CN105296503B - A method of cultivating the genetically modified plants of plant root system development enhancing and Delaying Leaf-Senescence - Google Patents

Abstract

The invention discloses a kind of cultivation plant root system development enhancing and the methods of the genetically modified plants of Delaying Leaf-Senescence, in the channel genes plant of falling purpose shown in SEQ ID NO:1 and expression and heredity will be stablized, obtains the genetically modified plants of the enhancing of with non-transgenic control plant compared with root system development and anti-aging.Genetically modified plants can be obviously promoted the growth and development of root system, and the photosynthesis of functional leaf is remarkably reinforced in late growth stage, postpone the degradation of leaf chlorophyll, delay the aging of transgenic plant, and then improve crop yield.

Description

A kind of genetically modified plants for cultivating plant root system development enhancing and Delaying Leaf-Senescence Method

Technical field

The present invention relates to gene engineering technology field, especially a kind of enhancing plant root growth develops and blade is delayed to decline The breeding method of old genetically modified plants.

Background technique

Wheat is that one of most important cereal crops, the gross area, total output and total trade occupy grain work in the world First of object.The raising of wheat yield has safely extremely important effect to world food.Nitrogen is nutrient necessary to crop, Since the 1980s, effective measure one of of investment of the nitrogenous fertilizer in Wheat Production as its volume increase, but a large amount of nitrogen The investment of fertilizer, not only reduces nitrogen utilization efficiency, while also bringing the waste of resource and the eutrophication of water body etc. one Serial environmental problem threatens human health and ecological environment security.People are adjusted to improve nitrogen at two aspects at present Utilization efficiency, first is that improving root plasma membrane to the absorbability of NH4+；Second is that improving the root structure high to nitrogen sensibility, that is, expand The absorption area of root.Crop overground part and growth ability are directly related with underground part root activity.Well developed root system, distribution it is deep and broad, Energetic, not early ageing be absorption of nutrient ingredients synthesis work well, the reliable guarantee that leaf color is verdant, millet straw ratio is high.Root system is living Power is strong, and the functional period of leaf duration is long, and chlorophyll content decrease speed is slow, is the reliable guarantee that crop finally obtains high yield. Therefore, flourishing root system plant is cultivated, is conducive to plant and obtains nutrient and moisture from soil, so that the growth of plant be promoted to send out It educates and yield composition.

Root system is both the organ that plant absorbs nutrient and moisture from soil, and many substance assimilations, conversion or synthesis Place, or with overground part carry out material exchange metabolic organ.Root system situation directly affects the growth hair of aerial part It educates and the formation of yield, is the key factor for restricting wheat yield potentiality and further playing.Exist for how to be improved using root system Moisture, nutrient and meteorological condition limitation are overcome in the production practices of this important cereal crops of wheat, give full play to wheat increase yield Potentiality, it has been discovered that being also extremely limited with knowledge, the technological means grasped.Glutamine synthelase (GS) is in higher plant nitrogen It plays an important role in metabolism, is the key enzyme being catalyzed during glutamic acid and ammonia formation glutamine progress nitrogen metabolism；It is high Equal plants glutamine synthetase is divided into plasmotype (GS1) and plastid type (GS2) two major classes；GS1 mainly assimilates to be inhaled from soil The primary ammonium of receipts and the ammonium for assimilating N circulation approach release in plant again, are widely present in plant tissue；GS2 can be same Change the ammonia that light respiration process is discharged, is primarily present in the plastids such as chloroplaset.At home and abroad there has been no about enhancing GS at present Expression enhancing root system development, delays the related technology reports of the technical solution of late growth stage plant senesecence in plant.

Summary of the invention

It is developed the technical problem to be solved in the present invention is to provide a kind of enhancing plant root growth and delays the base of its aging Cause and the method for utilizing this gene constructed genetically modified plants, genetically modified plants can be obviously promoted the growth and development of root system, and The photosynthesis of functional leaf is remarkably reinforced in late growth stage, postpones the degradation of leaf chlorophyll, delays declining for transgenic plant Always, and then crop yield is improved.

