CN101033469A - Dunaliella saline TPSP gene, encoding albumen and clone method thereof, and construction method for plant conversion carrier - Google Patents

Abstract

This invention relates to a DvTPSP gene, its encoded protein, cloning method and constructing method of transforming vector by plant. The gene has the base sequence shown by SEQ NO 1. This invention discovers DvTPSP gene and provides its full-length cDNA sequence for the first time, and after sequence analysis and contrast of TPS gene with other species, we know that this gene encodes trehalose-6-phosphatase acid synthetase and trehalose-6-phosphoric acid phosphatase esterase. However, this gene has great difference with the known Trehalose synthase gene, and is a new one found and cloned for the first time. When the gene is transferred into different yeast mutants for functional identification, it is found that the gene has some salt tolerance in the salt-sensitive mutant G19. In addition, the gene genetically transforms higher plants by Agrobacterium for molecular identification and adversity physiological analysis, the results show that the gene can increase the stress tolerance of plants, especially anti-drought ability, so the gene has value of application into the genetic engineering of plant anti-adversity.

Description

The construction process of Dunaliella salina TPSP gene, its proteins encoded and cloning process and plant conversion carrier

Technical field

The present invention relates to the construction process of a kind of Dunaliella salina TPSP gene, its proteins encoded and cloning process and plant conversion carrier.

Background technology

Dunaliella salina (Dunaliella viridis) is a kind of unicellular eucaryon algae, belongs to Chlorophyceae (Chlor-ophyceae), volvocales (Volvocales), Dunaliella salina section (Dunaliellaceae).Be distributed widely in salinity upper zone such as ocean, salt lake, the adventitia that acellular wall, protoplasma only are made up of one deck glycoprotein outward.

The salt frustule is lived in the extreme environment, can survive in containing the nutrient solution of 0.05-5.5mol/LNaCl, is the eukaryote of the salt tolerant found so far.The salt algae has very strong osmotic adjustment ability.It once can tolerate 3-4 osmotic pressure doubly and change; If change osmotic pressure by gradient, the osmotic pressure that it can tolerate more than 7 times changes.Simultaneously, the salt algae is to high salt, and environment stresses such as infiltration all have strong tolerance.Therefore the salt algae is that the idealized system of molecular mechanism is regulated in the tolerance of a research plant stress, is the fabulous biomaterial that people sought and tolerated the closely related gene of extreme environment.

Summary of the invention

One of purpose of the present invention is to provide a Dunaliella salina TPSP gene.

Two of purpose of the present invention is to provide the proteins encoded of this gene.

Three of purpose of the present invention is to provide the cloning process of this gene.

Four of purpose of the present invention is to provide the construction process of the plant expression vector of this gene.

For achieving the above object, the present invention adopts following technical scheme:

A kind of Dunaliella salina TPSP gene is characterized in that this gene has the base sequence shown in the SEQ NO 1.

A kind of above-mentioned Dunaliella salina TPSP gene coded protein is characterized in that having the aminoacid sequence shown in the SEQ NO 2.

A kind of cloning process of above-mentioned Dunaliella salina TPSP gene is characterized in that this method has following steps:

The primer sequence of three contig contig designs a. taking according to EST: P6 (+):

5 '-gtgtggtactacaaggatgcag-3 ', P6 (-): 5 '-atgtcctcatcactgcggtcgt-3 ', in the BAC library of salt algae, screen, obtain the segment of one section about 1.5kb; This section segment is checked order, and compare, learn that this section sequence strides an intron with the cDNA sequence of salt algae; Be forward primer with the P6 (+) that is positioned on the exon again, a design reverse primer P6 (2) on intron:

5 '-acgggcttattggacagcac-3 ', the BAC library of screening salt algae; Obtain four positive colonies, these four positive colonies are used Not I respectively, EcoR I and Hind III carry out enzyme spectrum analysis, first the maximum clone of segment who wherein comprises is chosen do the air gun library;

B. the clone that step a is chosen with Large Construct Kit (QIAGEN) extracting BAC plasmid, carries out the physics fragmentation with the BAC segment that obtains, the about 2～4kb of the segment that obtains; The rubber tapping of the segment of this 2～4kb is reclaimed, and carrier is to make with the pUC18 that Sma I enzyme cuts phosphorus; 2～4kp the segment that reclaims is connected into linearizing pUC18 checks order, sequencing primer is M13 (+) (-), has surveyed nine 96 orifice plates altogether, about 864 clones;

C. 864 above-mentioned clones raw data is imported the Linux program of Phred/Phrap, obtained 5 long contig, its length is respectively: 35945bp, 26502bp, 4913bp, 3076bp, 1407bp; Through the prediction of GENSCAN and FGENSH two fatware, wherein length is the complete sequence that the congtig of 35945bp is DvTPSP cDNA, and this sequence comprises the open reading frame of 3.2kb.

A kind of construction process of plant expression vector of above-mentioned Dunaliella salina TPSP gene is characterized in that the concrete steps of this method are:

A. the reconstruction of plasmid pBI121: according to the sequence of salt algae TPSP gene, the positive and negative adopted primer of design DvTPSP.

GUS(+)：5‘-atg cccgggaattctggtcagtcccttatg-3‘

Sma?I?EcoR?I

GUS(-)：5‘-atg gagctctcgaggtagcaattcccgag-3‘

Sac?I?Xho?I

TPSP(+)：5‘-ggagaacgggggatttgaggga-3‘

TPSP(-)：5‘-tgacaggaagtggcgagcgt-3‘

Cut, mend flat mode by enzyme and destroy the EcoR I enzyme point behind the NOS terminator among the pBI121, get carrier pBI121 (E ^-); Then, be template with pBI121, with GUS upstream and downstream primer PCR amplification gus gene, the PCR product reclaims the GUS segment, and is cloned into pBI121 (E with Sma I/Sac I double digestion ^-) SmaI/Sac I between, thereby at pBI121 (E ^-) gus gene upstream Sma I enzyme point back and downstream Sac I enzyme point before add EcoR I and Xho I enzyme point respectively, must carrier pBI121 (E ⁺/ X ⁺);

