CN1793365A - Salt resistant gene from salt mustard and its coding protein and application thereof - Google Patents

Abstract

The invention discloses one salt-proof gene from salt mustard and its coded protein and application. The gene can has one of the following sequences: SEQ ID No:1 DNA sequence; SEQ ID No:2 DNA sequence; SEQ ID No:1 defined DNA sequence crossing nucleotide sequence. Coding has one of the following amino acid residues sequence protein: SEQ ID No:2; the protein with the action of increasing plant saline resistance by replacing, faulting or adding one to ten amino acid residues of the SEQ ID No:2 amino acid residues sequence. The formed salt-proof gene can play an importance role in cultivating the plant with increased salt resistance such as rice, wheat.

Description

Derive from resistant gene of salt and proteins encoded and the application of salt mustard

Technical field

The present invention relates in the plant to coerce relevant gene and proteins encoded and application, particularly relate to resistant gene of salt and proteins encoded and its application in cultivating salt tolerance raising plant in the salt mustard with anti-.

Background technology

Studies show that some close relatives of Arabidopis thaliana have extremely strong resistance of reverse, and the suitable genetic manipulation that carries out.Wherein, halophytes-salt mustard is very similar to Arabidopis thaliana, and the gene order homology can reach 70-90%, not only has the gratifying form of genetics model plant, growth characteristics and genetic characteristics, and also the high salt concentration to seawater has stronger tolerance.Therefore, still not a kind of excellent hereditary pattern plant of salt mustard, a kind of especially resource plant that separates the excellent in resistance salt-related gene for research plant nature salt tolerant mechanism.

Summary of the invention

The resistant gene of salt and the proteins encoded thereof that the purpose of this invention is to provide a salt mustard.

Resistant gene of salt provided by the present invention, name is called ST225, derives from cress salt mustard (Thellungiella halophila), is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: 2 dna sequence dna;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.

The rigorous condition of described height be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.

SEQ ID № in the sequence table: 1 by 509 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 2 amino acid residue sequence from 5 ' end 74-424 bit base.

The albumen (ST225) that resistant gene of salt of the present invention is coded also belongs to protection scope of the present invention.It is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 2;

2) with SEQ ID № in the sequence table: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein that improves the plant salt endurance effect.

SEQ ID № in the sequence table: 2 are made up of 116 amino-acid residues.

Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification ST225.

Utilize plant expression vector, resistant gene of salt of the present invention is imported vegetable cell or tissue, can obtain high-salt tolerance power enhanced transgenic cell line and transfer-gen plant.

Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described expression of plants also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participated in mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of inducing (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean storage protein gene) 3 ' end to transcribe as the Agrobacterium crown-gall nodule all has similar functions.

When using ST225 to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, root specific expression promoter etc., they can use separately or be used in combination with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.

For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene of luminophor (gus gene, luciferase gene etc.) as adding the coding that in plant, to express, have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (as anti-weedkiller gene) etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Carry ST225 of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed cell or tissue is cultivated into plant.By the plant transformed host both can be monocotyledonss such as paddy rice, wheat, also can be dicotyledonss such as Arabidopis thaliana, soybean, rape,

Resistant gene of salt ST225 provided by the present invention and arabidopsis gene group gene are carried out sequence alignment, as a result in this gene and the arabidopsis gene group coding expressing protein gene (GenBank accession number: Atlg19530) higher similarity (75% similarity is arranged on the amino acid levels) is arranged, by resulting transgenic arabidopsis after transforming Ben Jiyin is carried out the environment stress test, prove the salt tolerance that can significantly improve transformed plant behind this gene transferred plant.The present invention lays a good foundation for degeneration-resistant and anti-retrocorrelation expression of gene in the artificial controlling plant, will play a significant role in cultivating resistance and resistance of reverse enhanced plant (particularly farm crop such as paddy rice, wheat, rape).

