CN102286494B - Porphyra yezoensis ueda TPS (trehalose-6-phosphate synthase) gene and application thereof in enhancing salt tolerance of rice - Google Patents

Abstract

The invention discloses a PyTPS (porphyra yezoensis ueda trehalose-6-phosphate synthase) gene which is cloned to construct a plant expression vector of the gene, and the plant expression vector is transferred into the agrobactrium tumefaciens strain, so that an engineering strain is obtained. A rice variety is transformed by utilizing agrobacterium-mediated transformation, so that genetically modified rice is obtained; after growing and acclimatization, a T0 individual plant is obtained, and a T1 seed is collected; a seedling germinated from the T1-generation genetically modified seed receives PCR (polymerase chain reaction) detection and a salt tolerance assay, so that a T1-generation genetically modified salt-resistant strain is obtained, a T2 generation receives a kanamycin resistance assay, PCR detection and Southern hybridization, and results indicate that the PyTPS gene is already integrated into the genome of the genetically modified strain. The PyTPS gene in the invention has a TPS domain as well as a TPP (thiamine pyrophosphate) domain; the PyTPS gene source is safe; the development of the plant morphology of the PyTPS genetically modified rice is normal; the PyTPS gene can be stably inherited in genetically modified plants; and moreover, the transformation system is highly effective.

Description

Yezoensis laver TPS gene and the application in improving the paddy rice salt tolerance thereof

Technical field

The present invention relates to biological technical field, relate in particular to yezoensis laver TPS gene and the application in improving the paddy rice salt tolerance thereof.

Background technology

Paddy rice is second largest in the world food crop, and rice is the staple food of making an appointment with half population in the world, is first food crop in China paddy rice.To the salt tolerant characteristics of paddy rice had basic elaboration ([1] Hu Shikai, Tao Hongjian, before the money, etc. the heredity of paddy rice salt tolerance and the progress of molecular breeding. Molecular Plant Breeding, 2010,8 (4): 629-640; [2] Zhang Suhong, Liu Lixin, Liu Zhongzhuo. research of paddy rice salt tolerant and breeding progress. rice in north china, 2009,39 (3): 118-121; [3] Qi Dongling, the writing brush dragon is planted, Zhang San unit. paddy rice salt tolerant/alkaline characterization and evaluation method. and plant genetic resources journal, 2005,6 (2): 226-231.).The salt tolerant characteristics of paddy rice are: 1) paddy rice is developed by the pantano plant, and its salt tolerance is relatively poor, and is relatively more responsive to salt stress; 2) paddy rice is the salt stress critical porion in seedling stage and reproductive stage; 3) different varieties salt tolerance difference is very big, and the kind that salt tolerance is high lacks very much.In fact the soil salinization is the main factor that restriction paddy rice cultivated area enlarges, and salt stress also is the most important environmental factor that causes the paddy rice underproduction in addition.Therefore, excavating resistant gene of salt, cultivating the salt water resistance rice varieties is the important channel that enlarges China's rice cropping area, increases rice yield, to improving China's grain yield, ensureing that grain security has positive meaning.

Trehalose has the effect that salt, arid etc. are coerced the protection biomass cells of resisting, and the trehalose synthesis related gene is imported recipient plant improve the focus that resistance such as its salt tolerant, drought resisting are plant genetic engineering research.Research is illustrated in intestinal bacteria, yeast and the higher plant, and the synthetic of trehalose accomplished by 6-phosphotrehalose UDP-transglucosylase synthetic enzyme (TPS) and 6-phosphotrehalose UDP-transglucosylase esterase (TPP) catalysis successively respectively.TPS or TPP gene are imported recipient plant separately can improve resistance such as its salt tolerant, the odd-shaped phenomenon occurs, do not meet actual needs but often follow transgenic plant to grow undesired, plant.

2002, American scholar Garg etc. merged the gene (otsA) of coding TPS enzyme in the intestinal bacteria and the gene (otsB) of coding TPP enzyme, and fusion gene (TPSP) is imported rice varieties; In tissue specificity expression promoter and down expression of the driving of coercing corresponding promotor; Obtained the transgenic paddy rice that salt tolerance improves respectively, the content of trehalose of transfer-gen plant can improve 3-10 doubly, and the performance of transgenic paddy rice seedling is good in the NaCl of 100mM solution; But not transfer-gen plant shows tangible salt damage characteristic ([4] GargA K; Kim J K, Owens T G, et al.Trehalose accumulation in rice plants confers hightolerance levels to different abiotic stresses [J] .Proc.Natl.Acad.Sci.; 2002,99:15898-15903.).2003; Korea S scholar Jang etc. adopt the similar techniques approach; The fusion gene that otsA and otsB are formed import paddy rice also obtained morphological development normal, to transgenic rice plant ([5] Jang I C of the NaCl performance resistance of 100mM; Oh S J; Seo J S; Et al.Expression of abifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate synthaseand trehalose-6-phosphate phosphatase in transgenic rice plants increases trehaloseaccumulation and abiotic stress tolerance without stunting growth [J] .Plant Physiol., 2003,131:516-524.).But the natural gene that does not also have TPS and TPP structural domain at present simultaneously transforms the report of plant.

Summary of the invention

Transform the situation of plant to paddy rice salt tolerance difference in the prior art and the natural gene that do not have TPS and TPP structural domain simultaneously; The invention provides yezoensis laver TPS gene and the application in improving the paddy rice salt tolerance thereof; The existing TPS structural domain of said TPS gene has the TPP structural domain again, is a natural geminus territory TPSP gene; The TPS gene is changed in the paddy rice, can significantly improve the salt tolerance of paddy rice.

For realizing the foregoing invention purpose, the present invention adopts following technical proposals to be achieved:

The invention provides yezoensis laver TPS gene, its sequence table is shown in SEQ ID No:1.

Said TPS gene has the TPS structural domain at 225-1650 base place, and there is the TPP structural domain at the place in the 1800-2475 base.

The primer of cloning said TPS gene has three pairs, and its title and sequence are following:

First pair of primer is TPSR1 (GACTCATATGACCCCCGGGCCTATCACTA) and 3Kpn I (CATGATGCTGTACAGCGCAAG), respectively should with this gene 1-22 base and 1339-1359 base pair;

Second pair of primer is Tre1 (CTACGCGCGTCACTTTCTCTC) and TPSa2 (CACTCCTTCGAATTCTTCTTG), respectively should with this gene 861-88 base 1 and 2034-2054 base pair;

The 3rd pair of primer is TPSb1 (CAAGAAGAATTCGAAGGAGTG) and TPSb2 (GACTAAGCTTCTACTGGCTCGGCAACGAGGAC), respectively should with this gene 2034-2054 and base 2706-2727 base pair.

The present invention also provides the plant expression vector of described yezoensis laver TPS gene, and further, said plant vector is pCAMBIA2300-PyTPS.

