WO2010127579A1 - 一种红树甜菜碱醛脱氢酶基因及其应用 - Google Patents

一种红树甜菜碱醛脱氢酶基因及其应用 Download PDF

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WO2010127579A1
WO2010127579A1 PCT/CN2010/071536 CN2010071536W WO2010127579A1 WO 2010127579 A1 WO2010127579 A1 WO 2010127579A1 CN 2010071536 W CN2010071536 W CN 2010071536W WO 2010127579 A1 WO2010127579 A1 WO 2010127579A1
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mangrove
aldehyde dehydrogenase
badh
dehydrogenase gene
betaine aldehyde
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PCT/CN2010/071536
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English (en)
French (fr)
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孙超
王君丹
陈文华
刘晓霞
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创世纪转基因技术有限公司
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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
    • 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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  • the invention relates to the field of plant genetic engineering, in particular to a mangrove betaine aldehyde dehydrogenase gene and application thereof in the development of salt-tolerant, cold-tolerant and drought-tolerant transgenic plants.
  • osmotic regulation which actively maintains osmotic balance by actively accumulating a large number of small molecular organic osmolytes, such as polyols, sugars, amino acids and their derivatives, in cells. Moisture in the body, these substances are called osmotic regulators.
  • Betaine is recognized as the most important cell-compatible substance in the protection of bacteria, plants, animals and other organisms. It is a kind of quaternary ammonium compound. Studies have shown that betaine is almost no longer synthesized. It is further metabolized and belongs to permanent or semi-permanent osmotic regulators. It has been paid more and more attention in the research of plant salt tolerance, drought tolerance and cold tolerance.
  • Betaine in plants can be divided into 4 types according to their structure and synthetic route, namely Gly betaine. , Pro betaine, Hydroxyproline betaine and Alanine betaine ( ⁇ -2 ⁇ -betaine) .
  • Gly betaine Pro betaine
  • Hydroxyproline betaine Hydroxyproline betaine
  • Alanine betaine ⁇ -2 ⁇ -betaine
  • Many studies have shown that the osmotic protection of glycine betaine is usually the strongest, and the glycine betaine is present in a variety of algae and seed plants of at least 10 families. Therefore, the existing studies mostly use glycine betaine. the Lord.
  • BADH Betaine aldehyde dehydrogenase
  • Arakawa et al. isolated and purified BADH from spinach leaves and prepared antibodies.
  • Weretilnyk et al. first isolated the mRNA of BADH from spinach, and proved that it was regulated by salt on the basis of successful expression in vitro.
  • Weretilnyk et al. first cloned BADH from spinach.
  • the cDNA of the gene which is 1819 bp in length, includes a 67 bp 5' non-coding region, a 1491 bp open reading frame and a 239 bp 3' non-coding region.
  • the entire BADH gene sequence of spinach and Chisui Valley, and all exon and intron regions of rice except for the 5' and 3' flanking sequences have also been cloned.
  • the BADH gene of higher plants has been studied to some extent in terms of structure, expression characteristics and its relationship with plant system evolution.
  • the object of the present invention is to provide a mangrove betaine aldehyde dehydrogenase A gene that increases the tolerance of a transgenic plant to abiotic stress caused by salinity, drought, and low temperature.
  • a mangrove betaine aldehyde dehydrogenase gene was cloned from mangrove with the nucleotide sequence shown in SEQ ID NO: 1.
  • a second object of the present invention is to provide a prokaryotic expression vector and a eukaryotic expression vector comprising a mangrove betaine aldehyde dehydrogenase gene.
  • the eukaryotic expression vector is a plant expression vector.
  • a plant cell, tissue or plant transformed with the plant expression vector A plant cell, tissue or plant transformed with the plant expression vector.
  • a third object of the present invention is to provide a betaine aldehyde dehydrogenase gene for use in the cultivation of salt tolerant, drought tolerant and cold tolerant plant varieties.
