CN115354035B

CN115354035B - Parthenium schneiderianum phenylalanine ammonia lyase SgPAL2 and encoding gene and application thereof

Info

Publication number: CN115354035B
Application number: CN202210462546.3A
Authority: CN
Inventors: 陈志坚; 李季肤; 邹晓燕; 李欣勇; 董荣书; 刘国道
Original assignee: Tropical Crops Genetic Resources Institute CATAS
Current assignee: Tropical Crops Genetic Resources Institute CATAS
Priority date: 2022-04-28
Filing date: 2022-04-28
Publication date: 2023-07-04
Anticipated expiration: 2042-04-28
Also published as: CN115354035A

Abstract

The invention discloses a stylosanthes guianensis phenylalanine ammonia lyase SgPAL2 and a coding gene and application thereof. The invention clones a phenylalanine ammonia lyase gene SgPAL2 with the expression level which is highly matched with the response process of the stylosanthes guianensis to the manganese toxicity, and proves that the expression of the SgPAL2 can promote the tolerance of transgenic arabidopsis to the manganese toxicity through a transgenic arabidopsis expression system, and simultaneously proves that the action mechanism of the gene is that the tolerance of plants to the manganese toxicity is improved through reducing the accumulation of manganese in plants, so that the gene can be used for preparing transgenic plants resistant to the manganese toxicity. The invention not only enriches the gene library of the plant resistant to the manganese poison, but also is beneficial to the cultivation of the crop variety resistant to the manganese poison.

Description

Parthenium schneiderianum phenylalanine ammonia lyase SgPAL2 and encoding gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering. More particularly relates to a stylosanthes guianensis phenylalanine ammonia lyase SgPAL2 and a coding gene and application thereof.

Background

Manganese (Mn) is one of the trace nutrient elements necessary for plant growth and development, and participates in a series of physiological and biochemical processes such as photosynthesis, respiration, protein synthesis and the like in plants. However, manganese is also one of heavy metal elements, and excessive accumulation of manganese due to soil pollution and the like can produce toxic effects on plants, and manganese toxicity is typically characterized in that brown spots, sallow and necrosis appear on mature leaves, and finally plant growth can be inhibited.

Phenylalanine ammonia lyase (Phenylalanine ammonia-lyase, PAL) is the rate-limiting enzyme of the plant phenylpropane metabolic pathway. The secondary metabolites such as phenols, flavonoids, lignin and the like formed by the phenylalanine through the conversion of the phenylpropane metabolic pathway play a key role in plant growth and development, disease resistance and stress resistance. It was found that metallomanganese stress affects plant phenylpropane metabolic pathways, thereby altering secondary metabolite content. For example, cowpea leaf phenolics and callose content increased significantly under excess manganese treatment conditions; similarly, lignin and flavonoid compounds in the rice leaves are increased under the treatment of excessive manganese, and the phenylalanine content in the poplar leaves is increased under the treatment of excessive manganese, so that the poison of manganese to plant bodies is relieved. At present, in the research on the relation of phenylalanine ammonia lyase and plant manganese toxic stress, most of the researches on whether phenylalanine ammonia lyase genes participate in alleviating plant metal manganese toxic stress at physiological level are not explicitly reported.

The coltsfoot (Stylosanthes guianensis) is a perennial leguminous herb, is native to Latin America, can be used as livestock forage grass, is used for intercropping of orchards, acid soil improvement and the like, and is excellent leguminous forage grass widely planted in tropical and subtropical areas. In the long-term natural evolution and artificial breeding process, the stylosanthes guianensis have stronger capability of adapting to the toxicity of metal manganese and aluminum in acid soil, which is possibly related to the in-vivo gene regulation and control, but still does not dig specific genes. Therefore, the related genes of the digging column flowers and plants adapting to the metal manganese toxicity stress have important significance for cultivating new varieties of crops with manganese toxicity resistance.

Disclosure of Invention

The invention aims to overcome the defect that the existing protein or gene which can not improve the manganese toxicity tolerance capability of plants is not available, and provides a stylosanthes guianensis phenylalanine ammonia lyase SgPAL2, and a coding gene and application thereof.

The first object of the invention is to provide a stylosanthes guianensis phenylalanine ammonia lyase SgPAL2.

