26基因在调控植物耐冷性中的应用 - Google Patents
GhMACPF26基因在调控植物耐冷性中的应用 Download PDFInfo
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- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- 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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Abstract
本发明公开了GhMACPF26基因在调控植物耐冷性中的应用。本发明提供了如下1)‑3)中任一种物质在调控植物耐冷性中的应用；1)蛋白GhMACPF26；2)编码蛋白GhMACPF26的核酸分子；3)含有编码蛋白GhMACPF26的核酸分子的重组载体、表达盒或重组菌；本发明从陆地棉冷处理后的叶片中克隆出棉花抗逆胁迫基因GhMACPF26，通过病毒介导的基因沉默技术沉默GhMACPF26基因后冷处理表明，沉默后的GhMACPF26棉花株系具有一定的抗冷特征，推测GhMACPF26基因是一类负调控的冷胁迫因子。
Description
技术领域
本发明属于生物技术领域，尤其涉及一种GhMACPF26基因在调控植物耐冷性中的应用。
背景技术
棉花是一类起源于热带或亚热带的经济作物，喜温不耐寒，对冷胁迫异常敏感。冷胁迫除了影响棉花种子萌发、生长发育，还能够影响棉花的产量和纤维品质等。随着近年来全球温度的不稳定性，低温频发的现象给农作物的生产带来严峻的挑战，常常导致大面积农作物受灾，给农业带来了巨大的损失，严重影响了国家的粮棉安全。此外，由于新疆等地区播种前期低温低的现状，长期使用地膜等播种模式，给当地带来了不可降解的白色污染等，为此，研究棉花对低温胁迫的响应机制，培育耐低温的无膜棉新品种具有重要的实际意义。
攻膜复合体/穿孔素超家族(Membrane attack complex/perforin superfamily,MACPF)结构域蛋白在高等真核生物中研究较多，而在植物中报道较少。在拟南芥中，含有MACPF结构域的蛋白功能的丧失能够通过激活水杨酸信号来诱导细胞死亡，并且能够改变叶片的内生微生物群落。
发明内容
本发明一个目的是提供如下1)-3)中任一种物质的应用。
本发明提供如下1)-3)中任一种物质在调控植物耐冷性中的应用；
1)蛋白GhMACPF26；
2)编码蛋白GhMACPF26的核酸分子；
3)含有编码蛋白GhMACPF26的核酸分子的重组载体、表达盒或重组菌；
所述蛋白GhMACPF26为如下(1)或(2)或(3)：
(1)由序列表中序列3所示的氨基酸序列组成的蛋白质；
(2)由序列表中序列3所示的氨基酸序列的末端添加标签序列组成的蛋白质；
(3)将序列表中序列3所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由(1)或(2)衍生的蛋白质。
上述应用中，所述编码蛋白GhMACPF26的核酸分子是如下1)-3)中任一种的DNA分子：
1)编码区为序列表中序列1所示的DNA分子；
2)在严格条件下与1)或2)限定的DNA序列杂交且编码具有相同功能蛋白质的DNA分子；
3)与1)限定的DNA序列至少具有70％、至少具有75％、至少具有80％、至少具有85％、至少具有90％、至少具有95％、至少具有96％、至少具有97％、至少具有98％或至少具有99％同源性且编码具有相同功能蛋白质的DNA分子。
抑制或降低蛋白GhMACPF26含量或活性的物质，或，抑制或沉默GhMACPF26基因表达的物质在提高植物耐冷性中的应用也是本发明保护的范围。
上述应用中，所述物质包括用于沉默GhMACPF26基因表达的VIGS载体。
在本发明的实施例中，用于沉默GhMACPF26基因表达的VIGS载体包括：pTRV1和pTRV2(在本发明中指重组载体pTRV2-GhMACPF26)；重组载体pTRV2-GhMACPF26，其为将序列表中序列2所示的GhMACPF26基因部分片段同源重组到pTRV2载体中，得到的载体：
上述抑制或降低蛋白GhMACPF26含量或活性，或，抑制或沉默GhMACPF26基因表达为将表达重组载体pTRV2-GhMACPF26的农杆菌和辅助载体pTRV1的农杆菌共同注入植物叶片，瞬时转染，实现抑制或降低蛋白GhMACPF26含量，或，抑制GhMACPF26基因表达。
上述提高植物耐冷性为与转入空载体棉花植株(pTRV2::00)相比，VIGS沉默GhMACPF26的转基因棉花(pTRV2::GhMACPF26)的耐冷性提高。
上述应用为：将表达重组载体pTRV2::GhMACPF26的农杆菌和表达辅助载体pTRV1的农杆菌共同注入植物叶片得到VIGS沉默GhMACPF26的转基因棉花(TRV::GhMACPF26)，且将表达载体pTRV1的农杆菌和表达pTRV2的农杆菌共同注入植物叶片得到转入空载体棉花植株(pTRV2::00)；冷胁迫VIGS沉默GhMACPF26的转基因棉花(pTRV2:GhMACPF26)和转入空载体棉花植株(pTRV2::00)，VIGS沉默GhMACPF26的转基因棉花(pTRV2::GhMACPF26)的耐冷性高于转入空载体棉花植株(pTRV2::00)。
