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CN101870983B - Wintersweet gene containing thioesterase functional domain and drought-resistant gene engineering application thereof - Google Patents

Wintersweet gene containing thioesterase functional domain and drought-resistant gene engineering application thereof Download PDF

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CN101870983B
CN101870983B CN2009102540003A CN200910254000A CN101870983B CN 101870983 B CN101870983 B CN 101870983B CN 2009102540003 A CN2009102540003 A CN 2009102540003A CN 200910254000 A CN200910254000 A CN 200910254000A CN 101870983 B CN101870983 B CN 101870983B
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gene
wintersweet
cpacyltase
thioesterase
tobacco
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CN101870983A (en
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张世宏
张莉弘
刘金亮
胡军
于慧美
殷君华
阳晓红
潘洪玉
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Jilin University
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Abstract

The invention provides a wintersweet gene containing a thioesterase functional domain and a drought-resistant gene engineering application thereof, belonging to the field of molecular biology and genetic engineering, and the invention provides a wintersweet gene and a protein containing the thioesterase functional domain, and simultaneously provides an expression vector and a host cell corresponding to the gene, and the invention has the beneficial effects that: the application of the wintersweet gene containing the thioesterase functional domain can improve the drought resistance of transgenic plants.

Description

包含硫酯酶功能域的腊梅基因及其抗旱基因工程应用Wintersweet gene containing thioesterase functional domain and its application in genetic engineering for drought resistance

技术领域 technical field

本发明属分子生物学与基因工程领域,具体涉及含硫酯酶功能域的腊梅基因克隆及其应用。The invention belongs to the field of molecular biology and genetic engineering, and in particular relates to the cloning of wintersweet gene containing thioesterase functional domain and its application.

背景技术 Background technique

在植物体中,质体的脂肪酸合成包括原核途径与真核途径。质体中的脂肪酸从头合成能被质体酰基转移酶终止,将酰基基团从acyl-ACP转至质体的甘油脂类中,这为原核途径;或者被acyl-ACP硫酯酶,将acyl-ACP水解,释放游离脂肪酸和ACP,然后将脂肪酸输出质体,被辅酶A重新酯化送到辅酶A池中。作为内质网甘油脂类的源出,这是真核途径。硫酯酶属酯酶家族酯酶,是水解酶的一种。可以将acyl-ACP水解,释放游离脂肪酸和ACP。In plants, fatty acid synthesis in plastids includes prokaryotic and eukaryotic pathways. De novo fatty acid synthesis in plastids can be terminated by plastid acyltransferase, which transfers the acyl group from acyl-ACP to glycerolipids in the plastid, which is the prokaryotic pathway; or by acyl-ACP thioesterase, which transfers acyl -ACP is hydrolyzed to release free fatty acids and ACP, and then the fatty acids are exported to the plastid and re-esterified by coenzyme A to the coenzyme A pool. As a source of glycerolipids from the endoplasmic reticulum, this is the eukaryotic pathway. Thioesterase belongs to the esterase family esterase, which is a kind of hydrolase. Acyl-ACP can be hydrolyzed to release free fatty acid and ACP.

脂肪酸的生物合成主要发生在质体的基质中,由乙酰辅酶A经一系列反应,生成不同链长(C8~C18)的脂肪酰ACP。在质体中发生的最后一步反应,是硫酯酶将游离的脂肪酸从ACP上释放出来,所以在脂肪酸从头合成中,acyl-ACP硫酯酶对于碳链的终止扮演重要角色。The biosynthesis of fatty acids mainly occurs in the matrix of plastids, and fatty acyl-ACPs of different chain lengths (C8-C18) are generated from acetyl-CoA through a series of reactions. The last reaction in the plastid is the release of free fatty acids from ACP by thioesterase, so in the de novo synthesis of fatty acids, acyl-ACP thioesterase plays an important role in the termination of the carbon chain.

根据氨基酸的序列比较和酶底物的特异性不同,植物中分为两种基本类型的酰基-ACP硫酯酶。一类命名为FatA,对于18:1Δ9-ACP活性最高,所以推测FatA决定着植物体内18:1输出到质体外的水平。第二类以16:0-ACP硫酯酶为代表,对较短链的饱和酰基-ACP活性最高。FatB对底物的催化活性为16:0-ACP>18:1Δ9-ACP>18:0-ACP>14:0-ACP。因为酰基ACP硫酯酶具有底物特异性,所以它的活性影响着各种甘油脂类中脂肪酸成分之间的比率,是决定脂肪酸的链长和饱和脂肪酸水平的主要因素。There are two basic types of acyl-ACP thioesterases in plants according to the sequence comparison of amino acids and the specificity of enzyme substrates. One class named FatA has the highest activity for 18:1 Δ9 -ACP, so it is speculated that FatA determines the level of 18:1 exported to the outside of the plastid in plants. The second class, represented by 16:0-ACP thioesterases, is most active towards shorter-chain saturated acyl-ACPs. The catalytic activity of FatB on substrates was 16:0-ACP>18: 1Δ9 -ACP>18:0-ACP>14:0-ACP. Because acyl-ACP thioesterase has substrate specificity, its activity affects the ratio between fatty acid components in various glycerolipids, and is the main factor determining the chain length of fatty acids and the level of saturated fatty acids.

最近研究发现FatB基因在植物体内是古老的,存在植物的各器官中,在花中表达量最高。推测FatA和FatB支系的分离比樟树植物的分离更早,估计分离约在1.5-3.0亿年前,FatA是由FatB衍生的。中链的硫酯酶是在被子植物进化过程中,由16:0硫酯酶经过几次进化过程演变而来的。Recent studies have found that the FatB gene is ancient in plants, exists in various organs of plants, and has the highest expression level in flowers. It is speculated that the separation of FatA and FatB clades was earlier than the separation of camphor plants, and the separation was estimated to be about 150-300 million years ago, and FatA was derived from FatB. Medium-chain thioesterases evolved from 16:0 thioesterases through several evolutionary processes during the evolution of angiosperms.

