CN103436540B

CN103436540B - Apocynum venetum eIF-5A gene and encoded protein thereof

Info

Publication number: CN103436540B
Application number: CN201310414921.8A
Authority: CN
Inventors: 王雷; 黄国庆; 李瑶; 吴琼; 曹涤非; 王东凯; 陈立娟
Original assignee: Institute of Advanced Technology of Heilongjiang Academy of Sciences
Current assignee: Institute of Advanced Technology of Heilongjiang Academy of Sciences
Priority date: 2013-09-12
Filing date: 2013-09-12
Publication date: 2015-04-08
Anticipated expiration: 2033-09-12
Also published as: CN103436540A

Abstract

The invention discloses an apocynum venetum eIF-5A gene and an encoded protein thereof. A nucleotide sequence of the apocynum venetum eIF-5A gene is shown in SEQ ID No:1; an amino acid sequence of the encoded protein of the apocynum venetum eIF-5A gene is shown in SEQ ID No:2. The eIF-5A gene is cloned from apocynum venetum which has extremely strong stress resistance, medicinal value and economic value; a complementary deoxyribonucleic acid (cDNA) sequence of the gene is 909bp; an open reading frame is 480bp; 159 amino acids are encoded. The expression quantity of the AveIF-5A gene ascends in roots, stems and leaves under the stress conditions of salt and alkali, drought and low temperature; the saline-alkali tolerance of saccharomycetes can be improved by the intact open reading frame (ORF) and deleted sub-AveIF-5A86-156aa of the AveIF-5A; the plant resistance can be improved and the peroxidization of plant membrane lipid can be relieved under the stress conditions of salt and alkali according to over-expression of the AveIF-5A gene in a plant.

Description

Kendir eIF-5A gene and proteins encoded thereof

Technical field

The present invention relates to kendir eIF-5A gene and proteins encoded thereof.

Background technology

Desertification is a global problem, and global ccd area is 4,000 ten thousand km ², and annual also with 5-7 ten thousand km ²speed expanded, threaten the soil of nearly 1/the 3rd and life of more than 10 hundred million people, the whole world.China is one of country that world's Desertification Soil area is larger, Desertification Soil area 262.2 ten thousand km ², account for 27.3% of territory total area, every year because desertification causes direct economic loss to reach 54,000,000,000 yuan.Desertification phenomenon in Western Region of Heilongjiang Province's is very serious, and area reaches 54079.27km ², account for the whole province total land area 45.48 ten thousand km ²11.89%.Desertification brings to work, agriculture, animal husbandry production and people's lives and has a strong impact on.Agro-ecology, production environment worsen, and farmland is subject to the harm of desertification, plough and meadow serious degradation.Face the reality, should actively take effective measures, control the condition that desertification produces, rely on science and technology to struggle with desertification, drought-enduring, Salt And Alkali Tolerance, effective utilization that is cold-resistant and anti-blown sand plant play huge effect in desertification of land control.

Kendir is a kind of perennial herb wild plant having Development volue, also known as bluish dogbane or bluish dogbane, it is Apocynaceae Apocynum, distribution is all had in provinces and regions such as Xinjiang of China, Qinghai, Gansu, Ningxia, the Inner Mongol, Shandong, most growth is on saline and alkaline sandy bare land, desert, cold-resistant, drought-enduring, salt tolerant, anti-blown sand, the improvement for desertification has positive effect.Kendir whole body is precious, it is a kind of wild excellent soil-and-water conservation effect integrating saline land greening value, fiber value, pharmaceutical use, ecological benefits and economic benefit have concurrently, can be described as one of the plant variety of saline and alkaline, desert, arid area most potentiality to be exploited and value.Research about kendir focuses mostly in the development and utilization of pharmaceutical use, and physiological change under saline and alkaline, drought stress conditions, the research of cultivation, Tissue Culture Regeneration System; Research in kendir molecular biology is less, for the clone of kendir anti contravariance related gene and functional analysis just less.

