CN115354035A

CN115354035A - Styrax stylosa phenylalanine ammonia lyase SgPAL2 as well as encoding gene and application thereof

Info

Publication number: CN115354035A
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: 2022-11-18
Anticipated expiration: 2042-04-28
Also published as: CN115354035B

Abstract

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

Description

Styrax stylosa phenylalanine ammonia lyase SgPAL2 as well as encoding gene and application thereof

Technical Field

The present invention belongs to the field of gene engineering technology. 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 essential micronutrients for plant growth and development, and participates in a series of physiological and biochemical processes such as photosynthesis, respiration and protein synthesis in plants. However, manganese is also one of heavy metal elements, excessive accumulation of manganese caused by soil pollution and the like can cause toxic effects on plants, and the manganese toxicity is typically characterized by brown spots, chlorosis and necrosis on mature leaves, and finally can inhibit the growth of the plants.

Phenylalanine Ammonia Lyase (PAL) is the rate-limiting enzyme of the Phenylalanine metabolic pathway in plants. Secondary metabolites such as phenols, flavonoids and lignin, which are formed by the conversion of phenylalanine through a phenylalanine metabolic pathway, play a key role in the growth and development, disease resistance and stress resistance of plants. Researches show that toxic stress of metal manganese can influence the phenylpropane metabolic pathway of plants, so that the content of secondary metabolites is changed. For example, under excess manganese treatment conditions, cowpea leaf phenolics and callose content increased significantly; similarly, lignin and flavonoid compounds in the rice leaves are increased under the excessive manganese treatment, and the content of phenylalanine in the poplar leaves is increased under the excessive manganese treatment, so that the toxicity of manganese to plants is relieved. At present, in researches related to the manganese toxicity stress of plants, most of the researches on whether phenylalanine ammonia lyase genes participate in the research of relieving the metal manganese toxicity stress of the plants at a physiological level have no clear report.

Stylosanthes guianensis is a perennial legume plant native to Latin America, which can be used as a livestock forage, for orchard intercropping and acid soil improvement, etc., and is a superior legume grass widely grown in tropical and subtropical regions. In the long-term natural evolution and artificial breeding processes, stylosanthes guianensis has strong capability of adapting to the toxicity of metal manganese and aluminum in acid soil, which may be related to the gene regulation in vivo, but specific genes are not excavated. Therefore, the mining of related genes of the stylosanthes guianensis which are suitable for the stress of the metal manganese toxicity has important significance for cultivating new varieties of manganese toxicity resistant crops.

Disclosure of Invention

The invention aims to overcome the defect that the existing protein or gene related to the capability of improving the tolerance of plants to manganese toxicity does not exist yet, and provides a stylosanthes guianensis phenylalanine ammonia lyase SgPAL2, and a coding gene and application thereof.

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

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

The third objective of the invention is to provide a recombinant expression vector.

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

The fifth purpose 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 bacterium in improving the tolerance of plants to manganese toxicity.

The sixth purpose 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 bacterium in preparing transgenic plants resistant to manganese toxicity.

The seventh purpose 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 bacterium in reducing the accumulation of plant manganese.

The above purpose of the invention is realized 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 the length is 2076bp.

The invention also provides a cloning primer of the stylosanthes guianensis 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 stylosanthes guianensis phenylalanine ammonia-lyase gene SgPAL2 shown in SEQ ID NO.1, namely, cDNA of the stylosanthes guianensis is taken as a template, and primers shown in SEQ ID NO.3 and SEQ ID NO.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 overexpression vector.

More specifically, the overexpression vector is pTF101s.

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

Specifically, the engineering bacteria are Escherichia coli Trans ₁ -T or Agrobacterium-competent cells GV3101.

The invention discovers that the transgenic plant over-expressing the SgPAL2 gene shown in SEQ ID No.1 has obviously enhanced tolerance to manganese poison compared with the wild type through over-expressing the SgPAL2 gene in the plant by transgenic technology, and confirms that the gene enhances the tolerance to manganese poison by reducing the accumulation of manganese in the plant.

