CN112522293A

CN112522293A - Populus nigra phosphatidylinositol specific phospholipase C coding gene PsnPI-PLC and application thereof

Info

Publication number: CN112522293A
Application number: CN202011573064.2A
Authority: CN
Inventors: 孙尧; 王雷; 李瑶; 黄国庆; 吴琼; 曹涤非; 薛佳莹; 孙鑫
Original assignee: Institute of Advanced Technology of Heilongjiang Academy of Sciences
Current assignee: Institute of Advanced Technology of Heilongjiang Academy of Sciences
Priority date: 2020-12-24
Filing date: 2020-12-24
Publication date: 2021-03-19
Anticipated expiration: 2040-12-24
Also published as: CN112522293B

Abstract

A coding gene PsnPI-PLC of the phosphatidylinositol specific phospholipase C of populus tremuloides and application thereof relate to the coding gene PsnPI-PLC of the phosphatidylinositol specific phospholipase C of populus tremuloides and application thereof. The aims are to provide a populus euphratica phosphatidylinositol specific phospholipase C coding gene PsnPI-PLC and application thereof. The nucleotide sequence of the coding gene PsnPI-PLC of the phosphatidylinositol-specific phospholipase C of populus tremuloides is shown in a sequence table Seq ID No: 1, the amino acid sequence is shown in a sequence table Seq ID No: 2, the gene is applied to improving the salt tolerance of plants. The salt tolerance of a PsnPI-PLC over-expressed tobacco plant is superior to that of a non-transgenic plant, the salt tolerance of tobacco can be improved by over-expression of the gene, and the gene has high application value in the field of forest salt tolerance breeding.

Description

Populus nigra phosphatidylinositol specific phospholipase C coding gene PsnPI-PLC and application thereof

Technical Field

The invention relates to a gene, a coding protein and application thereof, in particular to a populus euphratia phosphatidylinositol specific phospholipase C coding gene PsnPI-PLC.

Background

Lipid signaling is an important component of plant signal transduction, and organisms promote signal cascade and transmission through the change of lipid concentration, thereby regulating and controlling the vital activities of cells and individuals. Phospholipase C (PLC) is a member of the phospholipase family, and can hydrolyze phospholipids to produce a phosphate group-containing head and Diacylglycerol (DAG), and is classified into non-specific phospholipase C (NPC) and phosphatidylinositol-specific phospholipase C (PI-PLC) according to the substrate on which it acts.

The majority of PLCs in animals and plants are PI-PLC (programmable logic controller), which mainly comprises PH domain (N terminal), EF hand domain, X domain, Y domain and C2 domain (C terminal). Plants exist as a subset of PLCs, structurally close to the PLC delta subfamily, and lack PH domains, which are catalytically inactive but can help anchor the PLCs to substrates on the plasma membrane. In the case of PH domain deletion, PLC binds to PIP2 through a linker sequence between X-Y domains. The amino acid sequences of X and Y domains are most conserved, these two regions are composed of alternating alpha helices and beta sheets, and are the catalytic centers of the enzyme; EF hand domain has 1 typical interaction with Ca²⁺The combined helix-turn-helix structure, most plant PLCs contain only 2 EF hand domains (4 in animals)) Whether EF chiral domain deletion affects metal ion binding is not clear to date; in some plants, C2 domain regulates PLC binding to phospholipids, a process which is subject to Ca²⁺Regulation, which can still be accomplished in the absence of EF-hand domains, is an area that aids in the maintenance of catalytic activity.

The poplar has extremely high economic value and ecological value, but most poplar varieties have weak drought resistance and saline-alkali resistance. Populus deltoides (Populus simonixP. nigra) is a hybrid of Populus deltoides and Populus deltoides, has the advantages of fast growth, strong adaptability, cold resistance, drought resistance, barren resistance and slight saline-alkali resistance, and is an important greening and afforestation tree species. But in areas with water resource shortage and serious salinization, the growth and development of poplar trees are influenced. In order to cultivate new stress-resistant poplar species, a large number of stress-resistant genes are applied to the molecular breeding research of poplar to obtain high-efficiency and stable stress-resistant poplar plants, the action mechanism of related genes and complete upstream and downstream signal paths need to be determined, the breeding process is improved in a targeted manner on the basis, the foundation can be laid for the salt-tolerant mechanism research of poplar, and the method has important significance.