B. the clone of the gene induced type promoter element of Arabidopis thaliana rd29A/rd29B: according to adverse circumstance inducible genes rd29A in the Arabidopis thaliana of delivering on the document and the promoter sequence of rd29B, designed and synthesized two pairs of primers,

Prd29A(+)：5‘-agg aagcttatggaggagccatag-3‘

Hind?III

Prd29A(-)：5‘-agg tctagatgagtaaaacagaggaggg-3‘

Xba?I

Prd29B(+)：5‘-agg aagcttcgttgacagaaacagtc-3‘

Hind?III

Prd29B(-)：5‘-atg ggatccttcgtgtctctgagaa-3‘

BamH?I

By the method for PCR, promoter sequence Prd29A and the pRD29B of from the arabidopsis gene group, increase rd29A and rd29B; PRD29A and pRD29B use Hind III/Xba I and Hind III/BamH I double digestion respectively, reclaim two promotor segments, and are cloned into respectively among the pUC19 between Hind III/Xba I and Hind III/BamH I enzyme point, pUC29A and pUC29B;

C. the structure of plant conversion carrier: Prd29A and Prd29B are cut out from pUC29A and pUC29B respectively, and (the E that is cloned into pBI121 respectively ⁺/ X ⁺) in, replace original CaMV35S promotor, thereby obtain two adverse circumstance inducible plant expression vector pBI29A and pBI29B, then, TPSP is cut out from pBK-TPSP, and (the E that is cloned into pBI121 respectively ⁺/ X ⁺), among pBI29A and the pBI29B, replace original gus gene, thereby obtained three respectively by plant expression vector pBI35S-TPSP, pBI29A-TPSP and the pBI29B-TPSP of the salt algae TPSP gene of CaMV35S promotor, rd29A and rd29B control.

The present invention finds Dunaliella salina DvTPSP gene first, and Dunaliella salina DvTPSP is provided the full length cDNA sequence of gene first; Through sequential analysis, and with other species TPS gene pairs ratio, this gene encoding trehalose-6-phosphate synthetic enzyme and trehalose-6-phosphate phosphoesterase in the salt algae as can be known.Yet this gene and known trehalose synthesize enzyme gene have bigger different, are to find and be cloned into first a new trehalose synthesize enzyme gene in the salt algae first.This gene changed over to carry out Function Identification in the different yeast mutants, find that this gene has certain salt tolerance in salt sensitive mutant G19.In addition, this gene by Agrobacterium genetic transformation higher plant, has been carried out Molecular Identification and adversity physiological analysis, and the result proves that this gene can improve the stress tolerance of plant, particularly drought-resistant ability, so this gene has the engineered value of the plant stress-resistance of being applied to.

Description of drawings

Fig. 1 is that the yeast conversion carrier pYTPSP and the pATPSP double digestion of Dunaliella salina DvTPSP gene identified figure.

Fig. 2 is the influence figure of Dunaliella salina DvTPSP gene transformation yeast mutants G19 to growth.

Get about 500 G19/pYES2 (A) or G19/pYTPSP (B) cell separate application to containing different concns NaCl (1:150mM; 2:200mM; Solid AP 3:250mM) (Gal ,-Ura) plate (on 9 * 9cm), 30 ℃ cultivate 6d after, the growing state of transformant.

Fig. 3 is the influence figure of Dunaliella salina DvTPSP gene transformation yeast mutants G19 to growth.

The nutrient solution of G19/pYES2 and G19/pYTPSP is diluted to 10 respectively ⁵, 10 ⁴, 10 ³, 10 ²Four gradients of cells/ μ L, and respectively get 5 μ L drip to respectively the solid AP that do not contain NaCl and contain 150mM NaCl (Gal ,-Ura) on the plate, 30 ℃ cultivate 6d after, the growing state of transformant.

Fig. 4 is plasmid pBI121 (E ^-) enzyme cut evaluation figure.

Fig. 5 is plasmid pBI121 (E ⁺/ X ⁺) enzyme cut evaluation figure

M：1kb?Marker；1：pBI121/Sma?I+Sac?I；2：pBI121/EcoR?I+Xho?I；

3：pBI121(E ⁺/X ⁺)/Sma?I+Sac?I；4：pBI121(E ⁺/X ⁺)/EcoR?I+Xho?I。

Fig. 6 is the structure iron of carrier pBI121.

P：Promoter；T：Terminator；LB：Left?border；RB：Right?border

Fig. 7 is carrier pBI121 (E ⁺/ X ⁺) structure iron.

P：Promoter；T：Terminator；LB：Left?border；RB：Right?border

Fig. 8 is the pcr amplification figure of Prd29A and Prd29B.

M:1kb Marker; The PCR product of 1:Prd29A; The negative control of 2:1;

The PCR product of 3:Prd29B; The negative control of 4:3

Fig. 9 is that the PCR of pBI29A and pBI29B identifies figure

M:1kb Marker; 1:H ₂O (template)/Prd29A (+) (-) (primer); 2:pBI29B/Prd29A (+) (-);

3：pBI29A/Prd29A(+)(-)；4：H ₂O/Prd29B(+)(-)；5：pBI29A/Prd29B(+)(-)；

6：pBI29B/Prd29B(+)(-)

Figure 10 is that the enzyme of pBI35S-TPSP, pBI29A-TPSP and pBI29B-TPSP is cut evaluation figure.

M：1kb?Marker；1：pBI121/EcoR?I+Xho?I；2：pBI35S-TPSP/EcoR?I+Xho?I；

3：pBI29A-TPSP/EcoR?I+Xho?I；4：pBI29B-TPSP/EcoR?I+Xho?I

Figure 11 is plasmid pBI35S-TPSP, the pBI29A-TPSP that has inserted three different promoters of DvTPSP gene, the structure iron of pBI29B-TPSP.

P：Promoter；T：Terminator；LB：Left?border；RB：Right?border

Figure 12 .PCR identifies the transgenic seedling genomic dna

Lane1:H ₂O; Lane2:TPSP is over against photograph; Lane3, Lane4: wild-type Arabidopis thaliana;

Lane5, Lane6: the transgenosis contrast changes the control vector that pBI121 (E+/X+) does not promptly contain TPSP over to;

Lane7, Lane8: the transgenic seedling genomic dna that changes the pBI35S-TPSP plasmid;

Lane9, Lane10: the transgenic seedling genomic dna that changes the pBI29A-TPSP plasmid;

Lane11: the transgenic seedling genomic dna that changes the pBI29B-TPSP plasmid.

