WO2019128005A1 - 一种抗性基因及其用于制备转基因植物的应用 - Google Patents

一种抗性基因及其用于制备转基因植物的应用 Download PDF

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WO2019128005A1
WO2019128005A1 PCT/CN2018/084301 CN2018084301W WO2019128005A1 WO 2019128005 A1 WO2019128005 A1 WO 2019128005A1 CN 2018084301 W CN2018084301 W CN 2018084301W WO 2019128005 A1 WO2019128005 A1 WO 2019128005A1
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gene
cbdreb2al
plant
drought
transgenic
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岳修乐
安黎哲
占毅
张国艳
赵智星
蒲晓宏
隋孟君
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兰州大学
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Priority claimed from CN201711478626.3A external-priority patent/CN108018291B/zh
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • the invention belongs to the field of biotechnology, and particularly relates to a method for preparing a mountain ion mustard gene CbDREB2AL, a product thereof CbDREB2AL protein, a plant containing the antifreeze and drought tolerance gene CbDREB2AL, and the preparation thereof in the antifreeze and drought tolerant transgenic plants. application.
  • the annual grain output is caused by drought. The loss is very heavy, and China’s annual loss of food is 5 billion kilograms due to drought.
  • Cold-responsive genes encode a wide variety of proteins, such as plant respiration, carbon cycle, lipids, phenylpropanoids and enzymes involved in antioxidant metabolism processes, as well as transcription factors that regulate gene expression, antifreeze proteins, etc. [9-11].
  • Chorispora bungeana (also known as Chorispora exscapa) is a perennial herb of the genus Brassica, which is distributed on high-altitude subalpine meadows and gravel slopes. The environment is characterized by cold climate, drought, and strong radiation. . In China, it is mainly distributed in the streamstones of the source area of the Urumqi River. The gravel in the stream is poor in water retention, and the harsh environment often causes low temperature and drought stress of the mountain ion mustard. Alpine ion mustard has evolved an excellent adversity adaptation mechanism in order to adapt to adverse environmental conditions such as low temperature, strong ultraviolet, high wind and drought [12, 13].
  • the invention discloses an antifreeze and drought tolerance gene CbDREB2AL, which encodes a new protein CbDREB2AL which is induced by stress in a mountain ion mustard.
  • the protein encoded by this gene is closely related to the homologous genes of Amaranth (Es), Azolla (Nc), Arabidopsis thaliana (Al), Arabidopsis thaliana (At) and peanut (Ah). relationship.
  • the CbDREB2AL gene disclosed in the invention can be applied to the breeding of drought-tolerant and anti-freeze transgenic crops to improve its drought tolerance and frost resistance, and has broad application prospects.
  • Another object of the present invention is to provide a method for cultivating an antifreeze transgenic plant by transferring the gene into a plant to obtain a transgenic plant having antifreeze properties.
  • Still another object of the present invention is to provide a method for cultivating a drought-tolerant transgenic plant by transferring said coding gene into a plant to obtain a transgenic plant having drought-tolerant characteristics.
  • a further object of the present invention is to provide a use of the gene CbDREB2AL for transforming plants to produce antifreeze transgenic plants.
  • a further object of the present invention is to provide a use of the gene CbDREB2AL for transforming plants to produce drought tolerant transgenic plants.
  • Expression vectors, cell lines and host bacteria containing the gene of the present invention are all within the scope of the present invention.
  • the present invention clones the gene CbDREB2AL of the mountain ion mustard having the nucleotide sequence shown by SEQ ID NO: 1, wherein SEQ ID NO: 1 in the list consists of 1002 bases.
  • the gene CbDREB2AL is capable of encoding a CbDREB2AL protein having the amino acid sequence set forth in SEQ ID NO: 2, wherein SEQ ID NO: 2 in the list consists of 334 amino acids.
  • the protein encoded by the CbDREB2AL gene is the same as the dicotyledonous plant (Es), Azure (Nc), Arabidopsis thaliana (Al), Arabidopsis thaliana (At), and peanut (Ah).
  • the source gene has a close genetic relationship.