Disclosure of Invention

The invention aims to provide a populus euphratica phosphatidylinositol specific phospholipase C coding gene PsnPI-PLC and application thereof.

The nucleotide sequence of the coding gene PsnPI-PLC of the phosphatidylinositol-specific phospholipase C of populus tremuloides is shown in a sequence table Seq ID No: 1 is shown.

The coding gene PsnPI-PLC of the populus tremuloides phosphatidylinositol specific phospholipase C is applied to improving the salt tolerance of plants.

The coding gene PsnPI-PLC of the populus tremuloides phosphatidylinositol specific phospholipase C is applied to breeding salt-tolerant transgenic plants.

The invention has the beneficial effects that:

the coding gene of the phosphatidylinositol specific phospholipase C of populus tremuloides, namely the salt tolerance related gene PsnPI-PLC of populus tremuloides, provided by the invention can be used for improving the salt tolerance of plants or breeding salt tolerance transgenic plants. The transgenic tobacco is obtained by constructing a PsnPI-PLC overexpression vector and transfecting by a leaf disc method, after a transgenic strain system grows for 30 days in a MS culture medium of 150mM NaCl, the strain is higher than a non-transgenic strain, POD, SOD and proline are all obviously higher than the non-transgenic strain, and MDA is obviously lower than the non-transgenic strain. The results show that the salt tolerance of the PsnPI-PLC over-expressed tobacco plant is superior to that of a non-transgenic plant, the salt tolerance of the tobacco can be improved by over-expression of the gene, and the gene has high application value in the field of forest salt tolerance breeding.

Drawings

FIG. 1 is a diagram of the prediction of the conserved region of PsnPI-PLC;

FIG. 2 is a diagram of PsnPI-PLC domain element prediction;

FIG. 3 is a multiple sequence alignment chart of PsnPI-PLC with other 10 plant PI-PLC proteins;

FIG. 4 is a graph comparing plant height changes of PsnPI-PLC transgenic and non-transgenic tobacco after NaCl stress; wherein a is CK, b is P10, c is P20, d is P21;

FIG. 5 is a comparison graph of POD content determination after NaCl stress for PsnPI-PLC transgenic and non-transgenic tobacco; wherein a is CK, b is P10, c is P20, d is P21;

FIG. 6 is a view showing the assay of transgenic tobacco SOD content under NaCl stress; wherein a is CK, b is P10, c is P20, d is P21;

FIG. 7 is a view of assaying transgenic tobacco MDA content under NaCl stress; wherein a is CK, b is P10, c is P20, d is P21;

FIG. 8 is a graph comparing proline content determination after salt stress for PsnPI-PLC transgenic and non-transgenic tobacco; wherein a is CK, b is P10, c is P20, and d is P21.

Detailed Description

The first embodiment is as follows: the nucleotide sequence of the coding gene PsnPI-PLC of the phosphatidylinositol-specific phospholipase C of populus tremuloides is shown in a sequence table Seq ID No: 1 is shown.

The coding gene of the poplar phosphatidylinositol specific phospholipase C provided by the embodiment, namely the poplar salt tolerance related gene PsnPI-PLC, can be used for improving the salt tolerance of plants or breeding salt tolerance transgenic plants. The transgenic tobacco is obtained by constructing a PsnPI-PLC overexpression vector and transfecting by a leaf disc method, after a transgenic strain system grows for 30 days in a MS culture medium of 150mM NaCl, the strain is higher than a non-transgenic strain, POD, SOD and proline are all obviously higher than the non-transgenic strain, and MDA is obviously lower than the non-transgenic strain. The results show that the salt tolerance of the PsnPI-PLC over-expressed tobacco plant is superior to that of a non-transgenic plant, the salt tolerance of the tobacco can be improved by over-expression of the gene, and the gene has high application value in the field of forest salt tolerance breeding.

