CN107383179B

CN107383179B - A kind of and plant stress tolerance correlative protein GsSLAH3 and its encoding gene and application

Info

Publication number: CN107383179B
Application number: CN201710822313.9A
Authority: CN
Inventors: 孙晓丽; 朱延明; 段香波; 孙明哲; 贾博为; 于洋
Original assignee: Heilongjiang Bayi Agricultural University
Current assignee: Heilongjiang Bayi Agricultural University
Priority date: 2017-09-13
Filing date: 2017-09-13
Publication date: 2019-11-12
Anticipated expiration: 2037-09-13
Also published as: CN107383179A

Abstract

The invention discloses a kind of and plant stress tolerance correlative protein GsSLAH3 and its encoding gene and applications.The present invention is cloned from wild soybean obtains GsSLAH3 gene, carbonate suspension stress-inducing up-regulated expression；Tissue positioning analysis shows that GsSLAH3 gene is mainly expressed in the tissue such as root, stem, hypocotyl and fruit pod；By its transient expression in onion epidermis cell, discovery fusion protein fluorescence signal is mainly appeared in cell membrane；By GsSLAH3 gene overexpression in arabidopsis, discovery GsSLAH3 gene can enhance arabidopsis to the patience of carbonic acid salt stress.The present invention is to cultivate alkaline-resisting New Crop Varieties to have established Research foundation, to development and utilization saline alkali land resource important in inhibiting.

Description

A kind of and plant stress tolerance correlative protein GsSLAH3 and its encoding gene and application

Technical field

The invention belongs to field of biotechnology, and in particular to a kind of and plant stress tolerance correlative protein GsSLAH3 and its volume Code gene and application.

Background technique

Up to 1,500,000,000 mu of China's saline alkali land area, only just there is as many as fifty-five million mu in the Northeast.Saline and alkaline adverse circumstance seriously endangers The growth and development of crop are the significant problems for restricting China's agricultural production.Heilongjiang Province has number with ten million mu of salinization of soil Soil develops and uses these land resources, to improve China's farm output, Ensuring Food Safety have important practical significance and Strategic importance, while will also create huge ecological benefits and social benefit.Our province salt-soda soil predominantly contains Na₂CO₃And NaHCO₃ Basic soil, with neutral salt such as NaCl, Na₂SO₄Etc. comparing, carbonate is while causing osmotic stress and Ion toxicity, also It increases by CO₃ ^2-And HCO₃ ^-The high pH injury Deng caused by, to generate mixing toxic action.Therefore carbonic acid salt stress is than neutral Salt action mechanism is increasingly complex, endangers caused by plant bigger.

The hot and difficult issue that salt tolerance of crops has become current agricultural and the technical research of animal husbandry field is improved, and The significant problem of urgent need to resolve at present.In recent years, with the development of functional genomics and molecular biology, saline-alkali tolerant is excavated Key gene, being cultivated using technique for gene engineering, there are the New Crop Varieties of good saline-alkali tolerant character, which to have become, effectively improves work One of the means of object salt tolerant alkali ability.Wild soybean can survive in low temperature and height wetland with saline-alkaline, adaptable wide The features such as strong with resistance of reverse is the ideal donor material for obtaining saline-resisting and alkaline-resisting gene.

Anion channel wide participation plant organic acid secretion, film potential change, turgescence controls and the physiology such as crop tolerance to salt Process.It is incorporated in from the point of view of being studied in model plant arabidopsis and rice, wherein slow type anion channel (S-type anion Channel) in plant to playing critical function in the abiotic stress response such as light, arid, salt.Therefore, it excavates wild big Beans slow type anion channel protein gene will provide function significant genetic resources for the resistance to inverse molecular breeding of crop.

Summary of the invention

The technical problem to be solved by the present invention is to how regulate and control plant stress tolerance.

In order to solve the above technical problems, present invention firstly provides a kind of and plant stress tolerance correlative proteins.

Entitled GsSLAH3 provided by the present invention with plant stress tolerance correlative protein, for it is following a) or b) or c) or D) protein:

A) amino acid sequence is protein shown in sequence 2；

B) fused protein that the N-terminal of the protein shown in sequence 2 and/or C-terminal connection label obtain；

C) by amino acid sequence shown in sequence 2 by one or several amino acid residues substitution and/or missing and/or Add obtained protein with the same function；

D) homology with amino acid sequence shown in sequence 2 with 75% or 75% or more and egg with the same function White matter.

Wherein, sequence 2 is made of 561 amino acid residues.

In order to make protein in a) convenient for purifying, can in sequence table the amino terminal of protein shown in sequence 2 or Carboxyl terminal connects upper label as shown in Table 1.

The sequence of table 1, label

Label	Residue	Sequence
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

It is above-mentioned c) in protein G sSLAH3, the substitution of one or several amino acid residues and/or missing and/or It is added to the substitution and/or deletion and/or addition no more than 10 amino acid residues.

It is above-mentioned c) in protein G sSLAH3 can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression It obtains.

It is above-mentioned c) in protein G sSLAH3 encoding gene can by will in DNA sequence dna shown in sequence 1 lack one Or the codon of several amino acid residues, and/or carry out the missense mutation of one or several base-pairs, and/or its 5 ' end and/ Or 3 ' end connect the coded sequence of label shown in table 1 and obtain.

In order to solve the above technical problems, invention further provides biomaterials relevant to GsSLAH3 protein.

Biomaterial relevant to GsSLAH3 protein provided by the invention is following A 1) any one of to A12):

A1 the nucleic acid molecules of GsSLAH3 protein) are encoded；

A2) contain A1) expression cassettes of the nucleic acid molecules；

A3) contain A1) recombinant vectors of the nucleic acid molecules；

A4) contain A2) recombinant vector of the expression cassette；

A5) contain A1) recombinant microorganisms of the nucleic acid molecules；

A6) contain A2) recombinant microorganism of the expression cassette；

A7) contain A3) recombinant microorganism of the recombinant vector；

A8) contain A4) recombinant microorganism of the recombinant vector；

A9) contain A1) the transgenic plant cells systems of the nucleic acid molecules；

A10) contain A2) the transgenic plant cells system of the expression cassette；

A11) contain A3) the transgenic plant cells system of the recombinant vector；

A12) contain A4) the transgenic plant cells system of the recombinant vector.