  • a recombinant expression vector containing the CbDREB2AL gene can be constructed using an existing plant expression vector.
  • the plant expression vector includes a dual Agrobacterium vector and a vector which can be used for plant microprojectile bombardment, and the like, such as PMDC32, pCAMBIA3301, pCAMBIA1300 or other derivative plant expression vectors.
  • the plant expression vector carrying the gene CbDREB2AL of the present invention can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, Agrobacterium-mediated, and the like.
  • the transformed host plant may be a dicotyledonous plant such as Arabidopsis thaliana or tobacco.
  • the plant expression vector used may be processed, such as a gene (GFP gene, GUS gene, etc.) capable of expressing an enzyme or a luminescent compound capable of producing a color change in a plant, Resistant antibiotic markers (gentamicin markers, kanamycin markers, etc.) or anti-chemical marker genes (such as anti-fragrance genes). From the safety of transgenic plants, the transformed plants can be directly screened by adversity without any selectable marker genes.
  • a gene GFP gene, GUS gene, etc.
  • Resistant antibiotic markers gentamicin markers, kanamycin markers, etc.
  • anti-chemical marker genes such as anti-fragrance genes
  • the method for obtaining a transgenic plant provided by the present invention is to introduce the antifreeze protein of the above plant and the gene encoding the drought resistance protein CbDREB2AL into a plant to obtain an antifreeze and drought tolerant transgenic plant.
  • the cDNA of the CbDREB2AL gene is constructed downstream of the CaMV35s promoter to obtain an expression vector (the map is shown in FIG. 2), and the Agrobacterium strain containing the expression vector is obtained by electroporation, and the wild is transfected into the wild by the dip flower method.
  • Types of Arabidopsis thaliana are overexpressed transgenic plants.
  • the plant may be a dicotyledonous plant such as tobacco, Arabidopsis, and the like.
  • the present invention confirmed by comparison experiments that the transgenic plants containing the CbDREB2AL gene have antifreeze and drought tolerance characteristics.
  • the results in Fig. 3 and Fig. 4 show that the overexpression of CbDREB2AL can significantly enhance the antifreeze property of Arabidopsis thaliana after low temperature treatment. The survival rate of Arabidopsis is increased by about 20%.
  • the results in Figures 5 and 6 show that overexpression of CbDREB2AL can significantly enhance the drought tolerance of Arabidopsis, increasing the survival rate of Arabidopsis thaliana after drought treatment by about 45%.
  • CbDREB2AL can significantly enhance the drought tolerance and antifreeze characteristics of Arabidopsis thaliana, which has important theoretical and practical significance for cultivating excellent crop varieties, especially drought tolerant and freeze resistant crop varieties.
  • Figure 1 shows the evolutionary relationship of the amino acid sequence of the CbDREB2AL protein of the mountain ion mustard by homology alignment using MEGA software.
  • FIG. 1 Construction of the overexpression vector map, PMDC32-CbDREB2AL.
  • Chorispora bungeana also known as Chorispora exscapa
  • Arabidopsis thaliana Cold-0
  • Chorispora bungeana also known as Chorispora exscapa
  • Arabidopsis thaliana Cold-0
  • Agrobacterium GV3101 Pieris Biotechnology Co., Ltd.
  • plasmid PMDC32 American Arabidopsis Bioresource Center
  • RNA extraction and separation reagent Trizol, Invitrogen
  • the specific method was as follows: 100 mg of algal ion mustard regeneration seedlings were collected, and immediately ground into liquid powder in liquid nitrogen, and then 1 ml of Trizol reagent was added to fully mix.
  • the cDNA sequence of CbDREB2AL was amplified by RT-PCR according to the manual of PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa, No. 6210A): 4 ⁇ l of total RNA (about 3 ⁇ g) and some reagents in the kit were mixed.
  • the protein encoded by the CbDREB2AL gene in the present invention is the same as the dicotyledonous plant with the yam (Es), the azure blue (Nc), the Arabidopsis thaliana (Al), the Arabidopsis thaliana (At), and the peanut (Ah).