抑制或降低蛋白GhMACPF26含量或活性的物质，或，抑制或沉默GhMACPF26基因表达的物质在培育耐冷性植物中的应用也是本发明保护的范围。
本发明另一个目的是提供一种培育耐冷性提高的转基因植物的方法。
本发明提供的方法，为如下1)或2)：
1)所述的方法包括如下步骤：抑制或降低目的植物中蛋白GhMACPF26的含量和/或活性，得到转基因植物；
2)所述的方法包括如下步骤：抑制或降低目的植物中编码蛋白GhMACPF26的核酸分子的表达，得到转基因植物；
所述转基因植物的耐冷性高于所述目的植物；
所述蛋白GhMACPF26为如下(1)或(2)或(3)：
(1)由序列表中序列3所示的氨基酸序列组成的蛋白质；
(2)由序列表中序列3所示的氨基酸序列的末端添加标签序列组成的蛋白质；
(3)将序列表中序列3所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由(1)或(2)衍生的蛋白质。
上述方法中，所述抑制或降低目的植物中蛋白GhMACPF26的含量和/或活性，或，所述抑制或降低目的植物中编码蛋白GhMACPF26的核酸分子的表达，均是将沉默目的植物中编码蛋白GhMACPF26的核酸分子表达的物质导入所述目的植物中。在本发明的实施例中，是采用沉默GhMACPF26基因表达的VIGS载体导入目的植物。
上述中，所述植物为双子叶植物或单子叶植物。
上述抑制或降低蛋白GhMACPF26含量或活性的物质，或，抑制或沉默GhMACPF26基因表达的物质也是本发明保护的范围，包括用于沉默GhMACPF26基因表达的VIGS载体。
本发明从陆地棉冷处理后的叶片中克隆出棉花抗逆胁迫基因GhMACPF26，荧光定量结果表明GhMACPF26在棉花受冷胁迫12h能够高量表达。深入分析冷胁迫转录组数据表明GhMACPF26可以通过与WRKY、ERF等转录因子作用，参与棉花抗冷调控；进一步通过病毒介导的基因沉默技术沉默GhMACPF26基因后冷处理表明，沉默后的GhMACPF26棉花株系具有一定的抗冷特征，推测GhMACPF26基因是一类负调控的冷胁迫因子。
附图说明
图1为GhMACPF26在TM-1中不同冷胁迫时期的表达模式。
图2为pTRV2::GhMACPF26基因部分片段的PCR扩增产物；M泳道为MARKER，第1、2、3、4泳道为pTRV2::GhMACPF26的PCR目的片段，大小为300bp。
图3为pTRV2::GhMACPF26阳性植株PCR鉴定。M泳道为MARKER，1-8泳道为目的条带，大小为300bp。
图4为沉默株系与冷处理24h后表型观察和生理指标测定；(a)沉默株系阳性，TRV::CLA；(b)TRV::00冷处理前；(c)pTRV2::GhMACPF26冷处理前；(d)pTRV2::00冷处理24h；(e)pTRV2::GhMACPF26冷处理24h；(f)冷处理前RT-PCR表达量分析；(g)冷处理0h丙二醛含量测定；(h)冷处理24h丙二醛含量测定。
具体实施方式
下述实施例中所使用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、试剂等，如无特殊说明，均可从商业途径得到。
本实验选取的棉花材料为陆地棉遗传标准系TM-1品种(以下简称为野生型棉花)，种植于中国农业科学院棉花研究所棉花生物学国家重点实验室的植物光照培养室(16小时光照/8小时黑暗，25℃)。
下述实施例中试剂与耗材如下：
1、酶及试剂盒：GXL DNAPolymerase高保真酶、胶回收试剂盒购自Takara公司；RNA反转录试剂盒、KOD FX Neo酶(Code.No.KFX-201)购自Toyobo公司；Ultra One Step Cloning Kit试剂盒购自Vazyme公司；质粒少量提取试剂盒购自Magen公司；限制性内切酶(Xba I、Sac I)购自NEB公司；DNAMarker、植物总RNA提取试剂盒购自TIANGEN公司；荧光定量TransStart Top Green qPCR SuperMix购自TransGen公司。
2、其他药品：琼脂糖为西班牙原装产品，蛋白胨、酵母提取物、氯仿、异戊醇、乙醇、异丙醇、氯化钠、蔗糖、silwet L-77、间苯三酚等为国产分析纯，卡纳霉素、硫酸链霉素、氨苄青霉素等购自宝生物工程(大连)有限公司，大肠杆菌感受态细胞Trans5α购自北京全式金生物技术有限公司，农杆菌感受态细胞LBA4404购自上海唯地生物技术有限公司。
3、培养基：LB液体培养基：胰蛋白胨(Tryptone)10g/L、酵母提取物(Yeastextract)5g/L、氯化钠(NaCl)10g/L；LB固体培养基：胰蛋白胨(Tryptone)10g/L、酵母提取物(Yeast extract)5g/L、氯化钠(NaCl)10g/L、琼脂粉(Agar)15g/L，定容至1L；LB选择培养基：在LB铺平板前，待培养基高压灭菌冷却至55℃时加入相应浓度抗生素，摇匀后铺平板。本文中提到的但未列出的各种试剂溶液均按《分子克隆实验指南》第三版上的方法配制，生化试剂为分析纯或以上级。