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

Description of drawings

Fig. 1 is the physical map of carrier pCB2004

Fig. 2 A is that transformant (225) and wild-type Arabidopis thaliana (WT) are containing the growing state of sprouting after 12 days on the MS minimum medium of 0mM NaCl

Fig. 2 B is that transformant (225) and wild-type Arabidopis thaliana (WT) are containing the growing state of sprouting after 12 days on the MS minimum medium of 180mM NaCl

Fig. 2 C is the survival rate statistics of transformant (225) and wild-type Arabidopis thaliana (WT) after sprouting 12 days on the MS minimum medium that contains different concns (0mM, 180mM) NaCl

Fig. 3 is that the ST225 transformant (225) and the wild-type Arabidopis thaliana that are grown in the soil are coerced the growing state of processing after 5 days through different concns (0mM, 200mM, 250mM) NaCl

Fig. 4 is ST225 transformant (225) and the wild-type Arabidopis thaliana (WT) that is grown in the soil, coerces the lotus tongue diameter comparative result of processing after 8 days through different concns (0mM, 200mM, 250mM) NaCl

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment.

The acquisition of embodiment 1, salt mustard resistant gene of salt ST225

One, the structure in salt mustard cDNA library

Extract the total RNA of salt mustard, reverse transcription obtains the complete genomic cDNA of salt mustard, and (Marvin's is beautiful for Wang Zonggui, Zheng Wenling for the method (Gateway Technology) of employing high flux construction carrier; Gateway cloning system: the new development of DNA recombinant technology.Chinese biological engineering magazine (2003), the 23rd the 7th phase of volume) with salt mustard cDNA elder generation recombinant clone in pDONR207 carrier (Invitrogen company), obtain Entry cDNA library, and then shuttled back and forth to the plant overexpression binary vector pCB2004 (physical map as shown in Figure 1) that contains 35S promoter in whole cDNA library with the method for recombinant clone, obtain salt mustard cDNA library.Above-mentioned recombining reaction carries out according to the experiment guide that Invitrogen company provides fully.

Two, transform the wild-type Arabidopis thaliana with salt mustard cDNA library

Shuttled back and forth in binary vector pCB2004 in the salt mustard cDNA library that step 1 makes up, electricity is converted into Agrobacterium C58 again, the screening positive recombinant, adopt the Arabidopis thaliana titbit to soak the method (floral dip method) (StevenJ, Clough and Andrew F.Bent:Floral dip:a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana.The PlantJournal:Volume 16Issue 6Page 735-December 1998) of flower conversion with the extensive arabidopsis thaliana transformation of positive recombinant.

Three, the screening of arabidopsis thaliana salt-tolerance transformant

1, the establishment in Arabidopis thaliana transformant storehouse

The conversion that extensive sowing step 2 obtains in soil has the seed of the Arabidopis thaliana transformant in salt mustard cDNA library, behind seed germination about 5 days, surface sprinkling concentration is 0.2% weedicide (glufosinate ammonium, commercial Liberty by name, France Aventis crop science company) screening, is contrast with the wild-type Arabidopis thaliana.Can observe the transformant plant after about 1 week and the wild-type plant has significant difference, the wild-type plant can not survive under 0.2% weedicide glufosinate condition, and the transformant plant is unaffected, can normal growth.With 200 strain transformant plant is that kind is received by a unit.

2, high throughput method primary dcreening operation salt tolerant transformant plant

Used seed all carries out the surface sterilization processing with the following method in following each experiment: at room temperature sterilized 15 minutes with 50% thimerosal (Guangzhou Blue Moon Co., Ltd), use purified rinse water 4-5 time through sterilization again.

To place 3 days down at 4 ℃ through the seed of disinfectant step 1 results, so that the seed germination unanimity.Then planting seed is made its germination on MS minimum medium (containing 2% sucrose, 1.5% agar powder, pH value 5.8, high-temperature steam sterilization 15 minutes), the seed germination growth conditions is: 22 ℃, and illumination cultivation.After the germination, it is seeded in high flux screening salt tolerant transformant on the MS minimum medium that contains 220mM NaCl and 25mg/L weedicide glufosinate, the culture dish of each 15cm is broadcast 3000 seeds again.Treat plant strain growth after 10 days, the transformant plant (not only antiweed but also salt tolerant) that survives is transplanted in the soil, and the individual plant receipts are planted.

3, the multiple sieve of salt tolerant transformant

To each possible salt tolerant transformant, after choosing its about 25 to 30 seeds and carrying out surface sterilization, be seeded on the saliferous MS minimum medium of different concns with the method for above-mentioned steps 2, salt concn is between 0-220mM NaCl.Corresponding data is observed and write down to seed growth after the about week, observe after 10 days, the plant quantity of statistics survival, check ratio between survival plant quantity and the dead plant quantity, the immigration of survival plant is contained on the MS minimum medium of 25mg/L weedicide glufosinate 1 week back observation seedling survival condition.If seedling is all survived, show that the salt-tolerance character of above-mentioned salt-resistance strain and anti-herbicide gene may be chain, meet the screening requirement.At last, transplant above salt-resistance strain in soil, individual plant is received and is planted, and seed is preserved standby.