The second object of the invention is to provide a coding gene of the stylosanthes guianensis phenylalanine ammonia lyase SgPAL2.

It is a third object of the present invention to provide a recombinant expression vector.

The fourth object of the invention is to provide a recombinant engineering bacterium.

The fifth object of the invention is to provide the application of the P.stylosa phenylalanine ammonia lyase SgPAL2, the coding gene, the recombinant expression vector or the recombinant engineering bacteria in improving the manganese toxicity tolerance of plants.

The sixth object of the invention is to provide the application of the stylosanthes guianensis phenylalanine ammonia lyase SgPAL2, the coding gene, the recombinant expression vector or the recombinant engineering bacteria in preparing transgenic plants resistant to manganese poisoning.

The seventh object of the invention is to provide the application of the stylosanthes guianensis phenylalanine ammonia lyase SgPAL2, the coding gene, the recombinant expression vector or the recombinant engineering bacteria in reducing plant manganese accumulation.

The above object of the present invention is achieved by the following technical scheme:

the invention provides a stylosanthes guianensis phenylalanine ammonia lyase SgPAL2, the amino acid sequence of which is shown as SEQ ID NO. 2.

The invention also provides a coding gene of the phenylalanine ammonia lyase SgPAL2.

Specifically, the nucleotide sequence of the coding gene is shown as SEQ ID NO.1, and is 2076bp long.

The invention also provides a cloning primer of the P.stylosa phenylalanine ammonia lyase gene SgPAL2 shown in SEQ ID NO.1, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, and the sequence of the downstream primer is shown in SEQ ID NO. 4.

The invention also provides a cloning method of the colpitis stylosa phenylalanine ammonia lyase gene SgPAL2 shown in SEQ ID NO.1, namely, the colpitis stylosa cDNA is used as a template, and primers shown in SEQ ID NO.3 and 4 are used for amplification.

The invention also provides a recombinant expression vector which contains the coding gene of the phenylalanine ammonia lyase SgPAL2.

Specifically, the expression vector is an over-expression vector.

More specifically, the overexpression vector is pTF101s.

The invention also provides a recombinant engineering bacterium which contains the recombinant expression vector.

Specifically, the engineering bacteria are escherichia coli Trans ₁ T or Agrobacterium competent cell GV3101.

The invention discloses a method for over-expressing a stylosanthes guianensis phenylalanine ammonia lyase gene SgPAL2 shown in SEQ ID NO.1 in plants by a transgenic technology, which is characterized in that compared with a wild type, the transgenic plant over-expressing the SgPAL2 has obviously enhanced tolerance to manganese poisoning, and the gene is proved to enhance the tolerance to manganese poisoning by reducing the accumulation of manganese in the plant.

Therefore, the invention provides the application of the stylosanthes guianensis phenylalanine ammonia lyase SgPAL2, the coding gene of the stylosanthes guianensis phenylalanine ammonia lyase SgPAL2, the recombinant expression vector or the recombinant engineering bacteria in improving the manganese toxicity tolerance capability of plants.

In particular, the improvement of the manganese toxicity tolerance of plants is achieved by expressing the stylosanthes guianensis phenylalanine ammonia lyase SgPAL2 in plants.

The invention also applies for protecting the stylosanthes guianensis phenylalanine ammonia lyase SgPAL2, the coding gene of the stylosanthes guianensis phenylalanine ammonia lyase SgPAL2, the recombinant expression vector or the application of the recombinant engineering bacteria in preparing transgenic plants resistant to manganese poisoning.

The invention also applies for protecting the stylosanthes guianensis phenylalanine ammonia lyase SgPAL2, the coding gene of the stylosanthes guianensis phenylalanine ammonia lyase SgPAL2, and the application of the recombinant expression vector or the recombinant engineering bacteria in reducing plant manganese accumulation.

In particular, the plant is a dicot.

More specifically, the dicotyledonous plant is a plant of the genus coltsfoot or Arabidopsis.