硫酯酶具有酰基转移酶的功能域,可以将酰基从酰基-酰基载体蛋白上分离出来。Thioesterases possess the functional domain of acyltransferases, which can detach acyl groups from acyl-acyl carrier proteins.

脂酰-ACP硫酯酶催化FAS循环的终止,已从多种植物中纯化,编码基因也已从红花、油菜和拟南芥菜等多种植物中克隆。不同的脂酰-ACP需要不同的酶,如红花中克隆的硫酯酶对油酰-ACP特异,而拟南芥中克隆的硫酯酶对14~18碳饱和底物具特异性。Fatty acyl-ACP thioesterase catalyzes the termination of the FAS cycle. It has been purified from various plants, and its coding genes have also been cloned from various plants such as safflower, rapeseed and Arabidopsis. Different fatty acyl-ACPs require different enzymes. For example, the thioesterase cloned from safflower is specific to oleoyl-ACP, while the thioesterase cloned from Arabidopsis is specific to 14-18 carbon-saturated substrates.

酰基ACP硫酯酶成为脂类生物合成酶系中第一个经改造,并转入转基因植物以产生经济产品的酶。生物技术公司Calgene的工作首先证明了存在中链酰基ACP硫酯酶,并提供了生化证据。然后从加州月桂中克隆了12:0-ACP硫酯酶。将编码单个中链酰基ACP硫酯酶的基因转入植物可以明显改变种子油中所储存的脂肪酸的长度。除了月桂中的特异于月桂酸的酰基ACP硫酯酶以外,特异于其他链长的硫酯酶也已从不同植物中鉴定出来了。其他生产富含别种中链脂肪酸,如豆蔻酸和葵酸的转基因植物也已得到。Acyl-ACP thioesterase became the first enzyme in the lipid biosynthetic enzyme system to be modified and transferred into transgenic plants to produce economical products. Work by the biotechnology company Calgene was the first to demonstrate the existence of medium-chain acyl-ACP thioesterases and provide biochemical evidence. The 12:0-ACP thioesterase was then cloned from Laurel california. Transferring into plants a gene encoding a single medium-chain acyl-ACP thioesterase can significantly alter the length of fatty acids stored in seed oil. In addition to the lauric acid-specific acyl-ACP thioesterases in laurel, thioesterases specific for other chain lengths have also been identified from different plants. Other transgenic plants producing rich in other medium-chain fatty acids, such as myristic acid and capric acid, have also been obtained.

在研究拟南芥突变体(Fatb-ko)时,发现突变体的生长速率低于野生型拟南芥,生长4周以后,突变体鲜重与野生型植物相比较减少50%。而且突变体的种子发育能力低,形态学发生改变。在突变体中,在不同的组织中,饱和脂肪酸总的数量比野生型减少40%-50%,这种减少仅发生在细胞溶质中的脂肪酸组分、叶和茎表面的蜡层、种子中的三酰基甘油中,而且发现突变体在植株生长和种子萌发都受到影响。When studying the Arabidopsis mutant (Fatb-ko), it was found that the growth rate of the mutant was lower than that of the wild-type Arabidopsis, and after 4 weeks of growth, the fresh weight of the mutant was reduced by 50% compared with the wild-type plant. Moreover, the seed development ability of the mutant was low and the morphology changed. In the mutant, in different tissues, the total amount of saturated fatty acids was reduced by 40%-50% compared with the wild type, and this reduction occurred only in the fatty acid fraction in the cytosol, the wax layer on the surface of leaves and stems, and in seeds in triacylglycerols, and the mutants were found to be affected in both plant growth and seed germination.

腊梅是一种众所周知的多抗植物,其所具有的极强的抗低温、抗干旱、抗病虫能力,这与腊梅的叶片表面含有大量的蜡质层有关,而蜡质层是由长链脂肪酸构成。在众多的研究中,还没有看到有关含硫酯酶功能域的腊梅基因在植物的抗逆生理功能方面的相关报道。Wintersweet is a well-known multi-resistant plant, which has extremely strong resistance to low temperature, drought, and disease and insect pests. This is related to the fact that the surface of wintersweet leaves contains a large amount of waxy layer, and the waxy layer is composed of long chains. fatty acid composition. In numerous studies, there is no relevant report on the physiological function of the wintersweet gene containing the thioesterase domain in the stress resistance of plants.

发明内容 Contents of the invention

(1)提供一种DNA序列,其编码一种含硫酯酶功能域的腊梅基因,命名为CpAcylTase;(2)提供一种利用基因工程方法得到CpAcylTase编码的蛋白;(3)提供CpAcylTase基因在植物抗旱方面的应用。(1) provide a kind of DNA sequence, its coding a kind of wintersweet gene containing thioesterase functional domain, named after CpAcylTase; (2) provide a kind of protein that utilizes genetic engineering method to obtain CpAcylTase encoding; (3) provide CpAcylTase gene in Applications in plant drought resistance.

本发明提供了CpAcylTase基因,它具有如序列表中SEQ ID NO:1所示的核苷酸序列。The invention provides CpAcylTase gene, which has the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing.

从腊梅花冠cDNA文库中,利用PCR方法筛选克隆出CpAcylTase基因:利用RNAiso Reagent(购自大连Takara公司)提取腊梅花冠总RNA,按照SMARTTM cDNA Library ConstructionKit合成腊梅花冠cDNA文库,随机挑取1000个文库克隆测序和生物信息学分析,获得腊梅花冠抗性ESTs序列。从腊梅花ESTs序列中,发现编码CpAcylTase的cDNA序列,根据此序列设计上下游引物,并以腊梅花cDNA文库为模板进行PCR扩增,克隆出CpAcylTase的全长基因。The CpAcylTase gene was screened and cloned by PCR method from the cDNA library of the wintersweet corolla: the total RNA of the wintersweet corolla was extracted using RNAiso Reagent (purchased from Dalian Takara Company), the cDNA library of the wintersweet corolla was synthesized according to the SMARTTM cDNA Library Construction Kit, and 1000 library clones were randomly selected Sequencing and bioinformatics analysis were used to obtain the sequences of ESTs resistant to the wintersweet corolla. The cDNA sequence encoding CpAcylTase was found from the ESTs sequence of wintersweet flower, and the upstream and downstream primers were designed according to the sequence, and the full-length gene of CpAcylTase was cloned by PCR amplification using the wintersweet flower cDNA library as a template.