  • a fourth object of the present invention is to provide a protein sequence encoded by a mangrove betaine aldehyde dehydrogenase gene having SEQ ID NO: 3 The amino acid sequence shown.
  • the expression of the protein in the recipient plant is the key to the cultivation of the salt-tolerant, drought-tolerant and cold-tolerant plant varieties of the present invention.
  • the present invention clones the mangrove betaine aldehyde dehydrogenase (BADH) gene from the mangrove, the gene sequence thereof and the salt-tolerant BADH reported in the literature.
  • the gene has the highest homology, and the encoded protein has a conserved amino acid sequence unique to the same protein: QLFIDGE.
  • a prokaryotic expression vector and a binary plant expression vector were constructed using the mangrove betaine aldehyde dehydrogenase gene.
  • Mangrove betaine aldehyde dehydrogenase gene can be highly expressed in E. coli, and in high salt environment, transfer to BADH The E. coli growth of the gene is better than that of the untransferred BADH Gene E. coli.
  • the results of transgenic tobacco showed that the cloned mangrove betaine aldehyde dehydrogenase gene could be highly expressed in tobacco, and the transgenic tobacco obtained had obvious improvement in tolerance to salt stress, osmotic stress and low temperature stress.
  • Figure 1 is a schematic diagram of RNA detection of mangrove RNA
  • Figure 2 is a schematic diagram showing the results of 5'Race identification of the mangrove BADH gene
  • Figure 3 is a schematic diagram showing the results of 3'Race identification of the mangrove BADH gene
  • Figure 4 is a flow chart showing the construction of the prokaryotic expression vector EPSPS-pET30a;
  • Figure 5 is a schematic diagram showing the results of expression analysis of mangrove BADH gene in E. coli
  • Figure 6 is a schematic diagram showing the growth state of control Escherichia coli in LB medium with different NaCl concentrations
  • Figure 7 is a schematic diagram showing the growth state of BADH engineering bacteria in LB medium with different NaCl concentration
  • Figures 8a and 8b are flow diagrams showing the construction of the binary plant expression vector pCAMBIA300BADH
  • Figure 9 is a schematic diagram of the results of the seed germination test.
  • the whole idea of the present invention includes the following points: the betaine aldehyde dehydrogenase gene (BADH gene) is cloned from the salt-tolerant plant mangrove, and the prokaryotic expression vector and the binary plant expression vector are constructed, respectively transformed into E. coli and tobacco, and transferred to BADH.
  • BADH gene betaine aldehyde dehydrogenase gene
  • the high-salt culture of Escherichia coli was carried out, and the stress of BADH gene-positive tobacco was tested to verify the function of BADH gene.
  • the method comprises the following steps: 1. extracting total RAN of mangrove, preparing cDNA by reverse transcription; 2. amplifying the expression sequence tag of BADH gene from mangrove cDNA by PCR; 3. cloning the full length sequence of BADH gene by RACE method. The following is described in further detail:
  • the above degenerate primers P1 and P2 were used for PCR amplification to obtain an expression sequence tag (EST) of the BADH gene, and the base sequence thereof is shown in SEQ ID NO: 2.
  • the EST sequence was highly homologous to the known BADH gene, up to 88%.
  • the cloned sequence was determined to be the EST sequence of the mangrove BADH gene.
  • GSP-RT 5'-GGGTCCGATACTTTGATG-3'
  • GSP1 5'-CCGATACTTTGATGTTCTCAGAC-3'
  • GSP2 5'-CTCCAAAAGTTGTGCTGCAATGCTCTC-3'
  • AAP 5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3'
  • Cycle parameters 94 °C for 5 minutes; 94 °C for 30 seconds, 55 °C for 30 seconds, 72 °C 2 minutes; 30 cycles; 72 ° C for 7 minutes; 5 ° C;
  • Cycle parameters 94 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 2 minutes for 30 cycles; 72 °C 7 minutes; 5 °C.
  • the 5' end sequence of mangrove BADH gene was obtained by RACE method.