The second embodiment is as follows: the first difference between the present embodiment and the specific embodiment is: the amino acid sequence of the protein coded by the gene is shown in a sequence table Seq ID No: 2, respectively. The rest is the same as the first embodiment.

The third concrete implementation mode: the coding gene PsnPI-PLC of the populus euphratica phosphatidylinositol-specific phospholipase C is applied to improving the salt tolerance of plants.

The fourth concrete implementation mode: the coding gene PsnPI-PLC of the populus euphratica phosphatidylinositol-specific phospholipase C is applied to breeding salt-tolerant transgenic plants.

The embodiment is applied to salt-tolerant breeding of forest trees.

The effect of the invention was verified by the following experiments:

example 1:

1. cloning of Poplar PI-PLC Gene

Using Populus tremula cDNA sequence as a template, based on Populus PI-PLCORF sequence information in JGI database (https:// phytozome.jgi.doe.gov/pz/portal.html), a sequence as defined in Seq ID No: 3 and Seq ID No: 4 (orf7-1F and orf7-1R) to amplify the PsnPI-PLC gene fragment.

The PCR system was as follows:

ddH₂o make up the volume 10. mu.L.

The reaction program is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, renaturation at 52 ℃ for 30s, extension at 72 ℃ for 2min, 30 cycles; and (3) extending for 10min at 72 ℃, and recovering the target fragment by using a gel recovery kit.

Connecting the recovered gene fragment to a pMD19-T vector, transforming the connection product into escherichia coli competent cells, and picking single clones to be respectively expressed by Seq ID No: 5 and Seq ID No: pMD19-T universal primers M13+ and M13-shown in 6-were used for PCR in the following PCR system:

ddH₂o make up the volume 10. mu.L.

The reaction program is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, renaturation at 52.1 ℃ for 30s, extension at 72 ℃ for 2min, 30 cycles; and (3) extending at 72 ℃ for 10min, and sequencing and verifying the obtained specific fragment to finally obtain the sequence shown in Seq ID No: 1, and the sequence of the poplar PI-PLCcDNA.

2. Poplar PI-PLC gene structure domain verification and sequence analysis

The overall length of the poplar PI-PLCcDNA sequence obtained in the embodiment is 1764bp, and the code has the sequence shown in the sequence table as Seq ID No: 2 in the presence of a protease. The molecular weight of the protein is 66.6KDa and the theoretical isoelectric point is 5.82 as a result of analyzing related parameters of the protein by using ProtParam software (https:// web. expasy. org/ProtParam /). The conserved region of the protein is analyzed by the function of CD-search (https:// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb. cgi) of NCBI, the analysis result is shown in figure 1, the obtained poplar PI-PLC gene comprises a conserved PLC structural domain and belongs to phosphatidylinositol-specific phospholipase C encoding gene; the result of InterPro (http:// www.ebi.ac.uk/InterPro /) analysis shows that the poplar PI-PLC gene encoding protein obtained in the embodiment comprises EF hand type domain, X domain, Y domain and C2 domain (figure 2); these characteristics are in line with the basic characteristics of the phosphatidylinositol-specific phospholipase C family, and a new member of the phosphatidylinositol-specific phospholipase C gene family is obtained by preliminary determination and is named as PsnPI-PLC.

The NCBI Blast program (https:// Blast. NCBI. nlm. nih. gov/Blast. cgi) was used for homology sequence search, 11 other plant PI-PLC amino acid sequences were selected and subjected to multiple sequence alignment by mega software analysis.