In above-mentioned biomaterial, A1) nucleic acid molecules be it is following 1) or 2) or 3) shown in gene:

1) its coded sequence is cDNA molecule or genomic DNA molecule shown in sequence 1；

2) there is 75% or 75% or more identity with the nucleotide sequence 1) limited, and encodes GsSLAH3 protein CDNA molecule or genomic DNA molecule；

1) or 2) 3) and the cDNA of GsSLAH3 protein is encoded with the nucleotide sequence hybridization that limits under strict conditions Molecule or genomic DNA molecule.

Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also To be RNA, such as mRNA or hnRNA.

Wherein, sequence 1 is made of 1686 nucleotide, amino acid sequence shown in coded sequence 2.

Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation Method is mutated the nucleotide sequence of coding GsSLAH3 of the invention.Those have and the present invention by manually modified The nucleotide sequence 75% of isolated GsSLAH3 or the nucleotide of higher identity, as long as encoding GsSLAH3 and having Identical function is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.

Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair Amino acid sequence shown in bright coded sequence 2 composition protein nucleotide sequence have 75% or higher or 85% or Higher or 90% or higher or 95% or higher identity nucleotide sequence.Identity can with the naked eye or computer software It is evaluated.Using computer software, identity between two or more sequences can be indicated with percentage (%), can be with For evaluating the identity between correlated series.

Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90% or 95% or more identity.

In above-mentioned biomaterial, A2) described in the nucleic acid molecules containing coding GsSLAH3 expression cassette (GsSLAH3 gene Expression cassette), it is the DNA for referring to express GsSLAH3 in host cell, which not only may include that starting GsSLAH3 is transcribed Promoter may also include the terminator for terminating GsSLAH3 transcription.Further, the expression cassette may also include enhancer sequence.It can Include but is not limited to for promoter of the invention: constitutive promoter；It organizes, the promoter and induction that organ and development are special Type promoter.Suitable transcription terminator includes but is not limited to: Agrobacterium nopaline syntase terminator (NOS terminator), flower Cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine close Enzyme terminator.

The recombinant vector of the GsSLAH3 expression casette can be contained with existing expression vector establishment.The plant table It include double base agrobacterium vector and the carrier etc. that can be used for plant micropellet bombardment up to carrier.As pAHC25, pBin438, pCAMBIA1302、pCAMBIA2300、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、 PCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company) etc..The plant expression vector also may include foreign gene 3 ' end untranslated regions, i.e., comprising polyadenylation signals and it is any other participate in mRNA processing or gene expression DNA fragmentation. The bootable polyadenylic acid of polyadenylation signals is added to 3 ' ends of mRNA precursor, as Agrobacterium crown gall nodule induces (Ti) plasmid Gene (such as rouge alkali synthetase gene Nos), the non-translational region of the end of plant gene (such as soybean storage protein genes) 3 ' transcription are equal With similar functions.When using gene constructed plant expression vector of the invention, enhancer, including translational enhancer also can be used Or transcriptional enhancer, these enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must be with The reading frame of coded sequence is identical, to guarantee the correct translation of entire sequence.The translation control signal and initiation codon Source be it is extensive, can be natural, be also possible to synthesis.Translation initiation region can come from transcription initiation region or knot Structure gene.For the ease of transgenic plant cells or plant are identified and screened, plant expression vector used can be carried out Processing, as be added the coding that can be expressed in plant can produce color change enzyme or luminophor gene (gus gene, Luciferase genes etc.), the marker gene of antibiotic (as assigned the nptII gene to kanamycins and associated antibiotic resistance, The bar gene to herbicide phosphinothricin resistance is assigned, assigns the hph gene to antibiotic hygromycin resistance, and assign to ammonia The dhfr gene of methotrexate resistant is assigned to the EPSPS gene of glyphosate) or anti-chemical reagent marker gene etc. is (such as Anti- herbicide gene), provide metabolism mannose ability mannose-6-phosphate isomerase gene.From the safety of genetically modified plants Property consider, any selected marker can be not added, directly with adverse circumstance screen transformed plant.

In above-mentioned biomaterial, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.

In above-mentioned biomaterial, the microorganism can be yeast, bacterium, algae or fungi, such as Agrobacterium.

In above-mentioned biomaterial, the transgenic plant cells system does not include propagation material.

In order to solve the above technical problems, the present invention also provides GsSLAH3 protein or the new applications of above-mentioned biomaterial.

The present invention provides the application of GsSLAH3 protein or above-mentioned biomaterial in regulation plant stress tolerance.

It is described to be regulated to improve in above-mentioned application.

The present invention also provides GsSLAH3 protein or above-mentioned biomaterial in the genetically modified plants for cultivating resistance of reverse raising In application.

The present invention also provides the application of GsSLAH3 protein or above-mentioned biomaterial in plant breeding.

In above-mentioned application, the resistance of reverse is alkali resistance；The alkali resistance is specially the salt stress of resistance to carbonic acid；The resistance to carbonic acid Salt stress is specially resistance to NaHCO₃Stress.

In above-mentioned application, the plant is monocotyledon or dicotyledon, the dicotyledon concretely beans Section plant and/or crucifer and/or compositae plant；The leguminous plant can be yellow for soybean, crowtoe, clover or water Skin；The crucifer can be arabidopsis or rape；The compositae plant can be sunflower；The arabidopsis can be quasi- south Mustard (Columbia ecotype col-0).

In order to solve the above technical problems, the present invention finally provides a kind of side of genetically modified plants that cultivation resistance of reverse improves Method.

The method provided by the invention for cultivating the genetically modified plants that resistance of reverse improves includes improving GsSLAH3 in recipient plant The expression quantity and/or activity of protein, the step of obtaining genetically modified plants；The resistance of reverse of the genetically modified plants be higher than it is described by Body plant.

In the above method, it is described improve recipient plant in GsSLAH3 protein expression quantity and/or active method be GsSLAH3 protein is overexpressed in recipient plant.