  • the results of PCR identification are shown in Fig. 2.
  • PCR amplification conditions were: 94 ° C 5 minutes; 94 ° C 30 seconds, 52 ° C 30 seconds, 72 ° C 2 minutes 30 cycles; 72 ° C 7 minutes; 5 ° C.
  • the results of 5'Race and 3'Race were ligated with DNAman software to obtain the full-length sequence of mangrove BADH gene.
  • the base sequence is SEQ ID. NO: 1 is shown.
  • the full-length sequence of the BADH gene can be obtained by RT-PCR.
  • the primer is BADH5': 5'-CAAAACCCACTGAAGAGTCC-3'
  • the amplification conditions were: 94 ° C for 5 minutes; 94 ° C for 30 seconds, 56 ° C for 45 seconds, 72 ° C for 1 minute. 30 cycles; 72 degrees for 5 minutes.
  • the mangrove BADH gene carrying the 5'UTR and 3'UTR sequences has the base sequence shown in SEQ ID NO:4.
  • the betaine aldehyde dehydrogenase encoded by the mangrove BADH gene of the present application has an amino acid sequence of SEQ ID NO: 3 is shown.
  • the prokaryotic expression vector EPSPS-pET30a of PET system was constructed. The specific construction process is shown in Figure 4.
  • the E. coli DH5 ⁇ was transformed with the prokaryotic expression vector EPSPS-pET30a.
  • the BADH gene of mangrove specifically expressed a protein band of 55KD in E. coli DH5 ⁇ .
  • the results of SDS-PAGE electrophoresis identification are shown in Fig. 5.
  • E. coli DH5 ⁇ itself contains a substrate required for BADH gene metabolism, the function of the BADH gene was initially verified directly using Escherichia coli DH5 ⁇ . After induced by IPTG for 3 hours, E. coli DH5 ⁇ was directly transferred into LB medium with different NaCl concentration (1%, 3%, 5%, 7%), and its OD value (bacterial concentration) was measured at different times. The growth conditions are shown in Tables 1 and 2.
  • Table 1 Cell concentration (OD value) of control cells grown at different NaCl concentrations at different times
  • the binary plant expression vector pCAMBIA300BADH which drives the drought-inducible promoter RD29A to drive BADH gene expression, is shown in Figures 8a and 8b.
  • Sph I was digested with the vector Rd29A-BADH-Tnos-PMD and then digested with EcoRI to obtain a plant expression cassette fragment in which the Ra29A promoter drives BADH gene expression.
  • the fragment was inserted into the vector pCAMBIA2300, and the final vector was named pCAMBIA2300BADH.
  • the tobacco seeds were soaked in 75% alcohol for 30 s, and then immersed in 0.1% liters of mercury for 8 min for surface disinfection.
  • the sterile tobacco seeds were placed on MS medium (30 g/L sucrose) and sterilely germinated to prepare sterile seedlings.
  • the leaves of the sterile seedlings were cut into 5 mm ⁇ 5 mm leaf discs, and the non-transgenic tobacco leaf discs were transferred into 1.5%, 2%, 2.5%, and 3%, respectively.
  • the differentiation medium of NaCl a differentiation medium to which no NaCl was added was used as a control.
  • the transgenic T1 seed and non-transgenic seeds were cultured on MS medium containing 5% NaCl. The results are shown in Figure 9.
  • the transgenic T1 seed is seeded, at least 90% of the transgenic T1 seeds can be germinated, B plate
  • the middle is a control non-transgenic seed with a germination rate of about 15%, indicating that the transgenic seeds have better tolerance to salt stress.
  • Use 5% The PEG MS medium and the 4 °C low temperature treatment germination test also confirmed that the transgenic seeds also had good tolerance to osmotic stress and low temperature stress.