CpAcylTase的基因由1110个碱基组成,含有一个完整的开放阅读框架,为序列表中的SEQ ID NO:1序列。开放读码框的起始密码子为ATG,终止密码子为TAG。The gene of CpAcylTase consists of 1110 bases and contains a complete open reading frame, which is the sequence of SEQ ID NO: 1 in the sequence listing. The start codon of the open reading frame is ATG, and the stop codon is TAG.

本发明还提供了涉及CpAcylTase基因编码的蛋白,其具有序列表中SEQ ID NO:2中所示的氨基酸序列,是含有369个氨基酸残基,其理论分子量大小为41.7kDa,预测等电点为7.72。根据PSORT分析CpAcylTase蛋白前18个左右氨基酸为信号肽,该蛋白为稳定蛋白。The present invention also provides a protein encoded by the CpAcylTase gene, which has the amino acid sequence shown in SEQ ID NO: 2 in the sequence table, contains 369 amino acid residues, and has a theoretical molecular weight of 41.7kDa and a predicted isoelectric point of 7.72. According to PSORT analysis, the first 18 or so amino acids of CpAcylTase protein are signal peptides, and this protein is a stable protein.

重组原核表达载体含有CpAcylTase基因,重组原核表达载体转化的宿主细胞为大肠杆菌。The recombinant prokaryotic expression vector contains CpAcylTase gene, and the host cell transformed by the recombinant prokaryotic expression vector is Escherichia coli.

重组植物表达载体含有CpAcylTase基因,重组植物表达载体转化的宿主细胞为根瘤农杆菌,重组植物表达载体转化的转基因宿主细胞为植物细胞。The recombinant plant expression vector contains CpAcylTase gene, the host cell transformed by the recombinant plant expression vector is Agrobacterium tumefaciens, and the transgenic host cell transformed by the recombinant plant expression vector is a plant cell.

如实施例二所述,还可利用基因工程的方法,在高效大肠杆菌表达系统中表达出具有生物活性的CpAcylTase蛋白。As described in Example 2, the biologically active CpAcylTase protein can also be expressed in a high-efficiency Escherichia coli expression system by means of genetic engineering.

关于CpAcylTase基因在植物抗旱方面的应用:About the application of CpAcylTase gene in plant drought resistance:

CpAcylTase基因在培育抗逆性植物上的应用,包括用构建的表达载体转化植物细胞,将转化的植物细胞培育成植株。将CpAcylTase基因编码区克隆入植物表达载体pETV7中,通过农杆菌侵染法将其导入烟草叶片,获得过量表达的烟草株系,来提高转CpAcylTase基因烟草(简称转基因烟草)抗旱的能力。The application of the CpAcylTase gene in cultivating stress-resistant plants includes transforming plant cells with constructed expression vectors, and cultivating transformed plant cells into plants. Cloning the CpAcylTase gene coding region into the plant expression vector pETV7, and introducing it into tobacco leaves by Agrobacterium infection method to obtain overexpressed tobacco lines, to improve the ability of the CpAcylTase gene transgenic tobacco (abbreviated as transgenic tobacco) to resist drought.

在对野生烟草与转基因烟草进行保水性实验发现,转CpAcylTase基因烟草可以极大地降低水分的损失,其气孔关闭后的失水率相比野生烟草降低了30%。In the water retention experiment of wild tobacco and transgenic tobacco, it was found that the transgenic CpAcylTase gene tobacco can greatly reduce the loss of water, and the water loss rate after the stomata are closed is 30% lower than that of wild tobacco.

通过干旱胁迫,发现转基因烟草的抗干旱能力明显高于野生烟草,在干旱胁迫10天后,野生型烟草严重萎蔫达到100%,而转基因烟草严重萎蔫还不到2%,说明转基因烟草相对野生烟草表现出了较强的抗干旱胁迫损伤的能力。Through drought stress, it was found that the drought resistance of transgenic tobacco was significantly higher than that of wild tobacco. After 10 days of drought stress, the severe wilting of wild-type tobacco reached 100%, while that of transgenic tobacco was less than 2%. It has a strong ability to resist drought stress damage.

本发明的有益效果在于提供了一种CpAcylTase基因及蛋白,构建了植物表达载体,并获得转基因植物,CpAcylTase基因能提高转基因植物的抗旱能力。The invention has the beneficial effects of providing a CpAcylTase gene and protein, constructing a plant expression vector, and obtaining transgenic plants, and the CpAcylTase gene can improve the drought resistance of the transgenic plants.

附图说明 Description of drawings

图1为以腊梅花冠cDNA为模板,PCR扩增电泳图Figure 1 is an electrophoresis diagram of PCR amplification using the wintersweet corolla cDNA as a template

其中:M:marker自上到下大小为:1850bp、1470bp、1090bp、740bp、420bp、280bpAmong them: M: marker size from top to bottom: 1850bp, 1470bp, 1090bp, 740bp, 420bp, 280bp

1:目的条带1: destination strip

图2为SDS-PAGE原核表达全蛋白电泳图Figure 2 is the SDS-PAGE prokaryotic expression whole protein electrophoresis

其中:M:Marker,分别为66、43、31(KDa)Among them: M: Marker, respectively 66, 43, 31 (KDa)

1、2、3:含有重组克隆质粒pET-28a::CpAcylTase的BL21宿主菌全蛋白1, 2, 3: The whole protein of BL21 host bacteria containing the recombinant cloned plasmid pET-28a::CpAcylTase

CK:含有空载体质粒pET-28a的BL21宿主菌全蛋白CK: Whole protein of BL21 host bacteria containing empty vector plasmid pET-28a

图3为CpAcylTase植物表达载体的构建过程示意图Fig. 3 is the schematic diagram of the construction process of CpAcylTase plant expression vector

