CN100368543C - A Salt Tolerance Gene, Its Encoded Protein and Its Application from Salt Mustard - Google Patents

Abstract

The present invention discloses a salt resistant gene from salt mustard, a coded protein thereof and an application thereof. The aim of the present invention is to provide a salt resistant gene from salt mustard, a coded protein thereof and an application for the salt resistant gene in the process of breeding plants with enhanced salt resistance. The gene has one of the following nucleotide sequences: 1) a DNA sequence of SEQ ID No. 1 in a sequence list, 2) a DNA sequence for coding SEQ ID No. 2 in the sequence list, and 3) a nucleotide sequence which can be hybridized with the DNA sequence limited by SEQ ID No. 1 in the sequence list under the high precision condition. The coded protein has one of the following amino acid residue sequences: 1) SEQ ID No. 2 in a sequence list, and 2) a protein which is used for substituting, deleting or adding one to ten amino acid residues of an amino acid residue sequence of the SEQ ID No. 2 in the sequence list and has the function of enhancing the salt resistance of plants. The salt resistant gene of the present invention plays an important role in breeding plants with enhanced salt resistance (especially rice, wheat, etc.).

Description

A Salt Tolerance Gene, Its Encoded Protein and Its Application from Salt Mustard

technical field

The invention relates to a gene related to stress resistance in plants, its encoded protein and its application, in particular to a salt tolerance gene and its encoded protein in salt mustard and its application in cultivating plants with improved salt tolerance.

Background technique

Studies have shown that some close relatives of Arabidopsis have strong stress tolerance and are more suitable for genetic manipulation. Among them, the halophytic plant, Saltina thaliana, is very similar to Arabidopsis thaliana, with gene sequence homology up to 70-90%. Salt concentration has a strong tolerance. Therefore, Salt mustard is not only an excellent genetic model plant for studying the natural salt tolerance mechanism of plants, but also a resource plant for isolating genes related to excellent salt tolerance.

Contents of the invention

The purpose of the present invention is to provide a salt tolerance gene and its coded protein of the salt mustard.

The salt tolerance gene provided by the present invention, named ST63-2, is derived from the cruciferous plant Salt mustard (Thellungiella halophila), and is one of the following nucleotide sequences:

1) DNA sequence of SEQ ID No.: 1 in the sequence listing;

2) DNA sequence of SEQ ID No.: 2 in the coding sequence list;

3) A nucleotide sequence that can hybridize to the DNA sequence defined by SEQ ID No. 1 in the sequence listing under high stringency conditions.

The high stringency condition is to hybridize and wash the membrane at 65° C. in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

SEQ ID No. in the sequence listing: 1 is composed of 634 bases, and its coding sequence is the 116-388th base from the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID No.: 2 in the sequence listing.

The protein (ST63-2) encoded by the salt tolerance gene of the present invention also belongs to the protection scope of the present invention. It is a protein with one of the following amino acid residue sequences:

1) SEQ ID No. in the sequence listing: 2;

2) The amino acid residue sequence of SEQ ID No.: 2 in the sequence table is substituted, deleted or added with one to ten amino acid residues and has the effect of improving plant salt tolerance.

SEQ ID No. 2 in the sequence listing consists of 90 amino acid residues.

The expression vector, transgenic cell line and host bacteria containing the gene of the present invention all belong to the protection scope of the present invention.

The primer pair for amplifying any fragment in ST63-2 is also within the protection scope of the present invention.

The salt-tolerant gene of the present invention is introduced into plant cells or tissues by using plant expression vectors, and transgenic cell lines and transgenic plants with enhanced high-salt tolerance can be obtained.

The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. The plant expression may also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments that may participate in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopain synthase Nos gene), plant gene (such as soybean storage The untranslated region transcribed at the 3' end of protein gene) has similar functions.

When using ST63-2 to construct a plant expression vector, any enhanced promoter or inducible promoter can be added before its transcription start nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, root-specific expression Promoters, etc., they can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, these enhancer regions It can be ATG initiation codon or adjacent region initiation codon, etc., but must be the same as the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes (GUS genes, luciferase genes) that can express in plants and encode enzymes that can produce color changes or luminescent compounds etc.), resistant antibiotic markers (gentamicin markers, kanamycin markers, etc.), or chemical-resistant marker genes (such as herbicide resistance genes), etc. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

The plant expression vector carrying the ST63-2 of the present invention can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium-mediated, and Transformed plant cells or tissues are grown into plants. The transformed plant host can be monocotyledonous plants such as rice and wheat, or dicotyledonous plants such as Arabidopsis, soybean, rape, etc.

