CN102660555A - Peanut senescence gene for regulating programmed cell death, coding sequence and application - Google Patents

Abstract

The invention discloses a peanut senescence gene for regulating programmed cell death, a coding sequence and application. RNA (ribonucleic acid) is extracted from root tip of peanut, and is cloned to a peanut senescence gene AhSAG, and the ORF (open read frame) of the peanut senescence gene is a cDNA (complementary deoxyribonucleic acid) sequence with coding sequence of 474bp and codes of 157aa; the AhSAG comes from a peanut root tip meristematic tissue of aluminum-induced peanut root tip generating programmed cell death, and is a senescence induction enhanced gene to induce, activate and promote progeria of plants. The peanut senescence disclosed by the invention can be expressed in tobacco, and can be used for regulating the adaptability of plant cells to environment changes and maintaining environmental stability and synthesis capacity in the cell.

Description

The senility of peanuts gene of regulating cell programmed death and encoding sequence and application

Technical field

The invention belongs to the plant gene engineering technology field, be specifically related to senility of peanuts Gene A hSAG and the encoding sequence and the application of regulating cell programmed death.

Background technology

In plant, necrocytosis is a kind of common phenomena, is the part in the highly single-minded cell development process.In this process, vegetable cell receives stimulation external and internal information (external environment, growth signal, metabolism environment etc.), changes cell fate, causes necrocytosis.Necrocytosis shows as two types: necrocytosis (Cell necrosis) and programmed cell death (PCD).Necrocytosis normally because accidental extraneous factor extremely stimulates the lysosome particulate that causes to discharge like be heated or medicine etc., causes aqtocytolysis dead, is a kind of improper death.PCD is meant a series of orderly molecular events that receives genes encoding instruction regulation and control, is a kind of active, physiological process of cell death.Up to now; Many PCD incidents in higher plant, have been found; Comprise PCD and environmental induction PCD in the development of plants process; Mainly contain the anaphylaxis that the phytopathy original does to cause mutually (hypersensitive response, HR), the death of root tegumental cell, nucellar cell's degeneration, gluten cell degeneration, ventilating tissue's formation, somatic embryo generation, root cap necrocytosis, unisexual flower formation, megaspore disappearance, anther wall cracking etc.Plant PCD is similar with the zooblast apoptosis: being kept perfectly property of cytolemma and contraction, (Cysteine-containing Aspartate-specific proteases caspasess) participates in type L-Cysteine HCL Anhydrous, and DNA is divided into entosthoblast scalariform etc.But because there are marked difference in plant-animal on cellular form and physiological function, so the differing greatly of plant PCD and zooblast apoptosis.In plant, vacuole plays an important role in PCD, generally takes the mode of autophagy, but the most typical characteristic of zooblast apoptosis is to form apoptotic body, and by other cytophagy.

Cell aging is a kind of PCD phenomenon, and phalangeal cell is to the adaptive faculty of environmental change and keep cell homeostasis and the reduction of synthesis capability, like the orderly decline of chloroplast(id) on subcellular structure and other organoids; Macromolecular degraded, mobilization in the cell on the metaboilic level; The activation of SAG SAGs and expression on the gene level.The old and feeble last adjusting factor that is considered to growth and development of plants to a great extent, old and feeble speed directly has influence on plant-growth, influences crop yield, and blade early ageing is considered to the major reason of farm crop low yield.Environment stresses such as ultraviolet, temperature, arid, germ can promote plant that early ageing takes place; Thereby cause the reallocation (Kim H is regulation of leaf senescence.In senescence Processes in Plants J.2007.Molecular, and 231-255.Lim P is senescence.Annu Rev Plant Biology58:115-136 O.2007.Leaf) of the energy in the plant materials.Therefore, infer that environment stress is with old and feeble closely related.

Old and feeble up-regulated gene and old and feeble down-regulated gene have been comprised with old and feeble related gene.Old and feeble up-regulated gene or title SAG (senescence-associated genes; SAGs) be meant that some were not expressed or the gene of low expression level is activated in functional leaf between senescence phase; The mRNA level improves with leaf senile, i.e. up-regulated.Old and feeble down-regulated gene is meant and is suppressed and low expression level in the aging course, does not even express the genoid that the corresponding mRNA level descends.

Adopt differential screening and difference to subtract the mRNA construction cDNA library that hybridization detects greenery and old and feeble blade; Find that blade RNA total amount descends, and the rising gradually on the contrary of some expression of gene amount (clone of Liu Li .2008. rice leaf senescence-specific promotor and the evaluation of utilization and the early stage old and feeble rising expressing gene of sword-like leave. [academic dissertation]. Guangzhou: Agricultural University Of South China).In the full genome range of model plant Arabidopis thaliana, a large amount of SAG have been identified; With comprising 24; The Affymetrix chip hybridization Arabidopis thaliana of 000 gene transcription in old and feeble period; Find that wherein about 800 genes are rise expression (Gepstein S, Sabehi G, Carp MJ.2003.Large-scale identification of leaf senescence-associated genes.Plant Journal 36:629-642; Andersson A, Keskitalo J, Sjodin A..2004.A transcriptional timetable of autumn senescence.Genome Biology 5:R24; Lin J F, Wu S be events in senescencing Arabidopsis leaves.Plant Journal 39:612-638 H.2004.Molecular; Buchanan-Wollaston V; Page T, Harrison E. 2005.Comparative transcriptome analysis reveals significant differences in gene expression and signaling pathways between developmental and dark/starvation-induced senescence in Arabidopsis.Plant Journal 42 (4): 567-85; Van der Graff E, Schwacke be analysis of Arabidopsis membrane transporters and hormone pathways during developmental and induced leaf senescence.Plant Physiology 141:776-792 R.2006.Transcription; Guo F, Grawford N be nitric oxide synthasel is targeted to motochondria and protects against oxidative damage and dark-induced senescence.Plant Cell 17:3436-3450 M.2005.Arabidopsis).

Buchanan-Wollaston etc. have compared Arabidopsis leaf naturally-aged and the hungry expression that brings out suspension cell PCD on a large scale with gene chip; Find only to have in the gene that 827 naturally-ageds rise 326 to be that PCD in suspension cell strengthens and expresses (Buchanan-Wollaston V simultaneously; Page T, Harrison be transcriptome analysis reveals significant differences in gene expression and signaling pathways between developmental and dark/starvation-induced senescence in Arabidopsis.Plant Journal 42 (4) E.2005.Comparative: 567-85).Infer that the PCD process that necrocytosis comprises in the senile cell links to each other with the other types PCD in the plant growth and development process is staggered.Do not see that at present senility of peanuts Gene A hSAG announced.

Summary of the invention

The object of the invention:, a kind of senility of peanuts gene and encoding sequence and application of regulating cell programmed death are provided in order to regulate and control vegetable cell to the adaptive faculty of environmental change and keep cell homeostasis and synthesis capability.