图4为转基因烟草阳性苗RT-PCR鉴定电泳图Fig. 4 is the electrophoresis diagram of RT-PCR identification of transgenic tobacco positive seedlings

其中:M:marker自上到下大小为:2000bp、1000bp、750bp、500bp、250bpLine-1、Line-2、Line-3、Line-4:转基因烟草阳性苗RT-PCR W:野生型烟草RT-PCRAmong them: M: marker size from top to bottom: 2000bp, 1000bp, 750bp, 500bp, 250bp PCR

图5为干旱胁迫对转基因烟草与野生烟草的影响比较示意图Figure 5 is a schematic diagram of the comparison of the effects of drought stress on transgenic tobacco and wild tobacco

其中:W:野生烟草    T:转基因烟草Among them: W: wild tobacco T: transgenic tobacco

图6为野生烟草、转基因烟草保水性分析比较示意图Figure 6 is a schematic diagram of the water retention analysis comparison between wild tobacco and transgenic tobacco

具体实施方式 Detailed ways

实施例一:克隆CpAcylTase基因Example 1: Cloning of the CpAcylTase gene

1.提取RNA:1. Extract RNA:

取500mg腊梅花冠用RNAiso Reagent提取腊梅花总RNA。Take 500mg wintersweet corolla and use RNAiso Reagent to extract the total RNA of wintersweet flower.

2.构建cDNA文库:2. Construction of cDNA library:

取总RNA,按照SMARTTM cDNA Library Construction Kit合成腊梅花cDNA文库,随机挑取1000个文库克隆测序和生物信息学分析,获得腊梅花抗性ESTs序列。The total RNA was taken, and the cDNA library of wintersweet flower was synthesized according to the SMARTTM cDNA Library Construction Kit, and 1000 library clones were randomly selected for sequencing and bioinformatics analysis to obtain the sequence of wintersweet flower resistance ESTs.

3.克隆CpAcylTase基因片段的克隆:3. Clone the clone of CpAcylTase gene fragment:

根据腊梅花ESTs序列中找出的编码CpAcylTase的cDNA序列,设计上下游引物,并以腊梅花cDNA文库为模板进行PCR扩增。According to the cDNA sequence encoding CpAcylTase found in the ESTs sequence of wintersweet flower, the upstream and downstream primers were designed, and the PCR amplification was carried out with the cDNA library of wintersweet flower as a template.

特异性引物如下:Specific primers are as follows:

CpAcylTase-P1:5′-CCC

Figure G2009102540003D00031
CTAGTAATGAGCATGATTGCC-′3,斜体碱基为HindIII酶切位点;CpAcylTase-P1: 5′-CCC
Figure G2009102540003D00031
CTAGTAATGAGCATGATTGCC-′3, bases in italics are HindIII restriction sites;

CpAcylTase-P2:5′-CCG

Figure G2009102540003D00041
CTAATGCGATATGGAATTAGGGT-′3,斜体碱基为XhoI酶切位点。CpAcylTase-P2: 5′-CCG
Figure G2009102540003D00041
CTAATGCGATATGGAATTAGGGT-′3, bases in italics are XhoI restriction sites.

克隆PCR反应条件为:94℃预变性3min;94℃变性50sec;54℃退火30sec;72℃延伸1min,30个循环;72℃延伸10min。扩增产物经1%琼脂糖凝胶电泳检测,在1000bp位置上有一特异性的条带(图1)。进而按常规方法将片段克隆到pMD18-T Vector(购白大连Takara公司)中,并经上海生物工程公司进行测序。Cloning PCR reaction conditions were as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 50 sec; annealing at 54°C for 30 sec; extension at 72°C for 1 min, 30 cycles; extension at 72°C for 10 min. The amplified product was detected by 1% agarose gel electrophoresis, and there was a specific band at the position of 1000 bp (Fig. 1). Then, the fragment was cloned into pMD18-T Vector (purchased from Dalian Takara Company) according to conventional methods, and sequenced by Shanghai Bioengineering Company.

CpAcylTase基因的序列分析Sequence Analysis of CpAcylTase Gene

经测序该基因cDNA序列开放阅读框包含1110bp,含有一个完整的开放阅读框架,为序列表中的SEQ ID NO:1序列。编码369个氨基酸,具有序列表中SEQ ID NO:2中所示的氨基酸序列,分子量大小为41.7kDa,预测等电点为7.72。结果表明获得了全长基因的cDNA序列。After sequencing, the open reading frame of the cDNA sequence of the gene contains 1110 bp, which contains a complete open reading frame, which is the sequence of SEQ ID NO: 1 in the sequence listing. It encodes 369 amino acids, has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, has a molecular weight of 41.7kDa, and a predicted isoelectric point of 7.72. The results indicated that the cDNA sequence of the full-length gene was obtained.

利用NCBI(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)对该基因编码的氨基酸进行保守区域分析,结果表明:该蛋白属于acyl-ACP硫酯酶。Using NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to analyze the conserved region of amino acids encoded by the gene, the results showed that the protein belongs to acyl-ACP thioesterase.

在NCBI网站上对CpAcylTase基因编码的氨基酸序列进行BLAST,与已确定功能的其他物种氨基酸序列进行进化分析和同源性分析:CpAcylTase与肉豆蔻acyl-ACP thioesterase编码的氨基酸进化距离最近,与拟南芥同种氨基酸序列同源性为48%。Perform BLAST on the amino acid sequence encoded by the CpAcylTase gene on the NCBI website, and perform evolutionary analysis and homology analysis with the amino acid sequences of other species whose functions have been determined: CpAcylTase has the closest evolutionary distance to the amino acid encoded by acyl-ACP thioesterase in myristica, and is similar to that in Arabidopsis The amino acid sequence homology of mustard is 48%.

根据PSORT分析CpAcylTase前18个左右氨基酸为信号肽,是稳定蛋白。用SOPMA对CpAcylTase进行二级结构预测,发现含有39.84%的α螺旋(h),5.42%β转角(t),14.09%的延伸链(e),40.65%的随机卷曲(c)。According to PSORT analysis, the first 18 or so amino acids of CpAcylTase are signal peptides, which are stable proteins. Using SOPMA to predict the secondary structure of CpAcylTase, it was found that it contained 39.84% α-helix (h), 5.42% β-turn (t), 14.09% extended chain (e), and 40.65% random coil (c).

