CN107574170A - The albumen and application of asparagus resistant gene of salt AoCBL2 and its coding - Google Patents

Abstract

The invention discloses albumen and the application of a kind of asparagus resistant gene of salt AoCBL2 and its coding.Asparagus resistant gene of salt AoCBL2 CDS sequences such as SEQ ID NO:Shown in 1, the amino acid sequence such as SEQ ID NO of its encoding proteins:Shown in 2.Clone obtains resistant gene of salt AoCBL2 to the present invention from asparagus first, and prove that the gene is one of key gene in plant salt tolerance control path, using the method for genetic engineering, by AoCBL2 gene overexpressions into target plant, compared with control group, transfer-gen plant salt resistance ability significantly increases, and illustrates that the overexpression of AoCBL2 genes improves the resistance of plant pair salt adverse circumstance.AoCBL2 genes provide important theoretical foundation for research asparagus salt tolerant molecule mechanism and Breeding Application in the present invention, have broad application prospects and great economic value.

Description

The albumen and application of asparagus resistant gene of salt AoCBL2 and its coding

Technical field

The present invention relates to field of plant genetic, specifically, is related to asparagus resistant gene of salt AoCBL2 and its volume The albumen of code and application.

Background technology

According to statistics, China's salination and Wind-sand impacted lands account for the 1/5 of China's area, and as environmental degradation is expanding Greatly, management task is arduous.It was verified that the today increasingly sharpened in population, grain, soil contradiction, selection has Salt And Alkali Tolerance, resistance to Drought, impoverishment tolerant gene and the wild plant of potential age deduction and cultivated plant carry out adapting to plantation and promoted, and can not only change The ecological environment in kind salt-soda soil, land output can also be improved, increase people improve the enthusiasm in soil, are the synthesis of mobilizing the masses Administer a basic effective measures in salt-soda soil.The salinization of soil is to influence one of agricultural production key factor, and research plant is resistance to By mechanism and improve the problem of crop tolerance to salt alkalescence has increasingly turned into extensive concern.For a long time, people using morphology and The metabolic mechanism of the research methods such as physiology Preliminary study plant reply salt stress, and pass through gene cloning and heredity turns The technological means such as change, which identify, participates in part key gene in salt stress regulation process.However, at present to plant salt tolerance alkali mechanism The data of research is still very deficient, and the related resistant gene of salt and its mechanism of action of different plants are still unclear, perfect plant Thing salt tolerance mechanism is not yet established, and needs progress more in-depth study badly.

Asparagus, also known as asparagus (Asparagus officinalis L.), are the important industrial crops of Liliaceae, its is fresh Bamboo shoot matter is tender delicious, can be antitumor, anti-oxidant rich in the various active composition such as flavones, saponin(e, asparagine, selenium and plant polyose And reducing blood lipid, it is described as one of " kings of vegetables ", " ten big famous dish of the world ".Asparagus originates in Mediterranean, has anti-severe Saline and alkaline and drought characteristic, can 8 ‰ saline and alkaline aerial.Asparagus root system can be 3 meters as deep as underground, huge dynamic bulb group, Efficient needle-like intends leaf, drought-enduring, Salt And Alkali Tolerance, impoverishment tolerant, has good ecological functions, is successfully used for Wind-sand impacted lands and salt Administer alkali, realize that economic benefit is mutually unified with ecological benefits, disclosure satisfy that China salt-soda soil and the Wind-sand impacted lands comprehensive regulation Needs.In addition, asparagus secondary industry chain length, can produce the production of the high added values such as asparagus anticarcinogen, asparagus tea, wine and beverage Product, had a extensive future in food, medicine and other fields application and development.At present, China's planting asparagus and processing are quickly grown, it has also become generation The first big producting and exporting country of boundary, cultivated area account for the 43% of the whole world more than 95,000 hectares.

Forefathers' research shows that plant forms a series of stress response mechanism to carry during Saline Alkali Stress is adapted to The tolerance of high plant pair stress.Wherein, asparagus still lacks in molecular biology at present as a kind of important salt-tolerant plant Research of the aspect to its Mechanisms of Salt Resistance, therefore, the function of the exploration of resistant gene of salt in salt-tolerant plant asparagus, its encoding proteins is reflected The fixed Mechanisms of Salt Resistance for helping to disclose asparagus, it is significant to the genetic breeding and breed improvement of asparagus.

The content of the invention

It is an object of the invention to provide asparagus resistant gene of salt AoCBL2 and its albumen of coding.