The present invention is achieved in that

A kind of senility of peanuts gene and encoding sequence of regulating cell programmed death extract RNA from the peanut tip of a root and obtain aging gene AhSAG, and the base sequence of its ORF is 474bp, the cDNA sequence of coding 157aa, and its base sequence is shown in SEQ ID NO.1.SEQ IDNO.1 is:

ATAGGAAGAGCCGACATCGAAGGATCAAAAAGCAACGTCGCTATGAACGCTTGGCTG 60

CCACAAGCCAGTTATCCCTGTGGTAACTTTTCTGACACCTCTAGCTTCAAATTCCGAAGG 120

TCTAAAGGATCGTTAGGCCACGCTTTCACGGTTCGTATTCGTACTGGAAATCAGAATCAA 180

ACGAGCTTTTACCCTTCTGTTCCACACGAGATTTCTGTTCTCGTTGAGCTCATCTTAGGA 240

CACCTGCGTTATCTTTTAACAGATGTGCCGCCCCAGCCAAACTCCCCACCTGACAATGTC 300

TTCCGCCCGGATCGACCGGCCGAAGCCAGCCTTGGGTCCAAAAAGAGGGGCAATGCCCCG 360

CCTCCGATTCACGGAATAAGTAAAATAACGTTAAAAGTAGTGGTATTTCACTTTCGCCGT 420

TTCCGGCTCCCACTTATCCTACACCTCTCAAGTCATTTCACAAAGTCGGAC 474

Described AhSAG derives from the peanut tip of a root meristematic tissue that aluminium is induced peanut tip of a root generation programmed cell death, and the coded amino acid of AhSAG is listed as shown in SEQ ID NO.2.SEQ ID NO.2 is:

MIGRADIEGS?KSNVAMNAWL?PQASYPCGNF?SDTSSFKFRR?SKGSLGHAFT?VRIRTGNQNQ 60

TSFYPSVPHE?ISVLVELIL?G?HLRYLLTDVP?PQPNSPPDNV?FRPDRPAEAS?LGSKKRGNAP 120

PPIHGISKIT?LKVVVFHFRR?FRLPLILHLS?SHFTKSD 157

Described AhSAG is old and feeble induction-enhanced gene, and when concentration was handled less than 20 μ mol/LAl, the expression of AhSAG gene in anti-aluminium property kind of peanut and aluminium sensitive varieties was very low; When concentration was handled at 20～100 μ mol/L Al, the AhSAG gene expression amount significantly raise, and aluminium sensitive varieties expression amount is higher than anti-aluminium property kind expression amount; When concentration 100～400 μ mol/L Al handle, the AhSAG overexpression, induction of apoptosis all appears in anti-aluminium property kind and aluminium sensitive varieties, promotes plant early ageing.Spend No. 2 in peanut anti-aluminium kind 99-1507 in the present invention's test and the aluminium sensitive varieties, the testing program of delivering in " aluminium is to the influence of peanut root-tip cells morphological structure " at " Chinese oil crops journal " in March, 2008 according to the contriver obtains.

A kind of senility of peanuts gene of regulating cell programmed death and recombinant expression vector of coding protein sequence of comprising is to obtain recombinant expression vector through the AhSAG gene is imported carrier pBI121-GFP.

Said recombinant expression vector is sense expression vector or antisense expression vector; Be to utilize sense primer or antisense primer, be cloned into then on the carrier pTG19-T, cut through enzyme through pcr amplification Gene A hSAG; Import then among the carrier pBI121-GFP and obtain; It acts on plant and the just plant expression vector pBI121-GFP-AhSAG that obtains, antisense plant expression vector pBI121-GFP-anti-AhSAG, and the empirical tests sequence is correct; Be used for plant genetic and transform, carry out the AhSAG gene function and identify.

Described sense primer is:

The upper reaches: GCT CTAGAATGATAGGAAGAGCCGACATCGAAGG Xba I site

Downstream: TGC GGATCCCTAGTCCGACTTTGTGAAATGAC BamH I site

Described antisense primer is:

The upper reaches: TTGC GGATCCATGATAGGAAGAGCCGACATCGAAGG BamH I site

Downstream: GGCGT CTAGACTAGTCCGACTTTGTGAAATGAC Xba I site.

A kind of reorganization bacterium of recombinant expression vector of the senility of peanuts gene coded sequence that comprises the regulating cell programmed death; Described reorganization bacterium is to import agrobacterium tumefaciens bacterial strain EHA105 through just plant expression vector and antisense plant expression vector to obtain, and in the allos eukaryotic system, realizes functional expression.

The application of the transfer-gen plant of a kind of bacterium of recombinating, said transfer-gen plant are with in the justice that contains GFP (green fluorescent protein) and the antisense senility of peanuts Gene A hSAG importing tobacco of reorganization and obtain the tobacco transfer-gen plant.The mensuration of tobacco transfer-gen plant; It is GFP gene expression through the fluorescence microscope transformation of tobacco; Confirm expression and the location of AhSAG in tobacco; That in the form layers tissue of the center pillar of the tender tip of a root of children, root and stem, expresses is many, and only in cell walls and cytolemma, expresses.

Said tobacco transfer-gen plant aluminium is handled GFP (green fluorescent protein) expression down, is that aluminium is handled the expression of having induced senility of peanuts Gene A hSAG, and al concn is high more; Aging gene is expressed strong more; And the tobacco of the Yi Jiyin that under identical al concn, becomes a full member is more than the expression of antisense genetic tobacco, explains that aluminium coerces down, and tobacco tip of a root justice aging gene is expressed and strengthens; And tip of a root aging is accelerated in the expression of aging gene, produces programmed cell death.

Beneficial effect of the present invention:

The senility of peanuts Gene A hSAG of regulating cell programmed death of the present invention provides a kind of and helps to regulate and control vegetable cell to the adaptive faculty of environmental change and keep cell homeostasis and the method for synthesis capability, creates conditions for plant yield-increasing increases income.