4、主要仪器：PCR扩增仪(Eppendorf)、高速离心机(Eppendorf 5427R)、电泳设备(北京六一)、凝胶成像系统(BIO-RAD)、荧光定量PCR仪(ABI7500)、荧光显微镜(OlympusBX43)、恒温培养振荡器(上海智城)、人工气候试验箱(赛福)等。
下述实施例中GhMACPF26基因的核苷酸序列为序列1，GhMACPF26蛋白的氨基酸序列为序列表中序列3。其开放阅读框为1722bp，编码574个氨基酸，蛋白的相对分子量为38.298kDa，等电点为5.705。
实施例1、GhMACPF26基因的发现和克隆
一、GhMACPF26在不同开花时间陆地棉品种中的昼夜表达模式分析
1、采集样品
试验材料陆地棉TM-1种植于棉花生物学国家重点实验室的植物光照培养室(16小时光照/8小时黑暗，25℃)。在棉花三叶期的时候进行置于4°冷处理，然后每隔一段时间进行叶片取样，直至24h，所取材料迅速放入液氮中冷冻，保存于-80℃冰箱备用。
2、荧光定量检测
取上述不同材料的叶片样品，使用TIANGEN公司试剂盒提取样品总RNA，使用Toyobo的反转录试剂盒FSQ-201反转录总RNA获得cDNA，对不同时期中的GhMACPF26基因进行荧光定量来测定其表达量。
(1)RNA的提取步骤为：
1)匀浆处理：取适量纤维样品在液氮中迅速研磨成粉末，加入700μlSL(使用前加入β-巯基乙醇)，立即剧烈震荡使样品混匀；
2)12,000rpm离心2min；
3)将上清液转移至过滤柱CS上，12,000rpm离心2min，小心吸取收集管中的上清至新的RNase-Free的离心管中，吸头避免接触收集管中的细胞碎片；
4)加入0.4倍上清体积的无水乙醇，混匀，将混合物转入吸附柱CR3中，12,000rpm离心15sec，倒掉收集管中的废液，将吸附柱CR3放回收集管中；
5)向吸附柱CR3中加入350μl去蛋白液RW1，12,000rpm离心15sec，倒掉收集管中的废液，将吸附柱CR3放回收集管中；
6)DNaseI工作液：取10μlDNaseI储存液和70μlRDD溶液轻柔混匀；
7)向CR3中加入80μl的DNaseI工作液，室温静止15min；
8)静置完后，向CR3中加入350μl去蛋白液RW1，12,000rpm离心15sec，倒掉收集管中的废液，将吸附柱CR3放回收集管中；
9)向吸附柱CR3中加入500μl漂洗液RW(使用前加入乙醇)，12,000rpm离心15sec，倒掉收集管中的废液，将吸附柱CR3放回收集管中；
10)重复步骤9；
11)12,000rpm(～13,400×g)离心2min，将吸附柱CR3放入一个新的RNase-Free离心管中，向吸附膜的中间部位悬空滴加30-50μlRNase-FreeddH2O，室温放置2min，12,000rpm(～13,400×g)离心1min，得到RNA溶液。注意：洗脱缓冲液体积不应少于30μl，体积过小影响回收效率。RNA样品请在-70℃中保存。如果预期RNA得率大于30μg，可将步骤11中离心得到的RNA溶液再加入吸附柱CR3中，室温放置2min，12,000rpm(～13,400×g)离心1min，得到RNA溶液。
(2)cDNA的合成。将500ng RNA反转录为cDNA，采用Toyobo的反转录试剂盒FSQ-201，反转录体系为：
按下列组份配制RT反应液(反应液配制在冰上进行)：
表1为反转录体系
反转录反应条件如下：
37℃15min(反转录反应)，
98℃5s(反转录酶的失活反应)；
(3)实时荧光定量PCR。将反转录产物cDNA溶液稀释4倍作为qRT-PCR反应模板。
用GhUBQ7作为内参基因，根据Cottongen上GhMACPF26和GhUBQ7的参考CDS序列设计荧光定量PCR的引物如下：
表2为荧光定量PCR引物
冰上配制qRT-PCR反应体系，进行荧光定量PCR反应。
qRT-PCR反应体系为：
表3为荧光定量PCR反应体系
qRT-PCR反应程序：
表4为荧光定量PCR反应程序
qRT-PCR的结果如图1所示，对照(冷处理前)，冷处理2h、冷处理4h、冷处理6h、冷处理8h、冷处理12h、冷处理24h分别为冷胁迫不同时间的结果，可以看出，GhMACPF26在冷胁迫过程中表达量升高，并且在12h的时候表达量最高，说明GhMACPF26可能是一个能够参与棉花耐冷胁迫的关键基因。
二、棉花GhMACPF26基因部分片段的克隆
根据CottonOMICS(http://cotton.zju.edu.cn/index.htm)上GhMACPF26的参考CDS序列设计基因部分片段的克隆引物，
引物序列，扩增大小为300bp：
pTRV2::GhMACPF26-F:5′-AAGGTTACCGAATTCTCTAGAGACAAGGATGTTACAGTCATC-3′