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Description

一种红树甜菜碱醛脱氢酶基因及其应用
本发明涉及植物基因工程领域，尤其涉及一种红树甜菜碱醛脱氢酶基因及其在耐盐、耐寒、耐旱转基因植物研制方面的应用。
高盐、低温、干旱是影响植物生长和生产力最主要的非生物胁迫因子。高等植物适应环境胁迫的重要生理对策之一是渗透调节,通过在细胞中主动积累大量的小分子有机渗调物质,如多元醇类、糖类、氨基酸及其衍生物等,以维持渗透平衡和体内水分,这些物质被称为渗透调节剂。
甜菜碱是公认的在细菌、植物、动物等生物体中起着无毒渗透保护作用的最主要的细胞相溶性物质，它是一类季铵化合物，有研究表明，甜菜碱合成后几乎不再被进一步代谢，属于永久性或半永久性渗透调节剂，已在植物抗盐、耐旱、耐寒研究中越来越受到重视。
植物中的甜菜碱按其结构和合成途径不同可分为4种类型，即甘氨酸甜菜碱(Gly betaine) 、脯氨酸甜菜碱(Pro betaine) 、羟脯氨酸甜菜碱(Hydroxyproline betaine) 和丙氨酸甜菜碱(β-2α-betaine) 。许多研究表明，甘氨酸甜菜碱的渗透保护作用通常是最强的，并且所述甘氨酸甜菜碱在多种藻类和至少10个科的种子植物中存在，因些，现有研究多以甘氨酸甜菜碱为主。
在高等植物中，甜菜碱醛脱氢酶(BADH) 是合成甜菜碱的关键酶。自1981年从高等植物中分离到BADH后，有关甜菜碱合成酶分子生物学的研究很快引起了广泛关注。1987年，Arakawa等从菠菜叶中分离纯化了BADH并制备抗体。1989年，Weretilnyk等首先从菠菜中分离了BADH的mRNA，在成功实现其体外表达的基础上，证明其受盐调节。1990年，Weretilnyk等又从菠菜中首次克隆了BADH 基因的cDNA，此基因全长1819bp，包括67bp的5'端非编码区，1491bp开放阅读框架和239bp的3'端非编码区。目前，菠菜和千穗谷的BADH基因全序列、水稻除5'端和3'端侧翼序列以外的全部外显子区和内含子区也已被克隆。高等植物的BADH基因在结构、表达特性及其与植物系统进化的关系方面均在一定程度上得到了研究。
本发明的目的在于提供一种 红树甜菜碱醛脱氢酶 基因，其可以提高转基因植物的对盐碱、干旱和低温所造成非生物胁迫的耐受能力 。
从红树中克隆一种 红树甜菜碱醛脱氢酶 基因，具有 SEQ ID NO ： 1 所示的核苷酸序列。
本发明的第二个目的在于提供一种含有红树甜菜碱醛脱氢酶 基因 的原核表达载体和真核表达载体。
所述真核表达载体为植物表达载体。
用所述植物表达载体转化的植物细胞、组织或植株。
本发明的第三个目的在于提供红树甜菜碱醛脱氢酶 基因 在培育耐盐、耐旱、耐寒植物品种中的应用。
本发明的第四个目的在于提供一种红树甜菜碱醛脱氢酶基因编码的蛋白质序列，具有 SEQ ID NO ： 3 所示的氨基酸序列。
所述蛋白质在受体植物中的表达是本发明耐盐、耐旱、耐寒植物品种培育得以实现的关键。
本发明从红树中克隆了红树甜菜碱醛脱氢酶（ BADH ）基因，其基因序列和目前文献报导的耐盐类 BADH 基因同源性最高，其编码的蛋白质具有同类蛋白所特有的氨基酸保守序列： QLFIDGE 。 用红树甜菜碱醛脱氢酶基因构建原核表达载体和双元植物表达载体。红树甜菜碱醛脱氢酶基因可在大肠杆菌中高效表达，且在高盐环境下，转 BADH 基因的大肠杆菌生长状况要好于未转 BADH 基因的大肠杆菌。转基因烟草鉴定结果表明所克隆的的红树甜菜碱醛脱氢酶基因能在烟草内高效表达，获得的转基因烟草对盐胁迫、渗透胁迫和低温胁迫的耐受能力有明显改善
图 1 是红树 RNA 电泳检测示意图；