The present invention also provides the application of said yezoensis laver TPS gene in improving the paddy rice salt tolerance.

Further improvement to technical scheme: change yezoensis laver TPS gene over to paddy rice, the primer that the paddy rice transfer-gen plant is carried out the PCR detection is:

P1：5′-CTACGCGCGTCACTTTCTCTC-3′，

P2：5′-CATGATGCTGTACAGCGCAAG-3′；

Amplify one section sequence of the PyTPS gene of 499bp with primer P1 and P2, its sequence table is shown in SEQID No:2.

Further improvement to technical scheme: yezoensis laver TPS gene is changed over to paddy rice, and the primer that the paddy rice transfer-gen plant is carried out the PCR detection is:

P3：5′-AGGGAGGACGCACAATCCCACTAT-3′，

P4：5′-GCACGCAAGCGGAGAGAAAGTGACG-3′；

Amplify～one section sequence of 1000bp with primer P3 and P4, it includes the sequence of the PyTPS gene of the 892bp shown in SEQ ID No:3.

Further improvement to technical scheme: the reaction system of the paddy rice transfer-gen plant being carried out the PCR detection is 25 μ L; The dNTPs 1 μ L of 2.5mmol/L wherein; Concentration is each 1 μ L of two kinds of primers of upstream and downstream of 10 μ mol/L; 10 * PCR buffer, 2.5 μ L, template DNA 1 μ L, the Taq polysaccharase 0.2 μ L of 5U/ μ L;

The PCR response procedures is: 94 ℃ of sex change 4min; 94 ℃ of sex change 1min, 59 ℃ of annealing 1min, 72 ℃ are extended 1min; Last 72 ℃ are extended 10min; 4 ℃ of insulations, totally 35 circulations.

Further improvement to technical scheme: the anti-salt concn of paddy rice transfer-gen plant is 5 ‰-8 ‰.

Compared with prior art, advantage of the present invention and positively effect are:

1, the present invention has cloned TPS gene (PyTPS) from large edible marine alga yezoensis laver, and sequencing result shows the existing TPS structural domain of this gene, and the TPP structural domain is arranged again, is a natural geminus territory TPSP gene; And the TPSP gene of available technology adopting all is the fusion gene that forms through vitro recombination.

2, with the TPS gene transformation paddy rice of yezoensis laver, this TPS gene is cultured edible algae from producing, and the source of goal gene is safe, and its security is higher than the goal gene that domestic and international similar institute adopts far away.

3, the morphological development of TPS transgenic rice plant is normal, and anti-0.8% NaCl of transgenic paddy rice seedling coerces; Compare in the existing research only coercing of the NaCl of anti-100mM (0.58% NaCl) of transgenic paddy rice, the present invention can significantly improve its salt tolerance with the TPS transgenic applications in paddy rice.

4, the TPS gene in the transgenic paddy rice has been carried out the tracking in continuous 6 generations (T6) and detected, carried out hereditary transmission and inheritance stability Journal of Sex Research, the result shows the heredity stably in transfer-gen plant of TPS gene, and the result has stronger safety.The result in T2 generation and the result in T4 generation have only been reported in the prior art.

5, transformation system is very effective.Can obtain a large amount of transfer-gen plants in the short period of time, this is to obtain a large amount of transgenic seedlings and therefrom filter out the obviously important prerequisite of the transfer-gen plant of raising of salt tolerance.

After advantages embodiment of the present invention, other characteristics of the present invention and advantage will become clearer.

Description of drawings

Fig. 1 is the structure iron of pCAMBIA2300-PyTPS among the present invention.

Fig. 2 is indefinite bud and the plant that the callus differentiation obtains among the present invention, 1.G418 kanamycin-resistant callus tissue and indefinite bud; 2. regeneration plant.

Fig. 3 be among the present invention the T1 transfer-gen plant through 5 ‰ NaCl the result after handling, except contrast TP309, all the other all are the T1 transfer-gen plants among the figure.

Fig. 4 carries out the anti-salt gradient of 3-12 ‰ NaCl to T1 transgenic line H191 among the present invention to measure, and each handles used NaCl concentration: 1. water (contrast); 2.3 ‰; 3.6 ‰; 4.8 ‰; 5.10 ‰; 6.12 ‰..

Fig. 5 is that the kalamycin resistance of T2 transgenic line among the present invention is identified, 1. contrast TP309; 2. kalamycin resistance heterozygosis transgenic line; 3. the kalamycin resistance transgenic line that isozygotys.

Fig. 6 is the PCR detected result of T2 transgenic line among the present invention, and the A. primer is P1 and P2; B. primer is P3 and P4; C. primer is NPTII-F and NPTII-R, among the figure: M.DNA markerD2000; Pcr template DNA is: 1-4. transgenic line H155; 5-8. transgenic line H199; 9-12. transgenic line Y308; 13. contrast TP309; 14.pCAMBIA2300-PyTPS plasmid; 15. contrast (water).

Fig. 7 is the Southern results of hybridization of T2 transgenic line among the present invention, M. λ DNA/HindIII; 1-2.H155; 3-4.H191; 5-6.Y308; 7-8.TP309.

Fig. 8 is that the RT-PCR of T2 transgenic line among the present invention analyzes A.PyTPS; B.OsActinl, 1.TP309 is through water treatment; 2.TP309 NaCl solution-treated through 8 ‰; 3.H155 through water treatment; 4.H155 NaCl solution-treated through 8 ‰; 5.H191 through water treatment; 6.H191 NaCl solution-treated through 8 ‰; 7.Y308 through water treatment; 8.Y308 NaCl solution-treated through 8 ‰.

Embodiment

Below in conjunction with accompanying drawing and embodiment technical scheme of the present invention is done further detailed explanation.

Embodiment 1

One, experiment material

1. gene, bacterial classification and rice varieties

The present invention has cloned yezoensis laver 6-phosphotrehalose UDP-transglucosylase synthetic enzyme (TPS) gene (PyTPS); Bacillus coli DH 5 alpha, agrobacterium tumefaciens strains A GL1 are preserved by laboratory, Qingdao Agricultural University genetic research chamber, and genetically modified acceptor material is paddy rice (Oryza sativa L.) kind " Taibei 309 " (TP309) (Hunan Research Centre for Hybrid Rice provides).