  • the source gene has a close relationship, as shown in Figure 1.
  • Agrobacterium-mediated transformation The successfully constructed overexpression plasmid PMDC32-CbDREB2AL was transformed into Agrobacterium GV3101 by electric shock (voltage 2400V, capacitance 25 ⁇ F, impedance 200 ⁇ , electric shock cup 1mm), with 10mg/L rifampicin + Positive clones were screened on LB plates of 50 mg/L kanamycin.
  • Transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana 4 weeks after germination were placed in a low-temperature incubator as described in Example 2, and the temperature was lowered by 2 ° C per hour, and the temperature was reduced to -8 ° C. The treatment was started for 6 hours. After 2 days of recovery at 4 ° C, normal culture was performed, and the survival rate was counted. The results showed that overexpression of CbDREB2AL significantly enhanced the freeze resistance of Arabidopsis thaliana (Figure 3), increasing the survival rate of Arabidopsis thaliana after low temperature treatment by about 20% ( Figure 4).
  • the anti-freeze and drought-tolerant gene CbDREB2AL disclosed in the present invention can be applied to drought-tolerant and anti-freeze transgenic crop breeding, and obtain transgenic plants having drought-tolerant characteristics and anti-freezing characteristics.

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Abstract

提供了一种高山离子芥基因CbDREB2AL、其编码产物CbDREB2AL蛋白、含有抗冻及耐旱基因CbDREB2AL的植物的制备方法、及其在制备抗冻和耐旱转基因植物中的应用。所述的CbDREB2AL基因可应用到耐旱和抗冻转基因作物育种中以提高其耐旱性和抗冻性。

Description

一种抗性基因及其用于制备转基因植物的应用 技术领域
本发明属于生物技术领域，特别涉及一种高山离子芥基因CbDREB2AL、其编码产物CbDREB2AL蛋白、含有抗冻及耐旱基因CbDREB2AL的植物的制备方法、及其在制备抗冻和耐旱转基因植物中的应用。
背景技术
干旱严重影响着植物的生长发育，制约着作物的产量及种植范围，是目前影响农业生产的世界性问题。据统计：全球干旱、半干旱地区已超过土地总面积的1/3，在我国占土地总面积的47％，在耕地面积中干旱、半干旱地区已占51％，每年粮食产量因干旱而造成的损失非常惨重，我国每年因旱灾损失粮食达50亿公斤。目前，关于利用转基因技术提高植物的耐旱性已有不少报道，如：Mie Kasuga等人将拟南芥基因DREB1A转入烟草后能够提高转基因植株的耐旱性[1]，Honghong Hu等人将SNAC1基因在水稻中过表达能将转基因水稻在干旱胁迫下的结实率提高22–34％[2]，Bi‐Yan Zao等人将油菜中的BaABF2基因在拟南芥中过表达也能提高转基因植株的抗旱能力[3]。
低温也是影响植物地理分布和生长季节的最主要的环境因素之一，低温冻害严重地影响着农作物的生长状况和产量[4,5]。大量的研究表明，冷响应基因的表达对植物的抗冻有着至关重要的作用[6-8]。冷响应基因编码种类繁多的蛋白，如植物呼吸、碳循环、脂类、苯丙素和抗氧化代谢过程相关的酶，还有调控基因表达的转录因子，抗冻蛋白等[9-11]。
高山离子芥(Chorispora bungeana，又名Chorispora exscapa)是十字花科离子芥属多年生草本植物，分布在高海拔亚高山草甸和砾石质山坡上，该地区的环境特点是气候寒冷、干旱、强辐射。在我国主要分布于乌鲁木齐河源区流石碓，流石碓中砾石保水性差，恶劣环境时常造成高山离子芥低温和干旱胁迫。高山离子芥为了适应低温、强紫外、大风和干旱等不利的环境条件，进化出了优秀的逆境适应机制[12,13]。
本发明公开了一种抗冻及耐旱基因CbDREB2AL，该基因编码高山离子芥一个新的受逆境诱导而产生的蛋白CbDREB2AL。该基因编码的蛋白与山萮菜(Es)、天蓝遏蓝菜(Nc)、琴叶拟南芥(Al)、拟南芥(At)以及花生(Ah)的同源基因有较近的亲缘关系。本发明公开的CbDREB2AL基因可应用到耐旱和抗冻转基因作物育种中以提高其耐旱性和抗冻性，具有广阔的应用前景。
参考文献：
1.Kasuga,M.,et al.,A combination of the Arabidopsis DREB1A gene and stress-inducible rd29A promoter improved drought-and low-temperature stress tolerance in tobacco by gene transfer.Plant&cell physiology,2004.45(3):p.346-50.