In the above method, the method for the overexpression is that the encoding gene of GsSLAH3 protein is imported recipient plant；Institute The nucleotide sequence for stating the encoding gene of GsSLAH3 protein is DNA molecular shown in sequence 1.

In an embodiment of the invention, the encoding gene (i.e. nucleotide shown in sequence 1) of GsSLAH3 albumen is logical The recombinant vector pCAMBIA230035S-GsSLAH3 for crossing the expression cassette of the encoding gene containing GsSLAH3 albumen imports Agrobacterium In LBA4404.The recombinant vector pCAMBIA230035S-GsSLAH3 be by the SmaI of pCAMBIA230035S carrier and DNA fragmentation between XbaI enzyme cutting site replaces with GsSLAH3 gene shown in sequence 1 in sequence table, and keeps The carrier obtained after the other sequences of pCAMBIA230035S carrier are constant.The recombinant vector pCAMBIA230035S- GsSLAH3 expresses GsSLAH3 albumen.

In the above method, the resistance of reverse is alkali resistance；The alkali resistance is specially the salt stress of resistance to carbonic acid.

In the above method, the salt stress of resistance to carbonic acid is resistance to NaHCO₃Stress, is embodied as in NaHCO₃The condition of stress Under: the seed germination rate of genetically modified plants is higher than recipient plant and/or the root long of genetically modified plants is longer than recipient plant and/or turns The Aboveground Biomass of Young of gene plant is higher than recipient plant and/or the chlorophyll content of genetically modified plants is higher than recipient plant And/or the Electrolyte Leakage Rate of genetically modified plants is lower than recipient plant.The NaHCO₃Concentration concretely 4mM, 5mM, 7mM, 8mM and 150mM.

In the above method, the genetically modified plants are interpreted as not only including by the GsSLAH3 genetic transformation recipient plant Obtained first generation genetically modified plants also include its filial generation.For genetically modified plants, the gene can be bred in the species, The gene transfer can also be entered to other kinds of same species with traditional breeding techniques, particularly including in commercial variety.It is described Genetically modified plants include seed, callus, intact plant and cell.

In the above method, the recipient plant is monocotyledon or dicotyledon, and the dicotyledon specifically may be used For leguminous plant and/or crucifer and/or compositae plant；The leguminous plant can be soybean, crowtoe, clover or water Calusena lansium；The crucifer can be arabidopsis or rape；The compositae plant can be sunflower；The arabidopsis can be quasi- Southern mustard (Columbia ecotype col-0).

The present invention is cloned from wild soybean obtains GsSLAH3 gene, carbonate suspension stress-inducing up-regulated expression；Tissue Positioning analysis shows that GsSLAH3 gene is mainly expressed in the tissue such as root, stem, hypocotyl and fruit pod；By its transient expression in ocean In green onion epidermal cell, discovery fusion protein fluorescence signal is mainly appeared in cell membrane；By GsSLAH3 gene overexpression in quasi- south In mustard, discovery GsSLAH3 gene can enhance arabidopsis to the patience of carbonic acid salt stress.The present invention is to cultivate alkaline-resisting New Crop Varieties Research foundation is established, to development and utilization saline alkali land resource important in inhibiting.

Detailed description of the invention

Fig. 1 is GsSLAH3 gene in wild soybean root in 50mM NaHCO₃Expression pattern under processing.

Fig. 2 is the relative expression quantity of GsSLAH3 gene in wild soybean different tissues.

Fig. 3 is the Subcellular Localization of GsSLAH3.

Fig. 4 is the RT-PCR identification for turning GsSLAH3 arabidopsis.

Fig. 5 is to turn GsSLAH3 Arabidopsis plant in 7mM NaHCO₃With 8mM NaHCO₃Processing under germination period phenotype and sprout The statistical analysis of hair rate.

Fig. 6 is to turn GsSLAH3 gene Arabidopsis plant in 4mM NaHCO₃With 5mM NaHCO₃Seedling Stage phenotype under processing And the statistical analysis of root long and Aboveground Biomass of Young.

Fig. 7 is to turn GsSLAH3 gene Arabidopsis plant in 150mM NaHCO₃Seedling stage phenotype and chlorophyll under processing The statistical analysis of content and Electrolyte Leakage Rate.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Quantitative test in following embodiments, is respectively provided with three repeated experiments, and results are averaged.

Wild-type soy G07256 in following embodiments is recorded in following document: Mingzhe Sun, Xiaoli Sun, Yang Zhao,Hua Cai,Chaoyue Zhao,Wei Ji,Huizi DuanMu,Yang Yu,Yanming Zhu.Ectopic expression of GsPPCK3and SCMRP in Medicago sativa enhances plant Alkaline stress tolerance and methionine content.PLOS ONE 2014,9 (2): e89578, it is public Crowd can obtain at applicant (Heilongjiang Bayi Agricultural Reclamation University), the biomaterial only attach most importance to duplicate invention related experiment institute With not can be used as other purposes and use.

PBSK-35S-eGFP carrier in following embodiments is recorded in following document: Xiaoli Sun, Wei Ji, Xiaodong Ding,Xi Bai,Hua Cai,Shanshan Yang,Xue Qian,Mingzhe Sun,Yanming Zhu.GsVAMP72,a novel Glycine soja R-SNARE protein,is involved in regulating plant salt tolerance and ABA sensitivity.Plant Cell Tiss Organ Cult 2013,113: 199-215, the public can obtain at applicant (Heilongjiang Bayi Agricultural Reclamation University), the biomaterial only attach most importance to duplicate invention phase It closes used in experiment, not can be used as other purposes and use.

PCAMBIA230035S carrier in following embodiments is recorded in following document: Ailin Liu, Yang Yu, Xiangbo Duan,Xiaoli Sun,Huizi Duanmu,Yanming Zhu.GsSKP21,a Glycine soja Sphase kinase associated protein,mediates the regulation of plant alkaline Tolerance and ABA sensitivity.Plant Mol Biol (2015) 87:111-124, the public can be from applicant It is obtained at (Heilongjiang Bayi Agricultural Reclamation University), which only attaches most importance to used in the related experiment of duplicate invention, not can be used as it Its purposes uses.

