WO2010127579A1

WO2010127579A1 - Mangrove betaine aldehyde dehydrogenase gene and uses thereof

Info

Publication number: WO2010127579A1
Application number: PCT/CN2010/071536
Authority: WO
Inventors: 孙超; 王君丹; 陈文华; 刘晓霞
Original assignee: 创世纪转基因技术有限公司
Priority date: 2009-05-08
Filing date: 2010-04-02
Publication date: 2010-11-11
Also published as: CN101880678A; CN101880678B

Abstract

Mangrove Betaine aldehyde dehydrogenase gene and the uses thereof in the researches of breeding new plant species having salt, drought and low temperature tolerances are provided. The mangrove betaine aldehyde dehydrogenase gene has the nucleotide sequence as shown in SEQ ID NO: 1. Utilizing the said mangrove betaine aldehyde dehydrogenase gene to construct prokaryotic expression vectors and binary plant expression vectors, transforming E.coli and tobacco respectively with the vectors, culturing BADH transgenic E.coli under high salt conditions, and conducting abiotic stress experiments on BADH positive transgenic tobacco. The mangrove betaine aldehyde dehydrogenase gene is highly expressed in E.coli, and under high salt conditions, the growth of BADH transgenic E.coli is better than non-transgenic E.coli. Transgenic tobacco identification results show that the mangrove betaine aldehyde dehydrogenase gene cloned can be highly expressed in tobacco, and the transgenic tobacco obtained has good ability to tolerate some abiotic stress.

Description

Mangrove betaine aldehyde dehydrogenase gene and application thereof

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.

High salt, low temperature and drought are the most important abiotic stress factors affecting plant growth and productivity. One of the important physiological strategies for adapting higher plants to environmental stress is 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 (β-2α-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.

Betaine aldehyde dehydrogenase (BADH) in higher plants It is a key enzyme for the synthesis of betaine. Since the isolation of BADH from higher plants in 1981, studies on the molecular biology of betaine synthase have quickly attracted widespread attention. In 1987, Arakawa et al. isolated and purified BADH from spinach leaves and prepared antibodies. In 1989, 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. In 1990, 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. At present, 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 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. 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. Each of the above points is a separate embodiment, which will be further described in detail below in conjunction with the specific embodiments.

Example 1 Cloning of Mangrove Betaine Aldehyde Dehydrogenase (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:

1. Extracting total RNA from mangrove and preparing cDNA by reverse transcription

1) 0.1 g of leaves were ground with liquid nitrogen and added to a 1.5 ml centrifuge tube containing 1 ml of Trizol.

2) Place at room temperature for 5 min, add 200 μl of chloroform, mix, and centrifuge at 12000 g for 10 min.

3) The supernatant was added to an equal volume of chloroform, and the mixture was centrifuged at 12000 g for 10 min.

4) The supernatant was further added to an equal volume of isopropanol, precipitated, and centrifuged at 12000 g for 10 min.

5) The precipitate was washed with 75% ethanol, and air-dried with 500 ul of water.

6) Add an equal volume of phenol/chloroform (1:1) and mix for 5 min at room temperature and centrifuge for 10 min at 12000 g.

7) Take the supernatant and add an equal volume of chloroform, mix and centrifuge at 12000g for 5min.

8) The supernatant was further added to an equal volume of isopropanol and centrifuged at 12000 g for 10 min.

9) The precipitate was washed twice with 75% ethanol, air-dried, and dissolved with an appropriate amount of water.

10) Electrophoresis detection and reverse transcription to obtain cDNA.

Reverse transcription steps:

(1) In a 0.2 ml tube, add the following ingredients:

Total RNA (0.1 μg/μl) 2.0μl

Oligo(dT _12-18 )(2 μ M) 2.0μl

(2) 70 ° C water bath for 10 minutes. Immediately placed in an ice bath;

(3) Add the following ingredients: 2.0 μl 10×RT buffer; 2.0 μl 250 μM dNTP mix; 2.0 μl 100 mM DTT; 9.8 μl DEPC H ₂ O; 0.2 μl ₂₀₀ U μl/l SuperScript II;

(4) The following reactions were carried out: 42 ° C for 90 minutes; 70 ° C for 15 minutes; -20 ° C for storage.

The results of RNA RNA electrophoresis detection are shown in Figure 1.

2. Amplification of the expression sequence of BADH gene from mangrove cDNA by PCR

1) Design of degenerate primers

Degenerate primers based on the conserved regions of known BADH gene sequences published on Genebank,

P1: 5'-GGRCCAAGYCKGCADCCTTCYTC-3'

P2: 5'-TTcTGGACaAAYGGwCARATHTG-3'

2) Acquisition of mangrove BADH gene EST

Using the mangrove cDNA as a template, 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.

Through sequence comparison of genebank, 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.