2.Hu,H.H.,et al.,Overexpressing a NAM,ATAF,and CUC(NAC)transcription factor enhances drought resistance and salt tolerance in rice.Proceedings of the National Academy of Sciences of the United States of America,2006.103(35):p.12987-12992.
3.Zhao,B.Y.,et al.,BnaABF2,a bZIP transcription factor from rapeseed(Brassica napus L.),enhances drought and salt tolerance in transgenic Arabidopsis.Bot Stud,2016.57(1):p.12.
4.Thomashow,M.F.,PLANT COLD ACCLIMATION:Freezing Tolerance Genes and Regulatory Mechanisms.Annual Review of Plant Physiology and Plant Molecular Biology,1999.50(1):p.571-599.
5.Orvar,B.L.,et al.,Early steps in cold sensing by plant cells:the role of actin cytoskeleton and membrane fluidity.The Plant journal:for cell and molecular biology,2000.23(6):p.785-94.
6.Zhu,J.K.,Cell signaling under salt,water and cold stresses.Current Opinion in Plant Biology,2001.4(5):p.401-406.
7.Lee,B.h.,The Arabidopsis Cold-Responsive Transcriptome and Its Regulation by ICE1.The Plant Cell Online,2005.17(11):p.3155-3175.
8.Chinnusamy,V.,J.Zhu,and J.K.Zhu,Gene regulation during cold acclimation in plants.Physiologia Plantarum,2006.126(1):p.52-61.
9.Demiral,T.and I.Türkan,Exogenous glycinebetaine affects growth and proline accumulation and retards senescence in two rice cultivars under NaCl stress.Environmental and Experimental Botany,2006.56(1):p.72-79.
10.Uemura,M.,et al.,Responses of the plasma membrane to low temperatures.Physiologia Plantarum,2006.126(1):p.81-89.
11.Ruelland,E.and A.Zachowski,How plants sense temperature.Environmental and Experimental Botany,2010.69(3):p.225-232.
12.Zhao,Z.,et al.,Deep-sequencing transcriptome analysis of chilling tolerance mechanisms of a subnival alpine plant,Chorispora bungeana.BMC Plant Biology,2012.12(1):p.222.
13.Shi,Y.,X.Yue,and L.An,Integrated Regulation Triggered by a Cryophyte omega-3Desaturase Gene Confers Multiple-Stress Tolerance to Tobacco.J Exp Bot,2018.