Description of drawings

Fig. 1: be the base sequence figure of AhSAG gene ORF;

Fig. 2: be the Argine Monohydrochloride sequence chart of AhSAG gene ORF;

Fig. 3: be pcr amplification AhSAG gene intermediate outage swimming collection of illustrative plates; M is 0331maker, 1 negative contrast, and 2 is the AhSAG intermediate segment;

Fig. 4: be pcr amplification AhSAG gene 3 ' race electrophoretogram; M is 0331maker, and 1 is AhSAG 3 ' race, 2 negative contrasts;

Fig. 5: be pcr amplification AhSAG gene 5 ' race electrophoretogram; M is 0331maker, and 1 is other experiment picture, and 2 is AhSAG 5 ' race;

Fig. 6: be pcr amplification AhSAG full length gene cDNA electrophoretogram; M is 0331maker, 1 negative contrast, and 2 negative contrasts, 3 is the AhSAG full-length gene;

Fig. 7: for AhSAG at NCBI upper amino acid sequence homology analysis;

Fig. 8: for the enzyme of just plant expression vector pBI121-GFP-AhSAG and antisense plant expression vector pBI121-GFP-anti-AhSAG is cut checking; M is Maker, and 1 is pBI121-GFP-AhSAG/BamH I and XbaI, and 2 is pBI121-GFP-anti-AhSAG/BamH I and Xba I I;

Fig. 9: be the differential expression figure of AhSAG gene under the different aluminum treatment condition;

Figure 10: for changeing AhSAG genetic tobacco form, other provides its coloured picture is the examination as to substances bibliography; A is not genetically modified tobacco, and B is an antisense AhSAG tobacco, and C is the adopted AhSAG tobacco of becoming a full member;

Figure 11: for the PCR that changes the positive antisense genetic tobacco of AhSAG plant detects electrophoretogram; M is Maker DL-2000,1 negative contrast, and 2-3 is EHA105/pBI121-GFP-AhSAG, 4-5 is EHA105/pBI121-GFP-anti-AhSAG.;

Figure 12: be the fluorescence location collection of illustrative plates of AhSAG gene in the transgene tobacco; A is the tip of a root of transgene tobacco not; B is the tip of a root of adopted AhSAG genetic tobacco of becoming a full member; C is the tip of a root of antisense AhSAG genetic tobacco; D is transgene tobacco tender leaf leaf epidermal cell fluorogram not, and E is the adopted AhSAG genetic tobacco tender leaf epidermic cell fluorogram of becoming a full member, and F is an antisense AhSAG genetic tobacco tender leaf epidermic cell fluorogram;

Figure 13: GFP under aluminium is handled expresses for the transgene tobacco root-tip cells; A, E are the tip of a root fluorogram of not transgene tobacco; B-D is the adopted AhSAG genetic tobacco root-tip cells GFP expression under different al concns of becoming a full member; F-H antisense AhSAG genetic tobacco root-tip cells GFP under different al concns expresses, and B, F are 30 μ mol/LAlCl ₃, C, G are 90 μ mol/LAlCl ₃, D, H are 150 μ mol/LAlCl ₃(be pH4.2～4.3 and handle 24h).

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Implementation method among the following embodiment like no specified otherwise, is ordinary method.Used material among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.% among the following embodiment like no specified otherwise, is the quality percentage composition.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.

Implement to prepare

1, used peanut varieties: spend No. 2 in the peanut varieties of anti-aluminium 99-1507 and the aluminium sensitive varieties.

2, will supply the examination peanut seed to place 26 ℃ of vernalization 4d of rough sand, and urge the peanut of bud to remove kind of a skin, the Hoagland nutritive medium is cultivated, and every 2d changes one time of nutrition liquid.Peanut seedling is containing the CaCl of 0.5mmol/L after extracting second true leaf out ₂Pre-treatment 24h in the solution of (pH4.2～4.3).

3, select 0 (negative control), 20 (lower concentration is handled, and PCD does not all take place for both), 100 (spending generation PCD in having only No. 2), 400 μ mol/L aluminium to handle (PCD all takes place for both) and handle 4d, a situation arises to check PCD with DNA Ladder and TUNEL.

4, the bacterial strain, the plasmid that use in the research are seen table 1.

Table 1: plasmid that uses in this work and bacterial strain

5, peanut tip of a root RNA extracts in a small amount

Peanut tip of a root RNA extracts in a small amount and uses the Trizol method, and operation steps is following:

(1) prepares special-purpose rifle head and centrifuge tube, utensils such as mortar, mortar rod and iron spoon, autoclave sterilization 4h.

(2) in mortar, pour the liquid nitrogen precooling into, preserve the subsequent use about 0.2g of the tip of a root with-80 ℃ and pour in the mortar, pour the capacity liquid nitrogen into; Push sample gently with the alms bowl rod simultaneously, when treating that liquid nitrogen also has a small amount of the residue, rapid at once strong ground sample to white fine powder powder; Repeat 3～4 times; During liquid nitrogen grinding, do not let the liquid nitrogen volatilization clean, in case major structure becomes the degraded of endogenous RNase to RNA.

(3) scrape sample thief with the centrifuge tube of precooling, add the Trizol reagent of 1mL precooling at once, thermal agitation mixing up and down, room temperature leaves standstill 15min, till obvious layering.Attention will be selected suitable lysate, and amount is many, and cracking is wanted fully.

(4) 12,000rpm, centrifugal 5min draws supernatant, moves in the new 1.5mL centrifuge tube, adds 200 μ L chloroforms, vibration up and down, room temperature is placed 5min, and 12,000rpm, centrifugal 10min carefully draws the upper strata water to new 1.5mL centrifuge tube.

(5) add the Virahol of equal-volume precooling, mixing gently ,-20 ℃ leave standstill 30min, and 12,000rpm, 4 ℃ of centrifugal 15min abandon supernatant.

(6) 75% ethanol swing washes deposition 2～3 times, gentle vibration, and the deposition that suspends, 12,000rpm, centrifugal 5min carefully removes supernatant with the rifle head, dries to transparence.

(7) with 30 μ LDEPC-H ₂The O dissolution precipitation is got 2 μ L 1% and is carried out agarose electrophoresis.Detect qualified RNA sample and can directly descend the step test, or be stored in-80 ℃.

6, used primer in each step is seen table 2.

Table 2: the primer that this institute uses

Embodiment 1:RACE method obtains the AhSAG full-length gene

1, cDNA's is synthetic

According to different needs, carry out reverse transcription, obtain different cDNA, see table 3.

Segment, 3 ' end and 5 ' end cDNA's is synthetic in the middle of the table 3AhSAG

Template	The reverse transcription primer	Purposes
			99-1507400 μ mol/L Al handles 4d	OligoT18	Common templates
99-1507400 μ mol/L Al handles 4d	F	3 ' race template
			99-1507400 μ mol/L Al handles 4d	SAG 5 ' reverse transcription	5 ' race template

2, middle segment, 3 ' end and 5 ' end cDNA segment pcr amplification (50 μ L system)

(1) segment in the middle of

Reaction system:

Adopt operation on ice, the concrete operations condition is seen table 3.

Table 3: reaction system operational condition

Reactive system	Volume
			10 * Buffer (contains Mg 2+)	5μL
dNTP(2.5mM)	4μL
		P1:SAG last (10 μ M)	2μL
Under the P2:SAG (10 μ M)	2μL
		5 times of OligoT18 reverse transcription product dilutions	1μL
Ex-Taq	0.25μL
		ddH 2O	35.75μL
total	50μL

Reaction conditions:

Concrete operation condition is seen table 4.