In order to solve the above technical problems, the technical solution used in the present invention is as follows.

Gene relevant to plant root system development and anti-aging, the SEQ ID NO in nucleotide sequence such as sequence table: Shown in 1.

The protein of said gene coding, amino acid sequence is as shown in the SEQ ID NO:2 in sequence table.

The promoter of said gene, nucleotide sequence is as shown in the SEQ ID NO:3 in sequence table.

The amplimer of said gene, the nucleotide sequence of forward primer as shown in the SEQ ID NO:4 in sequence table, The nucleotide sequence of its reverse primer is as shown in the SEQ ID NO:5 in sequence table.

The amplimer of above-mentioned promoter, the nucleotide sequence such as the SEQ ID NO:6 institute in sequence table of forward primer Show, the nucleotide sequence of reverse primer is as shown in the SEQ ID NO:7 in sequence table.

The method of its aging is developed and delayed to enhancing plant root growth, and channel genes shown in SEQ ID NO:1 are fallen mesh Plant in and stablize expression and heredity, obtain the enhancing of with non-transgenic control plant compared with root system development and anti-aging Genetically modified plants.

As a kind of optimal technical scheme of the above method, first recombination of the building containing gene shown in SEQ ID NO:1 Expression vector, then utilizes gained recombinant expression carrier to construct purpose plant, and evaluation and screening positive plant obtains and normal plants Compared to root system development enhancing and the genetically modified plants that slow down of aging.

As a kind of optimal technical scheme of the above method, the method for constructing recombinant expression carrier is as follows: first wanting right TaGS2-A1 gene described in asking 1 is connected under the Ubiquitin promoter of expression vector pUBI:cas, then by TaGS2- A1 own promoter replaces Ubiquitin promoter, is finally configured to have own promoter+gene recombinant expression load Body pTaGS2::TaGS2-A1.

As a kind of optimal technical scheme of the above method, the method for constructing purpose plant is as follows: will correctly connect purpose The rataria of the method transformed wheat of the recombinant expression carrier of gene and the pAHC25 carrier biolistic bombardment with Bar gene, It by rataria induction, hypertonic culture, shoots and renewal cultivation process, the wheat immature embryo callus after conversion is placed in containing double third ammonia Differentiation screening is carried out on the screening and culturing medium of phosphorus, the young shoot with Herbicid resistant extends, takes root, practices transplantation of seedlings, obtains T0 generation Transgenic plant.

As a kind of optimal technical scheme of the above method, the method for evaluation and screening positive plant is as follows: according to recombination matter Sequence design amplimer on grain pTaGS2-TaGS2-A1 carrier, in the nucleotide sequence of forward primer such as sequence table Shown in SEQ ID NO:8, the nucleotide sequence of reverse primer is as shown in the SEQ ID NO:9 in sequence table；Reaction condition is 94 DEG C of 2 min, 98 DEG C of 10 s, 53 DEG C of 30 s, 68 DEG C of 3 min, 39 circulations, 68 DEG C of 7 min of extension pass through gel Electrophorogram evaluation and screening sun plant, then in PCR trace detection per generation, is selfed breeding, it is ensured that target gene stablizes heredity, obtains Stablize transgenic line to T3, T4 generation.

The beneficial effects of adopting the technical scheme are that genetically modified plants can be obviously promoted the growth of root system And development, and the photosynthesis of functional leaf is remarkably reinforced in late growth stage, postpone the degradation of leaf chlorophyll, delays transgenosis The aging of plant, and then improve crop yield.Detailed test data is referring to embodiment and its attached drawing.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of recombinant expression carrier pTaGS2-TaGS2.