Sequence comparison of the salt-tolerant gene ST63-2 provided by the present invention with the Arabidopsis genome gene shows that the gene has a high similarity with an unannotated open reading frame (ORF) in the Arabidopsis genome (amino acid level 81%), by carrying out the adversity stress test on the transgenic Arabidopsis obtained after transforming the gene, it is proved that the salt tolerance of the transformed plant can be significantly improved after the gene is transferred into the plant. The invention lays the foundation for artificially controlling the expression of stress resistance and stress tolerance related genes in plants, and will play an important role in cultivating plants with enhanced stress resistance and stress tolerance (especially rice, wheat, rape and other crops).

The present invention will be further described below in conjunction with specific embodiments.

Description of drawings

Figure 1 is the physical map of vector pCB2004

Figure 2A is the growth situation of transformants (63-2) and wild-type Arabidopsis (WT) after germination on the MS basic medium containing 0mM NaCl for 7 days

Figure 2B is the growth situation of transformants (63-2) and wild-type Arabidopsis (WT) after germination on the MS basic medium containing 100mM NaCl for 5 days

Figure 2C is the growth situation of transformants (63-2) and wild-type Arabidopsis (WT) after germination on the MS basic medium containing 150mM NaCl for 7 days

Figure 3 is the statistical result of the survival rate of transformants (63-2) and wild-type Arabidopsis (WT) after germination on MS basic medium containing different concentrations (0mM, 100mM and 150mM) NaCl for 7 days

Fig. 4 is the statistical result of fresh weight per unit plant of transformant (63-2) and wild-type Arabidopsis (WT) after germination on MS minimal medium containing different concentrations (0mM, 100mM and 150mM) NaCl for 7 days

Detailed ways

The methods used in the following examples are conventional methods unless otherwise specified.

Example 1. Acquisition of Salt Mustard Salt Tolerance Gene ST63-2

1. Construction of the Salt mustard cDNA library

Extract the total RNA of Saltina, reverse transcribe to obtain the cDNA of the whole genome of Saltina, and use the method of high-throughput vector construction (Gateway Technology) (Wang Zonggui, Zheng Wenling, Ma Wenli; Pathway cloning system: New progress in DNA recombination technology. China Biotechnology Magazine (2003), volume 23, No. 7) the salt mustard cDNA was recombined and cloned into the pDONR207 vector (Invitrogen Company) to obtain the Entry cDNA library, and then the whole cDNA library was shuttled to contain the 35S promoter by recombination cloning. The plant overexpressed the binary vector pCB2004 (the physical map is shown in Figure 1), and the Salina cDNA library was obtained. The above-mentioned recombination reactions were carried out in full compliance with the experimental guidelines provided by Invitrogen.

2. Transformation of wild-type Arabidopsis thaliana with the Salina cDNA library

The salt mustard cDNA library constructed in step 1 was shuttled into the binary vector pCB2004, and then electrotransformed into Agrobacterium C58, and the positive recombinants were screened, and the Arabidopsis floral dip method (floral dip method) was used (StevenJ, Clough and Andrew F.Bent: Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal: Volume 16 Issue 6 Page 735-December 1998) Large-scale transformation of positive recombinants into Arabidopsis thaliana.

3. Screening of Arabidopsis salt-tolerant transformants

1. Creation of Arabidopsis transformant library

The seeds of the Arabidopsis thaliana transformant transformed with the Salina cDNA library obtained in step 2 were sown in soil on a large scale, and the surface was sprayed with a concentration of 0.2% herbicide (glufosinate ammonium, commercially known as Liberty) about 5 days after the seeds germinated. , France Aventis Crop Science Company) was screened, using wild-type Arabidopsis as a control. After about 1 week, it can be observed that the transformant plants are significantly different from the wild-type plants. The wild-type plants cannot survive under the condition of 0.2% herbicide glufosinate, while the transformant plants are not affected and can grow normally. Harvest with 200 transformant plants as a unit.

2. Preliminary screening of salt-tolerant transformant plants by high-throughput method

The seeds used in each of the following experiments were all surface sterilized by the following method: sterilized at room temperature for 15 minutes with 50% disinfectant (Guangzhou Blue Moon Co., Ltd.), and then rinsed with sterilized pure water for 4-5 minutes. all over.