Figure 13 is that transgenic seedling is-20 ℃ of freezing treatment phenotypes.

Figure 14 is that arid is handled phenotype

A: the transgenosis contrast, withered; B: change pBI35S-TPSP, normal growth; C: change pBI29A-TPSP, normal growth; D: change pBI35B-TPSP, normal growth

Figure 15 is freezing treatment RT-PCR

TPSP is not expressed in the A:WT contrast; B: the transgenosis contrast, do not express TPSP;

C: change pBI35S-TPSP, constitutive expression TPSP.D:pBI29A-TPSP abduction delivering TPSP,

Begin to express at 1h, expression amount increases along with the increase of time;

The expression of E:pBI29B-TPSP is not obvious at this.

Figure 16 is that arid is handled RT-PCR

TPSP is not expressed in the A:WT contrast; B: the transgenosis contrast, do not express TPSP;

C, commentaries on classics pBI35S-TPSP, constitutive expression TPSP; D:pBI29A-TPSP abduction delivering TPSP;

E:pBI29B-TPSP expression amount under drought-induced condition changes to some extent.

Embodiment:

Embodiment one: the clone of Dunaliella salina TPSP gene

The primer sequence that three contigs (contig) of taking according to EST (Expressed Sequence Tag) design: P6 (+): 5 '-gtgtggtactacaaggatgcag-3 ', P6 (-): 5 '-atgtcctcatcactgcggtcgt-3 ', in the BAC library of salt algae, screen, obtain the segment of one section about 1.5kb.This section segment is checked order, and compare with the cDNA sequence of salt algae, this section sequence is striden an intron as can be known.Be forward primer with the P6 (+) that is positioned on the exon again, a design reverse primer P6 (2) on intron: 5 '-acgggcttattggacagcac-3 ', the BAC library of screening salt algae.Obtain four positive colonies, these four positive colonies are used Not I respectively, EcoR I and HindIII carry out enzyme spectrum analysis, can determine, first clone is the longest, and the segment that is comprised is maximum, therefore first clone are chosen and do the air gun library.

This clone is chosen, with Large Construct Kit (QIAGEN) extracting BAC plasmid.The BAC segment that obtains is carried out the physics fragmentation, the about 2～4kb of the segment that obtains.The segment rubber tapping of this 2～4kb is reclaimed.Carrier is to make with the pUC18 that Sma I enzyme cuts phosphorus.2～4kp the segment that reclaims is connected into linearizing pUC18 checks order, sequencing primer is M13 (+) (-).Nine 96 orifice plates have been surveyed altogether, about 864 clones.One of raw data input is cried in the Linux program of Phred/Phrap and is obtained 5 contig that grow, long respectively 35945bp, 26502bp, 4913bp, 3076bp, 1407bp.Wherein by GENSCAN ( Http:// genes.mit.edu/GENSCAN.html) and FGENSH ( Http:// www.softberry.com/berry.html) prediction of two fatware is the complete sequence that has comprised complete DvTPSPcDNA in the congtig of 35945bp as can be known in the longest contig, this sequence comprises the open reading frame of 3.2kb, by analysis as can be known this genes encoding 930 complete amino acid whose protein products.

Carry out domain analyses by the Blast program among the Genbank, find that this gene mainly comprises two structural domains, Asn50 is similar to OtsA (trehalose-6-phosphatesynthase) and the zymic TPS1 of about 500 amino acid regions of Arg542 and E.coli from the N end, belong to Glycosyltransferase family 20, participate in to form glycosidic link and can in the transportation of carbohydrate and metabolism, play a role.C end contains Trehalose_Ppase (592-910), i.e. trehalose-Phospholipid hydrolase structural domain, Phospholipid hydrolase structural domain are present in many Phospholipid hydrolases family and comprise among the zymic TPS2.The trehalose Phospholipid hydrolase can be with the trehalose-6-phosphate dephosphorylation, and generates trehalose and ortho-phosphoric acid.Because this gene has the structural domain of TPS and TPP, we are DvTPSP with this unnamed gene.

Compare homology that analyze to find salt algae DvTPSP and eukaryotic TPS with all known protein sequences and be higher than homology with prokaryotic cell prokaryocyte TPS.With Arabidopis thaliana TPS7,8,9 consistence reaches 50%, and similarity reaches 67%.With the consistence of the TPS of Seleginella lepidophylla and paddy rice (Oryza sativa, japonica cultivar-group) be respectively 34% and 50%.With the consistence of fission yeast and yeast saccharomyces cerevisiae TPS 39% and 35% (Fig. 3) arranged.DvTPSP and plant TPS height consistence have partly illustrated the conservative property in salt algae and higher plant sibship and the evolution.The TPP of DvTPSP and some species also has higher homology, and for example, with Candidaalbicans TPP, the consistence of the TPP of fission yeast TPP and yeast saccharomyces cerevisiae is respectively 31%, 29% and 31%.

Embodiment two: structure, yeast conversion and the functional analysis of DvTPSP yeast conversion carrier

In order to verify whether DvTPSP has the function of TPS and/or TPP, DvTPSP is changed in yeast tps1 deletion mutantion and the tps2 deletion mutantion carry out the zymic functional analysis.Consider that this gene takes from the salt algae, and the salt algae is a kind of eukaryote of salt tolerant extremely, in order to confirm further whether DvTPSP has anti-salt functional, this gene is changed among the zymic salt sensitizing mutation bacterial strain G19, observes this gene and whether has the salt tolerance.

1, the structure of yeast conversion carrier: for the method that has complementary functions by yeast verifies further whether DvTPSP has the function of TPS and/or TPP, we are gene constructed in Yeast expression carrier pAJ401 and pYES2 this, with EcoR I/Xho I double digestion pBK-TPSP, reclaim the DvTPSP fragment, be cloned into respectively among yeast shuttle vector pYES2 and the pAJ401 by EcoR I/XhoI enzyme point, get yeast conversion carrier pYTPSP and pATPSP, and identify by EcoR I/Xho I double digestion, referring to Fig. 1.