pTRV2::GhMACPF26-R:5′-GAGCTCGGTACCGGATCCAAGGGATGAACACACCGGTTC-3′
具体克隆基因的过程为：
(1)试验材料陆地棉品种TM-1种植于棉花生物学国家重点实验室的植物光照培养室(16小时光照/8小时黑暗，25℃)。将陆地棉TM-1样品生长处于三叶期的棉花进行冷胁迫(置于4°培育)，分别在冷胁迫0h，2h，4h，6h，12h和24h进行取样，所取材料迅速放入液氮中冷冻，保存于-80℃冰箱备用。使用TIANGEN公司试剂盒提取样品总RNA，使用Toyobo的反转录试剂盒FSQ-201反转录总RNA获得cDNA。
(2)PCR扩增目的基因
将上述反转录产物cDNA溶液稀释4倍作为PCR反应模板。在冰上配制以下体系，根据TaKaRaGXL DNA Polymerase高保真酶说明书，PCR反应体系如下：
表5为GXL高保真酶PCR扩增反应体系
PCR扩增程序为：
表6为高保真酶PCR扩增程序
反应结束后4℃保存，用1％的琼脂糖电泳进行检测。
结果如图2所示，M泳道为MARKER，第1-4泳道为pTRV2::GhMACPF26基因部分片段的PCR产物，可以看出，得到大小为300bp的条带，符合预期。
将上述PCR产物送去测序，结果该PCR产物具有序列表中序列2(GhMACPF26_target_cds)所示DNA片段，为GhMACPF26基因部分片段。
实施例2、抑制GhMACPF26基因在提高植物耐冷性中的应用
TRV-VIGS体系中的pTRV1载体、pTRV2载体和pTRV2-CLA-阳性对照(文献中名称为pTRV2-GhCLA1)均记载在如下文献中：TRV病毒介导的基因沉默体系在棉花中的建立及应用，王心宇，吕坤，蔡彩平，徐君，郭旺珍；作物学报Acta Agronomica Sinica，2014,40(8)：1356-1363。
一、重组载体的制备
将实施例1的PCR产物使用Ultra One Step Cloning Kit试剂盒连接到Xba I和Sac I双酶切后的pTRV2载体，得到重组载体pTRV2-GhMACPF26。
重组载体pTRV2-GhMACPF26为将序列表中序列2所示的GhMACPF26基因部分片段同源重组到pTRV2载体中，得到的载体。
二、重组菌的制备
利用冻融法转化根癌农杆菌LBA4404感受态细胞，具体转化过程如下：
(1)向上海唯地生物的根癌农杆菌LBA4404感受态细胞100μl中加入上述一制备的重组载体pTRV2-GhMACPF26 1μg(2-10μl)，混匀后冰浴30min；液氮速冻2-3min，37℃热激90s；
(2)冰浴5min，再加入800μl LB液体培养基；
(3)190rpm，28℃，培养4h后,4000rpm离心5分钟，吸去上清液至剩余400-500μl，反复吸打混匀后取200μL菌液涂在含有卡那霉素、硫酸链霉素和利福平的三抗筛选培养基上，28℃培养大约36-48h，抗性菌落可见；
(4)挑取单菌落在1ml的含有三抗的LB液体培养基中培养16h左右，直至浑浊；
(5)菌落PCR(引物为pTRV2::GhMACPF26-F和pTRV2::GhMACPF26-R，得到300bp的为阳性)和酶切(采用XbaI和SacI双酶切，得到300bp的为阳性)鉴定，筛选出阳性农杆菌株，20％甘油菌液于-80℃保存。
阳性农杆菌株命名为重组菌LBA4404/pTRV2-GhMACPF26。
将pTRV1(辅助质粒)转入根癌农杆菌LBA4404中，得到重组菌LBA4404/pTRV1；
将pTRV2-CLA-阳性对照转入根癌农杆菌LBA4404中，得到重组菌LBA4404/pTRV-阳性对照。
将pTRV2转入根癌农杆菌LBA4404中，得到重组菌LBA4404/pTRV2。
三、沉默GhMACPF26的转基因棉花
1、利用子叶注射的方法进行病毒沉默
(1)从LB培养基上挑取单克隆
将重组菌LBA4404/pTRV1和重组菌LBA4404/pTRV2-GhMACPF26分别于5ml LB培养基中(含卡那，庆大50ug/ml),28℃,50rpm过夜培养；
(2)加入45ml农杆菌induction cμlture solution,28℃,100rpm过夜,培养至OD600＝1.5-2.0；
(3)菌液回收：离心4000rpm,1 0min,收集沉淀，用infiltration solution调节OD600＝1.5-2.0，室温(25℃)静置3h～5h，得到重组菌LBA4404/pTRV1菌液和重组菌LBA4404/pTRV2-GhMACPF26菌液；
(4)将相同浓度的重组菌LBA4404/pTRV1菌液和重组菌LBA4404/pTRV2-GhMACPF26菌液按照体积比1:1混匀，再用针头划伤野生型棉花(陆地棉遗传标准系TM-1)子叶背面，用无针注射器将不同混合组的菌液注入，棉株黑暗过夜培养；
(5)将处理后的植株转到温度23℃,光强120μE m-2S-1light，光周期16/8h光照/黑暗的条件下培养，得到病毒沉默后棉花植株。
重复步骤(1)-(4)，按照上述方法，将重组菌LBA4404/pTRV1菌液和重组菌LBA4404/pTRV2菌液按照1:1混匀后注射入野生型棉花，得到转入空载体棉花植株(命名为TRV::00)。