Agrobacterium LBA4404 in following embodiments is recorded in following document: Ailin Liu, Yang Yu, Xiangbo Duan,Xiaoli Sun,Huizi Duanmu,Yanming Zhu.GsSKP21,a Glycine soja Sphase kinase associated protein,mediates the regulation of plant alkaline tolerance and ABA sensitivity.Plant Mol Biol (2015) 87:111-124, the public can (Heilungkiang Aug. 1st be land-reclaimable from applicant University) at obtain, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other purposes and uses.

The clone of embodiment 1, wild soybean GsSLAH3 gene

1, it selects full wild soybean G07256 seed and handles 10min in the concentrated sulfuric acid to remove to desilt film, the dense sulphur of evacuation It is placed on wet filter paper after after acid with aseptic water washing 3-4, the vernalization in 3 days of 25 DEG C of dark cultures grows to about 1-2cm to bud When, it is transferred into the alms bowl for filling Huo Gelan culture solution, is fixed with space wadding, immerse bud in culture solution, and placed It is cultivated in growth cabinet.

2, long to 3 week old to seedling, it takes its root 3cm to be put into EP pipe, is placed in -80 DEG C of preservations.The extraction reference of RNA RNAprep pure kit (TRANSGEN BIOTECH) specification carries out the synthesis of cDNA later.

3, using the cDNA of synthesis as template, PCR amplification is carried out with primer Primer-KS and Primer-KAS.Primer sequence It is as follows:

Primer-KS:5 '-TATAACCTGTGCTTCTGATAGTGTGT -3 '；

Primer-KAS:5 '-TGTTGGTTGCCTCATTATTTTCTT -3 '.

PCR amplification system (50 μ L): 4 μ L, 10 × PS buffer (Mg of cDNA²⁺) 10 μ L, dNTP Mixture (2.5mM) 411 μ L, Prime Star DNA Polymerase (TaKaRa) of μ L, Primer-KAS of μ L, Primer-KS 0.5 μ L, ddH₂O 29.5μL.PCR amplification condition are as follows: 98 DEG C of 8min；98 DEG C of 10s, 58 DEG C of 10s, 72 DEG C of 2min, 30 circulations；72℃10min；4℃ It terminates.

Pcr amplification product is subjected to 1.5% agarose gel electrophoresis detection, obtains the band that molecular weight is about 1.9Kb, is used Ago-Gel QIAquick Gel Extraction Kit (TRANSGEN BIOTECH) recycles pcr amplification product；By itself and pEASY-Blunt Simple carrier (TRANSGEN BIOTECH) connection, obtains recombinant plasmid pEASY-Blunt Simple-GsSLAH3.And it will It delivers sequencing after converting bacillus coli DH 5 alpha competent cell.

Sequencing result shows: PCR amplification obtains the amplified production that size is 1910bp, and it includes complete sizes to be The open reading frame (ORF) of 1686bp, size are the nucleotide sequence of the DNA fragmentation of 1686bp as shown in sequence 1 in sequence table, And by unnamed gene shown in sequence 1 be GsSLAH3 gene, GsSLAH3 gene coding amino acid sequence such as sequence table in sequence Shown in column 2, amino acid sequence shown in sequence 2 is named as GsSLAH3 albumen.

The expression characterization analysis of embodiment 2, GsSLAH3 gene

One, GsSLAH3 gene expression pattern analysis under alkaline stress in wild soybean root

1, it selects full wild soybean G07256 seed and handles 10min in the concentrated sulfuric acid and go to desilt film, the evacuation concentrated sulfuric acid It uses aseptic water washing 3-4 times, is placed on wet filter paper, 25 DEG C of dark culture 3d vernalization, removal when bud is about 1-2cm afterwards, Water planting is carried out with Huo Gelan fluid nutrient medium.

2, long to 3 week old to wild soybean seedling, in 50mM NaHCO₃(pH8.5) under the conditions of handle 0h, 1h, 3h, 6h, 9h, 12h, clip rapidly rear for 24 hours children tender, are placed in -80 DEG C of preservations.Total serum IgE is extracted, reverse transcription is that cDNA is stand-by as template.

3, using Real time-PCR method, using wild soybean GAPDH gene as reference gene, with untreated samples As control, the relative expression quantity of GsSLAH3 gene different time points after treatment is analyzed.The relative expression quantity of gene use than Compared with C_TMethod (Δ Δ C_T) calculate: 2^-ΔΔCT=2^{(Δ CT processing-Δ CT control)}=2^{[(CT processing-CT internal reference)-(CT control-CT internal reference)]}.GsGAPDH and GsSLAH3 gene primer difference is as follows:

GsGAPDH-S:5 '-GACTGGTATGGCATTCCGTGT -3 '；

GsGAPDH-AS:5 '-GCCCTCTGATTCCTCCTTGA -3 '；

GsSLAH3-S:5 '-AGAGCCAGCAAGCGGTGTC -3 '；

GsSLAH3-AS:5 '-GTAACTGTGGACCCTCCAATGTT -3 '.

PCR reaction condition: 95 DEG C of 2min → [95 DEG C of 15s → 60 DEG C 30s] × 40 → 95 DEG C of 1min → 55 DEG C 1min → 95 ℃30s。

As a result as shown in Figure 1.It can be seen from the figure that in 50mM NaHCO₃Under processing, the expression of GsSLAH3 gene upregulation, And reach peak in 6h, expression quantity relative reduction but opposite 0h still keeps higher level later.Illustrate that GsSLAH3 gene is one A alkaline stress response gene.

Two, the tissue expression pattern analysis of GsSLAH3 gene

1, it selects full wild soybean G07256 seed and handles 10min in the concentrated sulfuric acid and remove to desilt film, used after evacuation is dense It aseptic water washing 3-4 times, is placed on wet filter paper, 25 DEG C of dark culture 3d vernalization are shifted when bud is about 1-2cm Into the seedling-growing container for filling 30% turfy soil, 70% common soil, then it is placed in growth cabinet and cultivates.

2, wild soybean different tissues (root, stem, spire, climax leaves, hypocotyl, embryo, fruit pod and flower) is taken, is set respectively It is saved in -80 DEG C.