Therefore, the application of the invention ensures 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 poison tolerance of plants.

Specifically, the improvement of the manganese poison tolerance of the plant is realized by expressing the stylosanthes guianensis phenylalanine ammonia lyase SgPAL2 in the plant.

The invention also applies to protect 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 preparing transgenic plants resistant to manganese toxicity.

The invention also applies to protect 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 reducing the accumulation of plant manganese.

Specifically, the plant is a dicot.

More specifically, the dicotyledonous plant is stylosanthes guianensis or arabidopsis thaliana.

The invention has the following beneficial effects:

the phenylalanine ammonia lyase gene SgPAL2 with the expression level highly consistent with the response process of stylosanthes guianensis to manganese toxicity is cloned in the stylosanthes guianensis, the expression of the SgPAL2 is proved to be capable of promoting the tolerance capability of transgenic arabidopsis thaliana to manganese toxicity by a transgenic arabidopsis thaliana expression system, and the action mechanism of the gene is proved to be that the tolerance capability of the plant to manganese toxicity is improved by reducing the accumulation of manganese in the plant, so that the gene can be used for preparing the transgenic plant with the tolerance to manganese toxicity. The invention not only enriches the gene bank of the manganese poison resistance of the plants, but also is beneficial to the cultivation of the manganese poison resistance crop varieties.

Drawings

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

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

FIG. 3 shows the effect of overexpression of the SgPAL2 gene on the tolerance of Arabidopsis manganese poisoning; wherein, the graph A is the phenotype analysis result of over-expression Arabidopsis line and wild Arabidopsis under the manganese treatment condition with different concentrations, 0.1mM is the control treatment, 2mM and 4mM MnSO ₄ For the excess manganese treatment, WT is a wild type strain, OE1/OE2 is an excess expression strain, and the scale in the figure is 2cm; FIG. B is a comparison of biomass of overexpression Arabidopsis lines and wild type Arabidopsis, each treatment in the experiment was set with 4 biological replicates, and the columns in the figure are the mean and standard error of the 4 biological replicates; FIG. C is a comparison result of manganese concentrations in an overexpression Arabidopsis strain and a wild type Arabidopsis under the condition of manganese treatment with different concentrations; the number one in the figure indicates that the difference between the over-expression Arabidopsis thaliana strain and the wild Arabidopsis thaliana is significant, and P<0.05。

Detailed Description

The invention is further described with reference to the drawings and specific examples, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 cloning of Stylosanthes guianensis phenylalanine ammonia lyase Gene SgPAL2

1. Cloning of Stylosanthes guianensis phenylalanine ammonia lyase gene SgPAL2

The nucleotide sequence of the primers for cloning the Stylosanthes guianensis phenylalanine ammonia lyase gene SgPAL2 is as follows:

an upstream primer: 5' ATGGCAGCCATTCATCCACGTGA-

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

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

total RNA on the overground part of Stylosanthes guianensis No.5 is extracted by referring to TRNzol Universal total RNA extraction reagent (TIANGEN) instruction, then a cDNA First chain is synthesized according to the method of RevertAID First Strand cDNA Synthesis kit (Thermo Fisher), the obtained cDNA is used as a template, and PCR amplification is carried out by using the primer of the cloned Stylosanthes guianensis phenylalanine ammonia lyase gene SgPAL2.

The PCR reaction (20. Mu.L) was: 2 μ L10 XEx Taq Buffer,1.6 μ L dNTP mix, 1 μ L primer (10 μ M), 0.12 μ L Ex Taq,1 μ L cDNA template, add ddH ₂ O is complemented to 20 mu L; the reaction procedure is as follows: 94 ℃ for 1min,94 ℃ for 30s,58 ℃ for 30s,72 ℃ for 120s, cycle number 35, extension at 72 ℃ for 10min.