It is a further object of the present invention to provide application of the asparagus resistant gene of salt in plant salt endurance is improved.

The present invention is directed to the present situation that the basic research of salt tolerant molecular mechanism is weak in asparagus, and clone obtains the resistance to alkali of asparagus first Because of AoCBL2, and further analyze effect of the gene in logical gene engineering method improves plant salt endurance.

The present invention designs a pair of special primer (SEQ by analyzing the full-length genome high-flux sequence result of asparagus ID NO:3-4), to asparagus kind, ' the tender stem sample cDNA of well ridge 111 ' enters performing PCR amplification, obtains asparagus resistant gene of salt AoCBL2 CDS sequences (total length 681bp), Gene A oCBL2 CDS sequences are：

i)SEQ ID NO:Nucleotide sequence shown in 1；Or

ii)SEQ ID NO:Nucleotide sequence shown in 1 is substituted, lacks or added one or more nucleotides and expression The nucleotide sequence of identical function protein；Or

Iii) under strict conditions with SEQ ID NO:Sequence shown in 1 hybridizes and the nucleotides of expression identical function protein Sequence, the stringent condition are in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C Lower hybridization, and wash film with the solution；Or

Iv) and i), ii) or nucleotide sequence iii) there is more than 90% homology and express identical function protein Nucleotide sequence.

Present invention also offers the albumen of above-mentioned asparagus resistant gene of salt AoCBL2 codings, the amino acid sequence of the albumen is such as SEQ ID NO:Shown in 2；Or SEQ ID NO:Sequence shown in 2 be substituted, lack or add it is obtained by one or more amino acid, And the derived protein with identical function.

The present invention also provides the expression cassette containing asparagus resistant gene of salt AoCBL2, carrier, engineering bacteria and transgenic cell line.

Carrying the expression vector of the target gene can be turned by using Ti-plasmids, plant viral vector, direct DNA The standard biologic such as change, microinjection, electroporation technical method imports (Weissbach, 1998, Method in plant cell Plant Molecular Biology VIII, Academy Press, New York, the 411-463 pages；Geiserson and Corey, 1998, Plant Molecular Biology, 2nd Edition), and the plant tissue of conversion is cultivated into plant.

, can in the front end of its transcription initiation nucleotides when being building up to using the genetic fragment of the present invention in plant expression vector Add any one enhancing promoter or inducible promoter.For the ease of identifying transgenic plant cells or plant And screening, used carrier can be processed, such as add resistant antibiotic marker (such as kanamycins).Quilt The host of conversion is the various plants including arabidopsis, cultivates the floristics of different salt resistance abilities.

The present invention also provides asparagus resistant gene of salt AoCBL2 and is used to improve plant salt tolerance ability by gene engineering method Using.The salt resistance ability refers to plant in the case where soil salt content is higher, the ability of normal growth；The raising is resistance to Salt ability, refer to improve the soil salt content upper limit that plant is resistant to and keeps normal growth under this condition.

In the specific embodiment of the present invention, Gene A oCBL2 is building up on carrier pCAMBIA2301, uses institute Recombinant vector arabidopsis thaliana transformation is obtained, screens positive transgenic plant.

Preferably, using Agrobacterium-mediated genetic transformation method arabidopsis thaliana transformation, Agrobacterium used in conversion is EHA105.

The present invention further provides the improvement plant (salt resistance ability enhancing) obtained using said gene engineering technology in base Because of the application in engineering field.

Clone obtains resistant gene of salt AoCBL2 to the present invention from asparagus first, and proves that the gene regulates and controls road for plant salt tolerance One of key gene in footpath, using the method for genetic engineering, Gene A oCBL2 is transformed into target plant, is remarkably improved The salt resistance ability of transfer-gen plant, for from now on using technique for gene engineering improve plant salt tolerance provide it is important it is theoretical according to According to having broad application prospects and great economic value.

Brief description of the drawings

Fig. 1 is AoCBL2 gene cDNA total length PCR amplifications in the embodiment of the present invention 1；Wherein 1,2 represent respectively The PCR primer of AoCBL2 gene cDNAs, M represent molecular size range.

Fig. 2 is the testing result of AoCBL2 genes expression quantity in transgenic arabidopsis in the embodiment of the present invention 3；Wherein 1- 12 be that 12 T1 compare for transgenic line, WT for wildtype Arabidopsis thaliana ,+it is recombinant plasmid pCAMBIA2301-AoCBL2 ,-be Negative control H₂O, M are molecular size range.