3, it extracts the RNA of wild soybean different tissues and reverse transcription is that do template spare by cDNA.

4, the GsSLAH3 gene relative expression quantity in wild soybean different tissues is detected according to the method in the 3 of step 1.

As a result as shown in Figure 2.As can be seen from the figure: GsSLAH3 gene is mainly expressed in root, stem, hypocotyl and fruit pod In, wherein the expression quantity highest in hypocotyl；And GsSLAH3 gene expression amount is relatively low in flower, leaf and embryo.

The GsSLAH3 protein subcellular positioning analysis that embodiment 3, biolistic bombardment mediate

1, using the cDNA of wild soybean G07256 as template, PCR expansion is carried out using GsSLAH3-YS and GsSLAH3-YAS Increase, obtains GsSLAH3 gene.Primer sequence is following, and (the restriction enzyme site sequence that underscore mark introduces, left side are protection alkali Base):

GsSLAH3-YS:5 '-GGGTCGACATGGAAAACAACCTTAAC -3 '；

GsSLAH3-YAS:5 '-CGTCTAGATGGCTCCCAAGTCTTAAATG–3’。

PCR amplification system: 20 5 × PrimeSTAR of μ L^TMHS PCR buffer, 8 μ L dNTP mix are (A, G, T, C, each 2.5mM), 2 μ L upstream and downstream primer (10 μM), 1 μ L dilute the common template of 100 times (plasmid containing target gene), 1 μ L high-fidelity Enzyme [PrimeSTAR DNA Polymerase (TaKaRa)], sterile ddH₂O supplies volume (100 μ L of total volume).

PCR reaction condition: 98 DEG C of 8min；98 DEG C of 10s, 60 DEG C of 10s, 72 DEG C of 1.5min, 30 circulations；72℃10min；4 DEG C terminate reaction.

2, above-mentioned pcr amplification product and carrier pBSK-35S-eGFP are carried out with restriction enzyme SalI and XbaI double Digestion, connection, obtains recombinant plasmid.Coupled reaction system are as follows: 10 × Buffer 1 μ L, T₄1 μ L of DNA ligase, carrier are large stretch of The concentration ratio of section and target gene is 1:1, ddH₂O is supplied volume (10 μ L of total volume), and 16 DEG C of connections are overnight.

3, above-mentioned recombinant plasmid and empty carrier (pBSK-35S-eGFP) are bombarded by onion epidermis cell using particle bombardment (specific method is referring to U.S. Bio-Rad Bole's Helios gene gun system specification).After dark culture 12h, clip was bombarded Onion epidermis cell, load observes green fluorescence under laser confocal microscope.After the fluorescence for observing recombinant protein, Matter wall is separated with the sucrose solution processing load of 30% (w/v), further observes fluorescence again.

As a result as shown in Figure 3.Turning empty carrier fluorescence signal has expression in entire cell；And it is glimmering to turn recombinant plasmid green Optical signal is concentrated mainly on cell membrane, and after plasmolysis, and fluorescence signal enhances on film.Illustrate GsSLAH3 mainly as film Protein exhibits function.

Embodiment 4, the acquisition for turning GsSLAH3 Arabidopsis plant and its salt stress of resistance to carbonic acid analysis

One, turn the acquisition and Molecular Identification of GsSLAH3 arabidopsis

1, using the recombinant plasmid pEASY-Blunt Simple-GsSLAH3 in embodiment 1 as template, using GsSLAH3- ES and GsSLAH3-EAS primer carries out PCR amplification, obtains the area CDS of GsSLAH3 gene.Primer sequence is as follows:

GsSLAH3-ES:5 '-AGGCCCGGGATGGAAAACAACCTTAACCGT–3'；

GsSLAH3-EAS:5 '-TCGTCTAGATCATGGCTCCCAAGTCTTAAATGG–3’。

PCR amplification system (20 μ L): 0.2 2 μ L, dNTP Mixture of μ L, 10 × buffer of template 2 μ L, Mg²⁺1 μ L, 0.6 0.6 μ L, KOD DNA Polymerase (TaKaRa) of μ L, GsSLAH3-EAS of GsSLAH3-ES 0.4 μ L, ddH₂O 13.2 μL。

PCR amplification condition: 95 DEG C of 2min；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1.5min, 30 circulations；72℃10min；4 DEG C terminate reaction.

2, it is produced with the PCR amplification that Restriction enzyme Sma I and XbaI obtain pCAMBIA230035S carrier and step 1 Object carries out double digestion, and connection obtains recombinant plasmid pCAMBIA230035S-GsSLAH3.And sequence verification is carried out to it.

Sequencing result shows: recombinant vector pCAMBIA230035S-GsSLAH3 is by pCAMBIA230035S carrier DNA fragmentation between SmaI and XbaI enzyme cutting site replaces with GsSLAH3 gene shown in sequence 1 in sequence table, and keeps The carrier obtained after the other sequences of pCAMBIA230035S carrier are constant.Recombinant vector pCAMBIA230035S-GsSLAH3 table Up to GsSLAH3 protein shown in sequence 2.

3, recombinant vector pCAMBIA230035S-GsSLAH3 is converted in agrobacterium tumefaciens lba4404 using freeze-thaw method, It identifies to obtain positive transformant through PCR, for infecting Arabidopsis plant.

4, intended using dipping in colored method the Agrobacterium containing recombinant plasmid pCAMBIA230035S-GsSLAH3 is infected wild type Southern mustard (Columbia ecotype col-0).Harvest T₁For seed and in the 1/2MS culture medium of kanamycins containing 25mg/L (kana) Upper screening.The next generation generated to the seedling that screening obtains carries out kana screening again, so repeats, finally obtains T₃In generation, turns GsSLAH3 arabidopsis homozygous lines.

5, T is extracted₃In generation, turns GsSLAH3 Arabidopsis plant total serum IgE, passes through RT-PCR method qualitative detection GsSLAH3 gene Relative expression quantity, gene primer sequence is the same as embodiment 2.Simultaneously using Actin2 as reference gene, primer sequence is as follows:

Actin2-S:5 '-TTACCCGATGGGCAAGTC -3 '；

Actin2-AS:5 '-GCTCATACGGTCAGCGATAC -3 '.