EIF-5A (eukaryotic translation initiation factor, eIF-5A) be eukaryotic cell Protein translation initiation factor, ubiquity in eukaryotic cell, be made up of 154 amino-acid residues, relative molecular mass is about 16700, and iso-electric point is about 5.1, is the only protein containing carboxylic putrescine Methionin (hypusine) residue found at present, its precursor non-activity, becomes activated eIF-5A albumen after DHS enzyme catalysis, modification processing.The report of research in people and yeast cell about eIF-5A is more, has and reports that elF-5A is relevant with the many vital movements in cell, as cell proliferation, protein translation, mRNA degraded, the conversion of cell cycle and cell aging and apoptosis.The research of eIF-5A in plant is started late, although report and eIF-5A imported in Arabidopis thaliana, banana, willow, improve biomass and the resistance of transfer-gen plant, but the function and efficacy mechanism of this gene is not fully aware of, especially, under adverse circumstance environment, the research of this biology of gene function is just less.

Summary of the invention

The object of this invention is to provide kendir eIF-5A gene and proteins encoded thereof.

The nucleotide sequence of kendir eIF-5A gene of the present invention is as shown in SEQ ID No1.

The aminoacid sequence of the proteins encoded of kendir eIF-5A gene of the present invention is as shown in SEQIDNO:2.

The present invention is extremely strong and have the plant of pharmaceutical use and economic worth from resistance--clone eIF-5A gene kendir; The expression of this gene under low temperature, arid, Saline Alkali Stress condition; Complete for this gene ORF and difference in functionality district deleted that subsequence imports in yeast, by the analysis to transgenic yeast resistance, determines the relation of difference in functionality district and resistance; For verifying the function of eIF-5A gene further, build eIF-5A gene plant expression vector, transformation of tobacco, by transfer-gen plant molecular Biological Detection and resistance analysis, determine the impact of eIF-5A gene overexpression on stress resistance of plant, thus the function of this gene is identified.Lay the foundation, also for the application of this gene in adversity gene Engineering Breeding provides theoretical foundation and genetic resources for disclosing the biological function of kendir eIF-5A gene under adverse environmental factor further in the present invention.

The present invention clones and obtains eIF-5A gene cDNA sequence from kendir is 909bp, and opening code-reading frame is 480bp, 159 amino acid of encoding.Under saline and alkaline, arid, low temperature stress condition, AveIF-5A gene expression amount all raises in root, stem, leaf.The complete ORF of AveIF-5A and delete that sub-AveIF-5A86-156aa all can improve yeast and resist saline and alkaline ability.Under Saline Alkali Stress condition, AveIF-5A gene overexpression in plant can improve stress resistance of plant, alleviates the peroxidation of plant membrane lipid.

Accompanying drawing explanation

Fig. 1 be in the present invention kendir blade total serum IgE electrophorogram, wherein 1 and 2 be kendir blade RNA electrophoresis result;

Fig. 2 is the electrophorogram of AvelF-5A degenerated primer PCR result in the present invention, and wherein M is DL2O00, and 1 is degenerated primer PCR primer;

Fig. 3 is the electrophorogram of AvelF-5A5 ' RACE amplified production in the present invention, and wherein M is DL2000, and 1 is 5 ' RACE amplified production;

Fig. 4 is the electrophorogram of AveIF-5A3 ' RACE amplified production in the present invention, and wherein M is DL2O00, and 1 is sp5/anchorprimerPCR product, and 2 is sp4/anchorprimerPCR product;

Fig. 5 is that in the present invention, AveIF-5A guards functional area figure;

Fig. 6 is NaHCO in the present invention ₃coerce the histogram of the expression of lower kendir eIF-5A gene in root, stem and leaf, wherein for leaf, stem, is root;

Fig. 7 is the histogram of the expression of kendir eIF-5A gene in root, stem and leaf under drought stress in the present invention, wherein for leaf, stem, is root;

Fig. 8 is the histogram of the expression of kendir eIF-5A gene in root, stem and leaf under low temperature stress in the present invention, wherein for leaf, stem, is root;

Fig. 9 is the complete ORF of AveIF-5A and delete sub-schematic diagram in the present invention;

Figure 10 be in the present invention recombinant bacterium at NaHCO ₃the histogram of the survival rate under coercing;