Fig. 3 is that wildtype Arabidopsis thaliana is compareed in the embodiment of the present invention 4 with converting the Arabidopsis plant of AoCBL2 genes not With the response analysis under concentration salt stress adverse circumstance.Wherein, scheme A be the transgenic arabidopsis strain AoCBL2-1 that selects at random and For AoCBL2-6T1 for sprouting of the seed under various concentrations salt stress and growing state, WT is wildtype Arabidopsis thaliana control.Scheming B is Root length after being grown 15 days in the 1/2MS culture mediums of different salinity.

Embodiment

Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment According to conventional laboratory conditions, as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW, Molecular Cloning:A Laboratory Manual, 2001), or the condition according to manufacturer's specification suggestion.

The clone of the AoCBL2 genes of embodiment 1

By early stage to asparagus new varieties ' the full-length genome high-flux sequence result of well ridge 111 ' (JK111) is analyzed, Design special primer P1 forward primers:5 '-ATGTTGCAGTTCTTAGAGGG-3 ' and P2 reverse primers:5’- TCAGGTATCGTCGACTTGAG-3’(SEQ ID NO:3-4), using conventional CTAB method (references《Plant genetic engineering》, king General Guan Yu's Tomb, Fang Hongjun chief editors) from asparagus kind, ' well ridge 111 ' is middle to extract tender stem total serum IgE, and reverse transcription synthesis cDNA, utilization are above-mentioned Amplified in the cDNA that primer P1 and P2 obtains from RNA reverse transcriptions such as SEQ ID NO:Asparagus resistant gene of salt AoCBL2 shown in 1 CDS sequences, Gene A oCBL2 CDS sequences 681bp (Fig. 1).

Comprise the following steps that：

(1) into centrifuge tube add CTAB (cetyl trimethylammonium bromide) Extraction buffer [2% (W/V) CTAB, NaCl 1.4mol/L, EDTA (ethylenediamine tetra-acetic acid) 20mmol/L, TrisHCl 100mmol/L, 2% (W/V) PVP] and 10% beta -mercaptoethanol, is preheated in water-bath；

(2) asparagus spear is cooled down with liquid nitrogen and ground, added in extract solution, mixed, 65 DEG C of water-baths 10 minutes；

(3) isometric chloroform is added：Isoamyl alcohol (volume ratio 24：1) mixed liquor, overturn mix, stand 10min, 4 DEG C 12000g centrifuges 10min；

(4) supernatant, repeat step (3) are taken；

(5) supernatant is taken, adds final concentration of 2mol/L LiCl, ice bath 10-12 hours, 11000rpm, 4 DEG C of centrifugations 15min, supernatant is abandoned, precipitation is cleaned twice with 75% ethanol, be dissolved in stand-by in appropriate DEPC (pyrocarbonic acid diethyl ester) processing water；

(6) from asparagus kind, ' extraction tender stem total serum IgE is template in well ridge 111 ', and (Thermo is purchased from using reverse transcriptase Fisher Scientific companies) its reverse transcription synthesized into first chain of cDNA, reaction condition is：65℃5min,42℃ 50min, 70 DEG C of 10min；

(7) asparagus anthocyanidin synzyme base is amplified in the cDNA obtained using above-mentioned primer P1 and P2 from RNA reverse transcriptions Because of AoCBL2 CDS sequences；

Reaction condition：94 DEG C of pre-degeneration 4min；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 1min, 33 circulations；72 DEG C are prolonged Stretch 10min.The PCR primer for expanding acquisition is connected into pMD18-T carriers (being purchased from precious bioengineering Dalian Co., Ltd), conversion is big Enterobacteria competent cell, screening positive clone are simultaneously sequenced, and obtain required full-length gene.Extract and carry from positive colony The plasmid of AoCBL2 gene C DS sequences, is named as pMD18-AoCBL2 plasmids.

The structure and genetic transformation of the AoCBL2 gene overexpression carriers of embodiment 2

In order to preferably analyze Gene A oCBL2 biological function, further the gene was realized in arabidopsis Amount expression, the structure of AoCBL2 gene overexpression carriers and comprising the following steps that for genetic transformation：It will be obtained first in embodiment 1 PMD18-AoCBL2 plasmid SacI and the KnpI double digestions arrived, reclaim purpose fragment；Meanwhile carried with same method digestion Double tobacco mosaic virus promoter 35S genetic transformation carrier pCAMBIA2301.Digestion finishes, with including AoCBL2 genes Endonuclease bamhi and the pCAMBIA2301 carriers of digestion do coupled reaction, and (bacterial strain is purchased from precious bioengineering to conversion bacillus coli DH 5 alpha Dalian Co., Ltd).By digestion screening positive clone, recombinant vector is obtained, is named as pCAMBIA2301-AoCBL2.