Four, the acquisition of resistant gene of salt ST225

Insert the upstream and downstream sequences Design primer amplification cDNA sequence in site according to salt mustard cDNA in carrier pCB2004, primer sequence is as follows:

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

AttB2 (downstream primer): 5 '-TACAAGAAAGCTGGGTTTTTTTTTTTT-3 '

The genomic dna of the salt tolerant transformant that the extraction step three screening obtains, and as template, under the guiding of primer Omega and attB2, carry out pcr amplification, 50uL pcr amplification system is: 0.25uL ExTaq polysaccharase (precious biotechnology (Dalian) company limited), the 2uL genomic dna, 10 * PCR damping fluid 5uL, dNTPs 100uM, primer each 25uM in upstream and downstream is supplemented to 50uL with distilled water with reaction system again.The salt mustard cDNA that changes over to round pcr amplification.The PCR reaction conditions is: 95 ℃ of 1min, 66 ℃ of 1min, 72 ℃ of 2min, totally 40 circulations.After reaction finishes, the PCR product is checked order as sequencing primer with Omega, sequencing result shows that this gene has SEQ ID № in the sequence table: 1 nucleotide sequence, SEQ ID № in the sequence table: 1 by 509 based compositions, its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 2 amino acid residue sequence from 5 ' end 74-424 bit base.With this resistant gene of salt called after ST225, the albumen called after ST225 of this coded by said gene.

The salt tolerant experiment of the Arabidopis thaliana of embodiment 2, commentaries on classics ST225 gene on substratum

Make up the overexpression carrier of ST225 with the method for embodiment 1, ST225 is cloned among the overexpression binary vector pCB2004 that contains 35S promoter, obtain the overexpression carrier of ST225, called after pCB2004/ST225.After order-checking is identified correctly, pCB2004/ST225 is transformed Agrobacterium C58 with electrotransformation, the screening positive recombinant soaks method (floral dip method) the conversion wild-type Arabidopis thaliana plant that flower transforms with the Arabidopis thaliana titbit again.Then transfer-gen plant is carried out the salt tolerant experiment, method is: transgenic seed is seeded in respectively on the MS solid medium that contains 0mM, 120mM, 150mM, 180mM NaCl, with the wild-type Arabidopis thaliana is contrast, cultivates under normal condition, observes and record germination and growing state.The result is not containing on the common MS substratum of NaCl, sprouts after 12 days transformant (225) upgrowth situation and wild-type (WT) basically identical (shown in Fig. 2 A, the black line top is wild-type (WT), and the below is transformant (225)); Under 180mM NaCl stress conditions, to sprout after 12 days, the wild-type growth obviously is suppressed, and the upgrowth situation of transformant significantly is better than wild-type (shown in Fig. 2 B, the black line top is wild-type (WT), and the below is transformant (225)), show that salt tolerance strengthens.(* represents difference and remarkable to the survival rate statistics of transformant (225) and wild-type (WT) shown in Fig. 2 C after sprouting 12 days on the MS minimum medium that contains 0mM, 180mM NaCl, P＜0.05, n=143), containing on the MS solid medium of 180mM NaCl 12 days transformant (225) of growth) survival rate be higher than wild-type (WT).

The salt tolerant experiment of the Arabidopis thaliana of embodiment 3, commentaries on classics ST225 gene in soil

With the planting seed of the ST225 transfer-gen plant (225) that obtains among the embodiment 2 and wild-type Arabidopis thaliana (WT) in the MS minimum medium, cultivate under the same conditions, after treating that cotyledon is opened, two kinds of Arabidopis thaliana plant are moved to continued growth in the soil, grow on (about 10 leaves) about two weeks, three groups of each minutes, pouring contains the aqueous solution of 0mM NaCl (contrast), 200mMNaCl, 250mM NaCl respectively, observes and write down its growing state.(every the line top is wild-type (WT) to plant strain growth situation after above-mentioned different concns salt stress is handled 5 days as shown in Figure 3, the below is transformant (225)), under the normal growth condition (0mM NaCl), transformant (225) upgrowth situation and wild-type (WT) basically identical; Under condition of salt stress, transformant (225) is obviously fast than wild-type plant strain growth.After handling 8 days, above-mentioned salt stress measures the lotus tongue diameter of transformant (225) and wild-type (WT), (* represents that difference reaches significantly to the result as shown in Figure 4, P＜0.05, n=26), compare with transformant (225), the growth of wild-type (WT) plant obviously is suppressed, and its plant is less than transformant (225).