3. Cloning the mangrove BADH gene by RACE

1) 5'Race of mangrove BADH gene

Design and synthesize 5' RACE primers,

GSP-RT: 5'-GGGTCCGATACTTTGATG-3'

GSP1: 5'-CCGATACTTTGATGTTCTCAGAC-3'

GSP2: 5'-CTCCAAAAGTTGTGCTGCAATGCTCTC-3'

AAP: 5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3'

AUAP : 5'-GGCCACGCGTCGACTAGTAC-3'

5'RACE method steps:

A. Reverse transcription

(1) Add the following ingredients to a 0.5 ml centrifuge tube:

1 μl 10 μM GSP1; 12.5 μl (1-5 μg) Total RNA; 13.5 μl DEPC H ₂ O;

(2) Mix, denature at 70 °C for 5 minutes, place in an ice bath for at least 2 minutes, and centrifuge;

(3) Add the following ingredients according to the following table:

2.5 μl 10× PCR buffer; 2.5 μl 0.1 M DTT; 1.25 μl RNaseOUT (40 U/μl); 3 μl 25Mm MgCl ₂ ; 1.25 μl 10 mM dNTP, the above ingredients can be mixed in another tube at 45 °C heat;

(4) Mix various ingredients, centrifuge, and incubate at 42 °C for 1-2 minutes;

(5) Add 1 μl of Superscript II RT and mix at 42 °C for 10 minutes at 50 °C. 1-1.5 hours;

(6) 70 ° C for 15 minutes to terminate the reaction;

(7) Centrifuge for 10-20 seconds, incubate at 37 °C, add 1 μl RNase Mix and mix at 37 °C 30 minutes;

(8) Centrifugation, ice bath.

B. GlassMAX Purified cDNA

(1) equilibrating the binding solution to room temperature;

(2) Incubate with 100 μl of ddH ₂ O at 70 °C;

(3) Will 120 μl binding solution (6M NaI) is added to the first chain synthesis mixture and mixed;

(4) Transfer the cDNA/NaI mixture to a GlassMAX spin column and centrifuge at 13,000 g for 20 s;

(5) Remove the column from the tube, transfer the filtrate to another clean centrifuge tube, store the tube until the cDNA purification is complete, and return the spin column to the original centrifuge tube;

(6) Will be 0.4ml cold 1 × wash buffer Add to the column, centrifuge at 13000g for 20 seconds, and discard the filter solution for 3 times;

(7) Wash the column twice with 0.4 ml of cold 70% ethanol;

(8) Centrifuge at 13000g for 1 minute to completely remove residual ethanol;

(9) Transfer the column to another centrifuge tube, add 50 μl of pre-warmed ddH ₂ O at 65 ° C, centrifuge at 13000 g for 20 seconds;

C. cDNA tailing

(1) Add the following ingredients to a 0.5 ml centrifuge tube:

5.0 μl 5×tailing buffer; 2.5 μl 2 mM dCTP; 16.5 μl cDNA sample; 24.0 μl H ₂ O

(2) 94 ° C for 3 minutes, placed on ice for 1 minute, centrifuged;

(3) Add 1 μl of TdT, mix and mix at 37 °C for 10 minutes;

(4) Heat at 65 °C for 10 minutes, centrifuge, and store in an ice bath.

D. Tail-tail cDNA PCR amplification

(1) On ice, add the following ingredients to a 0.2 ml centrifuge tube:

5 μl 10× PCR buffer; 3.0 μl 25 mM MgCl ₂ ; 1.0 μl 10 mM dNTP; 2.0 μl 10 μM GSP2; 2.0 μl 10 μM AAP; 5.0 μl dC-tailed cDNA; 31.5 μl ddH ₂ O;

(2) Denaturation at 94 °C for 5 minutes;

(3) Add 0.5 μl of Taq enzyme and mix;

(4) 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;

(5) Take 5-20 μl electrophoresis test.

E. Nested PCR

(1) Take 5 μl of the first round of PCR product and add 495 μl of TE buffer;

(2) On ice, add the following ingredients to a 0.2 μl centrifuge tube:

(3) 94 °C for 5 minutes;

(4) Add 0.5 μl of Taq enzyme and mix;

(5) 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.

2) Mangrove BADH Gene 3' Race

Design and synthesize 3'RACE primers:

Universal primer: APT18: 5'-CGCTACGTAACGGCATGACAGTG(T)18-3'

AP: 5'-CGCTACGTAACGGCATGACAGTG-3'

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 PCR identification are shown in Figure 3.

3) Obtaining the full-length cDNA sequence of mangrove BADH gene

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'

BADH3': 5'-GTTCAGTCTGGCAGTAGAGG-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.

In the gene Blast in the bank indicates that the cloned mangrove BADH gene of this application has 82% homology with the already cloned mangrove BADH gene.

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.

Example 2 Construction of prokaryotic expression vector of mangrove BADH gene and its expression in Escherichia coli

The prokaryotic expression vector EPSPS-pET30a of PET system was constructed. The specific construction process is shown in Figure 4. The E. coli DH5α was transformed with the prokaryotic expression vector EPSPS-pET30a. The BADH gene of mangrove specifically expressed a protein band of 55KD in E. coli DH5α. The results of SDS-PAGE electrophoresis identification are shown in Fig. 5.