Fig. 2 is the PCR detection figure for being overexpressed TaGS2-A1 transgenic wheat；In figure: 1, transgenic line OEGS2-51,4, Transgenic line OEGS2-52,5, transgenic line OEGS2-53,8, transgenic line OEGS2-57,9, transgenic line OEGS2-61,10, transgenic line OEGS2-94,12, be negative control (unconverted section's agriculture 199), 13, for negative control it is (empty Carrier plant), 14, positive control (plasmid), M, marker.

Fig. 3 is at two kinds for being overexpressed TaGS2-A1 transgenic wheat GS activities present figure under nitrogen level.

Fig. 4 is transgenic wheat and to impinging upon normally for root growth situation map under nitrogen level.

Fig. 5 is transgenic wheat and to impinging upon normally for root growth situation map under nitrogen level.

Fig. 6 is transgenic wheat in low nitrogen (a, c) and normally for the lateral root and adventitious root life under nitrogen supply horizontal (b, d) Long situation map.

Fig. 7 is low nitrogen and normally for transgenosis system under nitrogen level and to impinging upon Post flowering boot leaf chlorophyll content situation of change Figure.

Fig. 8 is low nitrogen and normally for transgenosis system under nitrogen level and to impinging upon Post flowering boot leaf chlorophyll content situation of change Figure.

Fig. 9 is low nitrogen and normally for the photosynthesis figure of transgenosis system under nitrogen level and control boot leaf in the watery stage.

Figure 10 be low nitrogen and normally for transgenosis system under nitrogen level and control wheat yield performance figure.

Specific embodiment

The present invention is described in detail in following embodiment.Various raw materials used in the present invention and items of equipment are conventional city Product is sold, can be bought and be directly obtained by market.

The discovery of embodiment 1, TaGS2 albumen and its encoding gene

Overall length is spliced according to the est sequence of wheat GS2 Unigene Ta.24748 first, and is set according to its conserved sequence GS2 gene primer is counted, with small 54 genomic DNA as template of laying down, filters out the primer suitable for genome amplification, and amplification is produced Object is tentatively sequenced.Then according to obtained primers, for screening small 54 libraries BAC of laying down.Screening is obtained BAC monoclonal plasmid is sequenced, and is finally obtained 1 TaGS2 gene, is distributed on the A genome of wheat, is named as TaGS2- A1。

According to the genome sequence design primer of TaGS2-A1 gene, using wheat (wheat breed small lay down 54) cDNA as template Expand target fragment.The cDNA sequence (SEQ ID NO:1) that 1 treaty 1.3kb is obtained after sequencing, contains 1 complete ORF； Its polypeptide (SEQ ID NO:2) for encoding 427 amino acid residues composition.

The separation of embodiment 2, the clone of TaGS2-A1 gene and its promoter

2.1 gene clonings: with the Trizol reagent of Invetrogen company extract wheat breed it is small lay down 54 total serum IgE, After DNase I (RNase-free) processing, 4 μ g is taken to carry out reverse transcription synthesis with the M-MLV reverse transcriptase of Promega company The first chain of cDNA uses it to carry out PCR amplification as template, obtains pcr amplification product.

PCR primer (introducing BamH I and Kpn I restriction enzyme site in forward primer and reverse primer respectively) sequence is as follows:

Forward primer: 5 '-AGTGGATCCATGGCGCAGGCAGTGGTGCCGGCGATG-3 ' (SEQ ID NO:4)；

Reverse primer: 5 '-TCAGGTACCTCATACCTTCAGCGCCAGCTTCTTG-3 ' (SEQ ID NO:5).

PCR reaction system: 39.5 μ l of ultrapure water, 10 × PCR buffer, 5 μ l, 2 μ l of template, concentration are 10 μM positive and negative To 0.5 μ l of each 1 μ l, KOD enzyme (5u/ μ l) of primer, 1 μ 1 of dNTPs (10mM).

PCR reaction condition: 94 DEG C 4 minutes；94 DEG C 1 minute, 56 DEG C 1 minute, 72 DEG C 3 minutes, 35 circulation；72℃ 8 minutes.