实施例二:CpAcylTase在大肠杆菌中的高效表达Example 2: High expression of CpAcylTase in Escherichia coli

1.原核表达载体的构建:1. Construction of prokaryotic expression vector:

为了表明CpAcylTase基因的编码功能,将CpAcylTase基因克隆到pET-28a(+)的HindIII和BamH I位点之间,并转化大肠杆菌BL21,获得高效表达。In order to show the coding function of the CpAcylTase gene, the CpAcylTase gene was cloned between the HindIII and BamH I sites of pET-28a (+), and transformed into Escherichia coli BL21 to obtain high-level expression.

设计并合成一对特异性引物:Design and synthesize a pair of specific primers:

5′-CGCCTAGTAATGAGCATGATT-′3(斜体碱基为BamHI的酶切位点)5′-CGC CTAGTAATGAGCATGATT-′3 (bases in italics are restriction sites of BamHI)

5′-CCC

Figure G2009102540003D00043
GTAGTTCCTTATGCGATA-′3(斜体碱基为HindIII的酶切位点)5′-CCC
Figure G2009102540003D00043
GTAGTTCCTTATGCGATA-′3 (base in italics is HindIII restriction site)

按分子克隆常规实验程序,用PCR的方法扩增出两端带有特定酶切位点的基因编码序列,将基因和载体pMD18-T连接,验证正确。取pMD18::CpAcylTase质粒和pET-28a(+)质粒(购于上海鼎国生物公司)分别用BamHI、HindIII双酶切、再连接,将连接产物转化大肠杆菌BL21菌株(购于凯基生物公司)中,并用含100ug/ml卡那霉素的LB平板筛选重组子。经质粒酶切鉴定和PCR鉴定后,将筛出的连接正确的重组子进行测序,证明插入载体的DNA序列的阅读框架是正确的。把构建成带有CpAcylTase基因的表达载体,命名pET-28a::CpAcylTase,用于诱导表达分析。According to the routine experimental procedure of molecular cloning, the gene coding sequence with specific restriction sites at both ends was amplified by PCR method, and the gene was connected with the vector pMD18-T to verify that it was correct. Take the pMD18::CpAcylTase plasmid and the pET-28a(+) plasmid (purchased from Shanghai Dingguo Biotechnology Co., Ltd.) and digest them with BamHI and HindIII respectively, and then ligate them again. ), and screen recombinants with LB plate containing 100ug/ml kanamycin. After identification by plasmid digestion and PCR, the screened recombinants with correct connection were sequenced to prove that the reading frame of the DNA sequence inserted into the vector was correct. The expression vector constructed with CpAcylTase gene, named pET-28a::CpAcylTase, was used for induced expression analysis.

2.原核表达:2. Prokaryotic expression:

将含有pET-28a::CpAcylTase重组子的菌株,接种于LB培养基(1L:蛋白胨10g,酵母提取物5g,NaCL10克),37℃培养至菌液OD600=0.4-0.6。将BL21重组表达菌研磨及超声破碎,我们得到了含有重组克隆质粒pET-28a::CpAcylTase的BL21宿主菌粗酶液。进行12%SDS-PAGE电泳检测,从图2中可以看出,CpAcylTase基因在BL21中高效表达。The strain containing the pET-28a::CpAcylTase recombinant was inoculated in LB medium (1 L: 10 g of peptone, 5 g of yeast extract, 10 g of NaCL), and cultured at 37°C until the OD 600 of the bacterial solution was 0.4-0.6. Grinding and sonicating the BL21 recombinant expression bacteria, we obtained the crude enzyme solution of the BL21 host bacteria containing the recombinant cloning plasmid pET-28a::CpAcylTase. 12% SDS-PAGE electrophoresis detection was carried out. It can be seen from Figure 2 that the CpAcylTase gene is highly expressed in BL21.

实施例三:含有CpAcylTase基因的植物表达载体的构建,及转基因烟草的制备Embodiment three: the construction of the plant expression vector containing CpAcylTase gene, and the preparation of transgenic tobacco

将本发明的CpAcylTase基因编码区克隆入植物表达载体pTEV7(吉林大学植物分子生物学研究室保存,殷君华,腊梅花水通道蛋白CpTIP cDNA的克隆及功能研究,中国知网收录,2008.)中,通过农杆菌侵染法将其导入烟草中,制作过量表达的烟草株系,来提高烟草抗旱能力。The CpAcylTase gene coding region of the present invention is cloned into the plant expression vector pTEV7 (preserved by the Laboratory of Plant Molecular Biology, Jilin University, Yin Junhua, the cloning and functional research of wintersweet flower aquaporin CpTIP cDNA, included in China National Knowledge Network, 2008.), Introduce it into tobacco by Agrobacterium infection method to produce overexpressed tobacco strains to improve the drought resistance of tobacco.

1.植物表达载体的构建1. Construction of plant expression vectors

设计一对引物,分别引入HindIII和XhoI的酶切位点,Design a pair of primers to introduce restriction sites for HindIII and XhoI respectively,

CpAcylTase-P1:5′-CCC

Figure G2009102540003D00051
CTAGTAATGAGCATGATTGCC-′3,斜体碱基为HindIII酶切位点;CpAcylTase-P1: 5′-CCC
Figure G2009102540003D00051
CTAGTAATGAGCATGATTGCC-′3, bases in italics are HindIII restriction sites;

CpAcylTase-P2:5′-CCG

Figure G2009102540003D00052
CTAATGCGATATGGAATTAGGGT-′3,斜体碱基为XhoI酶切位点。CpAcylTase-P2: 5′-CCG
Figure G2009102540003D00052
CTAATGCGATATGGAATTAGGGT-′3, bases in italics are XhoI restriction sites.