Table 4: operation condition

Cycle	Temperature and time
		1	94℃，5min
35	94℃，30s；47℃，30s；72℃，45s
		1	72℃，10min

Detect: 1% agarose electrophoresis, 600～700bp purpose segment is arranged, negative control does not have band.

(2) 3 ' Race (3 ' end)

Reaction system:

Reaction system adopts operation on ice, and the concrete operations condition is seen table 5.

Table 5: reaction system operational condition

Reactive system	Volume
			10 * Buffer (contains Mg 2+)	5μL
dNTP(2.5mM)	4μL
		P1:SAG 3 ' upper reaches (10 μ M)	2μL
P2：M(10μM)	2μL
		5 times of 3 ' race reverse transcription product dilutions	0.5μL
Ex-Taq	0.25μL
		ddH 2O	36.25μL
total	50μL

Reaction conditions:

Concrete operation condition is seen table 6.

Table 6: operation condition

Cycle	Temperature and time
		1	94℃，5min
35	94℃，30s；52℃，30s；72℃，30s
		1	72℃，10min

Detect: 1.2% agarose electrophoresis, there is 200～300bp purpose segment to occur, negative control does not have band.

(3)5’Race

Use rapid amplifying cDNA 5 ' terminal test kit (5 ' RACE System for Rapid Amplification of cDNA Ends, Version2.0 (Catalog no.18374-058, Invitrogen), concrete operations are following:

(A) cDNA purifying

I, 1 μ L RNase Mix handle reverse transcription product 30min for 37 ℃.

Ii, will place a moment, add 120 μ L again in cDNA solution with cDNA binding soln (binging solution) room temperature, mixing, and be transferred in the purification column, 13,000rpm, centrifugal 20s.

Iii, remove pillar, the liquid at the pipe end is transferred in the new PCR pipe, preserve liquid and know and confirm that the cDNA collection finishes, and puts into pipe with pillar again.

1 * the washings (wash buffer) of iv, adding 0.4mL precooling, 13,000rpm, centrifugal 20s discards waste liquid.

V, repeating step iv three times.

Vi, add 70% ethanol of 400 μ L precoolings, 13,000rpm, centrifugal 20s discards waste liquid, and again 13,000rpm, centrifugal 1min discards waste liquid.

Vii, with posts transfer to the new collection tube, add the ddH of 50 μ L65 ℃ preheatings ₂O, 13,000rpm, centrifugal 20s, wash-out cDNA.

(B) cDNA tailing

I, in purified cDNA solution, add following reagent successively, of short duration centrifugal, mixing.The concrete operations condition is seen table 7.

Table 7: operation condition

Reactive system	Volume
		The cDNA of purifying	10μL
5×tailing?buffer	5μL
		2mMdCTP	2.5μL
ddH 2O	6.5μL
		total	24μL

Ii, 94 ℃ are hatched 2～3min, and ice bath 1min is of short duration centrifugal immediately.

Iii, in above-mentioned mixed liquid, add 1 μ L TdT, of short duration centrifugal, mixing, 37 ℃ of 10min, 65 ℃ of 10min place rapidly on ice ,-20 ℃ of preservations.

(C) pcr amplification (50 μ L system)

I, first round PCR

Reaction system:

Adopt operation on ice, the concrete operations condition is seen table 8.

Table 8: reaction system operational condition

Reaction system	Volume
			10 * Buffer (contains Mg 2+)	5μL
dNTP(2.5mM)	4μL
		P1:AAP (test kit provides)	2μL
P2：SAG?5’GSP1(10μM)	2μL
		CDNA stoste after the processing	1μL
Ex-Taq	0.25μL
		ddH 2O	35.75μL
total	50μL

Reaction conditions:

Concrete operation condition is seen table 9.

Table 9: operation condition

Cycle	Temperature and time
		1	94℃，5min
35	94℃，30s；55℃，30s；72℃，30s
		1	72℃，10min

Detect: 1% agarose electrophoresis, no purpose segment is carried out second and is taken turns PCR.

Ii, Nested PCR ^2ndAmplification (50 μ L system)

Reaction system:

Adopt operation on ice, the concrete operations condition is seen table 10.

Table 10: reaction system operational condition

Reactive system

Volume

10 * Buffer (contains Mg 2+)	5μL
		dNTP(2.5mM)	4μL
P1:AUAP (test kit provides)	2μL
		P2：SAG?5’GSP2(10μM)	2μL
First round PCR product stoste	1μL
		Ex-Taq	0.25μL
ddH 2O	35.75μL
		total	50μL

Reaction conditions:

Concrete operation condition is seen table 11.

Table 11: operation condition

Cycle	Temperature and time
		1	94℃，5min
35	94℃，30s；50℃，30s；72℃，30s
		1	72℃，10min

Detect: 1.2% agarose electrophoresis, 200～300bp purpose segment appears, and negative control does not have band.

(4) order-checking

With the recovery of dna segment, connection, conversion, plasmid are carried for a short time, enzyme is cut checking, worker's order-checking is given birth in puncture, Shanghai, use bioinformation Vector NTI 8.0 softwares, NCBI to carry out sequential analysis.

3, AhSAG full-length gene pcr amplification checking

Middle segment, 3 ' and 5 ' the pulsating sequencing result of AhSAG gene are analyzed, according to overlap splicing full-length cDNA.In order to reflect the verity of ORF, improve PCR product fidelity of reproduction, the design primer increases with the high-fidelity enzyme once more from known non-OFR district, template used should be from same transcript, the safety of assurance sequence.

(1) RT-PCR (50 μ L system)

Reaction system:

Adopt operation on ice, the concrete operations condition is seen table 12.

Table 12: reaction system operational condition

Reactive system	Volume
			10 * Buffer (contains Mg 2+)	5μL
dNTP(2.5mM)	4μL
		On the P1:SAG total length (10 μ M)	2μL
Under the P2:SAG total length (10 μ M)	2μL
		OligoT18 reverse transcription product stoste	1μL
Ex-Taq	0.25μL
		ddH 2O	35.75μL
total	50μL

Reaction conditions:

Concrete operation condition is seen table 13.

Table 13: operation condition

Cycle	Temperature and time
		1	94℃，5min
35	94℃，30s；50.6℃，30s；72℃，45s
		1	72℃，10min

Detect: 1% agarose electrophoresis, there is 700～800bp purpose segment to occur, negative control does not all have band.