2, yeast conversion: yeast conversion carrier transformed yeast tps1 (coding TPS) deletion mutant YSH290 and yeast tps2 (coding TPP) deletion mutant YSH450 with building, observe the recovery situation of mutant phenotype.Simultaneously, in order to confirm further whether DvTPSP has anti-salt functional, this gene transformation in yeast salt sensitive mutant G19, is observed the variation of salt sensitivity.

Utilize the PEG/LiAC method for transformation with pAJ401 and pATPSP difference transformed yeast YSH202 (wild-type yeast bacterial strain) and YSH290; With pYES2 and pYTPSP difference transformed yeast YSH202, YSH450 and G19, get yeast transformant YSH202/pAJ401, YSH290/pAJ401, YSH290/pATPSP, YSH202/pYES2, YSH450/pYES2, YSH450/pYTPSP, G19/pYES2, G19/pYTPSP.

The result shows, what DvTPSP can not complementary YSH290 with glucose is being the mutant phenotype that can not grow on the substratum of single carbon source, can not complementary YSH450 the 37 ℃ of temperature sensitive mutation phenotypes (result does not show) that can not grow down.But the salt sensitizing mutation phenotype of the complementary G19 of DvTPSP energy part makes G19 containing faint growth on the AP substratum of 200mMNaCl, referring to Fig. 2; And G19 is after changing DvTPSP over to, and the transformant of same cell number is better than contrast in the growing way that contains on the AP substratum of 150mM NaCl, referring to Fig. 3.This shows that DvTPSP has the function of part salt tolerant, but because salt algae and yeast have bigger codon preference gender gap, and DvTPSP and yeast TPS and TPP on the sequence homology relative a little less than, therefore also be not enough to the mutant phenotype of complementary YSH290 and YSH450.

Embodiment three: the structure that is used for the Plant Transformation expression vector:

In order to tackle genetic engineering modified middle some problems that exist of present trehalose, unfavorable factor in growth that brings as the TreP high expression level and the growth etc., in this research except adopting composing type CaMV 35S promoter, two adverse circumstance inductive rd29A/B promotors in Arabidopis thaliana, have been cloned in addition, and carry out carrier reconstruction and obtain three different promoters CaMV 35S, the DvTPSP expression vector of rd29A and rd29B control.

1, the reconstruction of plasmid pBI121: because the gus gene two ends available restriction enzyme site on the pBI121 is very limited, and DvTPSP gene itself is very long, enzyme point is more, downcut the DvTPSP gene from pBK-TPSP and be difficult to directly be inserted into the carrier and replace original gus gene, therefore need reconstruct changing carrier.For the needs of pBI121 reconstruction, according to the carrier sequence, the positive and negative adopted primer of design gus gene adds Sma I and EcoR I enzyme point at sense primer one end, adds Sac I and Xho I enzyme point at antisense primer one end.In addition, for whether the plant conversion carrier that detects structure successfully imports Agrobacterium, according to the sequence of salt algae TPSP gene, the positive and negative adopted primer of design DvTPSP.1

GUS(+)：5‘-atg cccgggaattctggtcagtcccttatg-3‘

Sma?I?EcoR?I

GUS(-)：5‘-atg gagctctcgaggtagcaattcccgag-3‘

Sac?I?Xho?I

TPSP(+)：5‘-ggagaacgggggatttgaggga-3‘

TPSP(-)：5‘-tgacaggaagtggcgagcgt-3‘

Cut, mend flat mode by enzyme and destroy the EcoR I enzyme point behind the NOS terminator among the pBI121, get carrier pBI121 (E ^-), from EcoR I/BamH I double digestion result, referring to Fig. 4, pBI121 (E as can be known ^-) in EcoR I enzyme point destroyed.Then, be template with pBI121, with GUS upstream and downstream primer PCR amplification gus gene, the PCR product reclaims the GUS segment, and is cloned into pBI121 (E with Sma I/Sac I double digestion ^-) Sma I/Sac I between, thereby at pBI121 (E ^-) gus gene upstream Sma I enzyme point back and downstream Sac I enzyme point before add EcoR I and Xho I enzyme point respectively, must carrier pBI121 (E ⁺/ X ⁺).Identify by Sma I/Sac I and EcoR I/Xho I double digestion, referring to Fig. 5 pBI121 (E as can be known ⁺/ X ⁺) in EcoR I and Xho I enzyme point add successfully, referring to Fig. 6 and Fig. 7, thereby cleared away obstacle for the TPSP gene insertion vector.

2, the clone of the gene induced type promoter element of Arabidopis thaliana rd29A/rd29B

According to adverse circumstance inducible genes rd29A in the Arabidopis thaliana of delivering on the document and the promoter sequence of rd29B, designed and synthesized two pairs of primers, positive and negative adopted primer one end of Prd29A has added Hind III and Xba I enzyme point respectively; Positive and negative adopted primer one end at Prd29B has added Hind III and BamH I enzyme point respectively.By the method for PCR, the promoter sequence of from the arabidopsis gene group, increase rd29A and rd29B.

Prd29A(+)：5‘-agg aagcttatggaggagccatag-3‘

Hind?III

Prd29A(-)：5‘-agg tctagatgagtaaaacagaggaggg-3‘

Xba?I

Prd29B(+)：5‘-agg aagcttcgttgacagaaacagtc-3‘

Hind?III

Prd29B(-)：5‘-atg ggatccttcgtgtctctgagaa-3‘

BamH?I

The CTAB method is extracted the total DNA of Arabidopis thaliana.94 ℃ of pre-sex change 4min; 94 ℃, 30S; 55 ℃, 30S; 72 ℃, 1min; 30 circulations; 72 ℃, from the total DNA of Arabidopis thaliana, the increase promotor Prd29A of rd29A of 5min.94 ℃ of pre-sex change 4min; 94 ℃, 30S; 60 ℃, 30S; 72 ℃, 1min; 30 circulations; 72 ℃, increase from the total DNA of the Arabidopis thaliana promotor pRD29B of rd29B of 5min is referring to Fig. 8.PRD29A and pRD29B use HindIII/Xba I and Hind III/BamH I double digestion respectively, reclaim two promotor segments, and are cloned into respectively among the pUC19 between HindIII/XbaI and Hind III/BamH I enzyme point, pUC29A and pUC29B.Order-checking is finished by Shanghai Bo Ya Bioisystech Co., Ltd.