The invention has the following beneficial effects:

the invention clones a phenylalanine ammonia lyase gene SgPAL2 with the expression level which is highly matched with the response process of the stylosanthes guianensis to the manganese toxicity, and proves that the expression of the SgPAL2 can promote the tolerance of transgenic arabidopsis to the manganese toxicity through a transgenic arabidopsis expression system, and simultaneously proves that the action mechanism of the gene is that the tolerance of plants to the manganese toxicity is improved through reducing the accumulation of manganese in plants, so that the gene can be used for preparing transgenic plants resistant to the manganese toxicity. The invention not only enriches the gene library of the plant resistant to the manganese poison, but also is beneficial to the cultivation of the crop variety resistant to the manganese poison.

Drawings

FIG. 1 shows the effect of manganese treatments of different metals and different concentrations on the expression of the SgPAL2 gene in the aerial parts of the flowers and plants; wherein, the graph A is the effect result of different metal treatments on the expression of SgPAL2 genes; FIG. B shows the effect of manganese treatment at various concentrations on the expression of the SgPAL2 gene; data in the figures are mean and standard error of 3 replicates, with the signs indicating significant differences between treatment and control, P <0.05.

FIG. 2 shows subcellular localization results of the SgPAL2 gene; the first column in the figure shows GFP fluorescence signal, the second column shows chloroplast autofluorescence signal, the third column shows bright field, and the fourth column shows fusion result of GFP and chloroplast autofluorescence signal, and the scale is 20 μm.

FIG. 3 is an overexpression ofThe effect of the SgPAL2 gene on the manganese poisoning tolerance of arabidopsis thaliana; wherein, FIG. A shows the results of phenotypic analysis of over-expressed Arabidopsis lines and wild type Arabidopsis under different concentrations of manganese treatment, 0.1mM control treatment, 2mM and 4mM MnSO ₄ For the manganese excess treatment, WT is the wild type strain, OE1/OE2 is the over-expressed strain, scale bar 2cm; panel B shows the results of a comparison of the biomass of the over-expressed Arabidopsis strain and the wild type Arabidopsis, with 4 biological replicates per treatment in the experiment, and the columns in the figure show the mean and standard error of the 4 biological replicates; panel C shows the comparison of manganese concentrations in over-expressed Arabidopsis lines and wild Arabidopsis under different manganese concentration treatment conditions; in the figure, the numbers indicate significant differences between the over-expressed Arabidopsis lines and wild Arabidopsis, P<0.05。

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 cloning of the Symphytum officinale phenylalanine ammonia-lyase Gene SgPAL2

1. Cloning of the phenylalanine ammonia lyase Gene SgPAL2 of Symphytum officinale

The nucleotide sequence of the primer for cloning the P.stylosa phenylalanine ammonia lyase gene SgPAL2 is as follows:

an upstream primer: 5'-ATGGCAGCCATTCATCCACGTGA-3' (SEQ ID NO. 3)

Downstream primer 5'-TAAACTGAGATGGGAACCCCGTT-3' (SEQ ID NO. 4)

The cloning method of the columna phenylalanine ammonia lyase gene SgPAL2 comprises the following steps:

extracting total RNA of the overground part of the thermal-ground No.5 stylosanthes guianensis by referring to the instruction book of TRNzol Universal total RNA extraction reagent (TIANGEN), then synthesizing a first strand of cDNA according to a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher) method, taking the obtained cDNA as a template, and carrying out PCR amplification by using the primer for cloning the stylosanthes guianensis phenylalanine ammonia lyase gene SgPAL2.

The PCR reaction system (20. Mu.L) was: 2. Mu.L of 10 XEx Taq Buffer, 1.6. Mu.L of dNTP mix, 1. Mu.L of primer (10. Mu.M), 0.12. Mu.L of Ex Taq, 1. Mu.L of cDNA template, and ddH ₂ O makes up 20. Mu.L; the reaction procedure is: 94℃1min,94℃30s,58℃30s,72℃120s, 35 cycles, 72℃extension for 10 minutes.

After the PCR amplification was completed, a 1% agarose gel (containing Golden view nucleic acid dye stain) was prepared, 6. Mu.L of the PCR product was added to 1. Mu.L of a 6×loading buffer for electrophoresis detection, and imaged on a gel imaging system. The PCR product was recovered to 2076bp in length using Sanprep column DNA gel recovery kit (Bio-technology).