The selected sequences are as follows:

AtPLC1 NP_568881；AtPLC2 NP_001030660；AtPLC3 NP_195565；AtPLC8 NP_001327564；AtPLC9 NP_190306；ZmPLC1 AAS45137；SlPLC4 NP_001234181；GmPLC12 XP_006601953；OsPLC1 XP_015646464；OsPLC2 XP_015628700；OsPLC3 XP_015618103。

the PLC protein sequences are respectively from plants such as arabidopsis thaliana and rice, and the multi-sequence alignment result is shown in figure 3, so that the homology between the PsnPI-PLC and the plant PLC is not high, but the selected genes have the conserved structural characteristics of the PI-PLC. At the same time, the differences between different PI-PLCs within the same species are significant. Although OsPLC1 and OsPLC3 are reported to participate in plant salt tolerance regulation, EF hand type domain can be determined to be deleted through conservative domain analysis, so that compared with Psn PI-PLC, the structure of the plant salt tolerance regulation is greatly different, and no relevant report exists at present aiming at the research of similar proteins with Psn PI-PLC in Blast results in the plant salt tolerance field.

Example 2: construction of poplar PsnPI-PLC gene overexpression vector

Designing a sequence shown in Seq ID No: 7 and Seq ID No: 8, cloning PsnPI-PLC gene by taking populus tremuloides cDNA as a template and using specific primers 7-1F-Sma I and 7-1R-Sac I as shown in the specification, wherein the reaction system is as follows:

ddH₂o make up the volume 10. mu.L.

The reaction program is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, renaturation at 60.5 ℃ for 30s, extension at 72 ℃ for 2min, 30 cycles; extension at 72 ℃ for 10min, recovery of amplified fragments using gel recovery kit, double cleavage of PCR product and pROKII vector with Sma I and Sac I, recovery of the products separately and ligation using T4 ligase, transformation of E.coli competent cells using a DNA fragment as described in Seq ID No: 7 and Seq ID No: 8, carrying out PCR verification on specific primers 7-1F-Sma I and 7-1R-Sac I, submitting to sequencing, wherein the sequencing primers are universal primers 35S and M13F, the positive clone is named as pROKII-PsnPLC, extracting a plasmid to transform an agrobacterium strain EHA105, and the constructed pROKII-PsnPLC overexpression agrobacterium strain is frozen at-80 ℃ for later use.

Example 3: obtaining of tobacco strain transformed with PsnPI-PLC gene and analysis of plant salt tolerance

The pROKII-PsnPLC overexpression vector in example 2 was used for transforming tobacco by the Agrobacterium-mediated leaf disc method, transgenic and non-transgenic tobacco T2 seeds were sterilized and sown in MS medium containing 0 and 150mM NaCl, transgenic and non-transgenic tobacco seedlings with 3-5d of the same size after germination were transferred to subculture medium containing 0 and 150mM NaCl and cultured using leaf DNA as a template using a DNA sequence such as Seq ID No: 9 and Seq ID No: 10, carrying out PCR verification on the specific primers p7-1F and p7-1R, determining positive plants through resistance screening and molecular detection, and selecting a wild type (ck) and three groups of transgenic lines (named as p10, p20 and p21 respectively, wherein each group has 6 biological repeats) to measure physiological and biochemical indexes of the plants after culturing the plants in a subculture medium for 30 days.

The determination of the physiological index includes: plant height, POD content, SOD content, MDA content, and proline content. Wherein POD content measurement uses POD determination kit produced by Nanjing institute of built bioengineering; pro content determination adopts a proline content kit produced by Suzhou Keming biotechnology limited; SOD content and MDA content determination adopts MDA detection kit and total SOD activity detection kit (NBT method) produced by Biyunnan biotechnology.

FIG. 4 shows the results of measurements of transgenic (p10, p20, p21) and non-transgenic (ck) tobacco plant heights under normal growth and 150mM NaCl conditions, wherein the transgenic plant and the ck plant have no significant difference under the normal growth conditions, the plant heights of all the plants under salt stress conditions are significantly lower than the normal growth conditions, and the plant heights of the transgenic plants are slightly higher than the ck, wherein the p20 plant height is 2 times that of the ck (p <0.01), and the p21 plant height is 1.66 times that of the ck (p <0.05), so that compared with the normal conditions, the growth of all the tobacco plants is affected by salt stress, but the transgenic tobacco is significantly less than the ck.