After the PCR amplification is completed, 1% agarose gel (containing Golden view nucleic acid dye stain) is prepared, 6 mu L of PCR product is added with 1 mu L of 6 × Loading buffer for electrophoresis detection, and imaging is carried out on a gel imaging system. The PCR product with a length of 2076bp was recovered using a Sanprep column DNA gel recovery kit (raw).

Cloning the recovered PCR product to Trans-T1 carrier for sequencing identification to obtain the full-length cDNA of the Stylosanthes guianensis phenylalanine ammonia lyase gene SgPAL2, 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 Stylosanthes guianensis phenylalanine ammonia lyase Gene SgPAL2

1. Construction of vectors

(1) Construction of overexpression vector:

amplifying an ORF fragment (2076 bp) of the SgPAL2 gene using the above-ground cDNA of the Stylosanthes guianensis obtained in example 1 as a template, and using an upstream specific primer 5-; after the PCR fragment is sequenced to confirm that no errors exist, the target vector is subjected to single enzyme digestion by restriction enzyme Xbal I, the SgPAL2 gene is connected to the target vector pTF101s and transformed into Escherichia coli Trans1-T (all-type gold), sequencing analysis is carried out, and the pTF101s-SgPAL2 recombinant overexpression vector is successfully obtained after the sequencing does not exist.

(2) Construction of expression vector for subcellular localization analysis:

using the cDNA of the aerial part of Cymbopogon flexuosus obtained in example 1 as a template, an ORF fragment of the SgPAL2 gene was amplified using an upstream specific primer 5 '-TCTAGCTACCGGTATGGCAGCCATTCATCCACG-3' (SEQ ID NO: 7) and a downstream specific primer 5 '-ATGGTGGCGACCGGTAACTGAGATGGAACCCCCG-3' (SEQ ID NO: 8), and after the sequencing of the PCR fragments was confirmed, the PCR-amplified fragment of the SgPAL2 gene was ligated to a vector pEGAD for single-restriction linearization of AgeI, to obtain an SgPAL2-GFP fusion expression vector (35S:: sgPAL 2-GFP) initiated from the 35S promoter.

2. Expression analysis of SgPAL2 Gene

The invention uses different metals and manganese with different concentrations to treat the stylosanthes guianensis seeds and observes the influence of different treatments on the SgPAL2 gene expression.

Specifically, selecting and heat grinding No.5 stylosanthes guianensis seed, removing seed coat, heating at 80 deg.C for 2min, placing in dark condition for germination for 2-3 d, transferring to Hoagland nutrient solution (culture solution containing 400 μ M NH) 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 pH 5.8) for two weeks, treated with 800 μ M Fe, 400 μ M Mn, 20 μ M Zn and 10 μ M Cu, respectively, and with different concentrations of manganese (0.5, 50, 400 and 800 μ M); harvesting the sample after two weeks of treatment; the RNA of the aerial part of each treated stylosanthes guianensis was extracted.

The RNA was reverse transcribed into cDNA, and the expression of SgPAL2 was further detected by quantitative PCR using the housekeeping gene SgEF α of Stylosanthes guianensis as an internal reference. The primers used for quantitative PCR detection of gene expression are shown below:

the quantitative primer of the Strychnos chinense SgEF alpha gene is 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 results of the effect of different metals and different concentrations of manganese treatment on the expression of SgPAL2 gene in the aerial part of stylosanthes are shown in fig. 1. Wherein, figure 1A shows 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 asterisks indicate that the difference between the treatment and the control is significant, P <0.05. As can be seen from the results shown in fig. 1A, the excess manganese treatment significantly increased the expression of SgPAL2 in the aerial part of stylosana compared to the control CK. Under the excessive manganese treatment, the SgPAL2 expression level of the overground part of the stylosana is 31.8 times of that of the control. While excessive Fe, zn and Cu treatment had no significant effect on SgPAL2 expression. The above results indicate that SgPAL2 is specifically induced by excessive manganese stress.