It is conducted into by agriculture bacillus mediated arabidopsis genetic transforming method in plan south, by infecting, co-culturing, sieving Transformation seedlings of the choosing with kalamycin resistance, then by taking root, practice the conventional steps (reference such as transplantation of seedlings：J. Pehanorm Brooker, EF Not Ritchie, T Mannies A Disi write；Huang Peitang, Wang Jiaxi etc. are translated；Molecular Cloning:A Laboratory guide (third edition)；Beijing, scientific publication Society；2002 editions), obtain transfer-gen plant.

The main agents and genetic transforming method used in the present embodiment are as follows：

(1) main agents

The abbreviation of used culture medium prescription and plant hormone represents as follows：The preparation reference of 1/2MS, MS culture medium Murashige T.and F.Skoog.Physiol.Plant,1962,15:Method disclosed in 473-497.6-BA(6- BenzylaminoPurine, 6-benzyl aminopurine)；NAA (Naphthalene acetic acid, methyl α-naphthyl acetate)；Kan (Kanamycin, kanamycins)；Cef (Cefotaxime, cephalosporin).Wherein, kanamycins and cephalosporin use 0.25 μm membrane filtration method sterilizing, in the above-mentioned culture medium in addition to Kan and Cef compositions through 121 DEG C of high pressure steam sterilization 20min Afterwards, when culture medium is cooled to 50-60 DEG C, corresponding antibiotic is added on superclean bench.

(2) Agrobacterium-mediated genetic transformation

1) culture of Agrobacterium

First, in solid LB media (10g/L peptones, 5g/L yeast extracts, 10g/ with corresponding resistance selection L sodium chloride, Kan 100mg/L, agar 1.5g/L) on preculture carry target gene AoCBL2 Agrobacterium EHA105 48 Hour, 28 DEG C of cultivation temperature；Picking preculture Agrobacterium single bacterium colony, it is inoculated in the LB liquid medium of corresponding resistance selection In (10g/L peptones, 5g/L yeast extracts, 10g/L sodium chloride, Kan 100mg/L), in 28 DEG C of 200rpm shaking table culture mistakes Night, to bacterial concentration OD₆₀₀Value about 0.8~1.0.

2) inflorescence method infects arabidopsis

Using the inflorescence infestation method arabidopsis thaliana transformation of improvement.Picking contains the Agrobacterium of pCAMBIA2301-AoCBL2 plasmids Single bacterium colony, it is transferred in 100mL LB fluid nutrient mediums, inserts 200r/min in 28 DEG C of shaking tables and be incubated overnight, make Agrobacterium bacterium solution dense Degree reaches OD₆₀₀At 0.8~1.0,4000r/min centrifugation 10min, after abandoning supernatant, contain sucrose 5g, SilwetL- with 100mL 77 50 μ L LB fluid nutrient mediums suspension Agrobacterium bacterium solution again.By arabidopsis flowerpot back-off in the beaker containing bacteria suspension, The inflorescence of arabidopsis is immersed in 10~20s in bacterium solution, after cultivating 24h under dark, is placed in 25 DEG C of temperature, humidity 60%, illumination Cultivated in the illumination box of intensity 3000~4000lx and 16h/8h photoperiod, water and cultivate plant every other day, treat Post flowering Harvest seed.By the arabidopsis seed of harvest with after sterilization 5min in 8%NaClO alcoholic solutions (95% alcohol), selecting Select and screened on culture medium (MS+50mg/L kanamycins+7g/L agar+30g/L sucrose).

The AoCBL2 gene transgenics T1 of embodiment 3 detects for seedling RT-PCR

In order to verify whether transgenic arabidopsis T1 is relevant with the AoCBL2 genes being transferred to for the change of strain salt resistance ability, AoCBL2 gene expressions in partial transgenic Arabidopsis plant are detected using RT-PCR method, as a result see Fig. 2.Specifically Step is as follows：