Purify pcr amplification product with QIAquick plastic recovery kit, with pMD18-T carrier (TAKARA, production number D101A it) is connected 8 hours at 16 DEG C, obtains recombinant plasmid pMD18-TaGS2-A1.Using 2mm pole cup, 2500V will be recombinated Plasmid pMD18-TaGS2-A1 converts bacillus coli DH 5 alpha (Quan Shijin, production number CD201), and conversion product is with ampicillin It is grown on LB plating medium, selects positive colony.Plasmid is extracted from positive colony, uses 3700 DNA of AbI PRISM points Analyzer (Perkin-Elmer/Applied Biosystem) is sequenced.Sequencing result shows to insert in pMD18-T carrier The TaGS2 gene shown in the nucleotide of 5 ' end the 1st to 1284 in the sequence 1 of sequence table.

2.2 isolation of promoter: extract wheat breed it is small lay down 54 genomic DNA；According to TaGS2-A1 genome sequence, if Meter expands the promoter primer of the gene；PCR amplification obtains PCR product, i.e., TaGS2-A1 promoter sequence (SEQ ID NO: 3).

PCR primer (introducing Pst I and BamH I restriction enzyme site in forward primer and reverse primer respectively) sequence is as follows:

Forward primer: 5 '-AGTCTGCAGTGGAGGGTGACTGCTCCAGAGTTC-3 ' (SEQ ID NO:6)；

Reverse primer: 5 '-TCAGGATCCCTTCCCCTTCAGCGCTAGCTGATC-3 ' (SEQ ID NO:7).

PCR reaction system: 39.5 μ l of ultrapure water, 10 × PCR buffer, 5 μ l, 2 μ l of template, concentration are 10 μM positive and negative To 0.5 μ l of each 1 μ l, KOD enzyme (5u/ μ l) of primer, 1 μ 1 of dNTPs (10mM).

PCR reaction condition: 94 DEG C 4 minutes；94 DEG C 1 minute, 56 DEG C 1 minute, 72 DEG C 2 minutes, 35 circulation；72℃ 8 minutes.

The recycling of PCR product and subsequent connection conversion are identical with method described in above-mentioned 1.1 with sequence verification method. PMD18-pTaGS2-A1 sequencing result is shown to insert in pMD18-T carrier in the sequence 3 of sequence table from 5 ' ends the 1st to TaGS2 gene promoter sequence shown in 1072 nucleotide.

The building of embodiment 3, recombinant expression carrier

It constructs the recombinant expression carrier of TaGS2-A1 overexpression in wheat: TaGS2 gene being first connected to expression and is carried Under the Ubiquitin promoter of body pUBI:cas, TaGS2-A1 own promoter is then replaced into Ubiquitin promoter, Finally it is configured to that there is own promoter+gene recombinant expression carrier pTaGS2::TaGS2.

Detailed process is as follows:

3.1, with restriction enzyme BamH I and Kpn I double digestion pMD18-TaGS2(incomplete digestion), recycling The segment (the ORF complete sequence comprising TaGS2-A1) of 1.3kb or so.

3.2, with restriction enzyme BamH I and Kpn I double digestion pUBI:cas, carrier framework is recycled.

3.3, the small fragment that step 1 recycles is connect with the carrier framework that step 2 recycles, obtains recombinant plasmid first (pUBI- TaGS2).

3.4, with restriction enzyme Pst I and BamH I double digestion pMD18-pTaGS2, the segment of 1.1kb or so is recycled (promoter sequence comprising TaGS2-A1).

3.5, with restriction enzyme Pst I and BamH I double digestion recombinant plasmid first (pUBI-TaGS2), recycling is about The carrier framework (including TaGS2-A1 gene and terminator, remove Ubiquitin promoter) of 4.7kb or so.

3.6, the segment that step 4 recycles is connected with the carrier framework that step 5 recycles, obtains recombinant plasmid second (pTaGS2- TaGS2-A1)；Its structural schematic diagram is shown in attached drawing 1.