Cloning the recovered PCR product onto a Trans-T1 (full-formula gold company) carrier for sequencing and identifying to obtain the full-length cDNA of the P.stylosa phenylalanine ammonia lyase gene SgPAL2, wherein the nucleotide sequence is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2.

EXAMPLE 2 subcellular localization and expression analysis of the phenylalanine ammonia lyase Gene SgPAL2 of P.stylosa

1. Construction of vectors

(1) Construction of the overexpression vector:

the ORF fragment (2076 bp) of the SgPAL2 gene was amplified using the aerial part cDNA of the P.stylosa obtained in example 1 as a template with the upstream specific primer 5'-CCGGGGATCCTCTAGAATGGCAGCCATTCATCCACG-3' (SEQ ID NO. 5) and the downstream specific primer 5'-GCAGGTCGACTCTAGATTAAACTGAGATGGGAACCCCG-3' (SEQ ID NO. 6); after the PCR fragment is confirmed to be error-free by sequencing, a target vector is subjected to single enzyme digestion by restriction enzyme Xbal I, sgPAL2 genes are connected to the target vector pTF101s and are transformed into escherichia coli Trans1-T (full gold), sequencing analysis is carried out, and the pTF101s-SgPAL2 recombinant overexpression vector is successfully obtained by sequencing without error.

(2) Construction of subcellular localization analysis expression vector:

using the above-ground portion cDNA of the P.stylosa obtained in example 1 as a template, the ORF fragment of the SgPAL2 gene was amplified using the upstream specific primer 5'-TCTAGCGCTACCGGTATGGCAGCCATTCATCCACG-3' (SEQ ID NO: 7) and the downstream specific primer 5'-ATGGTGGCGACCGGTAACTGAGATGGGAACCCCG-3' (SEQ ID NO: 8), and after the PCR fragment was sequenced without errors, the PCR amplified fragment of the SgPAL2 gene was ligated to the restriction enzyme AgeI single-cut linearization target vector pEGAD to obtain a SgPAL2-GFP fusion expression vector (35S: sgPAL 2-GFP) which was started by the 35S promoter.

2. Expression analysis of SgPAL2 Gene

According to the invention, after the stylosanthes guianensis seeds are treated by using different metals and manganese with different concentrations, the influence of different treatments on the expression of the SgPAL2 genes is observed.

Specifically, selecting seed of thermal grinding No.5 stylosanthes, removing seed coat, heating at 80deg.C for 2min, standing in dark for 2-3 d, and transferring to Hoagland nutrient solution (400 μM NH in culture solution) ₄ NO ₃ 、1500μM KNO ₃ 、500μM MgSO ₄ ·7H ₂ O、250μM KH ₂ PO ₄ 、300μM K ₂ SO ₄ 、1.5μM MnSO ₄ ·4H ₂ O、1μM ZnSO ₄ .7H ₂ O、2.5μM Na ₂ MoO ₄ ·2H ₂ O、0.5μM CuSO ₄ ·5H ₂ O、25μM MgCl ₂ 、40μM Fe-EDTA、1200μM Ca(NO ₃ ) ₂ Pre-incubation for two weeks at pH 5.8) with 800 μm Fe, 400 μm Mn, 20 μm Zn and 10 μm Cu, respectively, and with different concentrations of manganese (0.5, 50, 400 and 800 μm); harvesting the sample after two weeks of treatment; extracting the RNA of the aerial parts of the flowers and plants of each treatment.

The RNA was reverse transcribed into cDNA, and further the expression of SgPAL2 was detected by quantitative PCR, using the housekeeping gene SgEF. Alpha. Of P.stylosa as an internal reference. The primers used for quantitative PCR detection of gene expression levels are shown below, respectively:

the quantitative primers of the SgEF alpha gene of the stylosanthes guianensis are as follows:

SgEFαF：5’-CACTTCAGGACGTGTACAAGATC-3’(SEQ ID NO.9)

SgEFαR：5’-CTTGGAGAGCTTCATGGTGCA-3’(SEQ ID NO.10)

the quantitative primers of the SgPAL2 gene are as follows:

SgPAL2F：5’-AACTCTGTCAACGACAACCC-3’(SEQ ID NO.11)