图 2 是 红树BADH 基因的5'Race 鉴定结果示意图；
图 3 是 红树BADH 基因的3'Race 鉴定结果示意图；
图 4 是原核表达载体 EPSPS-pET30a 构建流程图；
图 5 是 红树BADH 基因在大肠杆菌中表达鉴定结果示意图；
图6是对照大肠杆菌在不同NaCl浓度LB培养基中的生长状况示意图；
图7是BADH工程菌在不同NaCl浓度LB培养基中的生长状况示意图；
图8a和8b是双元植物 表达载体 pCAMBIA300BADH 构建流程图；
图 9 是种子萌发测试实验结果示意图。
本发明整体思路包括以下几点：从耐盐植物红树中克隆甜菜碱醛脱氢酶基因（BADH基因），构建原核表达载体和双元植物表达载体，分别转化大肠杆菌和烟草，对转BADH基因大肠杆菌进行高盐培养，对转BADH基因阳性烟草进行逆境胁迫实验，验证BADH基因的功能。以上各点皆为单独实施例，以下将结合具体实施例做进一步详细说明。
实施例1 红树 甜菜碱醛脱氢酶（ BADH） 基因 的克隆
包括以下步骤：1、提取红树总RAN，反转录制备cDNA；2、用PCR方法从红树cDNA中扩增BADH基因的表达序列标签；3、用RACE方法克隆BADH基因全长序列。以下进一步详细描述：
1、提取红树总RNA 并反转录制备cDNA
1）用液氮研磨0.1g叶子加入到含有1毫升Trizol的1.5毫升离心管中。
2）室温放置5min，再加入200微升氯仿，混匀，12000g离心10min。
3）取上清加入等体积氯仿，混匀12000g离心10min。
4）再取上清加入等体积异丙醇，沉淀，12000g离心10min。
5）用75％乙醇洗涤沉淀，晾干用500ul水溶解。
6）加入等体积酚/氯仿（1：1）混匀，室温放置5min，12000g离心10min。
7）取上清加入等体积氯仿，混匀，12000g离心5min。
8）再取上清加入等体积异丙醇，12000g离心10min。
9）用75％乙醇洗涤沉淀2次，晾干，用适量的水溶解。
10)电泳检测，并做反转录获得cDNA。
反转录步骤：
（1） 在0.2ml tube中，加入下列成分：
总 RNA(0.1 μ g/ μl ) 2.0μl
Oligo(dT12-18)(2 μ M) 2.0μl
（2） 70℃水浴10分钟。立即放置在冰浴中；
（3） 加入下列成分：2.0 μl 10×RT buffer；2.0μl 250 μ M dNTP mix；2.0 μl 100mM DTT；9.8 μl DEPC H2O；0.2 μl 200U μ /l SuperScript II；
（4） 进行下列反应：42℃ 90分钟；70℃ 15分钟；-20℃保存。
红树RNA电泳检测结果见图1。
2、用PCR方法从红树cDNA中扩增BADH基因的表达序列标签
1）简并引物的设计
依据Genebank上发布的已知BADH基因序列保守区设计简并引物，
P1：5'－GGRCCAAGYCKGCADCCTTCYTC－3'
P2：5'－TTcTGGACaAAYGGwCARATHTG－3'
2）红树BADH基因EST的获得
以红树cDNA为模板，用上述简并引物P1和P2进行PCR扩增，获得BADH基因的表达序列标签（EST），其碱基序列如SEQ ID NO：2所示。
并通过genebank作序列对比发现这个EST序列与已知BADH基因同源性很高，最高达88％，确定克隆到的序列是红树BADH基因的EST序列。
  1. 3、 用RACE方法克隆红树BADH基因
1）红树BADH 基因的5'Race
设计并合成5'RACE引物，
GSP-RT: 5'-GGGTCCGATACTTTGATG-3'
GSP1: 5'-CCGATACTTTGATGTTCTCAGAC-3'