重复步骤(1)-(4)，按照上述方法，将重组菌LBA4404/pTRV1菌液和重组菌LBA4404/pTRV2-阳性对照菌液按照1:1混匀后注射入野生型棉花，得到沉默株系阳性对照(命名为TRV::CLA)。
2、沉默GhMACPF26的转基因棉花的PCR鉴定
1)将培养至三叶期的上述1得到的病毒沉默后棉花植株(注射3周后的植株叶片)叶片进行DNA提取。
2)对于沉默植株进行PCR扩增检测目标片段是否成功
以上述提取的DNA为模板，用引物TRV2::GhMACPF26-F和TRV2::GhMACPF26-R进行PCR扩增：
PCR的反应体系：
表7为Taq酶PCR扩增反应体系
PCR的扩增程序：
表8为Taq酶PCR扩增程序
分别取适量扩增产物在1％琼脂糖凝胶上进行电泳检测。
结果如图3所示，M泳道为MARKER，泳道1-8分别为病毒沉默后棉花植株，可以看出，得到大小为300bp的目的条带，说明TRV::GhMACPF26基因已经进入到棉花中。上述分子鉴定得到300bp的株系命名为VIGS沉默GhMACPF26的转基因棉花株系(TRV2::GhMACPF26)，共3个。
2、沉默GhMACPF26的转基因棉花的基因表达水平
提取VIGS沉默GhMACPF26的转基因棉花(图中记作pTRV2::GhMACPF26)的RNA作为模板，以GhMACPF26_F和GhMACPF26_R为荧光定量引物，内参引物为GhUBQ7_F和GhUBQ7_R，进行实时定量PCR检测。以转入空载体棉花植株(图中记作pTRV2::00)为对照。
结果如4f所示，可以看出，与转入空载体棉花植株相比，VIGS沉默GhMACPF26的转基因棉花中的GhMACPF26表达量降低，证明得到沉默转基因株系。
四、沉默GhMACPF26的转基因棉花的耐冷性分析
1、冷胁迫后表型观察
将生长至三叶期的TRV::GhMACPF26的棉花(图中记作pTRV2::GhMACPF26)和转入空载体棉花植株(图中记作pTRV2::00)进行4°冷处理24h。每个株系20株。以沉默株系阳性对照(图中记作pTRV2::CLA)为对照。
冷处理后表型观察，结果如图4a-4e所示，(a)pTRV2::CLA(白化对照)，(b)pTRV2::00冷处理前；(c)pTRV2::GhMACPF26冷处理前；(d)pTRV2::00冷处理24h；(e)pTRV2::GhMACPF26冷处理24h；可以看出，TRV::00冷处理后植株明显萎蔫；pTRV2::GhMACPF26冷胁迫后植株与胁迫前无显著变化，表明其抗冷性高；证明沉默GhMACPF26基因表达提高植物耐冷性。
2、丙二醛含量检测
使用南京建成生物工程研究所丙二醛检测试剂盒(序列号：A003-1-2；网站：http://www.njjcbio.com/products.asp？id＝287)对上述1冷处理前后的植株进行丙二醛的检测。
冷处理0h丙二醛含量测定和冷处理24h丙二醛含量测定结果如图4g和4h所示，可以看出，与pTRV2::00相比，pTRV2::GhMACPF26在冷胁迫24h后株系丙二醛的含量从极显著到显著。
因此，上述结果表明，推测GhMACPF26具有参与棉花的抗冷胁迫作用。
SEQUENCE LISTING
<110>中国农业科学院棉花研究所 邯郸市农业科学院
<120> GhMACPF26基因在调控植物耐冷性中的应用
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1722
<212> DNA
<213> Artificial sequence
<400> 1
atggaaaacc caagaacaag aagtggcagc cccttgtgct catcttctag cctagatgct 60
ttgactacaa cactaagcag ttcaattcaa gctctgggtc gtggctttga tgtcacttca 120
gatatacggc tgttatattg caaaggggca cctggttcaa ggttggttca gcttgacgaa 180
gatcataccg acgatcttgt tttgtctggt agggttattg tgcccaacgt gtctgccgat 240
atcaagtggt caatgggaaa gggtgacatc gagagaaaac cagtttgtag cttccatgag 300
atgtcaggat actttaatga gaaatctggt atagcagggc gtgttccgct gggaagcttt 360
aatgcaatgt tcaatttcac tggttcttca cgagttgatg cagctgctac aaaatccctt 420
gccatggttg gatacttaat tcccctatgc accgtcaaat tagctaagca gaatttaatt 480
ctgcatgaag atgtcaggcg cgctgttcct tacacttggg atcctgcagc tttggcaagt 540