PCR reaction system: 5 μ L 2 × Easy Taq DNA Polymerase, 0.8 μ L upstream primer (10 μM), 0.8 μ L Downstream primer (10 μM), 1 μ L dilute 100 times of cDNA, sterile ddH₂O supplies volume (10 μ L of total volume).

PCR amplification condition: GsSLAH3:94 DEG C of 10min → [94 DEG C of 30s → 60 DEG C 30s → 72 DEG C 90s] × 30 → 72 DEG C 10min → 4 DEG C terminate reaction；

Actin2:94 DEG C of 10min → [94 DEG C of 30s → 60 DEG C 30s → 72 DEG C 90s] × 28 → 72 DEG C of 10min → 4 DEG C terminate Reaction.

Agarose gel electrophoresis detection is carried out to PCR product, partial results are as shown in Figure 4.As can be seen from the figure: wild The RT-PCR of type Arabidopsis plant is without amplified production, and T₃Generation, which turns GsSLAH3 arabidopsis homozygous lines #14 and #15, can expand Increase purpose band out, show that foreign gene GsSLAH3 is not only smoothly integrated on the genome of arabidopsis, and can turn Normal transcription is expressed in gene arabidopsis.Choose T₃In generation, turns GsSLAH3 arabidopsis homozygous lines #14 and #15 for next step Phenotypic analysis.

Two, turn phenotypic analysis of the GsSLAH3 arabidopsis under alkaline stress

1, turn germination period phenotype and germination rate of the GsSLAH3 arabidopsis under alkali process

Choose full wildtype Arabidopsis thaliana and T₃In generation, turns the seed of GsSLAH3 arabidopsis homozygous lines #14 and #15, uses 5% hypochlorite disinfectant 5min, later with sterilizing ddH₂O is rinsed 3-5 times, is placed in 4 DEG C of processing 3d.Then seed is broadcast respectively In the NaHCO containing 0mM, 7mM and 8mM₃1/2MS culture medium on, 22 DEG C of culture 3d, daily observe phenotype simultaneously count seed sprouting Rate.In triplicate, every kind of each strain of processing uses 30 plants of plant for experiment.

As a result as shown in Figure 5.As can be seen from the figure: (0mM NaHCO under normal operation₃The control group of processing), it is wild Raw type arabidopsis and the germinating and germination rate for turning GsSLAH3 arabidopsis illustrate GsSLAH3 not to quasi- south without significant difference The seed sprouting of mustard has an impact；But in 7mM and 8mM NaHCO₃Under processing, wildtype Arabidopsis thaliana and turn GsSLAH3 arabidopsis Sprouting by a degree of inhibition, but relative to wildtype Arabidopsis thaliana, turn the seed germination rate of GsSLAH3 arabidopsis more Height is significantly higher than wild type.Therefore, overexpression GsSLAH3 gene improves patience of the arabidopsis in germination period to alkaline stress.

2, turn Seedling Stage phenotype and root long and fresh weight of the GsSLAH3 arabidopsis under alkali process

Choose full wildtype Arabidopsis thaliana and T₃In generation, turns the seed of GsSLAH3 arabidopsis homozygous lines #14 and #15, uses 5% hypochlorite disinfectant 5min, later with sterilizing ddH₂O is rinsed 3-5 times, is placed in 4 DEG C of Stratificated treatment 3d.Then seed is broadcast In 1/2MS solid medium.After 1 week, selects the consistent wildtype Arabidopsis thaliana of growing way and turn GsSLAH3 Arabidopsis thaliana Seedlings and turn respectively Move to the NaHCO containing 0mM, 4mM and 5mM₃Culture medium on cultivate vertically, observe alkaline stress processing group and control group arabidopsis Growing way counts root long and Aboveground Biomass of Young (fresh weight) after 7d.

As a result as shown in Figure 6.As can be seen from the figure: (0mM NaHCO under normal operation₃The control group of processing), it is wild It gives birth to type arabidopsis and turns growth conditions, root long and the fresh weight of GsSLAH3 arabidopsis #14, #15 without significant difference；And in 4mM and 5mM NaHCO₃Under processing, wildtype Arabidopsis thaliana and the growth for turning GsSLAH3 arabidopsis are suppressed, but wildtype Arabidopsis thaliana It is even more serious that suppressed degree ratio turns GsSLAH3 arabidopsis: root long and the Aboveground Biomass of Young for turning GsSLAH3 arabidopsis are obvious Higher than wild type.Therefore, overexpression GsSLAH3 gene improves patience of the arabidopsis in Seedling Stage to alkaline stress.

3, turn the measurement of seedling stage phenotype and physical signs of the GsSLAH3 arabidopsis under alkali process

Choose full wildtype Arabidopsis thaliana and T₃In generation, turns the seed of GsSLAH3 arabidopsis homozygous lines #14 and #15, and 4 DEG C Stratificated treatment 3d is sowed in nutritive cube (Nutrition Soil, kaffir lily soil, vermiculite are mixed by 1:1:1) later, is placed in a greenhouse culture (22 DEG C, illumination 16h/d).After 3 weeks, 3-4d every for the seedling of alkali process group pours a 150mM NaHCO₃Solution.20d observation The arabidopsis growing way of (alkali) processing group and untreated fish group, and measure outside the chlorophyll content and electrolyte of processing group and untreated fish group Infiltration rate (detection method referring to document " plant physiology experiment/Hao Zaibin etc. edit publishing house, Harbin Institute of Technology, 2004.9 ").