GSP2: 5'-CTCCAAAAGTTGTGCTGCAATGCTCTC-3'
AAP ： 5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3'
AUAP ： 5'-GGCCACGCGTCGACTAGTAC-3'
5'RACE方法步骤：
A．逆转录
(1) 在0.5ml离心管中加入以下成分：
1 μl 10μM GSP1；12.5 μl （1-5 μ g）Total RNA；13.5 μl DEPC H2O；
(2) 混匀，70 ℃ 变性5分钟，置于冰浴中至少2分钟，离心；
(3) 按下表加入以下成分：
2.5 μl 10×PCR buffer；2.5 μl 0.1M DTT；1.25 μl RNaseOUT（40U/μl）；3 μl 25Mm MgCl2；1.25 μl 10mM dNTP，以上各种成分可在另一管中混匀，在45 ℃ 预热；
(4) 混匀各种成分，离心，42 ℃ 温育1-2分钟；
(5) 加入1 μl Superscript Ⅱ RT 混匀，42 ℃ 10分钟，50 ℃ 1-1.5小时；
(6) 70 ℃ 15分钟，以终止反应；
　 (7) 离心10-20秒，37 ℃ 温育, 加入1 μl RNase Mix 混匀，37 ℃ 30分钟；
(8) 离心，冰浴。
B．GlassMAX 纯化cDNA
(1) 将 binding solution 平衡至室温；
(2) 取100 μl ddH2O 70 ℃ 温育；
(3) 将120 μl binding solution (6M NaI)加入到第一链合成混合物中，混匀；
(4) 将cDNA/NaI混合物转移到GlassMAX离心柱中，13000g离心20s；
(5) 从管中取出柱子，将滤过液转移至另一干净离心管中，保存此管直至cDNA纯化完成，将离心柱放回原来的离心管中；
(6) 将0.4ml冷1×wash buffer 加入到柱子中，13000g离心20秒,弃滤过液重复3次；
(7) 用0.4ml冷70%乙醇洗2次柱子；
(8) 13000g离心1分钟，以彻底除去残留的乙醇；
(9) 将柱子转移到另一个离心管中，加入50 μl 65 ℃ 预热的ddH2O, 13000g离心20秒；
C．cDNA加尾
(1) 在 0.5ml离心管中加入以下成分：
5.0 μl 5×tailing buffer; 2.5 μl 2mM dCTP;16.5μlcDNA sample;24.0 μl H2O
(2) 94 ℃ 3分钟，置于冰上 1分钟，离心；
(3) 加入 1 μl TdT，混匀，37 ℃ 10分钟；
(4) 65 ℃ 加热10分钟，离心，冰浴保存。
D．加尾cDNA PCR扩增
(1) 在冰上，向0.2ml离心管中加入以下成分：
5 μl 10×PCR buffer；3.0 μl 25mM MgCl2；1.0 μl 10mM dNTP；2.0 μl 10 μ M GSP2；2.0 μl 10 μ M AAP；5.0 μl dC-tailed cDNA；31.5 μl ddH2O；
(2) 94 ℃ 变性5分钟；
(3) 加入0.5 μl Taq酶，混匀；
(4) 循环参数：94 ℃ 5分钟；94 ℃ 30秒，55 ℃ 30秒，72 ℃ 2分钟；30个循环；72 ℃ 7分钟；5 ℃ ；
(5) 取5-20 μl 电泳检测。
E．巢式PCR
(1) 取5 μl 第一轮PCR产物加入 495 μl TE buffer；
(2) 在冰上，向0.2μl离心管中，加入以下成分：