Using TRIZOL reagents (being purchased from precious bioengineering Dalian Co., Ltd) from transgenic arabidopsis T1 for 1-12 strains The total serum IgE (extracting method is with reference to the operation of TRIZOL reagents specification) of plant is extracted in system, (Thermo is purchased from using reverse transcriptase Fisher Scientific companies) its reverse transcription synthesized into first chain of cDNA, reaction condition is 65 DEG C of 5min, 42 DEG C 50min, 70 DEG C of 10min.First the cDNA that reverse transcription obtains is detected with the reference gene Tubulin of report and concentration is adjusted It is whole, enter performing PCR detection, it is ensured that reference gene be able to can expand in control wildtype Arabidopsis thaliana and transgenic Arabidopsis plants Go out, and brightness is consistent.Then, according to the sequence of AoCBL2 genes, using primer P1 and P2, RT-PCR detections, reaction are carried out Condition is：94 DEG C of pre-degeneration 4min；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 1.5min, 33 circulations；72 DEG C of extension 10min. The agarose gel electrophoresis result of amplified production shows (Fig. 2), and the table of AoCBL2 genes is detected in 12 transgenic lines Reach.

The measure of 4 turns of AoCBL2 Arabidopsis plant salt tolerances of embodiment

In order to further analyze asparagus resistant gene of salt AoCBL2 biological function, we are by the gene in arabidopsis Overexpression is realized, and intends verifying that Gene A oCBL2 regulates and controls in salt stress from the phenotypic characteristic of transfer-gen plant salt resistant character On biological function.Specific experiment step is as follows：

(1) experiment material

The T1 that the AoCBL2 genetic transformation arabidopsis that screening obtains in early stage obtains chooses at random in positive transgenic strain The seed (AoCBL2-1 and AoCBL2-6) of each 2 strains is selected, control is used arabidopsis wild type seeds (WT), cultivated with 1/2MS 3 kinds of culture plates based on base, NaCl contents are respectively 0mM (1/2MS), 100mM (NaCl 100) and 150mM (NaCl 150) salt tolerance experiment, is carried out respectively.

(2) experimental method

The culture plate (square vertical flat plate with a scale) of 3 kinds of different NaCl contents is prepared respectively.2 selected at random The same position on every kind of flat board is broadcast after seed and the wild type seeds sterilization of individual transgenic line.Flat board is in a manner of near vertical It is placed on same illumination cultivation frame, light intensity is about 3600lx, light irradiation time 16h.Culture took out flat board after 10 days, took pictures Record, observes seed sprouting situation, and continued growth is to measuring root long after the 15th day.Setup Experiments repeat three times.

As a result show, 2 transgenic lines and the wild type seeds equal normal germination and growth of energy on 1/2MS culture mediums, say Bright seed quality is good.On addition 100mM NaCl 1/2MS culture mediums, wild type seeds sprout difficult, the budding time (5 My god) significantly it is later than transfer-gen plant (3 days), and root system does not extend substantially, it is slow-growing；And transfer-gen plant seed sprouts ability It is remarkably reinforced, and root system can extend, and grow and substantially accelerate compared with wild type.Especially when salinity increases to 150mM NaCl When, wild type seeds are not sprouted completely, and growth is suppressed completely, and transfer-gen plant seed still can be sprouted, and root system is still Extending, with wild type significant difference, this 2 transgenic line of explanation is remarkably reinforced with respect to wild type control salt resistance ability. In summary, in this example, the transfer-gen plant AoCBL2-6 selected at random compares control with AoCBL2-10 salt resistance ability Wildtype Arabidopsis thaliana (WT) greatly improves (Fig. 3).

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Sequence table

<110>Institute of Vegetables and Flowers, Jiangxi Academy of Agricultural Sciences

<120>The albumen and application of asparagus resistant gene of salt AoCBL2 and its coding