As a result as shown in Figure 7.As can be seen from the figure: in 150mM NaHCO₃Under processing, wildtype Arabidopsis thaliana is shown as Yellow leaf crimps, and wilts；And turn that GsSLAH3 arabidopsis chlorosis phenomenon is relatively fewer, and blade only slightly turns to be yellow.Chlorophyll contains Measure it is fixed the result shows that: in 150mM NaHCO₃After processing, although wildtype Arabidopsis thaliana and turning GsSLAH3 arabidopsis ' chlorophyll and containing Amount reduces, but turns GsSLAH3 arabidopsis reduction amplitude significantly lower than wild type, illustrates the photosynthetic system of wild-type Arabidopsis plants System receives bigger injury.Abiotic stress normally results in plant electrolyte extravasation, therefore Electrolyte Leakage Rate can be anti- Reflect the degree that plant sustains an injury.In 150mM NaHCO₃After processing, wildtype Arabidopsis thaliana and the electrolysis for turning GsSLAH3 arabidopsis Matter extravasation rate increases, and illustrates that all strains receive different degrees of injury, but compared with wildtype Arabidopsis thaliana, turns GsSLAH3 arabidopsis Electrolyte Leakage Rate is significantly lower than wild type, shows that it is relatively light by the extent of injury of alkaline stress.Cause This, overexpression GsSLAH3 gene improves patience of the arabidopsis in seedling stage to alkaline stress.

The above results show that GsSLAH3 gene overexpression significantly improves the resistance of reverse of plant, especially alkali resistance, GsSLAH3 gene being capable of the positive alkali resistance for adjusting arabidopsis.

Sequence table

<110>Heilongjiang Bayi Agricultural Reclamation University

<120>a kind of and plant stress tolerance correlative protein GsSLAH3 and its encoding gene and application