5 μl 10×PCR buffer；3.0 μl 25mM MgCl2；1.0 μl 10mM dNTP；2.0 μl 10 μ M GSP2；2.0 μl 10 μ M AAP；5.0 μl dC-tailed cDNA；31.5 μl ddH2O；
(3) 94 ℃ 5分钟；
(4) 加入 0.5μl Taq酶，混匀；
(5) 循环参数：94 ℃ 30秒，55 ℃ 30秒，72 ℃ 2分钟30个循环；72 ℃ 7分钟；5 ℃ 。
利用RACE方法获得红树BADH基因5'端序列，PCR鉴定结果见图2。
2）红树BADH 基因的3' Race
设计并合成3'RACE引物：
通用引物：APT18：5′-CGCTACGTAACGGCATGACAGTG(T)18-3′
AP：5′-CGCTACGTAACGGCATGACAGTG-3′
PCR扩增条件为：94℃ 5分；94℃ 30秒，52℃ 30秒，72℃ 2分钟30个循环；72℃ 7分钟；5℃。
PCR鉴定结果见图3。
3）红树BADH基因cDNA全长序列的获得
5'Race和3'Race的结果用DNAman软件拼接后获得红树BADH基因全长序列，其碱基序列如SEQ ID NO：1所示。BADH基因全长序列可用RT-PCR方法获得，引物为 BADH5': 5'-CAAAACCCACTGAAGAGTCC-3'
BADH3': 5'-GTTCAGTCTGGCAGTAGAGG-3'
扩增条件为：94℃ 5分钟；94℃ 30秒 56℃45秒 72℃ 1分钟 30循环；72度5分钟。
带有5'UTR和3'UTR序列的红树BADH基因，其碱基序列如SEQ ID NO：4所示。
在gene bank中Blast表明本申请克隆的红树BADH基因与已经克隆的一个红树BADH基因同源性为82％。
本申请所述红树BADH基因编码的 甜菜碱醛脱氢酶 ，其氨基酸序列如SEQ ID NO：3所示。
实施例 2 红树BADH基因原核表达载体的构建及其在大肠杆菌中的表达
构建PET系统原核表达载体EPSPS-pET30a，具体构建流程如图4，用原核表达载体EPSPS-pET30a转化大肠杆菌DH5α，红树BADH基因在大肠杆菌DH5α中特异表达出大小为55KD的蛋白质条带，其SDS-PAGE电泳鉴定结果见图5。
由于大肠杆菌DH5α自身含有BADH基因代谢所需的底物，所以直接使用大肠杆菌DH5α初步验证BADH基因的功能。大肠杆菌DH5α在IPTG诱导表达3小时后，直接转入不同NaCl浓度（1％，3％，5％，7％）的LB培养基中，分不同时间测其OD值（菌体浓度），监控其生长状况，结果见表1和表2。
表 1 ：不同 NaCl 浓度下对照菌体生长不同时间的菌体浓度（ OD 值）
Nacl 浓度
时间 (hr ）		 1% 3% 5% 7%
0 2.13 2.13 2.13 2.13
2 2.76 1.98 1.77 1.566
3.5 3.03 2.37 1.818 1.578
5.5 3.468 2.784 1.968 1.578
7 3.618 2.844 2.034 1.704
表 2 ：不同 NaCl 浓度下转 BADH 基因菌体生长不同时间的菌体浓度（ OD 值）
Nacl 浓度
时间 (hr ）		 1% 3% 5% 7%
0 2.922 2.922 2.922 2.922
2 3.36 3.144 2.784 2.484
3.5 3.456 3.24 3.054 2.712
5.5 3.534 3.492 3.186 2.658
7 3.75 3.744 3.198 2.652