2. plant culture

The substratum that adopts in the rice conversion has six kinds:

1) callus induction substratum: MS+2,4-D 2mg/L+CH 0.3g/L+ proline(Pro) 2.8g/L+ sucrose 45g/L+phytage 3.0g/L, pH5.8;

2) callus subculture medium: MS+2,4-D 2mg/L+CH 0.3g/L+ proline(Pro) 0.5g/L+ sucrose 30g/L+phytage 3.0g/L, pH 5.8;

3) be total to culture medium: CC+AS 20mg/L+CH 1g/L+ proline(Pro) 0.5g/L+KT0.8mg/L+NAA 0.2mg/L+ SANMALT-S 25mg/L+phytage 3.0g/L, pH 5.2;

4) screening culture medium: CC+2,4-D 2mg/L+ SANMALT-S 20mg/L+ N.F,USP MANNITOL 36.43mg/L+Cef400mg/L+G418100-150mg/L+phytage 3.0g/L, pH 5.8;

5) division culture medium: MS+NAA 0.2mg/L+TDZ 1mg/L+KT 1.6mg/L+CH 1g/L+ sorbyl alcohol 30g/L+ sucrose 30g/L+phytage 3.8g/L, pH 5.8;

6) strong plantlets and rootage substratum: 1/2MS+MET 2mg/L+ sucrose 20g/L+phytage 3.0g/L, pH 5.8.

Two, experimental technique

The present invention includes following concrete experimental procedure:

1.PyTPS the structure of gene plant expression vector

(1) amplification PyTPS gene

Yezoensis laver (Porphyra yezoensis) is provided by the Institute of Oceanology of the Chinese Academy of Sciences, increases, has cloned the PyTPS gene through RACE-PCR and RT-PCR, has obtained recombinant plasmid pET22b-PyTPS.When making up plant expression vector, be that template is carried out PCR acquisition PyTPS full-length gene with recombinant plasmid pET22b-PyTPS.Adopt P5 and P6 primer during PCR.

P5： TCTAGAATGACCCCCGGGCCTATCACTA(Xba?I)，(SEQ?ID?No：10)；

P6： GTCGACCTACTGGCTCGGCAACGAGGAC(Sal?I)，(SEQ?ID?No：11)。

PyTPS full length gene 2727bp, order-checking shows that the sequence of this gene is (SEQ ID No:1):