<160>2

<210>1

<211>1686bp

<212>DNA

<213>artificial sequence

<220>

<223>

<400>1

atggaaaaca accttaaccg tgaaacagaa gggcatggct tgcctaaaat tccatcacta 60

gttcaacata tatcatcaaa tgatgaagga aactttgaca atggtgactt gaaaaatcta 120

agctcatctt tcaaagagaa tgaaacaatc ttagcaggaa gccaaggtga tgaacatgca 180

gccattaatc atcggaataa gcattcggta tctatcaaca taccattctc ttgtgaagag 240

gttcagctgc ataacaccgt aagagttctc tatagtggcg aaaatgattt ctcttctcaa 300

tcaactacta cagactccaa gccaccatta ccatcacaat caatgccaaa tggcagtctg 360

cactcagagc cagcaagcgg tgtcaatttt aacaatcaag aaagcgtcat aagcttgaat 420

aataagagga ttgatttttt caaaacatgg tccagtaaac tagggggaca catatcagtt 480

atgagtggaa aggtacatac agaaagtgca gaagatgata acagcttatg caacactaat 540

aagcctttac ctgttgattt gttcttcaaa acattggagg gtccacagtt acaaactcct 600

aagaagtctt cagaagagat ggtgcttcct caggacaagc agtggccatt tcttcttcgg 660

tttccggttt catcttttgg tatttgtctt ggagttagca gtcaagcaat tctttggaaa 720

gcattggcca cgtctccttc cactgcattt cttcacataa cccctaaaat aaatttcatc 780

ctgtggttca tctccattgg tattgttgct actattttca ccacctatct cttcaaaata 840

attctccatt ttgaagcagt tcgtcgtgag taccaacatc cggttcgtgt taacttcttc 900

tttgcaccat ggatagccct tttgttccta gctcttggag ttcccccatc agttaccaag 960

gacttgcatc aagcagtttg gtacattcta atgattccat tgttctgcct taagctaaag 1020

atatatggac agtggatgtt tgggggcaaa agaatgctgt caaaggtggc caatccaaca 1080

aatctcttag caattgttgg aaattttgta ggagccttat tgggtgcatc aatgggccta 1140

aaagaagggc ctcttttctt ctttgctctt gggcttgctc actacatggt gttgtttgta 1200

actctctccc agatgcttcc aacaaataag accatcccaa aagacctcca tccagtgttc 1260

tttctttttg tggcaccgcc tagtgttgct gctatggcat gggctaagat tcagggttca 1320

tttcattatg aatcaaggat tttctatttc actgccatgt tcctatatat ttcactggct 1380

gtccgggtca atcttttcag aggattcaaa ttctcacttt catggtgggc ctacactttt 1440

ccaatgactg ctgcagcaat tgctaccata acctatacaa atcaagtcac aaatgtacta 1500

actcaagctt tgagtgtaat attgagtctc attgctacat tcacagtaac agcagtgctt 1560

gtctcgacta tagtgcatgc ctttgtccta cgagacctct ttcccaatga ccttgccatt 1620

gccacaagtg agagaaagca aaaaccacgc aggaaatggc tcccatttaa gacttgggag 1680

ccatga 1686

<210>2

<211>561

<212>PRT

<213>artificial sequence

<220>

<223>

<400>2

Met Glu Asn Asn Leu Asn Arg Glu Thr Glu Gly His Gly Leu Pro Lys

1 5 10 15

Ile Pro Ser Leu Val Gln His Ile Ser Ser Asn Asp Glu Gly Asn Phe

20 25 30

Asp Asn Gly Asp Leu Lys Asn Leu Ser Ser Ser Phe Lys Glu Asn Glu

35 40 45

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

50 55 60

Arg Asn Lys His Ser Val Ser Ile Asn Ile Pro Phe Ser Cys Glu Glu

65 70 75 80

Val Gln Leu His Asn Thr Val Arg Val Leu Tyr Ser Gly Glu Asn Asp

85 90 95

Phe Ser Ser Gln Ser Thr Thr Thr Asp Ser Lys Pro Pro Leu Pro Ser

100 105 110

Gln Ser Met Pro Asn Gly Ser Leu His Ser Glu Pro Ala Ser Gly Val

115 120 125

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

130 135 140

Asp Phe Phe Lys Thr Trp Ser Ser Lys Leu Gly Gly His Ile Ser Val

145 150 155 160

Met Ser Gly Lys Val His Thr Glu Ser Ala Glu Asp Asp Asn Ser Leu

165 170 175

Cys Asn Thr Asn Lys Pro Leu Pro Val Asp Leu Phe Phe Lys Thr Leu

180 185 190

Glu Gly Pro Gln Leu Gln Thr Pro Lys Lys Ser Ser Glu Glu Met Val

195 200 205

Leu Pro Gln Asp Lys Gln Trp Pro Phe Leu Leu Arg Phe Pro Val Ser

210 215 220

Ser Phe Gly Ile Cys Leu Gly Val Ser Ser Gln Ala Ile Leu Trp Lys

225 230 235 240

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

245 250 255

Ile Asn Phe Ile Leu Trp Phe Ile Ser Ile Gly Ile Val Ala Thr Ile

260 265 270

Phe Thr Thr Tyr Leu Phe Lys Ile Ile Leu His Phe Glu Ala Val Arg

275 280 285

Arg Glu Tyr Gln His Pro Val Arg Val Asn Phe Phe Phe Ala Pro Trp

290 295 300

Ile Ala Leu Leu Phe Leu Ala Leu Gly Val Pro Pro Ser Val Thr Lys

305 310 315 320

Asp Leu His Gln Ala Val Trp Tyr Ile Leu Met Ile Pro Leu Phe Cys

325 330 335

Leu Lys Leu Lys Ile Tyr Gly Gln Trp Met Phe Gly Gly Lys Arg Met

340 345 350

Leu Ser Lys Val Ala Asn Pro Thr Asn Leu Leu Ala Ile Val Gly Asn

355 360 365

Phe Val Gly Ala Leu Leu Gly Ala Ser Met Gly Leu Lys Glu Gly Pro

370 375 380

Leu Phe Phe Phe Ala Leu Gly Leu Ala His Tyr Met Val Leu Phe Val

385 390 395 400

Thr Leu Ser Gln Met Leu Pro Thr Asn Lys Thr Ile Pro Lys Asp Leu

405 410 415

His Pro Val Phe Phe Leu Phe Val Ala Pro Pro Ser Val Ala Ala Met

420 425 430

Ala Trp Ala Lys Ile Gln Gly Ser Phe His Tyr Glu Ser Arg Ile Phe

435 440 445

Tyr Phe Thr Ala Met Phe Leu Tyr Ile Ser Leu Ala Val Arg Val Asn

450 455 460

Leu Phe Arg Gly Phe Lys Phe Ser Leu Ser Trp Trp Ala Tyr Thr Phe

465 470 475 480

Pro Met Thr Ala Ala Ala Ile Ala Thr Ile Thr Tyr Thr Asn Gln Val

485 490 495

Thr Asn Val Leu Thr Gln Ala Leu Ser Val Ile Leu Ser Leu Ile Ala

500 505 510

Thr Phe Thr Val Thr Ala Val Leu Val Ser Thr Ile Val His Ala Phe

515 520 525

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

530 535 540

Arg Lys Gln Lys Pro Arg Arg Lys Trp Leu Pro Phe Lys Thr Trp Glu

545 550 555 560

Pro

Claims

1. following any application in regulation plant stress tolerance；The resistance of reverse is alkali resistance；

The alkali resistance is the salt stress of resistance to carbonic acid；

1) protein, amino acid sequence are protein shown in sequence 2；

2) coding 1) described in protein nucleic acid molecules；

3) contain the expression cassette of the 2) nucleic acid molecules；

4) contain the recombinant vector of the 2) nucleic acid molecules；

5) contain the recombinant vector of the 3) expression cassette；

6) contain the recombinant microorganism of the 2) nucleic acid molecules；

7) contain the recombinant microorganism of the 3) expression cassette；

8) contain the recombinant microorganism of the 4) recombinant vector；

9) contain the recombinant microorganism of the 5) recombinant vector.

2. following any application in the genetically modified plants for cultivating resistance of reverse raising；The resistance of reverse is alkali resistance；

The alkali resistance is the salt stress of resistance to carbonic acid；

1) protein, amino acid sequence are protein shown in sequence 2；

2) coding 1) described in protein nucleic acid molecules；

3) contain the expression cassette of the 2) nucleic acid molecules；

4) contain the recombinant vector of the 2) nucleic acid molecules；

5) contain the recombinant vector of the 3) expression cassette；

6) contain the recombinant microorganism of the 2) nucleic acid molecules；

7) contain the recombinant microorganism of the 3) expression cassette；

8) contain the recombinant microorganism of the 4) recombinant vector；

9) contain the recombinant microorganism of the 5) recombinant vector.

3. following any application in plant breeding；

1) protein, amino acid sequence are protein shown in sequence 2；

2) coding 1) described in protein nucleic acid molecules；

3) contain the expression cassette of the 2) nucleic acid molecules；

4) contain the recombinant vector of the 2) nucleic acid molecules；

5) contain the recombinant vector of the 3) expression cassette；

6) contain the recombinant microorganism of the 2) nucleic acid molecules；

7) contain the recombinant microorganism of the 3) expression cassette；

8) contain the recombinant microorganism of the 4) recombinant vector；

9) contain the recombinant microorganism of the 5) recombinant vector.

4. application according to claim 1 to 3, it is characterised in that: 2) coded sequence that the nucleic acid molecules are is sequence CDNA molecule or genomic DNA molecule shown in column 1.

5. a kind of method for cultivating the genetically modified plants that resistance of reverse improves, including improve in recipient plant described in claim 1 Protein expression quantity, the step of obtaining genetically modified plants；The resistance of reverse of the genetically modified plants is higher than the recipient plant； The resistance of reverse is alkali resistance；

The alkali resistance is the salt stress of resistance to carbonic acid.

6. according to the method described in claim 5, it is characterized by: the resistance of reverse of the genetically modified plants is planted higher than the receptor It is any in object present following (1)-(5):

(1) seed germination rate of genetically modified plants is higher than recipient plant；

(2) root long of genetically modified plants is longer than recipient plant；

(3) Aboveground Biomass of Young of genetically modified plants is higher than recipient plant；

(4) chlorophyll content of genetically modified plants is higher than recipient plant；

(5) Electrolyte Leakage Rate of genetically modified plants is lower than recipient plant.

7. method according to claim 5 or 6, it is characterised in that:

The method of the expression quantity of protein described in claim 1 is that table is crossed in recipient plant in the raising recipient plant Up to the protein described in claim 1.

8. method according to claim 5 or 6, it is characterised in that:

The method of the overexpression is that the encoding gene of protein described in claim 1 is imported recipient plant；

The nucleotide sequence of the encoding gene of the protein is DNA molecular shown in sequence 1.

9. method according to claim 5 or 6, it is characterised in that: the recipient plant is monocotyledon or dicotyledonous Plant.