由图6和图7中的生长数据可以看出随NaCl浓度的上升，菌体生长速度下降。在1％的基本NaCl浓度培养基中，转BADH基因的大肠杆菌的增长速度和对照大肠杆菌生长速度相当。在3％的NaCl浓度培养基中对比两个样品数据，BADH基因的表达明显改善了大肠杆菌的生长，使其在5.5hour培养后浓度达到接近1％NaCl浓度时的水平，而对照浓度明显低于1％的对照。在NaCl浓度为5％的培养基中，样品经过一段适应期后菌体浓度开始有所增加；而对照组却一直处于抑制生长状态。在NaCl浓度为7％的培养基中，样品和对照都出现明显的生长抑制，但是只有对照组中出现较明显的菌体死亡情况，样品组在经过一段时间生长后出现恢复生长的迹象。
实施例 3 红树BADH基因真核表达载体的构建
构建干旱诱导型启动子RD29A驱动BADH基因表达的双元植物表达载体pCAMBIA300BADH，参阅图8a和8b，具体构建流程：
1、Nco I和Sal I双酶切BaDH基因PCR产物和KTP-HT7-BT-PBS载体，将KTP-HT7-BT-PBS载体中的BT基因替换成BaDH基因，构建后载体命名为KTP-HT7-BADH-PBS；
2、Sal I酶切KTP-HT7-BADH-PBS后补平，再用BamH I进行酶切；将此片段插入Rd29A-TPS-Tnos-PMD载体中替换TPS片段，构建后载体命名为Rd29A-BADH-Tnos-PMD；
3、Sph I酶切载体Rd29A-BADH-Tnos-PMD后补平，再用EcoRI酶切，获得Ra29A启动子驱动BADH基因表达的植物表达盒片段，将此片段插入载体pCAMBIA2300，最终载体命名为pCAMBIA2300BADH。
实施例 4 红树 BADH 基因在烟草中的功能鉴定
对实验室现有非转基因烟草进行NaCl浓度的筛选，利用BADH具有耐盐功能的特点，直接以NaCl为筛选物质对转基因烟草进行筛选。
用75%酒精浸泡烟草种子30s，再用0.1%升汞浸泡8min，进行表面消毒。将消过毒的烟草种子置于MS培养基（加蔗糖30g/L）上无菌发芽，制备无菌苗。取无菌苗叶片剪成5mm×5mm大小的叶盘，将非转基因的烟草叶盘转入分别含1.5%、2%、2.5%、3% NaCl的分化培养基中，以不添加NaCl的分化培养基为对照。经观察，在含2.5% NaCl条件下的烟草叶盘已大面积枯黄，NaCl浓度达3%时烟草叶盘枯死，而用含有双元植物表达载体pCAMBIA300BADH的农杆菌浸染烟草叶盘后，将烟草叶盘转入含有5%NaCl的分化培养中，15天后观察发现，叶盘有分化现象，长出新芽。以上结果表明，转红树BADH基因的烟草具有较好的耐盐性。
将转基因T1代种子和非转基因种子，在含5%NaCl的MS培养基上培养，结果见图9，A平皿中是转基因T1代种子，其中至少90%的转基因T1代种子可以萌发，B平皿中是对照非转基因种子，萌发率约为15%，表明转基因种子对盐分胁迫具有更好的耐受能力。用含有5% PEG的MS培养基、以及4℃低温处理的萌发试验也验证了转基因种子对渗透胁迫、低温胁迫也具有较好耐受能力。

Claims (7)

1 、一种红树甜菜碱醛脱氢酶基因，其特征在于：所述基因具有 SEQ ID NO ： 1 所示的核苷酸序列。
2 、含有权利要求 1 所述红树甜菜碱醛脱氢酶基因的原核表达载体。
3 、含有权利要求 1 所述红树甜菜碱醛脱氢酶基因的真核表达载体。
4 、根据权利要求 3 所述的含有红树甜菜碱醛脱氢酶基因的真核表达载体，其特征在于：所述真核表达载体为植物表达载体。
5 、用权利要求 4 所述植物表达载体转化的植物细胞、组织或植株。
6 、权利要求 5 所述植物细胞、组织或植株在培育耐盐、耐旱、耐低温植物品种中的应用。
7 、权利要求 1 所述红树甜菜碱醛脱氢酶基因编码的蛋白质序列，其特征在于：所述蛋白质具有 SEQ ID NO ： 3 所示的氨基酸序列。
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