Place the sterilized seeds harvested from step 1 at 4 °C for 3 days to allow consistent seed germination. Then the seeds were sown on MS basic medium (containing 2% sucrose, 1.5% agar powder, pH value 5.8, sterilized by high temperature steam for 15 minutes) to germinate. The seed germination growth conditions were: 22° C., light culture. After germination, it was sown on the MS basic medium containing 220mM NaCl and 25mg/L herbicide glufosinate for high-throughput screening of salt-tolerant transformants, and 3000 seeds were sown in each 15cm petri dish. After the plants grew for 10 days, the surviving transformant plants (both herbicide-resistant and salt-tolerant) were transplanted into the soil, and single plants were harvested.

3. Re-screening of salt-tolerant transformants

For each possible salt-tolerant transformant, select its about 25 to 30 seeds and carry out surface sterilization with the method of the above-mentioned step 2, sow on the saline-containing MS basic medium of different concentrations, and the salt concentration is between 0-220mM NaCl between. After the seeds grow for about a week, observe and record the corresponding data. After 10 days of observation, count the number of surviving plants, check the ratio between the number of surviving plants and the number of dead plants, and transfer the surviving plants into MS basic medium containing 25mg/L herbicide glufosinate After 1 week, the survival of the seedlings was observed. If all the seedlings survive, it indicates that the salt-tolerant traits and herbicide-resistant genes of the above-mentioned salt-tolerant lines may be linked, which meets the screening requirements. Finally, transplant the above salt-tolerant strains into the soil, harvest the seeds from a single plant, and save the seeds for later use.

4. Acquisition of salt tolerance gene ST63-2

Design primers to amplify the cDNA sequence according to the upstream and downstream sequences of the insertion site of the salt mustard cDNA in the vector pCB2004. The primer sequences are as follows:

Omega (upstream primer): 5'-TTTTTACAACAATTACCAACAACAACAA-3'

attB2 (downstream primer): 5'-TACAAGAAAGCTGGGTTTTTTTTTTTT-3'

Extract the genomic DNA of the salt-tolerant transformant obtained in step 3 screening, and use this as a template to carry out PCR amplification under the guidance of primers Omega and attB2. The 50uL PCR amplification system is: 0.25uL ExTaq polymerase (Bao Biological Engineering ( Dalian) Co., Ltd.), 2uL of genomic DNA, 5uL of 10×PCR buffer, 100uM of dNTPs, 25uM of upstream and downstream primers, and then replenish the reaction system to 50uL with double distilled water. The transferred Salina cDNA was amplified by PCR technique. The PCR reaction conditions were: 95°C for 1min, 66°C for 1min, 72°C for 2min, a total of 40 cycles. After the reaction, the PCR product was sequenced with Omega as a sequencing primer. The sequencing results showed that the gene had the nucleotide sequence of SEQ ID No. 1 in the sequence listing, and SEQ ID No. 1 in the sequence listing consisted of 634 bases , its coding sequence is base 116-388 from the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID No.: 2 in the sequence listing. The salt tolerance gene was named ST63-2, and the protein encoded by the gene was named ST63-2.

Embodiment 2, the salt tolerance experiment of the Arabidopsis thaliana transgenic ST63-2 gene on the culture medium

The method of Example 1 was used to construct the overexpression vector of ST63-2, and clone ST63-2 into the overexpression binary vector pCB2004 containing the 35S promoter to obtain the overexpression vector of ST63-2, named as pCB2004/ST63-2 . After the correctness was identified by sequencing, pCB2004/ST63-2 was transformed into Agrobacterium C58 by electroporation, positive recombinants were screened, and wild-type Arabidopsis plants were transformed by the floral dip method. Then the transgenic plants were subjected to the salt tolerance experiment, the method was as follows: the transgenic seeds were respectively sown on MS solid medium containing 0mM NaCl, 100mM NaCl, and 150mM NaCl, and wild-type Arabidopsis was used as a control, cultivated under conventional conditions, and observed And record germination and growth. Results On the ordinary MS medium without NaCl, after 7 days of germination, the growth status of the transformant (63-2) was basically the same as that of the wild type (WT) (as shown in Figure 2A, the vertical region is the transformant (63-2) , the horizontal region is the wild type (WT)); under 100mM NaCl stress, after germination for 5 days, the germination of the wild type (WT) was inhibited, and the transformant (63-2) was almost unaffected (as shown in Figure 2B, The vertical area is the transformant (63-2), and the horizontal area is the wild type (WT)); under the stress condition of 150mM NaCl, after 7 days of germination, the growth of the wild type is inhibited, and the cotyledons basically do not open or die after opening, while the transformed The growth status of the body was significantly better than that of the wild type (as shown in Figure 2C, the vertical area is the transformant (63-2), and the horizontal area is the wild type (WT)), indicating that the salt tolerance is enhanced. The statistical results of the survival rate of the transformant (63-2) and wild type (WT) after germination on the MS basic medium containing the above-mentioned different concentrations of NaCl for 7 days are shown in Figure 3 (* indicates a significant difference, P<0.05, n =3), the statistic result of unit plant fresh weight is as shown in Figure 4 ( * represents difference significance, P＜0.05, n=3), the transformation of growing 7 days on the MS solid medium containing 100mM NaCl, 150mM NaCl The survival rate and fresh weight per unit plant of the somatic (ST63-2) were higher than those of the wild type (WT).