Salt stress causes the generation of active oxygen in cells, and Peroxidase (POD) and superoxide dismutase (SOD) can eliminate free radicals and reduce the damage of the cells caused by the salt stress. FIGS. 5 and 6 show the results of measurements of transgene and ckPOD and SOD content, respectively, where POD has no significant difference among the lines under normal conditions, and each transgenic line is significantly higher than ck after salt stress treatment; the SOD detection method is slightly changed, the reagent of the reaction system is halved, and the SOD enzyme activity in the reaction system is defined as 1 activity unit when the inhibition percentage in the xanthine oxidase coupling reaction is 50%. After salt stress treatment, the SOD content of each transgenic line is higher than ck. Therefore, the free radical scavenging capacity of the transgenic line is higher than that of ck, and the transgenic line has better salt tolerance.

The Malondialdehyde (MDA) content reflects the degree of damage caused by peroxidation of cell membrane lipids, and FIG. 7 shows that the MDA content of each transgenic line is lower than that of ck, wherein p10 is 0.41 times (p <0.01), p20 is 0.51 times (p <0.01) and p21 is 0.47 times (p <0.01) that of ck, as measured under 150mM NaCl. This shows that the damage of transgenic line is better than that of ck, and has better stress tolerance.

Small molecular substances such as proline (Pro) and soluble sugar can be used as osmoregulation substances to improve the salt tolerance of plants, and fig. 8 shows the determination results of the contents of transgenes and ck Pro under normal growth and 150mM NaCl conditions, the transgenic lines have no obvious difference from ck under normal conditions, and the Pro content of the transgenic lines is higher than ck after salt stress treatment.

In conclusion, the transgenic tobacco physiological index analysis is adopted to determine that the salt tolerance of plants can be obviously improved after the overexpression of the Populus tremula PI-PLC gene, and the transgenic tobacco PI-PLC gene has a good application prospect in the field of salt tolerance breeding of forest trees.

Sequence listing

<110> high technical research institute of academy of sciences of Heilongjiang province

<120> Populus tremuloides phosphatidylinositol specific phospholipase C coding gene PsnPI-PLC and application thereof