ATGACCCCCGGGCCTATCACTACCGATGCCGCTGCTGGTGTTGGCGTGGA

CACGATGGACGGCACGATGAAGTGGGAGCTCTTTGAGGAGAACCGCACC

CTTACTACCCGCTCCGGCCAAATGATTGTGAACGACGGGGAGTTTGGGAA

GTGGAATGATACTGATGAGGGGCGTGTCAAGTGCTTTCTGACGCCGGACG

AAGGCTTTGGTGACGCCATCGAGAGCCTAGTCATTGTGCTGTACCGACTG

CCTATCATTGCGAAGCGCGACGCGACCACGGGAGCTTGGAGCTTCAAATG

GGACGATGACGCCCTCTACCTAACTTCCACCGGTTTGCGGAAGGGCTTGG

AGCAGCTTAAGGTGGCACCTCTCTGGGTGGGCATATTGAACTCCGACGAC

GAGGTGCCCCGGCAAGAGCGTGAAGGGGTCGCCGACCGGCTGTTGGAG

GAGTTCAACTGCGTTCCCGTCTTCATCCCGCACGACACGCTGAAGCAGTT

ATACCAGGGCTTCTGCAAAGGCACCCTCTGGCCGCTTTTTCACATGGTGT

CGTCGGCGACAGACCACACGGAACACACTACCCGCTTTGACGACCGCCT

TTGGCGTGTTTACATGAACGTCAACCGGATGTTCCGGGACAAGGTGGTGG

AGGTGTACGATGGGGACCGAGCACTCATTTGGGTACACGACTACCACCTG

ATGCTTTTGCCGCAGGCATCGCGCTCGCGCCTGTCAGGTGTGAAGATTGG

CTTCTTTCTCCATATTCCGTGGCCTTCGTCAGAAGTGTACCGCGTGCTGCC

GTGGCGGAACGAACTGCTGAAGGGCATGCTTTCCGCGACGTTGCTGGGC

TTCCATCTTTTCGACTACGCGCGTCACTTTCTCTCCGCTTGCGTGCGGCTG

CTTAACCTGGAGCACGAGGCGAACAGGGGCTCCCTCGGTCTGGAGTACG

ACGGTCGCCACGTTATGCTGCGCGTGAGTCACATTGGCGTGGACCCAGAG

CGCTTTTCCGAGGGCCTGAGCGCGCCGTCGCTGACGGACCGGGTTGCCG

AGTTCAAGGAGCGGTTTGCGGATTGCACAGTCCTCGGTGCCGTGGACGA

CCTTGACCTCATCAAGGGCATTGCCCTCAAGCTGATGGGTTTCCAGCGGT

ATCTCGATTCCGCCCCCAAGATGCGAGGAAAGGTCGTACTTGTGCAAGTT

GCCATCCCAAAGGCGGCCCGCGTCAAGGAGTCTGTGCGTAACGAGATTC

GGGAGCTGGTGGCTGCCATCAACAACAAACACGGCGATGGGTCGGGGCG

GCGACCCGTATGGTACCTGGAAGAGAGCATCTCCTTTGAAAGCCGCCTTG

CGCTGTACAGCATCATGGATGCACTGGTGGTGACCCCCATCCGTGACGGC

CTCAACCTCATCCCATACGAGTACATTGTATCGACGTCCGAGGGGAAGGG

CCAGCTAATTCTTTCGGAGTTTACTGGCTGCTCGCGGGCGCTCTCATCTGC

GGAACGTGTCAACCCGTGGGACTTGGAGAAGCTGTCCAACGTCCTTGAC

ATGGTCGTTCAGAAGGCCATTGCCGCGGCGCCCGAGGTGGAGCTGAAGC

GTCGGGCGGACAAGGCCTACGTGTCGGCCCACTCGTCCCAGCAGTGGGC

GCAGTCGTTTTTGCATGACTTGAAGGAGGCGTCAGAGCCGGCACAGGTC

GTTGTCAAGGTGGGTCCGCTGGCCGGCTTGCCTGGTGTACTGGCCTACGA

CGAGTTCACCCTTCTCAACCGCTCGGCGGTTCTGCGCGCCTACAAGGCCG

CCAAGAGGCGCTTGTTCCTGTTTGACTATGATGGGACGCTGACCTCCATC

ACCGATCAGTCGTCCCAGATGGCCCACGCGTGGGCGCGGCCGAGCGAGG

CTGTGGCCGCCAACTTGGACACTTTGTCAAAGGACCCGCTCAACGATGTC

TACATCATGTCTGGCCGCAAGACTGAGGTGCTTGAAGCCGGCCTCAACAA

CTCCCCCACGATTGGGATTGCAGCTGAGCACGGCTTCTTCTACCGCAAGA

AGAATTCGAAGGAGTGGAACAGGCTGTTGGAGGATGCGGACCTGTCATG

GATGGAGTTGGCATTGCGTATCATGCTTATGTACACGGACCGTACGGACGG

ATCCTACGTGGAGCAGAAGAAGGCTGGCCTCGTGTGGCACTACCTGGAC

GCGGACCGGGAATTTGGCTCGTGGCAGGCGAAGGAGATGCGTGACCATC

TTGAGTCGCTGCTTTCTCCCTTCTCTGTGCAGGTGGTCACAGGCTATGGAT

GGCTTCAGGTCCGGATGGCTGCGATGAACAAGGGCGTTATGGTCGAGAC

GATTCTTCGCGATATGCCCGAGCCCCCGGACTTTGTGCTCTGCTGTGGCGA

CGACCGCACGGATGAGGACATGTTTGCGTATCTGGACACTCATCTGGACC

CTTCTGTGAAGCAGTTCACTTGCACAGTGGGCGTCAAGCCGAGCCACGC

GCGCTACTACCTGCACTCATCCAATGAGGTGGGCGCGCTTCTGGAGACGC

TGGTAACTGGAAGCTACCCGCGCGGCGTGCGCCCTCGCCCGCGTGGTGG

TTCTGTGTCCCTTGCGGACCTTGTACCCGACGACGAAGGCTCCTCCAGCA

CACCGGCGGTGCCGCAGAGGACCAACGGACCTCGGACAAGCGCGTCGG

GTCAGAAGAGACGAGGTCTGGCGTCCTCGTTGCCGAGCCAGTAG

(2) PyTPS gene and cloning vector pMD18-T and plant expression vector pCAMBIA2300-35S-OCS's is connected

Reclaim the PCR product, and under the effect of T4 dna ligase, be connected, connect the bacterium colony that product transformed into escherichia coli DH5 α has obtained anti-penbritin with carrier pMD18-T (purchase) in TaKaRa.Extract recombinant plasmid; Carry out double digestion with Sal I and Xba I; Recovery contains the endonuclease bamhi of PyTPS gene, and is cloned in the corresponding site of plant expression vector pCAMBIA2300-35S-OCS (available from Australian Cambia company), obtains the plant expression vector pCAMBIA2300-35S-OCS-PyTPS of this gene; Abbreviate pCAMBIA2300-PyTPS as, its structure iron is as shown in Figure 1.

2, the conversion of agrobacterium tumefaciens

When importing Agrobacterium AGL1, adopts pCAMBIA2300-PyTPS the liquid nitrogen flash freezer method.

3, the conversion of rice varieties TP309

3.1 inducing of paddy rice mature embryo callus

After the paddy rice mature seed removes clever shell, on super clean bench,, carry out surface sterilization 30min with 20% chlorine bleach liquor then with 70% ethanol disinfection, 1～2min; Rinsed with sterile water 3～4 times is placed on seed on the aseptic filter paper behind the suck dry moisture again, is placed on the substratum of callus induction; 30～40 seeds of every ware behind 26 ℃ of about 7～10d of dark cultivation, are removed the bud of sprouting; Peel the callus that the mature embryo scultellum grows, change the callus subculture medium over to, succeeding transfer culture is after one week under the same conditions; Compactness is good after selecting succeeding transfer culture, and the yellowish callus of color and luster is used for Agrobacterium-mediated Transformation.

3.2 the cultivation of Agrobacterium

Agrobacterium AGL1 (pCAMBIA2300-PyTPS) rules containing on the YEB flat board of 50mg/L kantlex, cultivates 48-72h for 28 ℃.Choose single colony inoculation in the YEB liquid nutrient medium that contains kantlex and each 50mg/L of Rifampin, in 28 ℃, 200rpm cultivates 24h, more than OD600=0.5, and centrifugal and collection agrobatcerium cell.After diluting above-mentioned agrobatcerium cell with 1ml AAM+AS (Syringylethanone, its final concentration are 20mg/L) substratum, be added in the AAM substratum of 100ml, in 28 ℃, 100rpm cultivates 2h.

3.3 the genetic transformation of agroinfection and paddy rice

The callus of succeeding transfer culture after one week put into aseptic triangular flask, add an amount of above-mentioned During Agrobacterium liquid, in 28 ℃, 80-100rpm cultivated 20 minutes.Outwell dip-dyeing solution, take out callus, on aseptic filter paper, inhale and remove unnecessary bacterium liquid; Be placed on the super clean bench and blow (about 2-3 hour), transfer to the solid that is covered with one deck aseptic filter paper and be total on the substratum, callus should be put neatly; Do not stack 28 ℃ of dark 2～3d that cultivate each other.

3.4 the screening of G418 kanamycin-resistant callus tissue and differentiation

Callus after cultivating altogether is seeded in screening culture medium (CC+2; 4-D 2mg/L+ SANMALT-S 20mg/L+ N.F,USP MANNITOL 36.43mg/L+Cef 400mg/L+G418 100mg/L+phytage 3.0g/LpH 5.8) on; Behind 28 ℃ of dark cultivation 20～25d; Change a sieve callus over to two sieve substratum (CC+2; 4-D 2mg/L+ SANMALT-S 20mg/L+ N.F,USP MANNITOL 36.43mg/L+Cef 400mg/L+G418150mg/L+phytage 3.0g/L pH 5.8), most of callus is brownization of beginning about screening 10d, chooses the milky resistant calli that grows again then at the edge of brownization tissue.

Good resistant calli goes to 26 ℃ from the resistant calli that after the screening of 2-3 wheel, grows, to select milk yellow compactness, on the division culture medium under the 16h/d illumination condition, generally passes through about 20d, has green point to occur, and further differentiates budlet behind the 30-40d.

3.5 plant regeneration, south numerous added-generation

When budlet grows to about 1cm; Move on on the root media, cultivate about two weeks, select high about 6-8cm; The seedling of well developed root system detects with the PCR that (P1 and P2) and (P3 and P4) two pairs of primers carry out the PyTPS gene by strain, obtains T0 for transfer-gen plant 793 strains (as shown in Figure 2).At 26 ℃, the 16h/d illumination condition was practiced seedling 2-3 days down with transfer-gen plant.With warm water flush away substratum, transplanting is buried, and all is transplanted in the greenhouse, when plant grows to 16-18cm, has selected 100 healthy and strong T0 individual plants, has planted the base, Hainan Island then, and south numerous added-generation has the work of 95 strains one-tenth and at the beginning of second, received seed (T1).

4. the PCR of transfer-gen plant detects

Each of transgenic paddy rice all must use from generation to generation the PCR of the PyTPS gene that (P1 and P2) and (P3 and P4) two pairs of primers import to detect evaluation.

The primer that transgenic rice plant is carried out the PCR detection has 3 pairs.