按分子克隆常规实验程序,用PCR的方法扩增出目的片段,将片段克隆到pMD18-T Vector、测序。将经测序鉴定正确的阳性重组质粒pMDl8::CpAcylTase和植物表达载体pETV7进行HindIII和XhoI双酶切。并将酶切产物回收、连接,构建成目的载体pTEV7::CpAcylTase(构建过程见图3)并转化到根瘤农杆菌EHA105中。According to the routine experimental procedure of molecular cloning, the target fragment was amplified by PCR method, and the fragment was cloned into pMD18-T Vector and sequenced. The correct positive recombinant plasmid pMD18::CpAcylTase and the plant expression vector pETV7 identified by sequencing were subjected to double digestion with HindIII and XhoI. The digested product was recovered, ligated, and constructed into the target vector pTEV7::CpAcylTase (see Figure 3 for the construction process) and transformed into Agrobacterium tumefaciens EHA105.

2.利用叶盘侵染法转化烟草2. Transformation of tobacco by leaf disk infection method

(1)挑取经鉴定的农杆菌接种到液体培养基中,28℃、250rpm振荡培养约48h,至对数生长后期,离心、弃上清,菌体用1/2MS液体培养液(1/2×MS大量元素,1×MS微量元素,1×MS维生素,1×铁盐,pH5.7-5.8)悬浮稀释至OD600=0.5左右;(1) Pick the identified Agrobacterium and inoculate it into the liquid medium, culture it with shaking at 28°C and 250rpm for about 48h, until the late logarithmic growth period, centrifuge, discard the supernatant, and use 1/2MS liquid culture medium (1/2 ×MS macroelement, 1×MS trace element, 1×MS vitamin, 1×iron salt, pH5.7-5.8) suspended and diluted to about OD600=0.5;

(2)取无菌烟草叶片,去掉主叶脉,将其剪成小块;(2) get the sterile tobacco blade, remove the main vein, and cut it into fritters;

(3)将剪好的烟草叶片置于MS分化培养基中(1×MS大量元素,1×MS微量元素,1×MS维生素,1×铁盐,蔗糖30g/L,6-BA 3mg/L,NAA 0.2mg/L,琼脂粉8g/L,pH5.7-5.8),28℃、光照时间16h/天、光照强度2000LX,培养2天;(3) Place the cut tobacco leaves in MS differentiation medium (1×MS macroelement, 1×MS trace element, 1×MS vitamin, 1× iron salt, sucrose 30g/L, 6-BA 3mg/L , NAA 0.2mg/L, agar powder 8g/L, pH5.7-5.8), 28°C, light time 16h/day, light intensity 2000LX, culture for 2 days;

(4)将培养2天的烟草叶片浸入第一步所述刚准备好的菌液中10min,在无菌滤纸上吸干菌液,将叶片置于MS培养基上,28℃暗培养2天;(4) Immerse the tobacco leaves that have been cultured for 2 days in the bacterial solution just prepared as described in the first step for 10 minutes, blot the bacterial solution on sterile filter paper, place the leaves on MS medium, and culture in the dark at 28°C for 2 days ;

(5)共培养后的叶片在其周围出现微菌落后先用含头孢霉素300mg/L的无菌水洗涤3次,再用含头孢霉素300mg/L的MS培养液洗1次,然后用无菌滤纸吸干,转入含有卡那霉素的MS筛选培养基上,恒温培养(条件同预培养),每15天更换一次培养基;(5) The leaves after the co-cultivation were washed with sterile water containing 300 mg/L of cephalosporin 3 times after microbacteria appeared around it, and then washed once with MS culture solution containing 300 mg/L of cephalosporin, and then Blot dry with sterile filter paper, transfer to MS screening medium containing kanamycin, cultivate at constant temperature (conditions are the same as pre-cultivation), and replace the medium every 15 days;

(6)待芽长至1cm左右时,切下并移入生根培养基中,促其生根。待根系发育后,对转化苗进行编号,并移入盛有无菌土的花盆中,用塑料薄膜保湿2天,室温常规管理。(6) When the bud grows to about 1 cm, cut it off and transfer it to the rooting medium to promote its rooting. After the root system developed, the transformed seedlings were numbered, and moved into flowerpots filled with sterile soil, kept moist with plastic film for 2 days, and managed at room temperature routinely.

以RT-PCR方法验证转基因烟草Verification of Transgenic Tobacco by RT-PCR

1.用常规方法从导入了CpAcylTase基因的烟草叶片中提取总RNA,以野生烟草为阴性对照,结果表明28S和18S条带型整齐,且28S条带明显亮于18S条带,说明总RNA完整性很好,没有发生降解,可以用作进一步的反转录鉴定。将提取的转CpAcylTase基因的烟草RNA以CpAcylTase-P2为引物经反转录得到单链cDNA。1. Extract total RNA from tobacco leaves introduced with CpAcylTase gene by conventional methods, and use wild tobacco as a negative control. The results show that the 28S and 18S bands are neat, and the 28S band is obviously brighter than the 18S band, indicating that the total RNA is complete The performance is very good, no degradation occurs, and can be used for further reverse transcription identification. The extracted tobacco RNA transcribed with CpAcylTase gene was reverse-transcribed with CpAcylTase-P2 as a primer to obtain single-stranded cDNA.

2.再以cDNA为模板,然后以CpAcylTase-P1、CpAcylTase-P2为引物进行PCR扩增。扩增产物经1%琼脂糖凝胶电泳检测,在相应位置上有一特异性的条带,与CpAcylTase基因部分片段大小一致,而在野生型植株阴性对照的泳道上没有该条带,说明检测的转基因植株为阳性。这初步证明了CpAcylTase基因已经整合入烟草基因组中,而且在RNA水平上得到了表达(如图4)。2. Then use cDNA as a template, and then use CpAcylTase-P1 and CpAcylTase-P2 as primers for PCR amplification. The amplified product was detected by 1% agarose gel electrophoresis, and there was a specific band at the corresponding position, which was consistent with the size of the partial fragment of the CpAcylTase gene, but there was no such band on the negative control lane of the wild-type plant, indicating that the detected Transgenic plants were positive. This preliminarily proves that the CpAcylTase gene has been integrated into the tobacco genome and expressed at the RNA level (as shown in Figure 4).