Figure 1B shows the effect of different concentrations of manganese treatment on SgPAL2 gene expression as the mean and standard error of 3 replicates, with a number indicating significant differences between the different manganese treatments and the control, P <0.05. As can be seen from the results shown in FIG. 1B, the expression of the SgPAL2 gene from aerial parts tended to increase and decrease with the increase of the manganese treatment concentration, and the highest expression level was 1.6 times that of the normal manganese treatment (0.5. Mu.M) in the 400. Mu.M manganese treatment.

3. Subcellular localization analysis of SgPAL2

Through an agrobacterium-mediated arabidopsis protoplast expression system, the constructed SgPAL2-GFP fusion expression vector and pEGAD empty vector are subjected to line transient expression in the arabidopsis protoplast, and then GFP fluorescence is observed by using a laser confocal microscope (Zeiss) to confirm the subcellular localization of SgPAL2.

The result of subcellular localization of the SgPAL2 gene is shown in FIG. 2, in which the first column shows the GFP fluorescence signal, the second column shows the chloroplast autofluorescence signal, the third column shows the bright field, and the fourth column shows the fusion result of the GFP and chloroplast autofluorescence signal, with a scale of 20 μm. As is clear from the results shown in FIG. 2, the Stylosanthes guianensis SgPAL2 protein is localized in the cytoplasm.

Example 3 transgenic experiments

1. Acquisition of transgenic Arabidopsis

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

2. Screening of transgenic Arabidopsis thaliana

Taking a proper amount of arabidopsis thaliana seeds in a 1.5mL centrifuge tube, adding 1mL of sterile water, soaking for 1min, then sterilizing for 1min by using 75% alcohol, washing for 3 times by using sterile water, finally adding 10% of sodium hypochlorite, sterilizing for 10min, then washing for 3 times by using sterile water, and continuously inverting the centrifuge tube in the process of sterilizing by using alcohol and sodium hypochlorite. After the seeds are sterilized, the seeds are placed in an MS (containing 5mg/L glufosinate ammonium) culture medium to be cultured for 7-10 d, arabidopsis thaliana which is herbicide-tolerant and normally grows is selected to be cultured in matrix soil, and after two weeks of culture, leaf DNA is extracted for PCR verification.

20 μ L PCR reaction: 10 μ L of 2 × Rapid Master Mix,0.6 μ L of the reverse primer (10 mol/L), 0.6 μ L of the forward primer (10 mol/L), 2 μ L of the DNA template, plus ddH ₂ O to 20 μ L; PCR amplification procedure: 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 finally extension at 72 ℃ for 10min.

And (4) keeping the arabidopsis thaliana with correct detection and continuously culturing until the seeds are mature, wherein the seeds are T1 generation seeds. And continuously screening for 3 times according to the method, obtaining T3 generation homozygous transgenic arabidopsis seeds, and reserving. The SgPAL2 gene T3 Arabidopsis seeds obtained by PCR confirmation are used for subsequent experiments.

3. Manganese treatment of transgenic arabidopsis thaliana:

for analyzing the manganese toxicity resistance of the transgenic arabidopsis, sterilized wild type and transgenic arabidopsis seeds are sown on an MS culture medium containing 0.8% agar for 7d, and then seedlings with consistent sizes are selected and transferred to a medium containing 0.1mM,2mM and 4mM MnSO respectively ₄ Treating for 7 days to obtain plants, and measuring the fresh weight and the manganese content of the plants.