First pair of primer:

P1 (like SEQ ID No:4): (5 '-CTACGCGCGTCACTTTCTCTC-3 '),

P2 (like SEQ ID No:5): (5 '-CATGATGCTGTACAGCGCAAG-3 ');

The first couple of primer P1 and P2, amplified production be～one section sequence of the PyTPS gene of 500bp, and its sequence table (like SEQ ID No:2) as follows:

CTACGCGCGTCACTTTCTCTCCGCTTGCGTGCGGCTGCTTAACCTGGAG

CACGAGGCGAACAGGGGCTCCCTCGGTCTGGAGTACGACGGTCGCCACGTT

ATGCTGCGCGTGAGTCACATTGGCGTGGACCCAGAGCGCTTTTCCGAGGGCC

TGAGCGCGCCGTCGCTGACGGACCGGGTTGCCGAGTTCAAGGAGCGGTTTG

CGGATTGCACAGTCCTCGGTGCCGTGGACGACCTTGACCTCATCAAGGGCAT

TGCCCTCAAGCTGATGGGTTTCCAGCGGTATCTCGATTCCGCCCCCAAGATG

CGAGGAAAGGTCGTACTTGTGCAAGTTGCCATCCCAAAGGCGGCCCGCGTC

AAGGAGTCTGTGCGTAACGAGATTCGGGAGCTGGTGGCTGCCATCAACAAC

AAACACGGCGATGGGTCGGGGCGGCGACCCGTATGGTACCTGGAAGAGAGC

ATCTCCTTTGAAAGCCGCCTTGCGCTGTACAGCATCATG。

Second pair of primer:

P3 (like SEQ ID No:6): (5 '-AGGGAGGACGCACAATCCCACTAT-3 ');

P4 (like SEQ ID No:7): (5 '-GCACGCAAGCGGAGAGAAAGTGACG-3 ');

The second couple of primer P3 and P4 are according to upstream region of gene 35S promoter and the design of PyTPS gene order; Amplified production is～fragment of 1000bp; This fragment comprises the sequence of PyTPS gene of carrier sequence and the 892bp of about 200bp, the sequence of the PyTPS gene of 892bp (like SEQ ID No:3) as follows:

ATGACCCCCGGGCCTATCACTACCGATGCCGCTGCTGGTGTTGGCGTG

GACACGATGGACGGCACGATGAAGTGGGAGCTCTTTGAGGAGAACCGCACC

CTTACTACCCGCTCCGGCCAAATGATTGTGAACGACGGGGAGTTTGGGAAG

TGGAATGATACTGATGAGGGGCGTGTCAAGTGCTTTCTGACGCCGGACGAA

GGCTTTGGTGACGCCATCGAGAGCCTAGTCATTGTGCTGTACCGACTGCCTA

TCATTGCGAAGCGCGACGCGACCACGGGAGCTTGGAGCTTCAAATGGGACG

ATGACGCCCTCTACCTAACTTCCACCGGTTTGCGGAAGGGCTTGGAGCAGCT

TAAGGTGGCACCTCTCTGGGTGGGCATATTGAACTCCGACGACGAGGTGCC

CCGGCAAGAGCGTGAAGGGGTCGCCGACCGGCTGTTGGAGGAGTTCAACTG

CGTTCCCGTCTTCATCCCGCACGACACGCTGAAGCAGTTATACCAGGGCTTC

TGCAAAGGCACCCTCTGGCCGCTTTTTCACATGGTGTCGTCGGCGACAGACC

ACACGGAACACACTACCCGCTTTGACGACCGCCTTTGGCGTGTTTACATGAA

CGTCAACCGGATGTTCCGGGACAAGGTGGTGGAGGTGTACGATGGGGACCG

AGCACTCATTTGGGTACACGACTACCACCTGATGCTTTTGCCGCAGGCATCG

CGCTCGCGCCTGTCAGGTGTGAAGATTGGCTTCTTTCTCCATATTCCGTGGC

CTTCGTCAGAAGTGTACCGCGTGCTGCCGTGGCGGAACGAACTGCTGAAGG

GCATGCTTTCCGCGACGTTGCTGGGCTTCCATCTTTTCGACTACGCGCGTC

ACTTTCTCTCCGCTTGCGTGC。

The 3rd pair of primer:

NPT II-F (like SEQ ID No:8): (5 '-TCCGGTGCCCTGAATGAACT-3 ');

NPT II-R (like SEQ ID No:9): (5 '-GGCGATACCGTAAAGCACGA-3 ').

The 3rd couple of primer NPT II-F and NPT II-R, amplified production be～the carrier pCAMBIA2300 of 580bp goes up the fragment of kalamycin resistance gene.

4.1 the extraction of the total DNA of rice leaf

Get about blade 0.2g, in the EP of 1.5mL pipe, add liquid nitrogen grinding, add extraction buffer (the 100mM Tris-HCl pH8.0 of 500 μ L65 ℃ preheatings then; 20mM EDTA; 500mM NaCl, 1.5% SDS), 65 ℃ of water-bath 30min (otherwise time rock gently).Add 200 μ L 5M Potassium ethanoates, ice bath 30min adds 500 μ L chloroforms then, gently behind the mixing, and ice bath 10min.9, the centrifugal 10min of 000rpm gets supernatant, adds the precooling Virahol of 0.6 times of volume, and mixing is placed 30min for-20 ℃, and 12, the centrifugal 10min deposit D of 000rpm NA, and wash twice with 70% ethanol, be dissolved in after drying up in the TE damping fluid of 50 μ L pH7.5.

When needing more a large amount of DNA (for example Southern hybridization analysis), available aforesaid method extracts in big centrifuge tube.

4.2 PCR reaction

PCR reaction TV is 25 μ L, dNTPs (2.5mmol/L) 1 μ L wherein, each 1 μ L of two kinds of primers of upstream and downstream (10 μ mol/L), 10 * PCR buffer, 2.5 μ L, template DNA 1 μ L (0.1-0.2 μ g), Taq polysaccharase (5U/ μ L) 0.2 μ L.The PCR response procedures is: 94 ℃ of sex change 4min; Next 35 circulations are: 94 ℃ of sex change 1min, and 59 ℃ of annealing 1min, 72 ℃ are extended 1min; Last 72 ℃ are extended 10min:4 ℃ of insulation.

5.T1 for the transgenic line selection of salt tolerance

Respectively get 50 from T1 for each strain system of seed, carry out vernalization and handle acquisition T1 for seedling strong sprout.Treatment process is: vernalization 3 days under 37 ℃ of conditions earlier; Carry out strong sprout after having gone out bud,, cultivated 12 days under the condition of 14 hours illumination/10 hour dark at 24 ℃.With the T1 that obtains for seedling earlier with the NaCl solution-treated of 3-5 ‰ 13 days, every NaCl solution that changed one time at a distance from 2 days, the concentration of maintenance NaCl solution is contrast with not genetically modified TP309 when carrying out selection of salt tolerance.Obtained anti-5 ‰ NaCl and 78 of plant of PCR male T1 (as shown in Figure 3).These 78 T1 are planted for the transgenic strain under normal operation, expand numerously, form strain system, gather in the crops seed (T2) respectively, preserve subsequent use.