(2) order-checking

With the recovery of dna segment, connection, conversion, plasmid are carried for a short time, enzyme is cut checking, worker's order-checking is given birth in puncture, Shanghai, use bioinformation Vector NTI 8.0 softwares, NCBI to carry out sequential analysis.The result that order-checking obtains:

Gene order:

TCAGTCATAATCCAACGCACGGTAGCTTCGCGCCACTGGCTTTTCAACCAAGCGCGATG

ACCAATTGTGCGAATCAACGGTTCCTCTCGTACTAGGTTGAATTACTATTGCGACACTATC

ATCAGTAGGGTAAAACTAACCTGTCTCACGACGGTCTAAACCCAGCTCACGTTCCCTATT

GGTGGGTGAACAATCCAACACTTGGTGAATTCTGCTTCACA

ATAGGAAGAGCCGA

CATCGAAGGATCAAAAAGCAACGTCGCTATGAACGCTTGGCTGCCACAAGCCAGT

TATCCCTGTGGTAACTTTTCTGACACCTCTAGCTTCAAATTCCGAAGGTCTAAAGG

ATCGTTAGGCCACGCTTTCACGGTTCGTATTCGTACTGGAAATCAGAATCAAACGA

GCTTTTACCCTTCTGTTCCACACGAGATTTCTGTTCTCGTTGAGCTCATCTTAGGA

CACCTGCGTTATCTTTTAACAGATGTGCCGCCCCAGCCAAACTCCCCACCTGACAA

TGTCTTCCGCCCGGATCGACCGGCCGAAGCCAGCCTTGGGTCCAAAAAGAGGGG

CAATGCCCCGCCTCCGATTCACGGAATAAGTAAAATAACGTTAAAAGTAGTGGTAT

TTCACTTTCGCCGTTTCCGGCTCCCACTTATCCTACACCTCTCAAGTCATTTCACAA

AGTCGGAC

AGTCAAGCTCAACAGG GTCTTCTTACCCCGCATCTCCA

Aminoacid sequence:

MIGRADIEGSKSNVAMNAWLPQASYPCGNFSDTSSFKFRRSKGSLGHAFTVRIRT

GNQNQTSFYPSVPHEISVLVELILGHLRYLLTDVPPQPNSPPDNVFRPDRPAEASLGSKK

RGNAPPPIHGISKITLKVVVFHFRRFRLPLILHLSSHFTKSD

The structure of embodiment 2, the positive and negative adopted plant expression vector of AhSAG cDNA

Utilize the material of enforcement preparation and the AhSAG gene that embodiment 1 obtains, between gus gene among the AhSAG gene insertion vector pBI121-GFP (containing the GFP GFP) and 35S promoter, and follow the NOS terminator to constitute a complete expression framework.Utilize sense primer and antisense primer ORFs (ORF), after the purifying and recovering, be cloned into empty carrier pTG19-T (worker is given birth in Shanghai), obtain just plasmid pTG19-AhSAG and antisense plasmid pTG19-anti-AhSAG through pcr amplification Gene A hSAG.The cloning vector of pTG19-AhSAG and pTG19-anti-AhSAG cDNA that will contain AhSAG is with BamH I and XbaI double digestion; Reclaim small segment respectively; Orientation is connected on the carrier pBI121-GFP of BamH I and XbaI double digestion, connects product Transformed E .coli DH5 α competent cell, alkaline process extraction plasmid.Justice carrier called after pBI121-GFP-AhSAG; Antisense vector called after pBI121-GFP-anti-AhSAG.

1, the clone of AhSAG gene coding region cDNA sequence

(1) RT-PCR (50 μ L system)

Reaction system:

Adopt operation on ice, the concrete operations condition is seen table 13.

Table 13: reaction system operational condition

Reactive system	Volume
			10 * Buffer (contains Mg 2+)	5μL
dNTP(2.5mM)	4μL
		P1:SAG justice/antisense the upper reaches (10 μ M)	2μL
P2:SAG justice/antisense downstream (10 μ M)	2μL
		OligoT18 reverse transcription product stoste	1μL
Ex-Taq	0.25μL
		ddH 2O	35.75μL
total	50μL

Reaction conditions:

Concrete operation condition is seen table 14.

Table 14: operation condition

Cycle	Temperature and time
		1	94℃，5min
35	94℃，30s；61℃，30s；72℃，40s
		1	72℃，10min

Detect: 1% agarose electrophoresis, there is 500bp purpose segment to occur, negative control does not all have band.

(2) order-checking

With the recovery of dna segment, connection, conversion, plasmid are carried for a short time, enzyme is cut checking, worker's order-checking is given birth in puncture, Shanghai, use bioinformation Vector NTI 8.0 softwares, NCBI to carry out sequential analysis.

2, the structure of the positive and negative adopted plant expression vector of AhSAG cDNA

(1) a large amount of preparations and the purifying of plasmid vector pBI121-GFP

Concrete operations are following:

The single colony inoculation of intestinal bacteria that (i) will contain plasmid contains the LB liquid nutrient medium of 100 μ g/mL Amp to 10mL, and 37 ℃, the 250rpm shaking culture is spent the night.

(ii) get the 2ml overnight culture and be inoculated into and contain corresponding antibiotic 100mL LB liquid nutrient medium, 37 ℃, the 250rpm shaking culture is spent the night; 4 ℃, the centrifugal 2min of 6000rpm collects thalline.

The solution I that (iii) adds the precooling of 4mL ice, vortex vibration mixing thalline, room temperature is placed 10min; Add the solution II (at present joining existing usefulness) that showing of 8mL join, mixing leaves standstill 2～5min gently, adds 6mL solution III (precooling), and gentle mixing leaves standstill 10min on ice.

(iv) 4 ℃, 12000rpm, centrifugal 10min gets supernatant; Add 1/10 volume 3mol/LNaAC (pH5.2) and equal-volume Virahol (precooling), mixing, room temperature is placed 10min; 4 ℃, 12000rpm, centrifugal 10min abandons supernatant, absolute ethanol washing deposition twice, control is done.

(v) 2mL TE dissolution precipitation, equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting once, 4 ℃, 12000rpm, centrifugal 3min.

(vi) get supernatant, repeating step v twice gets supernatant, adds the absolute ethyl alcohol of 1/10 volume 3mol/L NaAC and 2 times of volume precoolings, and-20 ℃ leave standstill more than the 30min behind the mixing; 4 ℃, 12000rpm, centrifugal 10min abandons supernatant; 70% washing with alcohol twice, control is done; 500 μ L TE (pH8.0) dissolving ,-20 ℃ of preservations are subsequent use.

(2) AhSAG gene and carrier pBI121-GFP's is connected

I, endonuclease reaction: use BamH I and Xba I double digestion;

II, glue reclaim;

III, linked system:

(i) forward linked system (making up pBI121-GFP-AhSAG):

The concrete operations condition is seen table 15.

Table 15: reaction system operational condition

Reactive system	Volume
			10×Ligase?buffer	2μL
The big fragment of pBI121-GFP that the enzyme switchback is received	2μL
		The pTG19AhSAG that the enzyme switchback is received	8μL
T 4DNA?lignase	1μL
		ddH 2O	7μL
Total	20μL

(ii) reverse linked system (making up pBI121-GFP-anti-AhSAG): the concrete operations condition is seen table 16.