sequence listing

<160>2

<210>1

<211>634

<212>DNA

<213> Cruciferous salt mustard (Thellungiella halophila)

<400>1

ggggatctcc tgaatcaata tattcaatct tctcttttat aataccaata aaaaaaaaaa 60

ctttgagaga ctcttcaata tttcgggttt tgttttgaag ctaaataaag ttagaatgga 120

aatgttacaa aaaaaatcgt caatagagac agagcccatg acgttgcact tcgatcaaat 180

aaaacgagct cgagaggaag caatgtatgt gaccaaaacc aagtcatttg aggaagctat 240

ggatattttc acaaaggaaa cgcaagagag cttaagggcc caggaaaaca gaggaagatg 300

ttcaaacatg aagggtgatg atgatgaaaa tgaacgatta atctttctta gtcctcatgg 360

ttgggacatt gcttcagctc ctttctaatt ttgaaactct tttgttctct gttttttgtt 420

ttttgttttt gtaacttctt tatgtaaata tctgaatacc tatagggttt tttgtttttg 480

tttttgtatc tcttttatgt aaacatctga atactttaaa ggatagatgt taactaaaca 540

gtatacttta caatcttcgt ctattataca aggaaggaaa ttcatttatga cataatcact 600

tgttttaaaa gaaaaaaaaa aaaaaaaaaa aaaa 634

<210>2

<211>90

<212>PRT

<213> Cruciferous salt mustard (Thellungiella halophila)

<400>2

Met Glu Met Leu Gln Lys Lys Ser Ser Ile Glu Thr Glu Pro Met Thr

1 5 10 15

Leu His Phe Asp Gln Ile Lys Arg Ala Arg Glu Glu Ala Met Tyr Val

20 25 30

Thr Lys Thr Lys Ser Phe Glu Glu Ala Met Asp Ile Phe Thr Lys Glu

35 40 45

Thr Gln Glu Ser Leu Arg Ala Gln Glu Asn Arg Gly Arg Cys Ser Asn

50 55 60

Met Lys Gly Asp Asp Asp Glu Asn Glu Arg Leu Ile Phe Leu Ser Pro

65 70 75 80

His Gly Trp Asp Ile Ala Ser Ala Pro Phe

85 90

Claims (8)

1. A salt tolerance gene of Salt mustard, which is one of the following nucleotide sequences: 1) The DNA sequence of SEQ ID №: 1 in the sequence listing; 2) The DNA sequence of SEQ ID №: 2 in the coding sequence list. 2. The salt tolerance gene according to claim 1, characterized in that: the nucleotide sequence of the gene is shown in SEQID №: 1. 3. The encoded protein of the salt-tolerant gene according to claim 1, its amino acid residue sequence is as shown in SEQ ID No.: 2. 4. An expression vector containing the salt tolerance gene of claim 1. 5. The transgenic plant cell line containing the salt tolerance gene of claim 1. 6. The host bacterium containing the salt tolerance gene of claim 1. 7. A method for cultivating salt-tolerant plants, which is to introduce the salt-tolerant gene according to claim 1 into plant cells or tissues using a plant expression vector to obtain transgenic cell lines and transgenic plants with enhanced high-salt tolerance. 8. The method according to claim 7, characterized in that: the plant is rice, wheat, soybean, rapeseed or Arabidopsis.

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