Sequencing result shows that two promotor length are respectively 870bp and 950bp, wherein Prd29A and reported sequence are in full accord, and Prd29B and reported sequence relatively have the difference of two bases, but these two bases are not in the functional area of promotor, wherein any change does not all take place in DRE cis-acting elements, ABA functional element ABREs, TATA frame and CAAT box, thereby can not influence promoter activity.

3, the structure of plant conversion carrier

Detect the activity of promotor for the ease of follow-up by GUS dyeing, at first Prd29A and Prd29B are cut out from pUC29A and pUC29B respectively, and (the E that is cloned into pBI121 respectively ⁺/ X ⁺) in, replacing original CaMV35S promotor, PCR detects, and referring to Fig. 9, show that Prd29A and Prd29B all successfully insert in the carrier, thereby obtain two adverse circumstance inducible plant expression vector pBI29A and pBI29B.Then, TPSP is cut out from pBK-TPSP, and (the E that is cloned into pBI121 respectively ⁺/ X ⁺), among pBI29A and the pBI29B, replace original gus gene, EcoR I/Xho I double digestion result, referring to Figure 10, show that DvTPSP successfully inserts in three carriers, thereby three have been obtained respectively by plant expression vector pBI35S-TPSP, pBI29A-TPSP and the pBI29B-TPSP of the salt algae TPSP gene of CaMV35S promotor, rd29A and rd29B control, referring to Figure 11.

Embodiment four: Agrobacterium-mediated Transformation Arabidopis thaliana and transfer-gen plant screening

The pBI35S-TPSP plasmid that builds is transformed importing Agrobacterium GV3101 by electric shock, and the picking male contains the Agrobacterium GV3101 mono-clonal of plasmid, is cultured to bacterium liquid OD ₆₀₀Be about 0.8, centrifugal collection thalline is abandoned supernatant, and precipitation infiltration substratum (1/2MS, 5% sucrose, 0.05%Silwet L-77, pH5.8) resuspended to bacterium liquid OD ₆₀₀About 0.8～1.0.The bacterium drop on Arabidopis thaliana is not bloomed inflorescence, is sealed maintenance humidity with preservative film, after 23 ℃ of dark overnight incubation, move to 23 ℃ of greenhouses again and normally cultivate.

Treat fruit pod maturation, collect seed, will plant on the 1/2MS substratum that contains 50mg/L Kana after the sterilization of Arabidopis thaliana seed, screening resistance seedling.Arabidopis thaliana is placed in 23 ℃ of illumination boxs, germinates in three days, takes out the resistance seedling about ten days, moves in the compost the following 23 ℃ of constant temperature culture of 16h/8h light/dark condition.

The screening of transfer-gen plant and evaluation: be sowed on the 1/2MS substratum that contains 50mg/L Kana screening resistance seedling after the seed disinfection sterilization.Place culture dish and in 23 ℃ of incubators, sprout, germinate after three days, week back observation.The transgenic seedling growth is normal, and the flavescence of non-transgenic seedling is withered.

Shift out positive seedling, treat that its blade is long to suitable size, extract genomic dna, PCR identifies that the result shows positive transgenic seedling, referring to Figure 12.

Embodiment five: the stress physiology of transfer-gen plant and Molecular Detection

1, freezing treatment:

Contain the pBI35S-TPSP resistance seedling that screens on the 1/2MS substratum of kana and move on on the 1/2MS that does not contain kana, handle preceding and wild-type WT indifference.-20 ℃ place 5h after, be positioned over 23 ℃ of illumination boxs and reply and cultivate, second day wild-type WT and the whole albefactions of pBI35S-TPSP; In the 5th day, there is the part transgenic seedling to reply; And replying does not appear in the wild-type WT that meanwhile handles, referring to Figure 13.

2, arid is handled:

With watering can 50ml water is squirted the exsiccant soil surface earlier, the resistance seedling that will screen from the 1/2MS substratum that contains kana shifts out and plants the compost of surface wettability again, moving the back covers with preservative film, after seedling is replied after one day, open preservative film, placed one day, after the moisture natural air drying on compost surface, plant is in drought status.Rehydration after one day, phenotype such as Figure 14: change unloaded transfer-gen plant and withered symptom occurs, fail to reply; And the transgenosis strain of other importing foreign gene DvTPSP is not withered, and answer occurred.

3, freezing treatment Molecular Detection:

The Arabidopis thaliana of long 8-10 sheet cotyledon placed under-7 ℃ coerce, respectively at 0h, 1h, 2h, 4h, 6h, 8h, 20h, 32h draws materials.Extract RNA respectively, RT-PCR detects, referring to Figure 15.The result shows: WT and transgenosis contrast Arabidopis thaliana are not expressed TPSP, and along with the equal no change of the increase in treatment time; Change pBI35S-TPSP Arabidopis thaliana constitutive expression TPSP; Change pBI29A-TPSP abduction delivering TPSP; Change pBI29B-TPSP because rd29B promotor abduction delivering when freezing is slower than rd29A, temporarily do not find out that in 32h the expression of TPSP changes.

4, arid is handled Molecular Detection:

Transfer-gen plant carries out drought stress, and the Arabidopis thaliana that will grow to 8～10 cotyledons carefully extracts with root from soil, is placed on the filter paper, keeping atmospheric moisture is 60%, respectively at 0h, 1h, 2h and 4h sampling, extract RNA, RT-PCR identifies, since WT, C, pBI35S-TPSP is the degraded of RNA sample behind 2hr, so do not adopt.Result's demonstration: the trehalose of WT and transgenosis contrast is not expressed; But constitutive expression in the Arabidopis thaliana that changes pBI35S-TPSP; The TPSP expression amount of pBI29A-TPSP transgenic seedling rose along with the treatment time; Changeing pBI29B-TPSP also has the expression of TPSP, referring to Figure 16.