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 681

<212> DNA

<213>Native sequences (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 1

atgttgcagt tcttagaggg agttaagcat ttgttagctg ctctgctgaa gtgctgcgat 60

cttgagatag gcaggcagcc caagggcctt gaagatcctg aacttttagc aagagagaca 120

gtgttcagcg taagtgaaat agaagcttta tatgagttgt ttaaaaagat cagcagtgct 180

gtgattgacg acgggctgat caacaaggaa gaattccagt tggcattatt caagactaac 240

aaaaaggaga gcttatttgc tgatcgggta tttgacttgt ttgatacaaa acacaatgga 300

attttaggtt ttgaggagtt tgcacgagcc ctatcagttt tccatccaaa tgctccaatt 360

gacgataaga ttgaattttc tttccaattg tatgatctga agcaacaagg attcattgaa 420

agacaagagg tgaaacaaat ggtagtggct acacttgccg agtctggaat gaacctttct 480

gatgaagtca tagagagtat cattgataag acatttgagg aagctgatac aaaacatgat 540

ggaaaaattg acaaggaaga gtggcggaat ttggtcttgc gacatccatc tttattaaag 600

aacatgaccc tccaatatct taaggacata acaacaacgt ttccaagctt tgtgtttcat 660

tctcaagtcg acgatacctg a 681

<210> 2

<211> 226

<212> PRT

<213>Native sequences (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 2

Met Leu Gln Phe Leu Glu Gly Val Lys His Leu Leu Ala Ala Leu Leu

1 5 10 15

Lys Cys Cys Asp Leu Glu Ile Gly Arg Gln Pro Lys Gly Leu Glu Asp

20 25 30

Pro Glu Leu Leu Ala Arg Glu Thr Val Phe Ser Val Ser Glu Ile Glu

35 40 45

Ala Leu Tyr Glu Leu Phe Lys Lys Ile Ser Ser Ala Val Ile Asp Asp

50 55 60

Gly Leu Ile Asn Lys Glu Glu Phe Gln Leu Ala Leu Phe Lys Thr Asn

65 70 75 80

Lys Lys Glu Ser Leu Phe Ala Asp Arg Val Phe Asp Leu Phe Asp Thr

85 90 95

Lys His Asn Gly Ile Leu Gly Phe Glu Glu Phe Ala Arg Ala Leu Ser

100 105 110

Val Phe His Pro Asn Ala Pro Ile Asp Asp Lys Ile Glu Phe Ser Phe

115 120 125

Gln Leu Tyr Asp Leu Lys Gln Gln Gly Phe Ile Glu Arg Gln Glu Val

130 135 140

Lys Gln Met Val Val Ala Thr Leu Ala Glu Ser Gly Met Asn Leu Ser

145 150 155 160

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

165 170 175

Thr Lys His Asp Gly Lys Ile Asp Lys Glu Glu Trp Arg Asn Leu Val

180 185 190

Leu Arg His Pro Ser Leu Leu Lys Asn Met Thr Leu Gln Tyr Leu Lys

195 200 205

Asp Ile Thr Thr Thr Phe Pro Ser Phe Val Phe His Ser Gln Val Asp

210 215 220

Asp Thr

225

<210> 3

<211> 20

<212> DNA

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 3

atgttgcagt tcttagaggg 20

<210> 4

<211> 20

<212> DNA

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 4

tcaggtatcg tcgacttgag 20

Claims (10)

1. asparagus resistant gene of salt AoCBL2, it is characterised in that Gene A oCBL2 CDS sequences are：

i)SEQ ID NO:Nucleotide sequence shown in 1；Or

ii)SEQ ID NO:Nucleotide sequence shown in 1 is substituted, lacks or added one or more nucleotides and expression is identical The nucleotide sequence of functional protein；Or

Iii) under strict conditions with SEQ ID NO:Sequence shown in 1 hybridizes and the nucleotides sequence of expression identical function protein Row, the stringent condition are in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C Hybridization, and wash film with the solution；Or

Iv) and i), ii) or nucleotide sequence iii) there is more than 90% homology and express the nucleosides of identical function protein Acid sequence.

2. the albumen that asparagus resistant gene of salt AoCBL2 described in claim 1 is encoded, it is characterised in that the amino acid sequence of the albumen Row such as SEQ ID NO:Shown in 2；Or SEQ ID NO:Sequence shown in 2 is substituted, lacks or added one or more amino acid institutes Derived protein obtaining and with identical function.

3. the expression cassette containing Gene A oCBL2 described in claim 1.

4. the carrier containing expression cassette described in Gene A oCBL2 described in claim 1 or claim 3.

5. the engineering bacteria containing expression cassette described in Gene A oCBL2 described in claim 1 or claim 3.

6. the engineering bacteria containing carrier described in claim 4.

7. Gene A oCBL2 described in claim 1 is used for the application that plant salt tolerance ability is improved by gene engineering method.

8. application according to claim 7, it is characterised in that the plant includes arabidopsis.

9. application according to claim 8, it is characterised in that the gene engineering method comprises the following steps：By gene AoCBL2 is building up on carrier pCAMBIA2301, with gained recombinant vector arabidopsis thaliana transformation, screens positive transgenic plant.

10. application according to claim 9, it is characterised in that the arabidopsis thaliana transformation, be to use agriculture bacillus mediated something lost Conversion method arabidopsis thaliana transformation is passed, Agrobacterium preferably used is EHA105.