Table 16: reaction system operational condition

Cycle	Temperature and time
		10×Ligase?buffer	2μL
The big fragment of pBI121-GFP that the enzyme switchback is received	2μL
		The pTG19AhantiSAG that the enzyme switchback is received	8μL
T 4DNA?lignase	1μL
		ddH 2O	7μL
Total	20μL

16 ℃ connect 1d.

The evaluation of IV, sense expression vector pBI121-GFP-AhSAG and antisense expression vector pBI121-GFP-anti-AhSAG will be connected product Transformed E .coli DH5 α competent cell, and plasmid is carried for a short time, and BamH I+Xba I enzyme is cut checking.

Result: through the PCR checking, successfully obtain sense expression vector pBI121-GFP-AhSAG and antisense expression vector pBI121-GFP-anti-AhSAG, can be used for the purposes such as Function Identification of this gene.

Embodiment 3:AhSAG gene is coerced the variation of expression amount down in different varieties and different aluminum

Select for use the Actin gene to do confidential reference items; Extract the RNA of 8 materials; With oligoT18 is that primer carries out reverse transcription; Obtain the cDNA of 8 materials, be designated as 99-0,99-20,99-100,99-400, middle 2-0, middle 2-20, middle 2-100 and middle 2-400 respectively, carry out RT-PCR (Real-time PCR) in real time.

(1) reaction system:

Adopt operation on ice, the concrete operations condition is seen table 17.

Table 17: reaction system operational condition

Reaction system	Volume
		?Hotstart?Fluo-PCR?Mix	12.5μL
?P1(10μM)	1μL
		?P2(10μM)	1μL
?cDNA(100mM)	0.5μL
		?ddH 2O	10μL
?total	25μL

(2) reaction conditions:

Concrete operation condition is seen table 18.

Table 18: operation condition

According to 2 ^{-△ △ t}Method is calculated relative expression quantity.

The result: in spend the trend basically identical with the expression amount of 99-1507 No. 2, just under lower concentration, in spend No. 2 expression amount to descend to some extent, might be the growth that the Al of lower concentration has promoted plant, cause the down-regulated expression of aging gene.But when when middle and high concentration is handled, the high abundance of aging gene is expressed.Thus, can think that the AhSAG gene belongs to old and feeble induction-enhanced gene, induce aging gene overexpressions such as AhSAG under the middle and high concentration, promote plant early ageing.

From AhSAG genetic expression and spend No. 2 AhSAG gene relative expressions with 99-1507 to take temperature; Spend No. 2 expression amounts under middle and high concentration Al handles to raise suddenly in the aluminium sensitive varieties; Extremely remarkable with anti-aluminium kind 99-1507 difference, especially under 100 μ mol/L Al handle, spend in having only to have produced the PCD phenomenon No. 2; And 99-1507 is when the 400 μ mol/LAl that produce PCD handle; AhSAG genetic expression is the highest, also can infer from this result: the generation of aging gene induced expression or promotion PCD, Al inductive aging gene is expressed and anti-aluminium property negative correlation.

Embodiment 4: change the acquisition of AhSAG genetic tobacco

1, plant recombination expression vector transforms agrobacterium tumefaciens

(1) preparation of agrobacterium tumefaciens competent cell

1. actication of culture: under sterile state, pick the bacterial classification that a small amount of glycerine preserves and transfer in 5mL YEP (50mg/LRif) liquid nutrient medium 200rpm, 28 ℃ of overnight cultures with the toothpick of sterilization.Pick a small amount of bacterium liquid with transfering loop, on YEP (Rif50mg/L) solid medium, draw broken line, be inverted for 28 ℃ and cultivate, bacterium colony 1-2d grows.

2. use the toothpick picking list colony inoculation of sterilizing to 5mL YEP (50mg/L Rif) liquid nutrient medium, 200rpm, 28 ℃ of overnight cultures.

3. the liquid-transfering gun bacterium liquid 2ml that pipettes overnight cultures forwards to and continues in the 50mL YEP liquid nutrient medium to cultivate, up to OD ₆₀₀Be 0.3-0.4.

4. ice bath 30min.

5. centrifugal 5min (4 ℃, 5000rpm) removes supernatant, stays deposition.

6. the 2mL 20mmol/L CaCl that adds sterilization ₂, fully shake up resuspended thalline.

7. 200 μ L, one pipe is distributed into tubule, immediately uses perhaps subsequent use with being stored in-80 ℃ behind the liquid nitrogen flash freezer.

(2) freeze-thaw method transforms

1. get plasmid pBI121-GFP-AhSAG and pBI121-GFP-anti-AhSAG 2 μ g (about 5-10 μ L) behind the purifying, join respectively in the 200 μ l competence EHAI05 cells of packing, fully mixing.

2. ice bath 30min.

3. after connecting leg one is reinstated the freezing 1-2min of liquid nitrogen, be put in water-bath 5min in 37 ℃ of water rapidly.

4. in pipe, add 800 μ L YEP liquid nutrient mediums respectively, 250rpm, 28 ℃ of prefigurations reach 5-6h.

5. be uniformly coated on 100-200 μ L bacterium liquid on YEP (Rif25mg/L, Str 25mg/L, the Km 100mg/L) solid medium with transfering loop, be inverted for 28 ℃ and cultivate, bacterium colony 2-3d is visible.

(3) identify that plasmid transforms Agrobacterium

1. extract the Agrobacterium DNA

(a) the toothpick picking list colony inoculation of using sterilization is cultivated 20-22h under 250rpm, 28 ℃ of conditions in 10mLYEP (Rif25mg/L, Str 25mg/L, Km 100mg/L) liquid nutrient medium.

(b) pipette the 2mL nutrient solution, the at room temperature centrifugal 10min of 5000rpm abandons supernatant, collects thalline.

(c) add lysozyme soln 0.5mL, the mixing that fully vibrates, water-bath 30min.

(d) add LS lysate 1mL, put upside down centrifuge tube rapidly 10 times, thorough mixing is even, and room temperature held 10min promptly obtains limpid heavy-gravity lysate.

(e) add TS solution 5mL, put upside down rapidly surplus the centrifuge tube 10 time, ice bath 60min has produced flocks.

(f) the centrifugal 15min of 12000rpm.

(g) pipette supernatant, add the absolute ethyl alcohol 2mL of precooling, fully behind the mixing, place half a hour on ice.The centrifugal 10min of 10000rpm abandons supernatant, collecting precipitation.

(h) add TES solution 0.1mL,, need flick tube wall for making resolution of precipitate.

(i) add chloroform 0.1mL, put upside down centrifuge tube for several times, thorough mixing is even, the centrifugal 3min of 10000rpm room temperature.