tttattgaaa attatggtac gcacattgtt acttctgcta caattggtgg gagagatgtt 600
gtttatgtca gacagcacca gtcatctcct ttatcgctaa cagacattga gaactatgtg 660
aaagacattg gggatcaaag gtttctggac tccaaaggcc agtcaagtgc tgcaccttta 720
aaatacaagg acaaggatgt tacagtcatc tttaggagga gaggaggtga tgaccttgag 780
cagagtcatg caaggtgggc agagactgtg caatcggcac cggatgtcat taacatgaca 840
tttatgccca ttgtttctct tcttgaagga gtgcctggta taaaacattt ggcacgtgca 900
atcgaactat acttggagta caagccacca atcgaggatt tacaatattt cctggatttt 960
caaattgctc gtgtttgggc tccagaacag agcaacattc aaaggaagga accggtgtgt 1020
tcatcccttc agtttagctt aatgggcccc aagctttata tcagcccaga tcaggtaaca 1080
gttggccgta aaccagttac tgggcttagg cttagcttag aaggcaacaa gcagaataga 1140
ctagcaattc atttgcagca cctagtgtcc cttccaaaaa tccttcaacc ccactgggat 1200
gtgcacatgg ccataggtgc acccaagtgg caaggtcctg aggagcaaga cagccgttgg 1260
tttgaaccca tcaagtggaa aaacttctcc catgtaagca ctgcaccaat agaacacact 1320
gacacttgca ttggagacct ctctggtgtc cacattgtca caggagcgca gcttggtgta 1380
tgggactttg gttccaaaaa cgtattacac ctgaaacttc tcttctctaa agtaccaggc 1440
tgcacaataa ggcgatctgt atgggatcat agtccatgca gcctttctac tgcacagagg 1500
accgacggat cttcatcatc agtttcgagt gagagaactt ctgacaataa aaaggaagat 1560
agttcaagcc atgttgggaa acttgcaaag attgtggatt caactgaaat gtcgaaagga 1620
ccacaggata gtccaggcca ttggttggtt acaggggcta agcttggggt ggacaagggg 1680
aagattgtat tgcgtattaa gtactcatta ctgaattact ga 1722
<210> 2
<211> 300
<212> DNA
<213> Artificial sequence
<400> 2
gacaaggatg ttacagtcat ctttaggagg agaggaggtg atgaccttga gcagagtcat 60
gcaaggtggg cagagactgt gcaatcggca ccggatgtca ttaacatgac atttatgccc 120
attgtttctc ttcttgaagg agtgcctggt ataaaacatt tggcacgtgc aatcgaacta 180
tacttggagt acaagccacc aatcgaggat ttacaatatt tcctggattt tcaaattgct 240
cgtgtttggg ctccagaaca gagcaacatt caaaggaagg aaccggtgtg ttcatccctt 300
<210> 3
<211> 573
<212> PRT
<213> Artificial sequence
<400> 3
Met Glu Asn Pro Arg Thr Arg Ser Gly Ser Pro Leu Cys Ser Ser Ser
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Ser Leu Asp Ala Leu Thr Thr Thr Leu Ser Ser Ser Ile Gln Ala Leu
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Gly Arg Gly Phe Asp Val Thr Ser Asp Ile Arg Leu Leu Tyr Cys Lys