发明内容
本发明的目的是提供一种基因CbDREB2AL及其编码蛋白。
本发明的另一个目的在于提供一种培育抗冻转基因植物的方法，是将所述的编码基因转入植物中，得到具有抗冻特性的转基因植物。
本发明的再一个目的在于提供一种培育耐旱转基因植物的方法，是将所述的编码基因转入植物中，得到具有耐旱特性的转基因植物。
本发明的又一个目的在于提供一种由所述基因CbDREB2AL用于转化植物以产生抗冻转基因植物的用途。
本发明的又一个目的在于提供一种由所述基因CbDREB2AL用于转化植物以产生耐旱转基因植物的用途。
含有本发明基因的表达载体、细胞系及宿主菌均属于本发明的保护范围。
为解决上述技术问题，本发明采用如下技术方案：
本发明克隆了高山离子芥的基因CbDREB2AL，所述基因CbDREB2AL具有SEQ ID NO:1 所示的核苷酸序列，其中，列表中的SEQ ID NO：1由1002个碱基组成。该基因CbDREB2AL能够编码CbDREB2AL蛋白，此种蛋白具有SEQ ID NO:2所示的氨基酸序列，其中，列表中的SEQ ID NO：2由334个氨基酸组成。
本发明中CbDREB2AL基因编码的蛋白与双子叶植物山萮菜(Es)、天蓝遏蓝菜(Nc)、琴叶拟南芥(Al)、拟南芥(At)以及花生(Ah)等的同源基因具有较近的亲缘关系。
可用现有的植物表达载体构建含有CbDREB2AL基因的重组表达载体。所述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等，如PMDC32、pCAMBIA3301、pCAMBIA1300或其它衍生植物表达载体。携带有本发明基因CbDREB2AL的植物表达载体可通过Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、显微注射、电导、农杆菌介导等常规生物学方法转化到植物细胞或组织中。被转化的宿主植物可以是拟南芥、烟草等双子叶植物。
为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用植物表达载体进行加工，如加入可在植物中表达可产生颜色变化的酶或发光化合物的基因(GFP基因、GUS基因等)、具有抗性的抗生素标记物(庆大霉素标记物、卡那霉素标记物等)或是抗化学试剂标记基因(如抗除芳剂基因)等。从转基因植物的安全性考虑，可不加任何选择性标记基因，直接以逆境筛选转化植株。
本发明所提供的获得转基因植物的方法，是将上述植物的抗冻蛋白和抗旱蛋白的编码基因CbDREB2AL导入植物中，得到抗冻和耐旱转基因植物。
本发明将CbDREB2AL基因的cDNA构建在CaMV35s启动子下游，得到表达载体(其图谱如图2所示)，利用电击转化的方法即得到含有该表达载体的农杆菌菌株，利用浸花法转染野生型拟南芥得到过表达的转基因植株。所述植物可为双子叶植物，如烟草、拟南芥等。
本发明通过对比试验验证了含有CbDREB2AL基因的转基因植株具有抗冻和耐旱特性，图3和图4中的结果显示，CbDREB2AL的过表达可以显著增强拟南芥的抗冻特性，使低温处理之后的拟南芥的存活率提高了20％左右。图5和图6中的结果显示，CbDREB2AL的过表达可以显著增强拟南芥的耐旱特性，使干旱处理之后的拟南芥的存活率提高了45％左右。
综上所述，CbDREB2AL的过表达可以显著增强拟南芥的耐旱及抗冻特性，这对于培养优良的作物品种特别是耐旱和抗冻的作物品种具有重要的理论及实践意义。
附图说明
图1高山离子芥CbDREB2AL蛋白的氨基酸序列经同源性比对，利用MEGA软件分析的进化关系。
图2构建的过表达载体图谱，PMDC32-CbDREB2AL。
图3对照(野生型WT为Col-0)和转基因植株(#2，#3)抗冻性表型比较。
图4对照(野生型WT为Col-0)和转基因植株(#2，#3)低温处理后的存活率比较，转基因植株的存活率和野生型相比差异极显著(***P<0.001,****P<0.0001,by Student’s t-test)。
图5对照(野生型WT为Col-0)和转基因植株(#2，#4)耐旱性表型比较。
图6对照(野生型WT为Col-0)和转基因植株(#2，#4)干旱处理后的存活率比较,转基因植株的存活率和野生型相比差异极显著(***P<0.001,by Student’s t-test)。
具体实施方式
下面结合具体实施例进一步详细描述本发明的技术方案，但本发明的保护范围不局限于以下所述。
在本发明的下述实施例中，所用的实验材料为高山离子芥(Chorispora bungeana，又名Chorispora exscapa)(西部地区特色植物种质资源库平台)和拟南芥(Arabidopsis thaliana，Col-0)(美国拟南芥生物资源中心)，农杆菌GV3101(普如汀生物技术有限公司),质粒PMDC32(美国拟南芥生物资源中心)。