Sequence table

＜110〉Shanghai University

＜120〉construction process of Dunaliella salina TPSP gene, its proteins encoded and cloning process and plant conversion carrier

<160>14

<210>1

<211>3204

<212>DNA

＜213〉Dunaliella salina (Dunaliella viridis)

<220>

<221>

<222>

<400>3204

1 GGCACGAGGA?GCCACCCAGC?AGCAGCCCAG?ATTATACAGG?AGGTGGTGC

51 ACTGTGCACA?GGCTGCAGCA?GGGGGCAGCA?TTCACCCAGC?ATTAAGCCAG

101 CCAGCATGCT?TAGCAGCCAT?GCCAAGTCCT?CCAGCTCTTC?CAGGAACCTG

151 GCAGGGATGC?TGGAGAACGG?GGGATTTGAG?GGAGAGGGCT?CTGGCAGTTG

201 CGGGGGAAGC?AGGCAGCAGG?CGCGCGACCC?TAGGCGCACC?AGCCTGAGTG

251 TGAACAGTGG?GCGAGTAATC?ATTGCGTCCA?ACCATCTGCC?GCTGCGGGTG

301 AAAAAGAGCT?CCACAGGAGA?GTGGCAGTTC?GAGTTCGATG?AGGACGCGCT

351 GACCGCGCAG?GCCAAGGATG?GCGTGCCGCG?CTCGCACTTC?CAGGAGGTGC

401 TGTATGTGGG?TGGCCTGCCC?GTGGACGTGG?AGCCTGAGGA?GCAGGAGAAC

451 ATCGCCATCA?AGCTGAAGGA?CCTGTACAAC?TGCTGCCCTG?TGTACCTGGA

501 CTCAGCTGTG?AAGGAACGTT?TCTACAAGGG?CTTCTGCAAG?CAGCAGCTGT

551 GGCCGCTCTT?CCACTACGTC?CTGCCTGGCT?CCCCCAGCAG?CTCCCAGCGC

601 TTCAATGTGG?AGTTCTGGCA?GGCGTATGTC?AAGGCCAACA?AGTGCTTTGC

651 GGACAAGATC?GTGGAGGAGA?GCCTCACAGA?CACCGAGTTT?GTGTGGATCC

701 ATGACTACCA?CCTGCTGGTC?CTGCCCTCCC?TCCTGCGCAA?GCGCTTCAAC

751 CGCATCCGTG?CCGGTGTCTT?CCTGCACTCA?CCCTTCCCCT?CGTCAGAGAT

801 CTTCAGGACC?TTCCCCAAAC?GCGAGGAGCT?GCTCCGCTCG?CTGCTGAATG

851 CAGATTTGAT?AGGTTTCCAC?ACGTTCGACT?ACGCTCGCCA?CTTCCTGTCA

901 TGCTGCTCGC?GCATGCTGGG?CCTGGAGCAT?GAGACTTCTA?GGGGCTCCAT

951 CACCATAGAC?TACTACGGCC?GGACAGTGGG?CATCAAGATC?ATGCCGACAG

1001?GTGTAAACCC?CAAGCGCTAC?CTGGACGGCT?TCTCGTGGGA?TGAGTTCAAG

1051?TGGAGGCGTG?GGGAGCTGGA?GGCGCAGTTC?AAGGGCATGA?CGGTGCTAGC

1101?GGGCGTAGAT?GACATGGACA?GCTTCAAGGG?CATCGACCTG?AAGCTGCAGG

1151?CGTTCGAGCG?GCTGCTAGAG?TACCATGCTG?AGTGGCGGGG?CAAGGTGGTG

1201?CTGGTCCAGG?TCACCAACCC?ACCGCGCTCC?ACGGGCCACG?ACATCACTGA

1251?GCTGCATGCC?TTTGTGACCA?ACCTGGTGGC?GTCCATCAAC?TACAAGTACG

1301?GCAACCCATC?CACCAACTAC?GTGCCAGTTC?ACTACCTGGA?GCGCCACGTG

1351?CCGCTGCATG?AGCGCATGGC?TTTCTACAGC?ATCGCGGACT?GTGTGGTGGT

1401?GACTGCGACG?CGCGACGGCA?TGAACCTTGT?GCCCTATGAG?TACATCGTGT

1451?GCCGGCAAGG?GTCGGATACC?ACTCCATGCG?CTGAGTCGAC?TGTGGAGTCG

1501?GGGCCCAGGG?ACTCCATGCT?GGTCGTGTCA?GAGTTCGTGG?GCTGCTCGCC

1551?ATCGCTCAGT?GGCGCCATCC?GCGTGAACCC?CTGGTCCATC?GACAGTGGCT

1601?ACAACGGCAT?GTACGCAGCC?ATCTGCATGC?CCCTGGAGCA?CCGCCGGCTG

1651?CGGCACCAGA?AGCACTGGCG?CTATGTCAGC?CAGCACACCG?TCGCATTCTG

1701?GGCGCAGTCA?TACCTCATGG?ACCTCGTGCG?CGTGACGCGC?AACCACGTGA

1751?CCATGAAGTG?CTACGGCCTT?GGCCTGGGCC?TGGACACCTT?CCGCATGGTG

1801?GCGCTGGATG?CCAACTTCCG?AAAGATGGAG?GAGTCGCAGG?TGGCCGCCAC

1851?ATACAAGAGC?TCCAAGAGCC?GTGTGTTCTT?CCTGGACTAC?GATGGCACCC

1901?TCACCGCGGG?CCAGCACACA?TCCATCACGC?TTGCTCCGCT?GGATGAGGTG

1951?CTGCAGGTCC?TGCGTGCGCT?GACTGCTGAG?CCGCTCAACA?AGGTGGTGCT

2001?CTTCAGCGGG?CGCCCCAAGG?CAGAGCTGCA?GGAGTGGTTC?GCTTCAGTGC

2051?CCAACCTCGC?GCTGGTGGCG?GAGAACGGCT?GCTACCTGCG?CCTAGGAGAG

2101?GGCCAGACCT?GGAGGTCGCA?CCTCACACCT?GGCCTGCAAG?TTGCTGACCA

2151?GTCGTGGGTC?AGCCTGGTGG?TGGCTGCGGA?CTTTGGGTGG?AAGAAGATGG

2201?CGCTGCCCAT?CCTGCAGCAG?TACCAGGAGA?GCACAGATGG?CAGCAGCATC

2251?GAGGCCAAGG?AGAGCGCGCT?GGTGTGGTAC?TACAAGGATG?CAGACCCCGA

2301?TTTCGGGAGC?TGGCAGGCCA?AGGAGCTGTT?GGATCACCTG?GAGGGCGTGC

2351?TGTCCAATAA?GCCCGTTGAG?ATTGTGGGCG?GCTCGTCGGC?TGTTGAGATC

2401?AAGCCGCAGG?GCGTGTCCAA?GGGTCAGGCA?GTGGAAAAGA?TGCTGGGGCA

2451?GTTGTATGGC?TACCCCGCGA?TCGCGGAACG?CCGCAGCAGC?AGCAGCAGGC

2501?CGATGGGGCG?GCCCAGCAGC?GACGCCAGCG?AGCGCAGACC?CTCCTCCTCC

2551?CGCTTCAGCA?TGAGTGACAG?AGGGCCCTCT?GCTGGCAGCG?CAGCAGTGCA

2601?GAGCGCTGCA?AGCTCAGTGC?AGGGTGGTGG?TGCTGAGGCG?GCAGCACAGC

2651?AAGAGCTCGC?TGCTGCAGGC?AAGGGCCCCG?ACTTTGTGCT?GTGCATCGGG

2701?GACGACCGCA?GTGATGAGGA?CATGTTCACG?AGCATAGAGA?TGATGCGCGC

2751?GTCGCCGCAG?ATGATGTCAT?CCGAGGTCTA?CGCATGCACA?GTGGGTCAAA

2801?AGCCCAGCCG?CGCCCCCTTC?TACCTGAACG?ACCCTGGAGA?AGTGTTGCAC

2851?CTCCTCGCGC?GCCTGGTGGG?CATCAACCTG?CCCAACCTGC?CCCTCTAGAT

2901?TTGGAGAGAG?CGAGCGTGTA?TGTGTGTGTG?CATCTGATCC?GTGCGAAACA

2951?GAAAACAGAA?AATGGATGCC?CTGCGATTCT?GGGTTGTTGG?GCACCATGCT

3001?TTCTTGGCTT?TTGTGTTCCT?TTTTTTGTTT?TTTAGGCAGT?TGCTGGGTGC

3051?AAGCACATTT?TTTTGGCTGT?TGTGCTGCAC?GTTTGCTTGT?CTGCTGAGCC

3101?ACTGGGCTCT?CACTTGCCCC?AAGGCTGCCA?TACACAGCTA?CAGCACCCTG

3151?ATTCGTGGGG?TGGTGTTCAT?GGCATGCCTA?TAAGCCTGGG?GCCTTTTTCC

3201?TTTC

<210>2

<211>930

＜212〉amino acid

＜213〉Dunaliella salina (Dunaliella viridis)