实施例四:干旱胁迫对转基因烟草与野生烟草的影响Example 4: Effects of Drought Stress on Transgenic Tobacco and Wild Tobacco

选取饱满野生和转基因烟草种子播撒在土中(蛭石∶草木炭为1∶1)中25℃、光照时间16h/天、光照强度2000LX。两周后,选择生长一致的小苗,移栽到盆中。恢复生长2周后,选取生长状况一致的健康植株对其共同进行停水干旱胁迫处理,8天后发现野生型烟草萎蔫达到100%时,转基因烟草却没有出现萎蔫情况(如图5)。Select plump wild and transgenic tobacco seeds and sow them in soil (vermiculite:grass charcoal ratio 1:1) at 25°C, light time 16h/day, and light intensity 2000LX. After two weeks, select seedlings with consistent growth and transplant them into pots. After 2 weeks of resuming growth, select healthy plants with the same growth status to be subjected to water deprivation and drought stress treatment. After 8 days, it was found that when the wilting of wild-type tobacco reached 100%, the transgenic tobacco did not wilt (as shown in Figure 5).

实施例五:离体叶片的失水速率测定Embodiment five: Dehydration rate determination of detached leaves

取同等培养条件下大小相近,质量相同的野生烟草及转基因烟草叶片,称取鲜重后放入28℃培养箱中。为使叶片气孔完全关闭,在150min之后开始测量水分减少情况,此后每隔1h称取叶片的质量共测定5次,计算两种叶片的失水率(如图6):Take wild tobacco and transgenic tobacco leaves of similar size and quality under the same culture conditions, weigh their fresh weight, and put them into an incubator at 28°C. In order to completely close the stomata of the leaves, the water loss was measured after 150 minutes, and the weight of the leaves was weighed every 1 hour for a total of 5 measurements, and the water loss rates of the two kinds of leaves were calculated (as shown in Figure 6):

失水率=(鲜重-失水后重量)/鲜重Water loss rate = (fresh weight - weight after water loss) / fresh weight

结果表明气孔关闭后转基因烟草单位时间内失水率相对野生烟草降低了30%。The results showed that after the stomata were closed, the water loss rate per unit time of the transgenic tobacco was reduced by 30% compared with that of the wild tobacco.

序列表sequence listing

SEQ ID NO.1的序列The sequence of SEQ ID NO.1

(i)序列特征:(A)长度:1110bp;(B)类型:核苷酸;(C)链性:单链。(i) Sequence features: (A) length: 1110bp; (B) type: nucleotide; (C) chain: single-stranded.

(ii)分子类型:核苷酸(ii) Molecule type: Nucleotide

(iii)序列描述:SEQ ID NO.1(iii) Sequence description: SEQ ID NO.1

1    ATGAGCATGA TTGCCAGCAG TGTAGGTGCT GCTTTTTTTC CAGCCCAAGG CATCATCAAG1 ATG AGCATGA TTGCCAGCAG TGTAGGTGCT GCTTTTTTTC CAGCCCAAGG CATCATCAAG

61   TCCAAGCCGG CTGGGTTGCA CGTGAAAGCA AATGGCCGAG CCTCCCCCAG CATTGACGGT61 TCCAAGCCGG CTGGGTTGCA CGTGAAAGCA AATGGCCGAG CCTCCCCCAG CATTGACGGT

121  CCGAAGGTGA CCGTCGGCCT AGAGGGCACG AACGCCTCGT CCACAAGGAA ATTCATGAAC121 CCGAAGGTGA CCGTCGGCCT AGAGGGCACG AACGCCTCGT CCACAAGGAA ATTCATGAAC

181  TTGTTGCCTG ACTGGAGCAT GCTACTTGCC GCCTTTACAA CCATCTTTGA GAAGCAGAAG181 TTGTTGCCTG ACTGGAGCAT GCTACTTGCC GCCTTTACAA CCATCTTTGA GAAGCAGAAG

241  GTTGTGGTCG ACCAGTTTCG ATTCGGCCAT GACAGGCTGG TTTACAGTGA GAATTTCACA241 GTTGTGGTCG ACCAGTTTCG ATTCGGCCAT GACAGGCTGG TTTACAGTGA GAATTTCACA

301  ATAAGGTCAT ATGAGATAGG TGCTGATCAG ACGGCATCAA TAGAGACAGT GATGAATCTT301 ATAAGGTCAT ATGAGATAGG TGCTGATCAG ACGGCATCAA TAGAGACAGT GATGAATCTT

361  TTGCAGGAGA CTGGAATCAA CTGTTTTAGG AGCCTTGGGC TTTTACTTGA TGGTTTTGAT361 TTGCAGGAGA CTGGAATCAA CTGTTTTAGG AGCCTTGGGC TTTTACTTGA TGGTTTTGAT

421  TCAACAGTGG AGATGTGTAA GAGAGATCTT ATATGGGTTG TGACTCGTAT GCAGGTTATC421 TCAACAGTGG AGATGTGTAA GAGAGATCTT ATATGGGTTG TGACTCGTAT GCAGGTTATC

481  GTTGATCACT ATCCTTCTAG GGGTGATACT GTTGAAGTAG AGACACACTG CGGTGCATAT481 GTTGATCACT ATCCTTCTAG GGGTGATACT GTTGAAGTAG AGACACACTG CGGTGCATAT

541  GGAAAGCATG GCCACCGCCG AGAATGGCTA ATCCGGAACA GCAAAACTGG TCAAATTCTT541 GGAAAGCATG GCCACCGCCG AGAATGGCTA ATCCGGAACA GCAAAACTGG TCAAATTCTT

601  ACACGAGCTA CCAGTGTTCT GGTGGTGATG AATAAGCGGA CGAGGAGATT GTCCATATTA601 ACACGAGCTA CCAGTGTTCT GGTGGTGATG AATAAGCGGA CGAGGAGATT GTCCATATTA