Figure 11 is the electrophorogram that transfer eIF-5A genetic tobacco PCR of the present invention detects, and wherein M is DL2000,1-16 is plant to be checked, and 17 is blank, and 18 is positive control;

Figure 12 is NaHCO in the present invention ₃coerce the histogram of the impact on tobacco POD activity, wherein is not for coerce, for coercing;

Figure 13 is NaHCO in the present invention ₃coerce the histogram of the impact on tobacco SOD activity, wherein is not for coerce, for coercing;

Figure 14 is NaHCO in the present invention ₃coerce the histogram of the impact on tobacco MDA content, wherein is not for coerce, for coercing.

Embodiment

Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.

Embodiment one: the nucleotide sequence of present embodiment kendir eIF-5A gene is as shown in SEQ ID No1.

In present embodiment, the cDNA sequence of kendir eIF-5A gene is 909bp, and opening code-reading frame is 480bp, 159 amino acid of encoding.

Embodiment two: the aminoacid sequence of the proteins encoded of present embodiment kendir eIF-5A gene is as shown in SEQ IDNO:2.

1, the preparation method of kendir eIF-5A gene is as follows:

Kendir eIF-5A gene clone: utilize SDS method to extract the total serum IgE of wild venetum blade, with Oligo d (T) ₁₈for primer, be cDNA with reference to precious biological Reverse TranscriptaseM-MLV (RNaseH-) the method reverse transcription in Dalian; On NCBI, retrieval obtains the eIF-5A gene order of eastern cottonwood (FJ032302), alfalfa (AF416338), potato (AB004823), grape (XM_002273229), Chinese rose (DQ345329), paragutta (AF516357), cassava (AF266464), carry out after sequence alignment at homology conserved regions design degenerated primer AveIF-5AJ-F and AveIF-5AJ-R, check order after carrying out PCR, obtain eIF-5A Gene Partial sequence, design 5 '/3 ' RACE primer SP1, SP2, SP3, SP4 and SP5.Hold according to method clone kendir eIF-5A gene 5 '/3 ' of Roche5 '/3 ' RACE Kit, 2nd Generation test kit

Above-mentioned primer sequence is in table 1:

Name	Primer sequence
		Oligo d(T) ₁₈	TTTTTTTTTTTTTTTTTT
AveIF-5AJ-F	CNCARCARGCNGGNACNA
		AveIF-5AJ-R	ATYTGYTCYTCNCCCAT
SP1	CTTTCCCTCAGCAAATCCATC
		SP2	TGGTAATCAACACGGTTCACAT
SP3	CTTGCCAGTCTTGGAAGTTGA
		SP4	AGATATTGTTCCCTCTTCCCAC
SP5	CTGAGGCTCCCAACAGATGA

The reaction system of above-mentioned RT-PCR is 20 μ l reaction systems, is made up of following ingredients:

Pcr amplification condition is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 54 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations; 72 DEG C extend 7min, 4 DEG C of insulations.

Result:

1. kendir Total RNAs extraction: as seen from Figure 1, the band of 285rRNA and 185rRNA of RNA is clear, and 28S rRNA brightness is apparently higher than 185rRNA, and the total serum IgE that application SDS method is extracted is without degraded, up-to-standard.Detect through nucleic acid-protein detector and show, the A260nm/A280nm of the RNA extracted, between 1.8 ~ 2.1, on average about 2.0, illustrates that the purity of RNA is higher.

2. kendir eIF-5A gene cDNA clone: utilize degenerated primer PCR to obtain the DNA fragmentation (see Fig. 2) of 388bp, find through BlastX comparison, this fragment has typical eIF-5A gene conserved sequence, to be AveIF-5A gene containing the unnamed gene of this fragment, to this cDNA fragment design special primer, adopt 5 '/3 ' end of the method amplification AveIF-5A gene of 5 '/3 ' RACE (rapidamplification of cDNA ends) respectively, 5 ' RACE obtains the fragment (Fig. 3) of a 283bp, 3 ' RACE obtains the band (Fig. 4) of a 500bp.