实施例1 高山离子芥蛋白编码基因CbDREB2AL序列的克隆：
利用RNA提取分离试剂(Trizol，Invitrogen)分离高山离子芥再生苗总RNA，其具体方法是：收集高山离子芥再生苗100mg，立即置于液氮中研磨成粉末，之后加入1ml Trizol试剂，充分混匀，吸入到一个1.5ml的离心管中；室温放置5min；加入0.2ml新鲜氯仿，剧烈振摇15s，室温静置3min；4℃，12000g离心15min；上清转移到一个新的1.5ml离心管中，加入0.5ml异丙醇混匀，4℃，12000g离心10min沉淀RNA；RNA沉淀用1ml 75％乙醇洗涤后溶于适量DEPC处理过的水中，-70℃保存备用。
根据快速末端扩增技术(RACE)获取该基因全长序列，然后设计以下引物进行编码区的克隆：
5’端引物：
Figure PCTCN2018084301-appb-000001
(其中划线序列为Invitrogen Gateway系统attB1序列)；
3’端引物：
Figure PCTCN2018084301-appb-000002
(其中划线序列Invitrogen Gateway系统attB2序列)。
通过RT-PCR扩增得到CbDREB2AL的cDNA序列，具体方法是：依据PrimeScript II 1st Strand cDNA Synthesis Kit(TaKaRa,No.6210A)的手册进行:4μl总RNA(约3μg)和试剂盒中的部分试剂混合(Oligo dT Primer 1μl,dNTP Mixture 1μl,RNase free Water 4μl)，65℃处理5min之后立刻冰上冷却，继续加入试剂混合(5×PrimeScript II Buffer 4μl，RNase Inhibitor 0.5μl，PrimeScript II RTase 1μl，RNase free Water 4.5μl)，混匀后，42℃处理60min；95℃处理5min，完成反转录反应。
吸取2μl上述反转录产物，作为模板进行PCR反应：94℃ 2min后进入扩增程序：94℃ 30s、56℃ 30s、72℃ 60s,30个循环后，72℃ 5min。扩增得到的SEQ ID NO：1序列总长1002个碱基，编码334个氨基酸,根据the 1997IUPAC standard atomic weights,assuming pH＝7.0计算分子量为37.294kDa,根据ExPASy's Compute pI/Mw program计算等电点为4.954。
本发明中CbDREB2AL基因编码的蛋白与双子叶植物与山萮菜(Es)、天蓝遏蓝菜(Nc)、琴叶拟南芥(Al)、拟南芥(At)以及花生(Ah)的同源基因有较近的亲缘关系，如图1所示。
实施例2 转CbDREB2AL基因植株的获得
1.高山离子芥CbDREB2AL基因植物过表达载体的构建：利用Invitrogen公司Gateway技术将测序验证过的实施例1得到的片段通过BP反应(BP
Figure PCTCN2018084301-appb-000003
II Enzyme mix，Invitrogen No.11789020)重组到pDONR/Zeocin载体(Invitrogen No.12535-035)，转化大肠杆菌DH5α感受态细胞中，经20mg/L的Zeocin筛选获得入门克隆，之后提取质粒再通过Gateway技术中的LR反应(LR
Figure PCTCN2018084301-appb-000004
II Enzyme mix，Invitrogen No.11791100)将CbDREB2AL基因重组到PMDC32载体上，转化大肠杆菌DH5α感受态细胞中，经50mg/L卡那霉素筛选得成功重组的过表达载体PMDC32-CbDREB2AL(如图2)。
2.农杆菌介导的转化：将构建成功的过表达质粒PMDC32-CbDREB2AL通过电击的方式(电压2400V，电容25μF，阻抗200Ω，电击杯1mm)转化农杆菌GV3101，用10mg/L利福平+50mg/L卡那霉素的LB平板筛选阳性克隆。将阳性克隆接种到YEP液体培养基中(含抗生素：链霉素25mg/L、利福平50mg/L、卡那霉素50mg/L)于恒温摇床上28℃，180rpm摇床培养至OD 600＝0.6-0.8，离心后用侵染培养基(1/2MS，5％sucrose，0.01％silwet L-77，pH5.7)重悬OD 600＝0.8-1。将拟南芥倒置使花苞浸没在侵染培养基溶液里30-60秒，用保鲜膜将侵染之后的拟南芥地上部分包裹，黑暗培养两天之后去掉保鲜膜，并用清水轻柔冲洗侵染部分，正常条件(25℃，16小时光照、8小时黑暗)继续培养直至收获种子。
3.转基因株系的筛选：将转基因拟南芥收获的种子，经表面消毒后，种在含有50mg/L潮霉素的MS平板上进一步筛选，经过传代，半定量PCR验证，最后得到纯合的CbDREB2AL过表达 的拟南芥T2代转基因株系。