Sequence table

<160>2

<210>1

<211>509

<212>DNA

＜213〉Cruciferae salt mustard (Thellungiella halophila)

<400>1

ggggaccgac?atccaagtct?tctacatact?tatcagaaaa?aaattaatta?gacaattgca 60

acgactaaga?gcaatgggtt?ctctaatgtc?aggatgggac?tctccaccag?cggatcctaa 225

ttcagtgaaa?aggtgcaagt?cactaacaag?agaagacatc?gacgctttct?ggaaagaaaa 180

gaagaaaaat?gaagaagaac?atgttcaagc?catttccaag?ttggtagttc?aggaggttgc 240

ggaaagtcaa?gcgcaagagg?agaagataga?agacgatcct?tatgaaaacc?aaagcaagaa 300

aagtggatgg?tggcaaagaa?gcacctgggc?gttcttgaat?gagccgaggg?aagaagaggg 360

tcggccgaac?aagtacgtgt?cgcagttcaa?agtggctcac?atcgccaaaa?gcgtgggcca 420

gtaatgatac?tattatgtaa?tcactggcta?atcacatgca?tgtctacctg?tgtgtgtgtg 480

tgaattgtga?ttagtcgtcc?tttctggtg 509

<210>2

<211>116

<212>PRT

＜213〉Cruciferae salt mustard (Thellungiella halophila)

<400>2

Met?Gly?Ser?Leu?Met?Ser?Gly?Trp?Asp?Ser?Pro?Pro?Ala?Asp?Pro?Asn

1 5 10 15

Ser?Val?Lys?Arg?Cys?Lys?Ser?Leu?Thr?Arg?Glu?Asp?Ile?Asp?Ala?Phe

20 25 30

Trp?Lys?Glu?Lys?Lys?Lys?Asn?Glu?Glu?Glu?His?Val?Gln?Ala?Ile?Ser

35 40 45

Lys?Leu?Val?Val?Gln?Glu?Val?Ala?Glu?Ser?Gln?Ala?Gln?Glu?Glu?Lys

50 55 60

Ile?Glu?Asp?Asp?Pro?Tyr?Glu?Asn?Gln?Ser?Lys?Lys?Ser?Gly?Trp?Trp

65 70 75 80

Gln?Arg?Ser?Thr?Trp?Ala?Phe?Leu?Asn?Glu?Pro?Arg?Glu?Glu?Glu?Gly

85 90 95

Arg?Pro?Asn?Lys?Tyr?Val?Ser?Gln?Phe?Lys?Val?Ala?His?Ile?Ala?Lys

100 105 110

Ser?Val?Gly?Gln

115

Claims (9)

1, the salt mustard resistant gene of salt is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: 2 dna sequence dna;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.

2, resistant gene of salt according to claim 1 is characterized in that: described gene has SEQ ID № in the sequence table: 1 dna sequence dna.

3, the proteins encoded of the described resistant gene of salt of claim 1 is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 2;

2) with SEQ ID № in the sequence table: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein that improves the plant salt endurance effect.

4, albumen according to claim 3 is characterized in that: described albumen has SEQ ID № in the sequence table: 2 amino acid residue sequence.

5, the expression vector that contains the described resistant gene of salt of claim 1.

6, the transgenic cell line that contains the described resistant gene of salt of claim 1.

7, the host bacterium that contains the described resistant gene of salt of claim 1.

8, a kind of method of cultivating salt-tolerant plant is to utilize plant expression vector that the described resistant gene of salt of claim 1 is imported vegetable cell or tissue, obtains high-salt tolerance power enhanced transgenic cell line and transfer-gen plant.

9, method according to claim 8 is characterized in that: described is paddy rice, wheat, soybean, rape or Arabidopis thaliana by the plant transformed host.

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