To top these 78 transgenic lines that just filter out anti-5 ‰ NaCl; Other gets T1 and carries out further salt tolerant gradient with the NaCl solution of 6-12 ‰ for 50 in seed again by the primary dcreening operation same procedure and measure, though there are 5 strains to tie up to that seedling height has reduction slightly under 8 ‰ the NaCl solution-treated, it is normal basically to grow; The concentration of NaCl is high again; Plant is injured seriously (as shown in Figure 4), and is difficult to after the rehydration restore normal growth, so we think that the anti-salt concn of these 5 transgenic lines is 8 ‰.Strain system to the minority high resistance salt that filters out further identifies.

6. to the T2 of high resistance salt further evaluation for transgenic line

To the process selection of salt tolerance, the minority T2 of the high resistance salt that obtains at last further once identifies for transgenic line.The anti-salt T2 transgenic line that top screening is obtained has carried out kalamycin resistance, PCR, Southern and RT-PCR analysis

6.1 kalamycin resistance is measured

To high resistance salt and the positive T2 transgenic line of PyTPS gene PCR detected result that obtains; Respectively get 60 in its seed; Presoaking and germinating 72h in 37 ℃ of incubators places petridish (12h illumination: 12h the is dark) cultivation in 28 ℃ of greenhouses that contains aqua sterilisa then.When treating that bud grows to about 1cm left and right sides, add kantlex (reaching concentration is 100mg/L) seedling is carried out the evaluation of kalamycin resistance performance, and be contrast with not genetically modified TP309.

With the 100mg/L kantlex 5 T2 are handled for the transgenic paddy rice seedling; Changed not obvious in preceding four days; And the middle part of time contrast and non-resistance seedling stem obviously bleaches to 5d, and the resistance seedling still keeps green, stops processing during to 7d; And use aqua sterilisa instead and cultivate, observations behind the continuation cultivation 5d.5 T2 are green for all seedling that three strain systems (H155, H191 and Y308) are arranged in the transgenic line all, show as the resistance of isozygotying (as shown in Figure 5); Two other strain system (H759 and Y371) adularescent plant occurs, and show as the heterozygosis resistance, and all seedling of contrast TP309 all becomes white, do not have resistance.

6.2 PCR detects

The field is transplanted in the strain system that measures kalamycin resistance the pure and mild resistance of performance; After growing to the completion of branch evil; Get blade and extract the genomic dna of transgenic line and not genetically modified contrast strain system by front 4.1 said methods; Be dissolved among the TE of pH7.5, be used for PCR and detect and the Southern hybridization analysis.

The isozygoty strain system of resistance of kantlex performance is carried out comprehensive PCR detection to three that obtain with three pairs of PCR primers that preceding flooring intermediary continues to PyTPS and the NPTII gene that imports; The result shows that all primers have all amplified the product of expection in these three strain systems, and promptly these three T2 transgenic lines all contain the goal gene PyTPS and the microbiotic selection gene NPTII (as shown in Figure 6) of importing.

6.3 Southern hybridization analysis

When carrying out the Southern hybridization analysis.Every kind of DNA sample 10 μ g that take a sample cut with 37 ℃ of enzymes of restriction enzyme EcoRI and to spend the night.Behind the complete degestion, under the voltage of 50V, run and carried out electrophoretic separation in 0.8% sepharose 10-12 hour.Behind the electrophoresis with gel with 0.25M HCl depurination 10min, sex change 30min in 0.4M NaOH solution, with the capillary transfer method with DNA go to Hybond N+ nylon membrane (AmershamPharmaria Biothech, USA) on, be used for Southern hybridization.One section pcr amplification product that comprises about 500bp of plasmid top 35S promoter and part target gene sequences, adopt random priming with α-32P-dCTP mark after, be used as probe.65 ℃ of incubation 20h carry out the Southern hybridization analysis.With 2 * SSC/0.1% SDS, 1 * SSC/0.1% SDS and 0.5 * SSC/0.1% SDS film washing liquid, the rinsing of under 65 ℃ condition, vibrating successively.After washing film and finishing, nylon membrane is blotted with filter paper, the preservative film parcel, (Amersham Pharmacia Biotech carries out radioautograph in USA) to place the phosphorus screen.

Top 3 T2 transgenic lines that obtain are carried out the Southern hybridization analysis; The result demonstrates among the contrast TP309 does not all have hybridization signal; And all transfer-gen plants all have hybridization signal, and H155 has two hybrid belts, and H191 and Y308 have only a hybrid belt; This shows that foreign gene has two copies in H155, have only a copy in H191 and Y308.The Southern results of hybridization shows that the PyTPS gene has been incorporated in the genome of these 3 transgenic lines (as shown in Figure 7).

6.4 RT-PCR analyzes

Detect the seed and the not genetically modified contrast seed germination of the minority T2 transgenic line that filters out through front each item; Seedling is coerced processing sampling after 12 hours with 8 ‰ NaCl solution; With reference to Zhang etc. (Plant Physiol, 2004, method 134:1500-1513) is extracted total RNA.Get 2 μ g and carry out reverse transcription through total RNA sample that DNAaseI handles, reaction volume 25 μ l obtain the first chain cDNA.Getting 1 μ L, the first chain cDNA product is template; Carry out the RT-PCR amplification with primer 3NGSP2 and TPS4 (table 1); Carry out expression analysis; Increase simultaneously with paddy rice Actin1 gene conserved sequence designed primer OsActin1-F and OsActin1-R (table 1), with the expression intensity of its amplified production (446bp) as internal reference.

3 T2 transgenic line seedling that obtain through top screening are carried out result's demonstration that RT-PCR analyzes: under water treatment and condition of salt stress, all do not have amplified production at contrast TP309 under the experiment condition; 3 T2 transgenic lines all amplify corresponding purpose fragment under water treatment and salt stress; PyTPS gene that this explanation imports has all obtained expression on transcriptional level under salt stress and non-stress conditions, but under condition of salt stress than expression amount under the non-stress conditions high (as shown in Figure 8).

Above-mentioned 3 T2 transgenic lines have arrived T6 generation now through cultivation, the Screening and Identification of continuous multi-generation subsequently, and are basicly stable, isozygoty.

Keep the strain system that kalamycin resistance, PCR detect, Southern is hybridized and the RT-PCR analysis all is positive in continued growth, results seed (T3).

Table 1:T2 detects the PCR primer and the amplified production thereof of usefulness for transfer-gen plant RT-PCR

The RT-PCR amplification condition is: 94 ℃ of 4min at first, and 30 circulations (94 ℃ of 50s, 58 ℃ of 50s, 72 ℃ of 1min) then, last 72 ℃ are extended 10min.Pcr amplification product separates through 1.2% Agarose gel electrophoresis; With Imaging Densito Meter (Model GS-670; Bio-Rad) carry out quantitatively, the expression level of goal gene, test repetition 3 times are represented in the comparison of the concentration of purpose band and contrast band concentration.

Above embodiment is only in order to explaining technical scheme of the present invention, but not limits it; Although the present invention has been carried out detailed explanation with reference to previous embodiment, for the person of ordinary skill of the art, still can make amendment to the technical scheme that previous embodiment is put down in writing, perhaps part technical characterictic wherein is equal to replacement; And these modifications or replacement do not make the essence of relevant art scheme break away from the spirit and the scope of the present invention's technical scheme required for protection.