2, the bioinformatic analysis of kendir eIF-5A gene and derivation proteins encoded thereof:

The ORF program in NCBI and conservative functional zone routine analyzer is adopted to infer 5 '/3 ' non-translational region of this gene, ORF district and conservative functional zone, obtain the amino acid sequence of the proteins encoded of kendir eIF-5A gene, ProtParam calculates this gene coded protein molecular weight and iso-electric point, and SignalP3.0 predicts the signal peptide of this albumen.

Result:

OAveIF-5A full length cDNA sequence is analyzed: the full length cDNA sequence obtained with PolyA through electronic splicing is 909bp.Adopt the ORF programdisplay 5 ' non-translational region in NCBI to be 8gbp, 3 non-translational regions are 340bp, and opening code-reading frame is 480bp, 159 amino acid of encoding.The protein molecular weight calculating AveIF-5A genes encoding through ProtParam is 17.48kDa, and theoretical iso-electric point is 5.61, and in 159 amino acid of this genes encoding, Asp is maximum, accounts for 9.4% of amino acid sum.Electronegative amino acid (Asp+Glu) has 26, and positively charged amino acid (Arg+Lys) has 20.Unstability index is 26.79, and this albumen is stabilize proteins.

2. the prediction in AveIF-5A Subcellular Localization and specific function site: by the conserved regions of conservative functional area (conserveddomains) the routine analyzer predicted protein of NCBI, result shows that this albumen has eIF-5A conserved sequence, and conserved regions is (see figure 5) between 86th ~ 156 amino acid.PSORT ( http:// psort.nibb.ac.jp/) Subcellular Localization prediction result display: it is 0.65 that AveIF-5A is positioned cytoplasmic confidence level, and the confidence level be positioned in mitochondrial matrix is 0.1, and the confidence level being positioned endoplasmic reticulum is 0.

3. the prediction of AveIF-5A signal peptide: SignalP3.0 predicts that this albumen does not have signal peptide, is nonsecreting type albumen.

3, the expression analysis of kendir eIF-5A gene: use 0.4mol/L NaHCO respectively ₃with 20% (W/V) PEG6000 solution, Stress treatment is carried out to kendir seedling, carry out low temperature stress the artificial climate indoor of 4 DEG C.Coercing 0,12,24,48 and 72h, get treatment group and control group root of Apocynum venetum L., stem, leaf, the expression of eIF-5A gene in mRNA level in-site is detected by Real-time PCR method, kendir GAPDH gene is as reference gene (be shown in table 2), utilize Δ Δ CT method to calculate the relative expression quantity of goal gene, the data obtained utilizes SpASS11.0 statistical software to carry out one-way analysis of variance Duncan inspection.

Table 2Real-timePCR primer sequence

The reaction system of above-mentioned PCR is 20 μ l reaction systems, is made up of following ingredients:

PC amplification condition is: 94 DEG C of denaturation 30s; 94 DEG C of sex change 12s, 58 DEG C of annealing 30s, 72 DEG C extend 40s, and read plate after 81 DEG C of ls, 44 take turns circulation; 55 ~ 99 DEG C of melt curve analysis analyses.

Result:

See Fig. 6,7,8; Under normal growth state) AveIF-5A gene all expresses in root, stem, leaf.At NaHCO ₃, arid, under low temperature stress condition, AveIF-5A gene expression amount all raises in root, stem, leaf, the expression amount change in root of this gene is not remarkable.Along with the prolongation of stress time, the expression amount of AveIF-5A gene in stem and leaf raises gradually.

4, the eukaryotic expression in eIF-5A gene difference in functionality district and resistance analysis: according to the Bioinformatics Prediction of kendir eIF-5A gene; in both sides, ORF district and delete that sub-both sides (see figure 9) adds Xba I and Xho I restriction enzyme site and protection base; design primer and PCR; obtain the fragment with restriction enzyme site, Primer is respectively AveIF-5A and AveIF-SAD(in table 3).Complete for this gene ORF and correlation function district deleted that sub-cDNA is building up on expression vector, respectively transformed yeast (INVScl) (method is shown in the working method in Invitrogen company pYES2), molecular Biological Detection and NaHCO are carried out to transgenic yeast ₃resistance is analyzed, to determine the relation in eIF-5A difference in functionality district and resistance.