(j) pipette supernatant, in supernatant, add the absolute ethyl alcohol of 2 times of volumes then, the centrifugal 10min of 12000rpm; Stay deposition; Precipitate 2 times with 70% absolute ethyl alcohol rinsing, after the drying at room temperature, add TE solution (RNase of 2 μ g/mL) 50 μ L dissolution precipitations; 37 ℃ of water-bath half a hour ,-20 ℃ of preservations are subsequent use.

2. PCR detects

Get 1 μ L plasmid as template, with special primer SAG justice, antisense upstream and downstream are carried out the PCR detection to EHA105/pBI121-GFP-AhSAG and EHA105/pBI121-GFP-anti-AhSAG respectively, and preserve through being accredited as the bacterium colony of positive colony.

2, agriculture bacillus mediated genetic transformation

(1) acquisition of tobacco aseptic seedling

The seed of tobacco K326 was used 0.1%HgCl then with 70% alcohol-pickled 30 seconds ₂Sterilization 5-7min is with rinsed with sterile water 3-5 time, after the filter paper that usefulness is sterilized blots the moisture of seed-coat; Be inoculated on the MS substratum, at 26 ℃, the 2000Lx state is cultivated down; Seed one week, sprouted the back, and aseptic seedling grew after two weeks, and the blade of treating seedling is used for transforming when growing to the 5-6 sheet.

(2) the screening culture medium selective pressure confirms

The aseptic seedling tobacco leaf that grows fine is inoculated into the MS that contains Km ₁On the division culture medium; If the concentration gradient of Km is 0mg/L, 25mg/L, 50mg/L, 75mg/L, 100mg/L, 125mg/L; Observe the differentiation situation of blade budlet; In order to the selective pressure of the selective marker of confirming optimal concentration, evaluation index: the optimum concn that is divided into Km that can suppress indefinite bud fully.

(3) leaf dish method transforms

1. the preparation of leaf dish and cultivation in advance

The little seedling leaf of tobacco that clip is aseptic removes middle arteries and veins, and the defoliation edge is cut into the fritter of 0.8 * 0.8cm then, is seeded in MS ₁Cultivate in advance on the tobacco division culture medium.After the preparatory cultivation of 2-3d, treat to carry out when the paddle cutout place has just begun to expand Agrobacterium and infect.

2. the preparation of Agrobacterium engineering bacteria liquid

YEP (Rif 50mg/L with sterilization; Km 100mg/L) liquid nutrient medium; Cultivate the agrobacterium tumefaciens EHA105 of band pBI121-GFP-AhSAG and pBI121-GFP-anti-AhSAG expression vector, the logarithmic phase of 240rpm, 28 ℃ of shaking culture to Agrobacteriums (about 24h).With the ratio of bacterium liquid in 1: 50, change in 50mLYEP (Rif 50mg/L, the Km 100mg/L) liquid nutrient medium then, 240rpm, 28 ℃ continue to be cultured to OD ₆₀₀=0.3-0.6.With centrifugal 10 minutes of bacterium liquid 8000rpm, collect thalline, use MS ₀Resuspended bacterium liquid makes OD ₆₀₀=0.6.

3. infect together and cultivate

(a) tobacco leaf disc that will cultivate is in advance put OD ₆₀₀In=0.6 the resuspended bacterium liquid, 28 ℃, soak 10min on the 190rpm shaking table;

(b) abandon bacteria-removing liquid,, be put into and blot leaf panel surface bacterium liquid on the aseptic filter paper with tweezers folder leafing dish;

(c) with suction go surperficial bacterium liquid the leaf dish be seeded in MS ₁On (adding lid layer filter paper) division culture medium, lucifuge under 26 ℃ of situation, is cultivated about 1-2d altogether.

4. induced bundle is sprouted

(a) after common the cultivation, the Agrobacterium of flush away leaf panel surface often shakes therebetween according to the following steps, makes the leaf dish fully contact solution:

A, sterilized water 15min;

B, sterilized water+cephamycin (500mg/L) 15min;

C, MS ₀+ cephamycin (500mg/L) 20min.

(b) with tweezers folder leafing dish, be placed on the sterilization filter paper, blot the moisture of leaf panel surface.

(c) be seeded in MS to the leaf dish ₂Cultivate on the substratum, gently press the leaf plate edge in substratum.2000Lx, alternately cultivates with light dark 16/8h by 26 ℃.

(d) subculture whenever biweekly.

5. root induction

When treating that the blade budlet grows to 2-3cm, cut leaf dish and young shoot from base portion, the budlet of cutting-out is put into MS ₃Carry out root induction in the substratum, 26-28 ℃, alternately cultivate with light dark 16/8h.Induce 2-3 after week, genetically modified budlet has root to grow, and treats that root grows to 3-4cm, gets final product acclimatization and transplants.

3, the transplanting of regeneration plant

Aseptic seedling band culturing bottle is moved apart culturing room together, be placed on the illumination condition and one week of temperature environment lower refining seedling of nature, treat the seedling robust growth; Root system than prosperity after, take out aseptic seedling, flush away substratum gently; Aseptic seedling is moved in the bucket that contains vegetable garden soil the observation of regularly watering.

4, Molecular Detection transfer-gen plant

(1) PCR detects

1. extract the total DNA of resistance tobacco plant;

2. PCR detects.

Result: successfully obtain transgenic tobacco plant.Behind the AhSAG transformation of tobacco, pBI121-GFP-AhSAG expresses enhancing, can quicken the aging of tobacco, and pBI121-GFP-anti-AhSAG can delay the aging of tobacco; The tobacco early growth that shows justice is fast, blooms early, and just tobacco leaf yellow of the same period is fast, and the tobacco early growth of antisense is slow, blooms late, and senility of peanuts Gene A hSAG has realized the functional expression in tobacco.The effect that has proved senility of peanuts Gene A hSAG is exactly to promote The Plant Senescence.So the AhSAG gene belongs to old and feeble induction-enhanced gene.

Embodiment 5: the observation that the transgene tobacco green fluorescence protein gene is expressed

This test has made up green fluorescent protein (green fluorescentprotein; GFP) the enhancing expression vector pBI121-GFP-AhSAG and the pBI121-GFP-anti-AhSAG of gene and AhSAG gene fusion; Agrobacterium-mediated transformation with optimizing transforms normal tobacco, obtains the transgene tobacco that PCR is positive.Transfer-gen plant has been observed the expression of green fluorescent protein in tobacco tissue and the cell under fluorescent microscope, carry out thus genetic expression Primary Location and quantitative observation.