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Gly Ala Pro Gly Ser Arg Leu Val Gln Leu Asp Glu Asp His Thr Asp
50 55 60
Asp Leu Val Leu Ser Gly Arg Val Ile Val Pro Asn Val Ser Ala Asp
65 70 75 80
Ile Lys Trp Ser Met Gly Lys Gly Asp Ile Glu Arg Lys Pro Val Cys
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Ser Phe His Glu Met Ser Gly Tyr Phe Asn Glu Lys Ser Gly Ile Ala
100 105 110
Gly Arg Val Pro Leu Gly Ser Phe Asn Ala Met Phe Asn Phe Thr Gly
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Ser Ser Arg Val Asp Ala Ala Ala Thr Lys Ser Leu Ala Met Val Gly
130 135 140
Tyr Leu Ile Pro Leu Cys Thr Val Lys Leu Ala Lys Gln Asn Leu Ile
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Leu His Glu Asp Val Arg Arg Ala Val Pro Tyr Thr Trp Asp Pro Ala
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Ala Leu Ala Ser Phe Ile Glu Asn Tyr Gly Thr His Ile Val Thr Ser
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Ala Thr Ile Gly Gly Arg Asp Val Val Tyr Val Arg Gln His Gln Ser
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Ser Pro Leu Ser Leu Thr Asp Ile Glu Asn Tyr Val Lys Asp Ile Gly
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Asp Gln Arg Phe Leu Asp Ser Lys Gly Gln Ser Ser Ala Ala Pro Leu
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Lys Tyr Lys Asp Lys Asp Val Thr Val Ile Phe Arg Arg Arg Gly Gly
245 250 255
Asp Asp Leu Glu Gln Ser His Ala Arg Trp Ala Glu Thr Val Gln Ser
260 265 270
Ala Pro Asp Val Ile Asn Met Thr Phe Met Pro Ile Val Ser Leu Leu
275 280 285
Glu Gly Val Pro Gly Ile Lys His Leu Ala Arg Ala Ile Glu Leu Tyr
290 295 300
Leu Glu Tyr Lys Pro Pro Ile Glu Asp Leu Gln Tyr Phe Leu Asp Phe
305 310 315 320
Gln Ile Ala Arg Val Trp Ala Pro Glu Gln Ser Asn Ile Gln Arg Lys
325 330 335
Glu Pro Val Cys Ser Ser Leu Gln Phe Ser Leu Met Gly Pro Lys Leu
340 345 350
Tyr Ile Ser Pro Asp Gln Val Thr Val Gly Arg Lys Pro Val Thr Gly
355 360 365
Leu Arg Leu Ser Leu Glu Gly Asn Lys Gln Asn Arg Leu Ala Ile His
370 375 380
Leu Gln His Leu Val Ser Leu Pro Lys Ile Leu Gln Pro His Trp Asp
385 390 395 400
Val His Met Ala Ile Gly Ala Pro Lys Trp Gln Gly Pro Glu Glu Gln
405 410 415
Asp Ser Arg Trp Phe Glu Pro Ile Lys Trp Lys Asn Phe Ser His Val