Table 3 vector construction the primer

Above-mentioned PCR system is as follows:

Pcr amplification condition is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations; 72 DEG C extend 7min, 4 DEG C of insulations.

Enzyme tangent condition is: 37 DEG C of enzymes cut through night.

The system of above-mentioned connection is as follows:

Be connected with goal gene digestion products pYES2 plasmid enzyme restriction product with Promega T4 DNA Ligase, linked system is as follows:

Result:

By the contrast bacterium INVScl/pYES2 and two of same cell density kind of recombinant bacterium INVScl/pYES2-AveIF-5A, INVScl/pYES2-AveIF-5A _86-156aabe coated on normal and coerce on substratum.After cultivating for some time, the bacterial plaque number on statistics substratum.Calculate the survival rate (Figure 10) of each bacterial strain.NaHCO ₃coerce down, the survival rate of INVScl/pYES2-AveIF-5A recombinant bacterium is higher than contrast and delete sub-INVScl/pYES2-AveIF-5A _86-156aa, delete sub-INVScl/pYES2-AveIF-5A _86-156aasurvival rate higher than contrast.

5, eIF-5A gene function checking: by gene constructed for eIF-5A on plant expression vector pKYLX-71, by Agrobacterium-mediated transformation tobacco SR-I (Nicotiana tabacum L.cv.Petit HavanaSR-I), PCR detection is carried out to transfer-gen plant.Detection non-transgenic and transgene tobacco are at normal growth and NaHCO respectively ₃pOD under stress state is active, SOD is active, MDA content, to determine the impact of eIF-5A gene overexpression on stress resistance of plant.

PC amplification condition is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 1min, and 30 take turns circulation; 72 DEG C extend 7min, 4 DEG C of insulations.

Result:

By kendir eIF-5A gene clone on plant expression vector, transformation Agrobacterium, utilizes bacillus mediated method transformation of tobacco, and Figure 11 is the result of transgene tobacco PCR.Use 0.25MNaHCO ₃after coercing one week, transgene tobacco physical signs is detected, the results are shown in Figure 12,13,14.In normal growth situation, transfer-gen plant POD is active, SOD is active in contrast, and MDA content is a little less than contrast; Under condition of salt stress, nontransgenic plants POD is active, the active rapid enhancing of SOD, and MDA content is higher than transfer-gen plant.This illustrates that eIF-5A gene overexpression can improve the resistance of plant, has certain alleviation to the peroxidation of plant membrane lipid.

Conclusion:

1. from kendir, clone obtains eIF-5A gene cDNA sequence is 909bp, and opening code-reading frame is 480bp, 159 amino acid of encoding.

2., under saline and alkaline, arid, low temperature stress condition, AveIF-5A gene expression amount all raises in root, stem, leaf.

3.AveIF-5A full-length cDNA and delete that sub-AveIF-5A86-156aa all can improve yeast and resist saline and alkaline ability.

4. under Saline Alkali Stress condition, AveIF-5A gene overexpression in plant can improve the resistance of plant, alleviates the peroxidation of plant membrane lipid.

Claims

1. kendir deletes sub-AveIF-5A86-156aa gene, it is characterized in that kendir deletes that the nucleotide sequence of sub-AveIF-5A86-156aa gene is as follows:

GTGAACCGTGTTGATTACCAGCTGATTGACATCTCTGAGGACGGCTTTGTTAGCCTTCTCACTGATAATGGTGAAACTAAGGATGACCTGAGGCTCCCAACAGATGAGAATCTGCTTAAACAGATCAAGGATGGATTTGCTGAGGGAAAGGATCTGATTGTGTCAGTGATGTCATCAATGGGGGAGGAGCAGATCTGCGCCCTCAAGGATATT。

2. kendir deletes the proteins encoded of sub-AveIF-5A86-156aa gene, it is characterized in that kendir deletes that the aminoacid sequence of the proteins encoded of sub-AveIF-5A86-156aa gene is as shown in 86th ~ 156 base acid sequences in SEQIDNO:2.