<220>

<221>

<222>

<400>930

1 MLSSHAKSSS?SSRNLAGMLE?NGGFEGEGSG?SCGGSRQQAR?DPRRTSLSVN

51 SGRVIIASNH?LPLRVKKSST?GEWQFEFDED?ALTAQAKDGV?PRSHFQEVLY

101?VGGLPVDVEP?EEQENIAIKL?KDLYNCCPVY?LDSAVKERFY?KGFCKQQLWP

151?LFHYVLPGSP?SSSQRFNVEF?WQAYVKANKC?FADKIVEESL?TDTEFVWIHD

201?YHLLVLPSLL?RKRFNRIRAG?VFLHSPFPSS?EIFRTFPKRE?ELLRSLLNAD

251?LIGFHTFDYA?RHFLSCCSRM?LGLEHETSRG?SITIDYYGRT?VGIKIMPTGV

301?NPKRYLDGFS?WDEFKWRRGE?LEAQFKGMTV?LAGVDDMDSF?KGIDLKLQAF

351?ERLLEYHAEW?RGKVVLVQVT?NPPRSTGHDI?TELHAFVTNL?VASINYKYGN

401?PSTNYVPVHY?LERHVPLHER?MAFYSIADCV?VVTATRDGMN?LVPYEYIVCR

451?QGSDTTPCAE?STVESGPRDS?MLVVSEFVGC?SPSLSGAIRV?NPWSIDSGYN

501?GMYAAICMPL?EHRRLRHQKH?WRYVSQHTVA?FWAQSYLMDL?VRVTRNHVTM

551?KCYGLGLGLD?TFRMVALDAN?FRKMEESQVA?ATYKSSKSRV?FFLDYDGTLT

601?AGQHTSITLA?PLDEVLQVLR?ALTAEPLNKV?VLFSGRPKAE?LQEWFASVPN

651?LALVAENGCY?LRLGEGQTWR?SHLTPGLQVA?DQSWVSLVVA?ADFGWKKMAL

701?PILQQYQEST?DGSSIEAKES?ALVWYYKDAD?PDFGSWQAKE?LLDHLEGVLS

751?NKPVEIVGGS?SAVEIKPQGV?SKGQAVEKML?GQLYGYPAIA?ERRSSSSRPM

801?GRPSSDASER?RPSSSRFSMS?DRGPSAGSAA?VQSAASSVQG?GGAEAAAQQE

851?LAAAGKGPDF?VLCIGDDRSD?EDMFTSIEMM?RASPQMMSSE?VYACTVGQKP

901?SRAPFYLNDP?GEVLHLLARL?VGINLPNLPL

<210>3

<211>22

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

＜223〉primer

<400>3

GTGTGGTACTACAAGGATGCAG 22

<210>4

<211>22

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

＜223〉primer

<400>4

ATGTCCTCATCACTGCGGTCGT 22

<210>5

<211>20

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

＜223〉primer

<400>5

ACGGGCTTATTGGACAGCAC 20

<210>6

<211>30

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

＜223〉primer

<400>6

ATGCCCGGGAATTCTGGTCAGTCCCTTATG 30

<210>7

<211>29

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

＜223〉primer

<400>7

ATGGAGCTCTCGAGGTAGCAATTCCCGAG 29

<210>8

<211>22

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

＜223〉primer

<400>8

GGAGAACGGGGGATTTGAGGGA 22

<210>9

<211>20

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

＜223〉primer

<400>9

TGACAGGAAGTGGCGAGCGT 20

<210>10

<211>24

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

＜223〉primer

<400>10

AGGAAGCTTATGGAGGAGCCATAG 24

<210>11

<211>28

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

＜223〉primer

<400>11

AGGTCTAGATGAGTAAAACAGAGGAGGG 28

<210>12

<211>26

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

＜223〉primer

<400>12

AGGAAGCTTCGTTGACAGAAACAGTC 26

<210>13

<211>25

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

＜223〉primer

<400>13

ATGGGATCCTTCGTGTCTCTGAGAA 25

<210>14

<211>25

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

＜223〉primer

<400>14

ATGGGATCCTTCGTGTCTCTGAGAA 25

Claims (4)

1. a Dunaliella salina TPSP gene is characterized in that this gene has the base sequence shown in the SEQ NO 1.

2. a Dunaliella salina TPSP gene coded protein according to claim 1 is characterized in that having the aminoacid sequence shown in the SEQ NO2.

3. the cloning process of a Dunaliella salina TPSP gene according to claim 1 is characterized in that this method has following steps:

The primer sequence of three contig contig designs a. taking according to EST: P6 (+): 5 '-gtgtggtactacaaggatgcag-3 ', P6 (-): 5 '-atgtcctcatcactgcggtcgt-3 ', in the BAC library of salt algae, screen, obtain the segment of one section about 1.5kb; This section segment is checked order, and compare, learn that this section sequence strides an intron with the cDNA sequence of salt algae; Be forward primer with the P6 (+) that is positioned on the exon again, a design reverse primer P6 (2) on intron: 5 '-acgggcttattggacagcac-3 ', the BAC library of screening salt algae; Obtain four positive colonies, these four positive colonies are used Not I respectively, EcoR I and HindIII carry out enzyme spectrum analysis, first the maximum clone of segment who wherein comprises is chosen do the air gun library;