Since E. coli DH5α itself contains a substrate required for BADH gene metabolism, the function of the BADH gene was initially verified directly using Escherichia coli DH5α. After induced by IPTG for 3 hours, E. coli DH5α 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

Nacl 浓度时间 (hr ）Nacl concentration time (hr )	1% 1%	3% 3%	5% 5%	7% 7%
0 0	2.13 2.13	2.13 2.13	2.13 2.13	2.13 2.13
2 2	2.76 2.76	1.98 1.98	1.77 1.77	1.566 1.566
3.5 3.5	3.03 3.03	2.37 2.37	1.818 1.818	1.578 1.578
5.5 5.5	3.468 3.468	2.784 2.784	1.968 1.968	1.578 1.578
7 7	3.618 3.618	2.844 2.844	2.034 2.034	1.704 1.704

Table 2: Cell concentration (OD value) of BADH gene growth at different NaCl concentrations at different times

Nacl 浓度时间 (hr ）Nacl concentration time (hr )	1% 1%	3% 3%	5% 5%	7% 7%
0 0	2.922 2.922	2.922 2.922	2.922 2.922	2.922 2.922
2 2	3.36 3.36	3.144 3.144	2.784 2.784	2.484 2.484
3.5 3.5	3.456 3.456	3.24 3.24	3.054 3.054	2.712 2.712
5.5 5.5	3.534 3.534	3.492 3.492	3.186 3.186	2.658 2.658
7 7	3.75 3.75	3.744 3.744	3.198 3.198	2.652 2.652

It can be seen from the growth data in Fig. 6 and Fig. 7 that the growth rate of the cells decreases as the NaCl concentration increases. In the 1% basic NaCl concentration medium, the growth rate of E. coli transgenic to BADH gene was comparable to that of control E. coli. Comparing the two sample data in 3% NaCl concentration medium, the expression of BADH gene significantly improved the growth of E. coli, and the concentration reached a level close to 1% NaCl concentration after 5.5 hours of culture, while the control concentration was significantly lower. At 1% control. In the medium with a NaCl concentration of 5%, the concentration of the bacteria began to increase after a period of adaptation; while the control group was always inhibited. In the medium with a NaCl concentration of 7%, both the sample and the control showed significant growth inhibition, but only the obvious cell death occurred in the control group, and the sample group showed signs of restoring growth after a period of growth.

Example 3 Construction of Eukaryotic Expression Vector of Mangrove BADH Gene

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.

1, Nco I and Sal I double-digested the BaDH gene PCR product and KTP-HT7-BT-PBS vector, and replaced the BT gene in the KTP-HT7-BT-PBS vector with the BaDH gene. The constructed vector was named KTP-HT7-BADH-PBS;

2, Sal I cut KTP-HT7-BADH-PBS and fill it up, then use BamH I was digested; the fragment was inserted into the Rd29A-TPS-Tnos-PMD vector to replace the TPS fragment, and the vector was named Rd29A-BADH-Tnos-PMD.

3, 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.

Example 4 Functional Identification of Mangrove BADH Gene in Tobacco

The screening of NaCl concentration in the laboratory's existing non-transgenic tobacco, using the characteristics of BADH with salt tolerance, directly screening the transgenic tobacco with NaCl as a screening material.

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. In the differentiation medium of NaCl, a differentiation medium to which no NaCl was added was used as a control. Observed, containing 2.5% The tobacco leaf discs under NaCl conditions have a large area of yellow, and the tobacco leaf discs die when the NaCl concentration reaches 3%. After the tobacco leaf discs are infiltrated with the Agrobacterium containing the binary plant expression vector pCAMBIA300BADH, the tobacco leaf discs are transferred to 5%. In the differentiation culture of NaCl, it was observed after 15 days that the leaf discs were differentiated and sprouted. The above results indicate that tobacco with BADH gene has better salt tolerance.

The transgenic T1 seed and non-transgenic seeds were cultured on MS medium containing 5% NaCl. The results are shown in Figure 9. In the A plate, 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.

Claims

A mangrove betaine aldehyde dehydrogenase gene, characterized in that the gene has SEQ ID NO: 1 The nucleotide sequence shown.

2. A prokaryotic expression vector comprising the mangrove betaine aldehyde dehydrogenase gene of claim 1.

3. An eukaryotic expression vector comprising the mangrove betaine aldehyde dehydrogenase gene of claim 1.

The eukaryotic expression vector containing the mangrove betaine aldehyde dehydrogenase gene according to claim 3, wherein the eukaryotic expression vector is a plant expression vector.

5. A plant cell, tissue or plant transformed with the plant expression vector of claim 4.

6. The use of the plant cell, tissue or plant according to claim 5 for cultivating a salt-tolerant, drought-tolerant, low-temperature resistant plant variety.

7. The protein sequence encoded by the mangrove betaine aldehyde dehydrogenase gene according to claim 1, wherein the protein has SEQ ID NO: 3 The amino acid sequence shown.