SgPAL2 R：5’-TGGATGGCAAACCGTTGTTA-3’(SEQ ID NO.12)

the effect of different metals and different concentrations of manganese treatment on the expression of the SgPAL2 gene in the aerial parts of the stylosanthes guianensis is shown in figure 1. Wherein, fig. 1A is the effect of different metal treatments on SgPAL2 gene expression, the data in the figure are the mean and standard error of 3 replicates, the signs indicate significant differences between the treatments and the controls, and P <0.05. As can be seen from the results shown in FIG. 1A, the excessive manganese treatment significantly increased the expression of SgPAL2 in the aerial parts of the flowers and plants compared to the control CK. Under the condition of excessive manganese treatment, the expression quantity of the SgPAL2 of the overground part of the stylosanthes guianensis is 31.8 times of that of the control. Whereas the effect of excessive Fe, zn and Cu treatment on SgPAL2 expression is not obvious. The above results demonstrate that SgPAL2 is specifically induced to express by excessive manganese stress.

Fig. 1B is a graph showing the effect of manganese treatment at different concentrations on SgPAL2 gene expression, the data in the graph are the mean and standard error of 3 replicates, the signs indicate significant differences between the different manganese treatments and the control, and P <0.05. As shown in FIG. 1B, the expression of the SgPAL2 gene of the stylosanthes guianensis tended to increase and decrease in the aerial parts with increasing concentration of manganese treatment, and the expression level was highest at 400. Mu.M manganese treatment and 1.6 times that of the normal manganese treatment (0.5. Mu.M).

3. Subcellular localization analysis of SgPAL2

The constructed SgPAL2-GFP fusion expression vector and pEGAD empty vector are subjected to transient expression in the Arabidopsis protoplast through an agrobacterium-mediated Arabidopsis protoplast expression system, and then GFP fluorescence is observed by a laser confocal microscope (Zeiss), so that subcellular localization of the SgPAL2 is confirmed.

The subcellular localization result of the SgPAL2 gene is shown in FIG. 2, wherein the first column is GFP fluorescence signal, the second column is chloroplast autofluorescence signal, the third column is bright field, the fourth column is fusion result of GFP and chloroplast autofluorescence signal, and the scale is 20 μm. From the results shown in FIG. 2, the P.stylosa SgPAL2 protein is localized in the cytoplasm.

Example 3 transgenic experiments

1. Obtaining transgenic Arabidopsis thaliana

The overexpression vector (pTF 101s-SgPAL 2) constructed in example 2 was transformed into Agrobacterium tumefaciens competent cell GV3101 (Plasmodium only), and Arabidopsis thaliana transformation was performed by Agrobacterium-mediated Arabidopsis inflorescence infection transformation to obtain transgenic Arabidopsis thaliana.

2. Screening of transgenic Arabidopsis thaliana

Taking a proper amount of Arabidopsis seeds, putting the Arabidopsis seeds into a 1.5mL centrifuge tube, adding 1mL of sterile water for soaking for 1min, then sterilizing with 75% alcohol for 1min, washing with sterile water for 3 times, finally adding 10% sodium hypochlorite for sterilizing for 10min, washing with sterile water for 3 times, and continuously inverting the centrifuge tube in the sterilization process of the alcohol and the sodium hypochlorite. After seed sterilization, placing the seeds in an MS (containing 5mg/L glufosinate) culture medium for 7-10 d, selecting arabidopsis thaliana which is tolerant to herbicide and grows normally, culturing the arabidopsis thaliana in matrix soil, and extracting leaf DNA after culturing for two weeks for PCR verification.

20. Mu.L PCR reaction system: 10. Mu.L of 2 XRapid Master Mix, 0.6. Mu.L of downstream primer (10 mol/L), 0.6. Mu.L of upstream primer (10 mol/L), 2. Mu.L of DNA template, and ddH ₂ O to 20. Mu.L; PCR amplification procedure: the pre-denaturation at 95℃for 1min, denaturation at 95℃for 15s, annealing at 58℃for 30s, annealing at 72℃for 120s for 35 cycles, and extension at 72℃for 10min.

The Arabidopsis thaliana, which remains correctly detected, continues to be cultivated until the seeds are mature, at which point the seeds are T1 generation seeds. The T3 generation homozygous transgenic Arabidopsis seeds are obtained by continuous screening for 3 times according to the method. PCR confirms that SgPAL2 gene T3 Arabidopsis seeds are obtained for subsequent experiments.