SEQUENCE?LISTING

 

< 110>Qingdao Agricultural University

<120>Yezoensis laver TPSGene and the application in improving the paddy rice salt tolerance thereof

<160> 15

<170> PatentIn?version?3.3

<210> 1

<211> 2727

<212> DNA

< 213>yezoensis laver

<400> 1

atgacccccg?ggcctatcac?taccgatgcc?gctgctggtg?ttggcgtgga?cacgatggac 60

ggcacgatga?agtgggagct?ctttgaggag?aaccgcaccc?ttactacccg?ctccggccaa 120

atgattgtga?acgacgggga?gtttgggaag?tggaatgata?ctgatgaggg?gcgtgtcaag 180

tgctttctga?cgccggacga?aggctttggt?gacgccatcg?agagcctagt?cattgtgctg 240

taccgactgc?ctatcattgc?gaagcgcgac?gcgaccacgg?gagcttggag?cttcaaatgg 300

gacgatgacg?ccctctacct?aacttccacc?ggtttgcgga?agggcttgga?gcagcttaag 360

gtggcacctc?tctgggtggg?catattgaac?tccgacgacg?aggtgccccg?gcaagagcgt 420

gaaggggtcg?ccgaccggct?gttggaggag?ttcaactgcg?ttcccgtctt?catcccgcac 480

gacacgctga?agcagttata?ccagggcttc?tgcaaaggca?ccctctggcc?gctttttcac 540

atggtgtcgt?cggcgacaga?ccacacggaa?cacactaccc?gctttgacga?ccgcctttgg 600

cgtgtttaca?tgaacgtcaa?ccggatgttc?cgggacaagg?tggtggaggt?gtacgatggg 660

gaccgagcac?tcatttgggt?acacgactac?cacctgatgc?ttttgccgca?ggcatcgcgc 720

tcgcgcctgt?caggtgtgaa?gattggcttc?tttctccata?ttccgtggcc?ttcgtcagaa 780

gtgtaccgcg?tgctgccgtg?gcggaacgaa?ctgctgaagg?gcatgctttc?cgcgacgttg 840

ctgggcttcc?atcttttcga?ctacgcgcgt?cactttctct?ccgcttgcgt?gcggctgctt 900

aacctggagc?acgaggcgaa?caggggctcc?ctcggtctgg?agtacgacgg?tcgccacgtt 960

atgctgcgcg?tgagtcacat?tggcgtggac?ccagagcgct?tttccgaggg?cctgagcgcg 1020

ccgtcgctga?cggaccgggt?tgccgagttc?aaggagcggt?ttgcggattg?cacagtcctc 1080

ggtgccgtgg?acgaccttga?cctcatcaag?ggcattgccc?tcaagctgat?gggtttccag 1140

cggtatctcg?attccgcccc?caagatgcga?ggaaaggtcg?tacttgtgca?agttgccatc 1200

ccaaaggcgg?cccgcgtcaa?ggagtctgtg?cgtaacgaga?ttcgggagct?ggtggctgcc 1260

atcaacaaca?aacacggcga?tgggtcgggg?cggcgacccg?tatggtacct?ggaagagagc 1320

atctcctttg?aaagccgcct?tgcgctgtac?agcatcatgg?atgcactggt?ggtgaccccc 1380

atccgtgacg?gcctcaacct?catcccatac?gagtacattg?tatcgacgtc?cgaggggaag 1440

ggccagctaa?ttctttcgga?gtttactggc?tgctcgcggg?cgctctcatc?tgcggaacgt 1500

gtcaacccgt?gggacttgga?gaagctgtcc?aacgtccttg?acatggtcgt?tcagaaggcc 1560

attgccgcgg?cgcccgaggt?ggagctgaag?cgtcgggcgg?acaaggccta?cgtgtcggcc 1620

cactcgtccc?agcagtgggc?gcagtcgttt?ttgcatgact?tgaaggaggc?gtcagagccg 1680

gcacaggtcg?ttgtcaaggt?gggtccgctg?gccggcttgc?ctggtgtact?ggcctacgac 1740

gagttcaccc?ttctcaaccg?ctcggcggtt?ctgcgcgcct?acaaggccgc?caagaggcgc 1800

ttgttcctgt?ttgactatga?tgggacgctg?acctccatca?ccgatcagtc?gtcccagatg 1860

gcccacgcgt?gggcgcggcc?gagcgaggct?gtggccgcca?acttggacac?tttgtcaaag 1920

gacccgctca?acgatgtcta?catcatgtct?ggccgcaaga?ctgaggtgct?tgaagccggc 1980

ctcaacaact?cccccacgat?tgggattgca?gctgagcacg?gcttcttcta?ccgcaagaag 2040

aattcgaagg?agtggaacag?gctgttggag?gatgcggacc?tgtcatggat?ggagttggca 2100

ttgcgtatca?tgcttatgta?cacggaccgt?acggacggat?cctacgtgga?gcagaagaag 2160

gctggcctcg?tgtggcacta?cctggacgcg?gaccgggaat?ttggctcgtg?gcaggcgaag 2220

gagatgcgtg?accatcttga?gtcgctgctt?tctcccttct?ctgtgcaggt?ggtcacaggc 2280

tatggatggc?ttcaggtccg?gatggctgcg?atgaacaagg?gcgttatggt?cgagacgatt 2340

cttcgcgata?tgcccgagcc?cccggacttt?gtgctctgct?gtggcgacga?ccgcacggat 2400

gaggacatgt?ttgcgtatct?ggacactcat?ctggaccctt?ctgtgaagca?gttcacttgc 2460

acagtgggcg?tcaagccgag?ccacgcgcgc?tactacctgc?actcatccaa?tgaggtgggc 2520

gcgcttctgg?agacgctggt?aactggaagc?tacccgcgcg?gcgtgcgccc?tcgcccgcgt 2580

ggtggttctg?tgtcccttgc?ggaccttgta?cccgacgacg?aaggctcctc?cagcacaccg 2640

gcggtgccgc?agaggaccaa?cggacctcgg?acaagcgcgt?cgggtcagaa?gagacgaggt 2700

ctggcgtcct?cgttgccgag?ccagtag 2727

 