420 425 430
Ser Thr Ala Pro Ile Glu His Thr Asp Thr Cys Ile Gly Asp Leu Ser
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Gly Val His Ile Val Thr Gly Ala Gln Leu Gly Val Trp Asp Phe Gly
450 455 460
Ser Lys Asn Val Leu His Leu Lys Leu Leu Phe Ser Lys Val Pro Gly
465 470 475 480
Cys Thr Ile Arg Arg Ser Val Trp Asp His Ser Pro Cys Ser Leu Ser
485 490 495
Thr Ala Gln Arg Thr Asp Gly Ser Ser Ser Ser Val Ser Ser Glu Arg
500 505 510
Thr Ser Asp Asn Lys Lys Glu Asp Ser Ser Ser His Val Gly Lys Leu
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Ala Lys Ile Val Asp Ser Thr Glu Met Ser Lys Gly Pro Gln Asp Ser
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Pro Gly His Trp Leu Val Thr Gly Ala Lys Leu Gly Val Asp Lys Gly
545 550 555 560
Lys Ile Val Leu Arg Ile Lys Tyr Ser Leu Leu Asn Tyr
565 570
Claims (8)
1.如下1)-3)中任一种物质在调控植物耐冷性中的应用；
1)蛋白GhMACPF26；
2)编码蛋白GhMACPF26的核酸分子；
3)含有编码蛋白GhMACPF26的核酸分子的重组载体、表达盒或重组菌；
所述蛋白GhMACPF26为如下(1)或(2)或(3)：
(1)由序列表中序列3所示的氨基酸序列组成的蛋白质；
(2)由序列表中序列3所示的氨基酸序列的末端添加标签序列组成的蛋白质；
(3)将序列表中序列3所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由(1)或(2)衍生的蛋白质。
2.根据权利要求1所述的应用，其特征在于：
所述编码蛋白GhMACPF26的核酸分子是如下1)-3)中任一种的DNA分子：
1)编码区为序列表中序列1所示的DNA分子；
2)在严格条件下与1)或2)限定的DNA序列杂交且编码具有相同功能蛋白质的DNA分子；
3)与1)限定的DNA序列至少具有70％、至少具有75％、至少具有80％、至少具有85％、至少具有90％、至少具有95％、至少具有96％、至少具有97％、至少具有98％或至少具有99％同源性且编码具有相同功能蛋白质的DNA分子。
3.抑制或降低蛋白GhMACPF26含量或活性的物质，或，抑制或沉默GhMACPF26基因表达的物质在提高植物耐冷性中的应用；
或，抑制或降低蛋白GhMACPF26含量或活性的物质，或，抑制或沉默GhMACPF26基因表达的物质在培育耐冷性植物中的应用。
4.根据权利要求3所述的应用，其特征在于：所述物质包括用于沉默GhMACPF26基因表达的VIGS载体。
5.一种培育耐冷性提高的转基因植物的方法，为如下1)或2)：
1)所述的方法包括如下步骤：抑制或降低目的植物中蛋白GhMACPF26的含量和/或活性，得到转基因植物；
2)所述的方法包括如下步骤：抑制或降低目的植物中编码蛋白GhMACPF26的核酸分子的表达，得到转基因植物；
所述转基因植物的耐冷性高于所述目的植物；
所述蛋白GhMACPF26为如下(1)或(2)或(3)：
(1)由序列表中序列3所示的氨基酸序列组成的蛋白质；
(2)由序列表中序列3所示的氨基酸序列的末端添加标签序列组成的蛋白质；
(3)将序列表中序列3所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由(1)或(2)衍生的蛋白质。
6.根据权利要求5所述的方法，其特征在于：
所述抑制或降低目的植物中蛋白GhMACPF26的含量和/或活性，或，所述抑制或降低目的植物中编码蛋白GhMACPF26的核酸分子的表达，均是将沉默目的植物中编码蛋白GhMACPF26的核酸分子表达的物质导入所述目的植物中。
7.根据权利要求3或4所述的应用或权利要求5或6所述的方法，其特征在于：所述植物为双子叶植物或单子叶植物。
8.抑制或降低蛋白GhMACPF26含量或活性的物质，或，抑制或沉默GhMACPF26基因表达的物质，包括用于沉默GhMACPF26基因表达的VIGS载体。
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