661  CCTGATGAAG TTAGAAGGGA ATTAGAGCCT TATTTCATGG AGAATCTTAG TGTGATGAAG661 CCTGATGAAG TTAGAAGGGA ATTAGAGCCT TATTTCATGG AGAATCTTAG TGTGATGAAG

721  GACCAAGGCA GAAAACTTCC CAAGGTCGAT CATAGCATTG CAGATTACGT CCGACAAGGG721 GACCAAGGCA GAAAACTTCC CAAGGTCGAT CATAGCATTG CAGATTACGT CCGACAAGGG

781  TTGACTTGTC AATGGAGTGA TTTGGATATC AATCAGCATG TAAACCATAT CAAATACGTT781 TTGACTTGTC AATGGAGTGA TTTGGATATC AATCAGCATG TAAACCATAT CAAATACGTT

841  AAATGGATTT TTGAGAGTGT TCCGGTTTCT ATCTTAGAAA GTCACGAGAT TTCCAGCATG841 AAATGGATTT TTGAGAGTGT TCCGGTTTCT ATCTTAGAAA GTCACGAGAT TTCCAGCATG

901  ACTCTTGAAT TTAAGAGAGA GTGCGGCAAG GATAGCATGT TGCAGTCTCT GACTGCCGTC901 ACTCTTGAAT TTAAGAGAGA GTGCGGCAAG GATAGCATGT TGCAGTCTCT GACTGCCGTC

961  GTGTCCGGTC GCAGGGTTGA CGGATCAGTA GAAGAAACTG ACGTTGAATT TCAGCACTTG961 GTGTCCGGTC GCAGGGTTGA CGGATCAGTA GAAGAAACTG ACGTTGAATT TCAGCACTTG

1021 CTCCAGCTTG AAGATGGGCC TGAGGTCATG AGGGGAACAA CAAAGTGGAG ACCCAAGAGT1021 CTCCAGCTTG AAGATGGGCC TGAGGTCATG AGGGGAACAA CAAAGTGGAG ACCCAAGAGT

1081 ACCCTGTTCC CTAATTCCAT ATCGCATTAG 1081 ACCCTGTTCC CTAATTCCAT ATCGCA TTAG

SEQ ID NO.2的序列The sequence of SEQ ID NO.2

(i)序列特征:(A)长度:369个氨基酸;(B)类型:氨基酸;(C)链性:单链。(i) Sequence features: (A) length: 369 amino acids; (B) type: amino acid; (C) chain: single chain.

(ii)分子类型:多肽(ii) Molecular type: polypeptide

(iii)序列描述:SEQ ID NO.2(iii) Sequence description: SEQ ID NO.2

1    MSMIASSVGA AFFPAQGIIK SKPAGLHVKA NGRASPSIDG1 MSMIASSVGA AFFPAQGIIK SKPAGLHVKA NGRASPSIDG

41   PKVTVGLEGT NASSTRKFMN LLPDWSMLLA AFTTIFEKQK41 PKVTVGLEGT NASSTRKFMN LLPDWSMLLA AFTTIFEKQK

81   VVVDQFRFGH DRLVYSENFT IRSYEIGADQ TASIETVMNL81 VVVDQFRFGH DRLVYSENFT IRSYEIGADQ TASIETVMNL

121  LQETGINCFR SLGLLLDGFD STVEMCKRDL IWVVTRMQVI121 LQETGINCFR SLGLLLDGFD STVEMCKRDL IWVVTRMQVI

161    VDHYPSRGDT VEVETHCGAY GKHGHRREWL IRNSKTGQIL161 VDHYPSRGDT VEVETHCGAY GKHGHRREWL IRNSKTGQIL

201    TRATSVLVVM NKRTRRLSIL PDEVRRELEP YFMENLSVMK201 TRATSVLVVM NKRTRRLSIL PDEVRRELEP YFMENLSVMK

241    DQGRKLPKVD HSIADYVRQG LTCQWSDLDI NQHVNHIKYV241 DQGRKLPKVD HSIADYVRQG LTCQWSDLDI NQHVNHIKYV

281    KWIFESVPVS ILESHEISSM TLEFKRECGK DSMLQSLTAV281 KWIFESVPVS ILESHEISSM TLEFKRECGK DSMLQSLTAV

321    VSGRRVDGSV EETDVEFQHL LQLEDGPEVM RGTTKWRPKS321 VSGRRVDGSV EETDVEFQHL LQLEDGPEVM RGTTKWRPKS

361    TLFPNSISH*361 TLFPNSISH*

Claims (7)

1. a wintersweet gene that comprises the thioesterase domain is characterized in that it is the nucleotide sequence shown in the SEQ ID NO:1 in the sequence table.
2. albumen is characterized in that it is by the aminoacid sequence shown in the SEQ ID NO:2 in the described wintersweet gene order coding that comprises the thioesterase domain of claim 1, the sequence table.
3. a recombinant prokaryotic expression vector is characterized in that containing the wintersweet gene that comprises the thioesterase domain as claimed in claim 1.
4. a recombinant plant expression vector is characterized in that containing the wintersweet gene that comprises the thioesterase domain as claimed in claim 1.
5. recombinant prokaryotic expression vector transformed host cells with claim 3, described host cell is intestinal bacteria.
6. recombinant plant expression vector transformed host cells with claim 4, described host cell is an agrobacterium tumefaciens.
7. the described application of wintersweet gene aspect the transgene tobacco drought resisting that comprises the thioesterase domain of claim 1.
CN2009102540003A 2009-12-09 2009-12-09 Wintersweet gene containing thioesterase functional domain and drought-resistant gene engineering application thereof Expired - Fee Related CN101870983B (en)

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Aubrey Jones等.Palmitoyl-Acyl Carrier Protein (ACP) Thioesterase and the Evolutidnary-Origin of Plant ACyl-ACP Thioesterases.《The Plant Cell》.1995,第7卷359-371. *
元冬娟等.高等植物的酰基- ACP硫酯酶研究进展.《中国油料作物学报》.2009,第31卷(第1期),103-108. *
王威浩等.植物脂酰-酰基载体蛋白硫酯酶研究综述.《经济林研究》.2009,第27卷(第2期),118-124. *

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