<160> 10

<210> 1

<211> 1764

<212> DNA

<213> Populus X of Populus tremula

<400> 1

atgtctaaac agacctacag agtttgcttg tgctttagca ggaggttcaa gcttgcagtg 60

gcagaggcac cggaggagat cagggctttg tttaatcaat actcggataa tggcatcatg 120

accgacagtc atctccacag gttcttgatc gaggctcaaa aacaagaaaa ggctactctc 180

gaggaagcac aagccatcat cgaaagcctc aaacatttgg ccatttttca ccgtaaaggg 240

ctcaatcttg aggccttttt taagtatctt tttggtgata ataaccctcc tcttgatctt 300

aaacttgggg cgcaccatga tatgacagct cccatttcac actatttcat ttataccggc 360

cacaattcat atctaactgg gaatcagccc agtagcgatt gcagcgatgt tcccatcata 420

aatgcactga agaaagatgt gagagtaatt gaattggata tatggccaaa ttccgacaag 480

gatgatgtgg aggtacttca tgggaggact ttgacaactc cggtgcaact tatcaaatgt 540

ttgaggtcaa tcaaggagca tgcttttact gcatctgaat tccctgttgt tataactcta 600

gaagatcacc ttactccaga tctccagtct aaagtgtcta agatggttac tcaaacattt 660

ggagacacac tgttttctcc tggctcggaa tgcttgaagg aattcccttc ccctgagtca 720

ttgaagagac gtattatcat atcaacaaaa ccaccgaagg agtaccttga ggcaaaggaa 780

attaaggata aagagagtga ttcccagaag ggtaatgttg ctcccgatga agaagcttgg 840

gggaaagaaa tccttaatct taaaggcgct gatgacaaga atgaattgga cgacgatgat 900

aatgatgccg aggaagatcc tggtgaagga gaccacaagt tgccgcatga tatagcacca 960

gaatataaac gtttaattgc tattcctgct gggaagccta aaggtggatt agaagaatgt 1020

ttgaaagagg attctgataa agcgaggcgt cttagcttaa gcgagcaaca acttgaaaat 1080

gctgcagaga cccatggaaa agaaattgtc aggtttaccc agcggaatat actcagggtg 1140

tatccaaagg gtattcgtgt gaactcatcc aactacaacc cactaattgg atggatgcat 1200

ggtgctcaaa tggttgcttt taatatgcag ggatatggaa gatcgctctg gatgatgcaa 1260

ggaatgttcg gagccaatgg tggttgtggt tttgtgaaga aaccggattt tcttctgaag 1320

tctggtcctc atggcgaggt atttgatccc aaagccaagt tacctgtgca aaaaactttg 1380

aaggtgaaaa tatacatggg tgaaggatgg tattatgatt tccatcacac acattttgat 1440

gcatattccc caccagattt ctatgtgagg gttgggattg ctggggtccc tgcagatact 1500

gggatgaaga aaacaagaac tctggaggac aattggatac ctgtttggga tgaggagttt 1560

gagtttccat taactgttcc agctctagct ctgctccgga ttgaagttca tgagtatgac 1620

atgtcagaaa aggatgactt tggtggtcaa acatgccttc ctgtgcggga gttgagagaa 1680

gggattcgag cagttccact ccatgaccgc aagggagaga aatacaattc tgtaaagctc 1740

cttgtgcgtc tcgaatttgt ttga 1764

<210> 2

<211> 587

<212> PRT

<213> Populus X of Populus tremula

<400> 2

Met Ser Lys Gln Thr Tyr Arg Val Cys Leu Cys Phe Ser Arg Arg

1 5 10 15

Phe Lys Leu Ala Val Ala Glu Ala Pro Glu Glu Ile Arg Ala Leu

20 25 30

Phe Asn Gln Tyr Ser Asp Asn Gly Ile Met Thr Asp Ser His Leu

35 40 45

His Arg Phe Leu Ile Glu Ala Gln Lys Gln Glu Lys Ala Thr Leu

50 55 60

Glu Glu Ala Gln Ala Ile Ile Glu Ser Leu Lys His Leu Ala Ile

65 70 75

Phe His Arg Lys Gly Leu Asn Leu Glu Ala Phe Phe Lys Tyr Leu

80 85 90

Phe Gly Asp Asn Asn Pro Pro Leu Asp Leu Lys Leu Gly Ala His

95 100 105

His Asp Met Thr Ala Pro Ile Ser His Tyr Phe Ile Tyr Thr Gly

110 115 120

His Asn Ser Tyr Leu Thr Gly Asn Gln Pro Ser Ser Asp Cys Ser

125 130 135

Asp Val Pro Ile Ile Asn Ala Leu Lys Lys Gly Val Arg Val Ile

140 145 150

Glu Leu Asp Ile Trp Pro Asn Ser Asp Lys Asp Asp Val Glu Val

155 160 165

Leu His Gly Arg Thr Leu Thr Thr Pro Val Gln Leu Ile Lys Cys

170 175 180

Leu Arg Ser Ile Lys Glu His Ala Phe Thr Ala Ser Glu Phe Pro

185 190 195

Val Val Ile Thr Leu Glu Asp His Leu Thr Pro Asp Leu Gln Ser

200 205 210

Lys Val Ser Lys Met Val Thr Gln Thr Phe Gly Asp Thr Leu Phe

215 220 225

Ser Pro Gly Ser Glu Cys Leu Lys Glu Phe Pro Ser Pro Glu Ser

230 235 240

Leu Lys Arg Arg Ile Ile Ile Ser Thr Lys Pro Pro Lys Glu Tyr

245 250 255

Leu Glu Ala Lys Glu Ile Lys Asp Lys Glu Ser Asp Ser Gln Lys