<210> 2

<211> 499

<212> DNA

< 213>yezoensis laver

<400> 2

ctacgcgcgt?cactttctct?ccgcttgcgt?gcggctgctt?aacctggagc?acgaggcgaa 60

caggggctcc?ctcggtctgg?agtacgacgg?tcgccacgtt?atgctgcgcg?tgagtcacat 120

tggcgtggac?ccagagcgct?tttccgaggg?cctgagcgcg?ccgtcgctga?cggaccgggt 180

tgccgagttc?aaggagcggt?ttgcggattg?cacagtcctc?ggtgccgtgg?acgaccttga 240

cctcatcaag?ggcattgccc?tcaagctgat?gggtttccag?cggtatctcg?attccgcccc 300

caagatgcga?ggaaaggtcg?tacttgtgca?agttgccatc?ccaaaggcgg?cccgcgtcaa 360

ggagtctgtg?cgtaacgaga?ttcgggagct?ggtggctgcc?atcaacaaca?aacacggcga 420

tgggtcgggg?cggcgacccg?tatggtacct?ggaagagagc?atctcctttg?aaagccgcct 480

tgcgctgtac?agcatcatg 499

 

<210> 3

<211> 892

<212> DNA

< 213>yezoensis laver

<400> 3

atgacccccg?ggcctatcac?taccgatgcc?gctgctggtg?ttggcgtgga?cacgatggac 60

ggcacgatga?agtgggagct?ctttgaggag?aaccgcaccc?ttactacccg?ctccggccaa 120

atgattgtga?acgacgggga?gtttgggaag?tggaatgata?ctgatgaggg?gcgtgtcaag 180

tgctttctga?cgccggacga?aggctttggt?gacgccatcg?agagcctagt?cattgtgctg 240

taccgactgc?ctatcattgc?gaagcgcgac?gcgaccacgg?gagcttggag?cttcaaatgg 300

gacgatgacg?ccctctacct?aacttccacc?ggtttgcgga?agggcttgga?gcagcttaag 360

gtggcacctc?tctgggtggg?catattgaac?tccgacgacg?aggtgccccg?gcaagagcgt 420

gaaggggtcg?ccgaccggct?gttggaggag?ttcaactgcg?ttcccgtctt?catcccgcac 480

gacacgctga?agcagttata?ccagggcttc?tgcaaaggca?ccctctggcc?gctttttcac 540

atggtgtcgt?cggcgacaga?ccacacggaa?cacactaccc?gctttgacga?ccgcctttgg 600

cgtgtttaca?tgaacgtcaa?ccggatgttc?cgggacaagg?tggtggaggt?gtacgatggg 660

gaccgagcac?tcatttgggt?acacgactac?cacctgatgc?ttttgccgca?ggcatcgcgc 720

tcgcgcctgt?caggtgtgaa?gattggcttc?tttctccata?ttccgtggcc?ttcgtcagaa 780

gtgtaccgcg?tgctgccgtg?gcggaacgaa?ctgctgaagg?gcatgctttc?cgcgacgttg 840

ctgggcttcc?atcttttcga?ctacgcgcgt?cactttctct?ccgcttgcgt?gc 892

 

<210> 4

<211> 21

<212> DNA

< 213>artificial sequence

<400> 4

ctacgcgcgt?cactttctct?c 21

 

<210> 5

<211> 21

<212> DNA

< 213>artificial sequence

<400> 5

catgatgctg?tacagcgcaa?g 21

 

<210> 6

<211> 24

<212> DNA

< 213>artificial sequence

<400> 6

agggaggacg?cacaatccca?ctat 24

 

<210> 7

<211> 25

<212> DNA

< 213>artificial sequence

<400> 7

gcacgcaagc?ggagagaaag?tgacg 25

 

<210> 8

<211> 20

<212> DNA

< 213>artificial sequence

<400> 8

tccggtgccc?tgaatgaact 20

 

 

<210> 9

<211> 20

<212> DNA

< 213>artificial sequence

<400> 9

ggcgataccg?taaagcacga 20

 

 

<210> 10

<211> 28

<212> DNA

< 213>artificial sequence

<400> 10

tctagaatga?cccccgggcc?tatcacta 28

 

 

<210> 11

<211> 28

<212> DNA

< 213>artificial sequence

<400> 11

gtcgacctac?tggctcggca?acgaggac 28

 

 

<210> 12

<211> 21

<212> DNA

< 213>artificial sequence

<400> 12

agccagaaag?ggggaggaaa?g 21

 

<210> 13

<211> 27

<212> DNA

< 213>artificial sequence

<400> 13

gacctgtcat?ggatggagtt?ggcattg 27

 

<210> 14

<211> 23

<212> DNA

< 213>artificial sequence

<400> 14

gaggcgcagt?ccaagagggg?tat 23

 

<210> 15

<211> 24

<212> DNA

< 213>artificial sequence

<400> 15

tcggccgttg?tggtgaatga?gtaa 24

Claims (8)

1. yezoensis laver TPS gene, its sequence table is shown in SEQ ID No:1.

2. the plant expression vector that contains the described yezoensis laver TPS of claim 1 gene.

3. the plant expression vector of yezoensis laver TPS gene according to claim 2 is characterized in that said plant vector is pCAMBIA2300-PyTPS.

4. the application of yezoensis laver TPS gene according to claim 1 in improving the paddy rice salt tolerance.

5. the application of yezoensis laver TPS gene according to claim 4 in improving the paddy rice salt tolerance is characterized in that changing yezoensis laver TPS gene over to paddy rice, and the primer that the paddy rice transfer-gen plant is carried out the PCR detection is:

P1：5′-CTACGCGCGTCACTTTCTCTC-3′，

P2：5′-CATGATGCTGTACAGCGCAAG-3′；

Amplify one section sequence of the PyTPS gene of 499bp with primer P1 and P2, its sequence table is shown in SEQID No:2.

6. the application of yezoensis laver TPS gene according to claim 4 in improving the paddy rice salt tolerance is characterized in that changing laver TPS gene over to paddy rice, and the primer that the paddy rice transfer-gen plant is carried out the PCR detection is:

P3：5′-AGGGAGGACGCACAATCCCACTAT-3′，

P4：5′-GCACGCAAGCGGAGAGAAAGTGACG-3′；

Amplify one section sequence of the PyTPS gene of 892bp with primer P3 and P4, its sequence table is shown in SEQID No:3.

7. according to claim 5 or the application of 6 described yezoensis laver TPS genes in improving the paddy rice salt tolerance; It is characterized in that it is 25 μ L that the paddy rice transfer-gen plant is carried out the reaction system that PCR detects; The dNTPs 1 μ L of 2.5mmol/L wherein, concentration is each 1 μ L of two kinds of primers of upstream and downstream of 10 μ mol/L, 10 * PCR buffer, 2.5 μ L; Template DNA 1 μ L, the Taq polysaccharase 0.2 μ L of 5U/ μ L;

The PCR response procedures is: 94 ℃ of sex change 4min; 94 ℃ of sex change 1min, 59 ℃ of annealing 1min, 72 ℃ are extended 1min; Last 72 ℃ are extended 10min; 4 ℃ of insulations, totally 35 circulations.

8. according to claim 5 or the application of 6 described yezoensis laver TPS genes in improving the paddy rice salt tolerance, the anti-salt concn that it is characterized in that the paddy rice transfer-gen plant is 5 ‰-8 ‰.

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