3. Manganese treatment of transgenic arabidopsis:

for manganese poisoning resistance analysis of transgenic Arabidopsis, sterilized wild type and transgenic Arabidopsis seeds were sown on MS medium containing 0.8% agar for 7d, and seedlings of uniform size were selected and transferred to a medium containing 0.1mM,2mM and 4mM MnSO, respectively ₄ After 7d treatment, plants were harvested and the fresh weight and manganese content of the plants were determined.

The effect of over-expression of the SgPAL2 gene on the manganese poisoning tolerance of Arabidopsis is shown in FIG. 3. Wherein FIG. 3A is an over-expressed Arabidopsis line (i.e., transgenic Arabidopsis lineArabidopsis lines) and wild type Arabidopsis under manganese treatment conditions at different concentrations, 0.1mM control treatment, 2mM and 4mM MnSO ₄ For the manganese excess treatment, WT is the wild type strain, OE1/OE2 is the over-expressed strain, scale 2cm in the figure. As can be seen from the results shown in FIG. 3A, the WT, OE1 and OE2 transgenic lines grew consistently under control of 0.1mM manganese; under 2mM and 4mM excessive manganese treatment, the plant growth conditions of the OE1 and OE2 transgenic lines are obviously better than those of the WT, which shows that the excessive expression of SgPAL2 can relieve the metal manganese poison of the Arabidopsis and improve the tolerance of the Arabidopsis to the manganese poison.

Fig. 3B is a comparison of the biomass of the overexpressed arabidopsis strain and the wild-type arabidopsis, 4 biological replicates per treatment in the experiment, the mean and standard error of the 4 biological replicates in the column, the number indicates significant differences between the overexpressed strain and the wild-type control strain, P <0.05. As can be seen from the results shown in FIG. 3B, the fresh weight difference between the overexpressed SgPAL2 transgenic lines (OE 1 and OE 2) and the WT wild-type was not apparent under the control 0.1mM treatment conditions; while under 2mM and 4mM manganese treatments, the fresh weight of OE1 and OE2 plants was significantly higher than that of wild-type plants. Under 2mM excess manganese treatment, the fresh weights of OE1 and OE2 plants were 1.38 and 1.4 times that of WT, respectively; under 4mM manganese treatment, the fresh weights of OE1 and OE2 plants were 2.1 and 1.8 times that of WT, respectively.

FIG. 3C is a comparison of manganese concentrations in over-expressed Arabidopsis lines and wild type Arabidopsis under different manganese concentration treatment conditions, where WT is wild type Arabidopsis, OE1/OE2 is over-expressed, where the number indicates significant differences between over-expressed Arabidopsis lines and wild type Arabidopsis, and P <0.05. From the results shown in FIG. 3C, it can be seen that over-expression of SgPAL2 significantly reduced the manganese concentration in Arabidopsis plants compared to wild type Arabidopsis WT at 2mM and 4mM manganese superphosphate treatment.

Taken together, the results show that over-expression of the SgPAL2 gene in arabidopsis thaliana can increase the tolerance of arabidopsis thaliana to manganese poisoning, which is to increase the tolerance of plants to manganese by reducing the accumulation of manganese in the plants.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Sequence listing

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Glu Pro Asp Pro Leu Gln Lys Pro Lys Lys Asp Arg Tyr Ala Leu Gln