The result: green fluorescence protein gene is all expressed at the root of become a full member justice and antisense AhSAG genetic tobacco, but in the tip of a root, expresses at most, and is many than cortex, epidermis in the center pillar expression of the tip of a root.Relative other positions that form layers is expressed in the stem are many, in leaf, the flower expression arranged all, but flower than leaf express less relatively.Male and female stamen, pollen granule are not expressed.Express manyly than Lao Ye in the young leaflet tablet, and in pore, express manyly than mesophyll cell, transforming just AhSAG genetic tobacco leaf expression, to transform antisense AhSAG genetic tobacco many.

Embodiment 6: aluminium is handled transgene tobacco GFP expression down

Behind the little seedling and propagating of genetically modified tobacco, a part of seedling is shifted out when 5-6 sheet true leaf is arranged the substratum borough chief of sprouting and taking root, and the substratum of flush away root is gently cultivated with the Hoagland nutritive medium, and every 2d changes one time of nutrition liquid.The Hoagland nutritive medium is containing the CaCl of 0.5mmol/L after cultivating 2d ₂Pre-treatment 24h in the nutritive medium of (pH4.2～4.3).With different aluminum concentration solution 0,30,90,150 μ mol/L AlCl ₃24h is handled in (pH4.2～4.3), with the root of the careful gripping tobacco of tweezers seedling, uses the filter paper suck dry moisture, makes interim slide.The genetically modified tobacco seedling of a part refines seedling when 5-6 sheet true leaf is arranged the substratum borough chief of sprouting and taking root and transplants then in bucket; Positions such as the root of clip tobacco seedling, stem, leaf, flower, gently flush away earth and dust are used the filter paper suck dry moisture; Make interim slide; Inverted phase contrast microscope is observed down, according to the fluorescence inverted phase contrast microscope (Leica, model: explanation DMI3000B) is observed material and is taken pictures; Select B (IF-490) exciter filter for use, the full-automatic photomicrographic apparatus of PM-30.Compare with not genetically modified plant.

The result: aluminium is handled the expression of having induced aging gene AhSAG; And be that the high more fluorescence of concentration is bright more; Explain that al concn is big more; The expression of senility of peanuts Gene A hSAG is many more, and under identical al concn, the tobacco tip of a root that the expression that transforms the just AhSAG genetic tobacco tip of a root transforms antisense AhSAG gene is many.Aluminium handle back GFP mainly in the elongation zone, the maturation zone expresses, and can judge the aluminium poison extent of injury of the tip of a root according to the black situation that the tip of a root shows.

Claims (10)

1. the senility of peanuts gene and the encoding sequence of a regulating cell programmed death is characterized in that: extract RNA from the peanut tip of a root and obtain aging gene AhSAG, its ORF encoding sequence is 474bp, the cDNA sequence of coding 157aa, and its base sequence is shown in SEQ ID NO.1.

2. the senility of peanuts gene and the encoding sequence of regulating cell programmed death according to claim 1 is characterized in that: said AhSAGDerive from aluminium and induce the peanut tip of a root meristematic tissue of peanut tip of a root generation programmed cell death.

3. the senility of peanuts gene and the encoding sequence of regulating cell programmed death according to claim 1 is characterized in that: said AhSAGEncoding amino acid sequence shown in SEQ ID NO.2.

4. according to the senility of peanuts gene and the encoding sequence of claim 1 or 3 described regulating cell programmed deaths, it is characterized in that: said AhSAGBe old and feeble induction-enhanced gene, when concentration is handled less than 20 μ mol/L Al, AhSAGThe expression of gene in anti-aluminium property kind of peanut and aluminium sensitive varieties is very low; When concentration is handled at 20 ~ 100 μ mol/L Al, AhSAGGene expression amount significantly raises, and aluminium sensitive varieties expression amount is higher than anti-aluminium property kind expression amount; When concentration 100 ~ 400 μ mol/L Al handle, AhSAGOverexpression, induction of apoptosis all appears in anti-aluminium property kind and aluminium sensitive varieties, promotes plant early ageing.

5. one kind comprises claim 1 or the senility of peanuts gene of 3 described regulating cell programmed deaths and the recombinant expression vector of coding protein sequence, it is characterized in that: said recombinant expression vector is through inciting somebody to action AhSAGGene imports carrier pBI121-GFP and obtains.

6. the senility of peanuts gene of regulating cell programmed death and the recombinant expression vector of encoding sequence of comprising according to claim 5; It is characterized in that: said recombinant expression vector is just carrier or antisense vector, is to utilize sense primer or antisense primer through the pcr amplification gene AhSAG, be cloned into then on the carrier pTG19-T, cut the back through enzyme and import among the carrier pBI121-GFP and obtain, be used for the just plant expression vector pBI121-GFP of plant -AhSAG, antisense plant expression vector pBI121-GFP -anti-AhSAG, the empirical tests sequence is correct, is used for plant genetic and transforms, and carries out AhSAGGene function is identified;

Described sense primer is:

The upper reaches: GCT CTAGAATGATAGGAAGAGCCGACATCGAAGG XbaThe I site

Downstream: TGC GGATCCCTAGTCCGACTTTGTGAAATGAC BamH I site

Described antisense primer is:

The upper reaches: TTGC GGATCCATGATAGGAAGAGCCGACATCGAAGG BamHThe I site

Downstream: GGCGT CTAGACTAGTCCGACTTTGTGAAATGAC XbaThe I site.

7. the reorganization bacterium of the recombinant expression vector of senility of peanuts gene that comprises claim 5 or 6 described regulating cell programmed deaths and encoding sequence; It is characterized in that: said reorganization bacterium is to import agrobacterium tumefaciens bacterial strain EHA105 through just plant expression vector and antisense plant expression vector to obtain, and in the allos eukaryotic system, realizes functional expression.

8. application that comprises the transfer-gen plant of the described reorganization of claim 7 bacterium, it is characterized in that: said transfer-gen plant is justice that contains GFP and the antisense senility of peanuts gene with reorganization AhSAGImport in the tobacco and obtain the tobacco transfer-gen plant.

9. the application of transfer-gen plant according to claim 8 is characterized in that: the mensuration of said tobacco transfer-gen plant is the GFP gene expression through the fluorescence microscope transformation of tobacco, confirms AhSAGExpression in tobacco and location, that in the form layers tissue of the center pillar of the tender tip of a root of children, root and stem, expresses is many, and only in cell walls and cytolemma, expresses.

10. the application of transfer-gen plant according to claim 8 is characterized in that: said tobacco transfer-gen plant aluminium is handled GFP expression down, is that the senility of peanuts gene is induced in the aluminium processing AhSAGExpression, al concn is high more, AhSAGExpress strongly more, and under identical al concn, the tobacco of the Yi Jiyin that becomes a full member is more than the expression of antisense genetic tobacco; Explain that aluminium coerces down; Tobacco tip of a root justice aging gene is expressed and strengthens, and tip of a root aging is accelerated in the expression of aging gene, produces programmed cell death.

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