The results of the effect of overexpression of the SgPAL2 gene on the tolerance to manganese poisoning of Arabidopsis are shown in FIG. 3. Wherein FIG. 3A shows the results of phenotypic analysis of over-expressed Arabidopsis lines (i.e., transgenic Arabidopsis lines) and wild type Arabidopsis under manganese treatment conditions of different concentrations, 0.1mM for control treatment, 2mM and 4mM MnSO ₄ For the excess manganese treatment, WT was the wild type strain and OE1/OE2 was the over-expression strain with a scale of 2cm. As can be seen from the results shown in FIG. 3A, the growth of WT, OE1 and OE2 transgenic lines was consistent under the control 0.1mM manganese; under the treatment of 2mM and 4mM excess manganese, the growth conditions of plants of OE1 and OE2 transgenic lines are obviously better than that of WT, and the over-expression of SgPAL2 can relieve the metal manganese toxicity of Arabidopsis and improve the tolerance of the Arabidopsis to the manganese toxicity.

FIG. 3B is a comparison of biomass of overexpression Arabidopsis lines and wild type Arabidopsis, each treatment was set to 4 biological replicates in the experiment, the columns in the figure are the mean and standard error of the 4 biological replicates, the asterisks indicate significant differences between the overexpression lines and the wild type control lines, P <0.05. As can be seen from the results shown in FIG. 3B, the fresh weight difference between the overexpression SgPAL2 transgenic lines (OE 1 and OE 2) and the WT wild type was not significant under the control 0.1mM treatment condition; whereas, the fresh weights of OE1 and OE2 plants were significantly higher than wild type plants under 2mM and 4mM manganese treatment. OE1 and OE2 plants were 1.38 and 1.4 times more shoot weight than WT, respectively, under 2mM excess manganese treatment; OE1 and OE2 plants were 2.1 and 1.8 times as bright as WT, respectively, under 4mM manganese treatment.

FIG. 3C is a comparison result of manganese concentrations in an overexpression Arabidopsis strain and a wild Arabidopsis under the condition of manganese treatment with different concentrations, WT is a wild Arabidopsis, OE1/OE2 is an overexpression strain, and the number in the figure indicates that the difference between the overexpression Arabidopsis strain and the wild Arabidopsis is significant, and P is less than 0.05. From the results shown in fig. 3C, it can be seen that the overexpression of SgPAL2 can significantly reduce the manganese concentration in arabidopsis plants compared to wild-type arabidopsis WT under the treatment of 2mM and 4mM of hypermanganese.

The results show that the tolerance of arabidopsis thaliana to manganese poison can be improved by over-expressing the SgPAL2 gene in arabidopsis thaliana, and the tolerance of the arabidopsis thaliana to manganese is enhanced by reducing the accumulation of manganese in the plant.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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Ala Leu Gln Lys Glu Met Val Arg Phe Leu Asn Cys Ala Ile Phe Gly

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His Glu Ser Glu Leu Ser His Asn Arg Leu Pro Lys Ser Ala Thr Arg

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Ala Ala Met Leu Val Arg Val Asn Thr Leu Leu Gln Gly Tyr Ser Gly

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Leu Ser Ala Val Phe Thr Gln Val Met Gln Gly Asn Pro Glu Phe Thr

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His His Leu Ile His Lys Leu Lys Tyr His Pro Gly Gln Ile Glu Ala

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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. A Stylosanthes guianensis phenylalanine ammonia lyase SgPAL2 is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.

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

3. The encoding gene of claim 2, wherein the nucleotide sequence is represented by SEQ ID No. 1.

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

5. A recombinant engineered 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 tolerance of plants to manganese toxicity.

7. The use according to claim 6, wherein said improvement of the tolerance of a plant to manganese poisoning is achieved by expressing the Stylosanthes guianensis Phenylalanine ammonialyase SgPAL2 in the plant.

8. Use of the Stylosanthes guianensis phenylalanine ammonia lyase SgPAL2 as claimed in claim 1, the coding gene as claimed in claim 2 or 3, the recombinant expression vector as claimed in claim 4 or the recombinant engineered bacterium as claimed in claim 5 for preparing transgenic plants resistant to manganese poisoning.

9. The use of the stylosanetam roseum 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 reducing the accumulation of manganese in plants.

10. The use according to any one of claims 6 to 9, wherein the plant is a dicotyledonous plant.