B. the clone that step a is chosen with Large Construct Kit (QIAGEN) extracting BAC plasmid, carries out the physics fragmentation with the BAC segment that obtains, the about 2～4kb of the segment that obtains; The rubber tapping of the segment of this 2～4kb is reclaimed, and carrier is to make with the pUC18 that Sma I enzyme cuts phosphorus; 2～4kp the segment that reclaims is connected into linearizing pUC18 checks order, sequencing primer is M13 (+) (-), has surveyed nine 96 orifice plates altogether, about 864 clones;

C. 864 above-mentioned clones raw data is imported the Linux program of Phred/Phrap, obtained 5 long contig, its length is respectively: 35945bp, 26502bp, 4913bp, 3076bp, 1407bp; Through the prediction of GENSCAN and FGENSH two fatware, wherein length is the complete sequence that the congtig of 35945bp is DvTPSP cDNA, and this sequence comprises the open reading frame of 3.2kb.

4. the construction process of the plant expression vector of a Dunaliella salina TPSP gene according to claim 1 is characterized in that the concrete steps of this method are:

A. the reconstruction of plasmid pBI121: according to the sequence of salt algae TPSP gene, the positive and negative adopted primer of design DvTPSP.

GUS(+)：5‘-atg cccgggaattctggtcagtcccttatg-3‘

SmaI EcoRI

GUS(-)：5‘-atg gagctctcgaggtagcaattcccgag-3‘

SacI XhoI

TPSP(+)：5‘-ggagaacgggggatttgaggga-3‘

TPSP(-)：5‘-tgacaggaagtggcgagcgt-3‘

Cut, mend flat mode by enzyme and destroy the EcoR I enzyme point behind the NOS terminator among the pBI121, get carrier pBI121 (E ^-); Then, be template with pBI121, with GUS upstream and downstream primer PCR amplification gus gene, the PCR product reclaims the GUS segment, and is cloned into pBI121 (E with Sma I/Sac I double digestion ^-) SmaI/Sac I between, thereby at pBI121 (E ^-) gus gene upstream Sma I enzyme point back and downstream Sac I enzyme point before add EcoR I and Xho I enzyme point respectively, must carrier pBI121 (E ⁺/ X ⁺);

B. the clone of the gene induced type promoter element of Arabidopis thaliana rd29A/rd29B: according to adverse circumstance inducible genes rd29A in the Arabidopis thaliana of delivering on the document and the promoter sequence of rd29B, designed and synthesized two pairs of primers,

Prd29A(+)：5‘-agg aagcttatggaggagccatag-3‘

Hind?III

Prd29A(-)：5‘-agg tctagatgagtaaaacagaggaggg-3‘

Xba?I

Prd29B(+)：5‘-agg aagcttcgttgacagaaacagtc-3‘

Hind?III

Prd29B(-)：5‘-atg ggatccttcgtgtctctgagaa-3‘

BamH?I

By the method for PCR, promoter sequence Prd29A and the pRD29B of from the arabidopsis gene group, increase rd29A and rd29B; PRD29A and pRD29B use HindIII/Xba I and HindIII/BamH I double digestion respectively, reclaim two promotor segments, and are cloned into respectively among the pUC19 between HindIII/Xba I and HindIII/BamH I enzyme point, pUC29A and pUC29B;

C. the structure of plant conversion carrier: Prd29A and Prd29B are cut out from pUC29A and pUC29B respectively, and (the E that is cloned into pBI121 respectively ⁺/ X ⁺) in, replace original CaMV35S promotor, thereby obtain two adverse circumstance inducible plant expression vector pBI29A and pBI29B, then, TPSP is cut out from pBK-TPSP, and (the E that is cloned into pBI121 respectively ⁺/ X ⁺), among pBI29A and the pBI29B, replace original gus gene, thereby obtained three respectively by plant expression vector pBI35S-TPSP, pBI29A-TPSP and the pBI29B-TPSP of the salt algae TPSP gene of CaMV35S promotor, rd29A and rd29B control.

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