260 265 270

Gly Asn Val Ala Pro Asp Glu Glu Ala Trp Gly Lys Glu Ile Leu

275 280 285

Asn Leu Lys Gly Ala Asp Asp Lys Asn Glu Leu Asp Asp Asp Asp

290 295 300

Asn Asp Ala Glu Glu Asp Pro Gly Glu Gly Asp His Lys Leu Pro

305 310 315

His Asp Ile Ala Pro Glu Tyr Lys Arg Leu Ile Ala Ile Pro Ala

320 325 330

Gly Lys Pro Lys Gly Gly Leu Glu Glu Cys Leu Lys Glu Asp Pro

335 340 345

Asp Lys Ala Arg Arg Leu Ser Leu Ser Glu Gln Gln Leu Glu Asn

350 355 360

Ala Ala Glu Thr His Gly Lys Glu Ile Val Arg Phe Thr Gln Arg

365 370 375

Asn Ile Leu Arg Val Tyr Pro Lys Gly Ile Arg Val Asn Ser Ser

380 385 390

Asn Tyr Asn Pro Leu Ile Gly Trp Met His Gly Ala Gln Met Val

395 400 405

Ala Phe Asn Met Gln Gly Tyr Gly Arg Ser Leu Trp Met Met Gln

410 415 420

Gly Met Phe Gly Ala Asn Gly Gly Cys Gly Phe Val Lys Lys Pro

425 430 435

Asp Phe Leu Leu Lys Ser Gly Pro His Gly Glu Val Phe Asp Pro

440 445 450

Lys Ala Lys Leu Pro Val Gln Lys Thr Leu Lys Val Lys Ile Tyr

455 460 465

Met Gly Glu Gly Trp Tyr Tyr Asp Phe His His Thr His Phe Asp

470 475 480

Ala Tyr Ser Pro Pro Asp Phe Tyr Val Arg Val Gly Ile Ala Gly

485 490 495

Val Pro Ala Asp Thr Gly Met Lys Lys Thr Arg Thr Leu Glu Asp

500 505 510

Asn Trp Ile Pro Val Trp Asp Glu Glu Phe Glu Phe Pro Leu Thr

515 520 525

Val Pro Ala Leu Ala Leu Leu Arg Ile Glu Val His Glu Tyr Asp

530 535 540

Met Ser Glu Lys Asp Asp Phe Gly Gly Gln Thr Cys Leu Pro Val

545 550 555

Arg Glu Leu Arg Glu Gly Ile Arg Ala Val Pro Leu His Asp Arg

560 565 570

Lys Gly Glu Lys Tyr Asn Ser Val Lys Leu Leu Val Arg Leu Glu

575 580 585

Phe Val

587

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence of PCR primer orf 7-1F.

<400> 3

gacaagacac gaacaacact c 21

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence of PCR primer orf 7-1R.

<400> 4

caacaaatgg acttcacaga t 21

<210> 5

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence of PCR primer M13 +.

<400> 5

gtaaaacgac ggccag 16

<210> 6

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence of PCR primer M13-.

<400> 6

caggaaacag ctatgac 17

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence of PCR primer 7-1F-Sma I.

<400> 7

tcccccggga cgaacaacac t 21

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence of PCR primer 7-1R-Sac I.

<400> 8

cgagctccaa caaatggact tcaca 25

<210> 9

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence of PCR primer p 7-1F.

<400> 9

aactcatcca actacaaccc acta 24

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence of PCR primer p 7-1R.

<400> 10

agagtccttg ttttcttcat ccc 23

Claims

1. The populus euphratica phosphatidylinositol specific phospholipase C coding gene PsnPI-PLC is characterized in that the nucleotide sequence of the gene is shown in a sequence table Seq ID No: 1 is shown.

2. The populus tremuloides phosphatidylinositol-specific phospholipase C encoding gene PsnPI-PLC according to claim 1, wherein the amino acid sequence of the protein encoded by the gene is shown in the sequence table Seq ID No: 2, respectively.

3. The use of the cottonwood phosphatidylinositol-specific phospholipase C encoding gene PsnPI-PLC according to claim 1, wherein the cottonwood phosphatidylinositol-specific phospholipase C encoding gene PsnPI-PLC is used for improving salt tolerance of plants.

4. The use of the Populus nigra phosphatidylinositol-specific phospholipase C encoding gene PsnPI-PLC as claimed in claim 1, wherein the Populus nigra phosphatidylinositol-specific phospholipase C encoding gene PsnPI-PLC is used for breeding salt tolerant transgenic plants.