实施例3 转基因CbDREB2AL拟南芥株系的抗冻性实验测定
如实施例2所述，将萌发后4周的转基因拟南芥和野生型拟南芥放入低温培养箱，梯度降温，每小时降2℃，降至-8℃开始计时，处理6小时，4℃恢复2天后正常培养，统计存活率。结果显示CbDREB2AL的过表达可以显著增强拟南芥的抗冻性(如图3)，使低温处理之后的拟南芥的存活率提高了20％左右(如图4)。
实施例4 转基因CbDREB2AL拟南芥株系的耐旱性实验测定：
如实施例2所述，将萌发后4周的转基因拟南芥和非转基因拟南芥同时停止浇水，干旱处理15天，然后恢复浇水5天，统计存活率。结果显示CbDREB2AL的过表达可以显著增强拟南芥的耐旱性(如图5)，使干旱处理之后的拟南芥存活率提高了45％左右(如图6)。
综上所述，本发明公开的一种抗冻及耐旱基因CbDREB2AL，可应用到耐旱和抗冻的转基因作物育种，得到具有耐旱特性和抗冻特性的转基因植株。

Claims (8)

  1. 一种高山离子芥基因CbDREB2AL，其特征在于，该基因的核苷酸序列如SEQ ID NO：1所示。
  2. 一种如权利要求1所述的基因CbDREB2AL编码的蛋白质CbDREB2AL，其特征在于，其氨基酸序列如SEQ ID NO：2所示。
  3. 一种含有如权利要求1所述的基因CbDREB2AL的表达载体。
  4. 一种含有如权利要求1所述的基因CbDREB2AL的细胞系。
  5. 一种含有如权利要求1所述的基因CbDREB2AL的宿主菌。
  6. 一种如权利要求1所述基因CbDREB2AL用于转化植物以产生抗冻转基因植物的用途。
  7. 一种如权利要求1所述基因CbDREB2AL用于转化植物以产生耐旱转基因植物的用途。
  8. 一种含有如权利要求1所述的基因CbDREB2AL的植物的制备方法，其特征在于，所述制备方法包括如下步骤：(1)表达载体的构建；(2)农杆菌介导的转化；(3)转基因株系的筛选。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113061613A (zh) * 2019-12-30 2021-07-02 兰州大学 CbDREB2AL基因在制备耐盐转基因植物中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999038977A2 (en) * 1998-02-03 1999-08-05 Mendel Biotechnology, Inc. Plant having altered environmental stress tolerance
CN1544626A (zh) * 2003-11-20 2004-11-10 北京林业大学 用转基因技术提高林木抗旱性的方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999038977A2 (en) * 1998-02-03 1999-08-05 Mendel Biotechnology, Inc. Plant having altered environmental stress tolerance
CN1544626A (zh) * 2003-11-20 2004-11-10 北京林业大学 用转基因技术提高林木抗旱性的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide 11 May 2017 (2017-05-11), "Predicted:Arabidopsis lyrata subsp. lyrata dehydra- tion-responsive element-binding protein 2A(LOC9307227), mRNA", XP055623267, retrieved from NCBI Database accession no. XM_021020347. 1 *
DATABASE Protein 20 March 2017 (2017-03-20), TABATA, S.: "DRE-binding protein 2A[Arabidopsis thaliana", XP055623272, retrieved from NCBI Database accession no. NP_196160.1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113061613A (zh) * 2019-12-30 2021-07-02 兰州大学 CbDREB2AL基因在制备耐盐转基因植物中的应用

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