325 330 335

Thr Ser Pro Gln Trp Leu Gly Pro Gln Ile Glu Val Ile Arg Tyr Ser

340 345 350

Thr Lys Ser Ile Glu Arg Glu Ile Asn Ser Val Asn Asp Asn Pro Leu

355 360 365

Ile Asp Val Thr Ser Asn Lys Ala Leu Asn Gly Gly Asn Phe Gln Gly

370 375 380

Thr Pro Ile Gly Val Ser Met Asp Asn Ala Arg Leu Ala Val Ala Ser

385 390 395 400

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

405 410 415

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

420 425 430

Leu Asp Tyr Gly Phe Lys Ala Ser Glu Val Ala Met Ala Ala Tyr Cys

435 440 445

Ser Glu Leu Gln Tyr Leu Ala Asn Pro Val Thr Ser His Val Gln Ser

450 455 460

Ala Glu Gln His Asn Gln Asp Val Asn Ser Leu Gly Leu Ile Ser Ala

465 470 475 480

Trp Lys Thr Val Glu Ala Val Glu Ile Leu Lys Leu Met Cys Ser Thr

485 490 495

Tyr Leu Val Ala Leu Cys Gln Ala Ile Asp Leu Arg His Leu Glu Glu

500 505 510

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

515 520 525

Thr Leu Ser Thr Asp Leu Phe Gly Ser Cys Glu Arg Asp Leu Leu Lys

530 535 540

Val Val Asp Arg Glu Tyr Val Phe Ser Tyr Ile Asp Asp Pro Phe Asn

545 550 555 560

Val Arg Tyr Pro Leu Met Pro Lys Leu Lys Gln Ile Leu Tyr Glu Glu

565 570 575

Ala His Ala Gly Ala Ile Ser Glu Lys Asn Thr Ser Val Leu Val Phe

580 585 590

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

595 600 605

Glu Val Glu Ser Ala Arg Val Ala Tyr Glu Asn Gly Asn Pro Thr Leu

610 615 620

Pro Asn Arg Ile Gln Glu Ser Arg Ser Tyr Pro Leu Tyr Lys Phe Val

625 630 635 640

Arg Glu Glu Leu Gly Val Gly Leu Leu Thr Gly Glu Lys Thr Ala Ser

645 650 655

Pro Asp Glu Glu Phe Glu Lys Val Phe Thr Ala Ile Cys Glu Gly Lys

660 665 670

Ile Phe Asp Ser Ile Leu Glu Cys Phe Lys Gln Trp Asn Gly Val Pro

675 680 685

Ile Ser Val

690

<210> 3

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atggcagcca ttcatccacg tga 23

<210> 4

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

taaactgaga tgggaacccc gtt 23

<210> 5

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

ccggggatcc tctagaatgg cagccattca tccacg 36

<210> 6

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

gcaggtcgac tctagattaa actgagatgg gaaccccg 38

<210> 7

<211> 35

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

tctagcgcta ccggtatggc agccattcat ccacg 35

<210> 8

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

atggtggcga ccggtaactg agatgggaac cccg 34

<210> 9

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

cacttcagga cgtgtacaag atc 23

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

cttggagagc ttcatggtgc a 21

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

aactctgtca acgacaaccc 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

tggatggcaa accgttgtta 20

Claims

1. The colpitis phenylalanine ammonia lyase SgPAL2 is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.

2. A gene encoding phenylalanine ammonia lyase SgPAL2 according to claim 1.

3. The coding gene according to claim 2, wherein the nucleotide sequence is shown in SEQ ID NO. 1.

4. A recombinant expression vector comprising the gene encoding phenylalanine ammonia-lyase SgPAL2 according to claim 2 or 3.

5. A recombinant engineering bacterium comprising the recombinant expression vector of claim 4.

6. The use of the stylosanthes guianensis phenylalanine ammonia lyase SgPAL2 of claim 1, the coding gene of claim 2 or 3, the recombinant expression vector of claim 4 or the recombinant engineering bacterium of claim 5 for improving the manganese toxicity tolerance of plants, characterized in that the plants are arabidopsis thaliana or stylosanthes guianensis; the improvement of the manganese toxicity tolerance of the plants is realized by expressing the stylosanthes guianensis phenylalanine ammonia lyase SgPAL2 in the plants.

7. The use of the stylosanthes guianensis phenylalanine ammonia lyase SgPAL2 according to claim 1, the coding gene according to claim 2 or 3, the recombinant expression vector according to claim 4 or the recombinant engineering bacterium according to claim 5 for preparing transgenic plants resistant to manganese poisoning, characterized in that the plants are arabidopsis thaliana or stylosanthes guianensis.

8. Use of the argininoacetamide ammonia lyase SgPAL2 according to claim 1, the coding gene according to claim 2 or 3, the recombinant expression vector according to claim 4 or the recombinant engineering bacterium according to claim 5 for reducing manganese accumulation in plants, characterized in that the plants are arabidopsis thaliana or argininoaceae.