The acquisition and its identification of embodiment 4, transgenic plant

4.1, method for transformation: Gene Knock-out Mice is utilized, by recombinant plasmid pTaGS2-TaGS2-A1 and as blank control PACH25 plasmid importing wheat breed Ji 5265 in.

Step of converting are as follows: by the correctly plasmid of connection target gene and the pAHC25 carrier particle gun with Bar gene The rataria of the method transformed wheat kind Ji 5265 of bombardment, while setting is transferred to pAHC25 empty carrier as control, lures by rataria It leads, hypertonic culture, shoot and the processes such as renewal cultivation, the wheat immature embryo callus after conversion is placed in the screening containing double third ammonia phosphorus Differentiation screening is carried out on culture medium, the young shoot with Herbicid resistant extends, takes root, practices transplantation of seedlings, obtains T0 and plants for transgenosis Strain.

More specific experimental working technique are as follows: the immature seed for taking 5265 Post flowering 12-14 d of Ji, with 10% chlorine After sour sodium disinfects 15 min, clear water is cleaned, and aseptically strips out rataria, scultellum tissue sets induced medium upwards On, each culture dish is inoculated with about 60-80 rataria, sets 22 DEG C, under dark condition after preculture 3-4d, goes to hypertonic culture After cultivating 4-6 h on base, biolistic bombardment is carried out, goes on recovery media and cultivates 2-3 weeks after 16-18 h；Return again to 5mg/L It is cultivated 6-8 weeks on differential medium under PPT selection pressure, carries out culture of rootage when shoot growth is at least 2-4 cm long, When stem length, which goes out, is more than 2 roots, plant is lightly taken out from culture medium, rinses root with tap water to remove culture medium. Seedling is transplanted in the basin (diameter 19cm) equipped with matrix, seedling grow in the incubator 2-3 weeks (24 DEG C, 18/6hr illumination Intensity), it is primary to pour Hoagland nutrient solution weekly, and seedling is then transferred to hot-house culture to solid maturation.

4.2, the identification of transgenic plant: transformed wheat kind Ji 5265 obtains transgenosis single plant T0 for plant, uses PCR Detection obtains positive transgenic wheat.Transgenic plant PCR detection primer is according to recombinant plasmid pTaGS2-TaGS2-A1 carrier On sequence, use SP6 and T7 as the primer of PCR, primer sequence is as follows:

SP6:5 '-ATTTAGGTGACACTATAG-3 ' (SEQ ID NO:8)；

T7:5 '-TAATACGACTCACTATAGGG-3 ' (SEQ ID NO:9)；

The primer can be amplified comprising pTaGS2 promoter, the ORF overall length of TaGS2-A1 gene and 3 ' NOS terminators Sequence, expanding fragment length are about 3 kb, and reaction condition is 94 DEG C of 2 min, 98 DEG C of 10 s, 53 DEG C of 30 s, and 68 DEG C 3 Min, 39 circulations, 68 DEG C of 7 min of extension.As a result referring to attached drawing 2；Verifying obtains stable transgenic plant.And into In row selfing breeding per generation, all carries out PCR trace detection, it is ensured that target gene stablizes heredity, obtains T3 and T4 generation stabilization and turns base Because of strain.We therefrom choose tri- independent transgenic lines of OEGS2-53, OEGS2-57 and OEGS2-94 and non-transgenic Negative control carries out subsequent test.

4.3, transgenic plant glutamine synthelase (GS) activity identification: low nitrogen (amount of nitrogen was taken at Post flowering 10 days For 45 Kg N ha^-1) and normally for nitrogen, (amount of nitrogen is 180 Kg N ha^-1) the lower boot leaf compareed with three transgenosis systems of level Fresh 0.1 g of sample is placed in the centrifuge tube of 2 mL, at once with after liquid nitrogen flash freezer, with plant tissue homogenate system (QIAGEN, TissueLyser II) it is ground into powder, sample cell is placed on ice, quickly adds 800 μ l to extract into each sample cell (extracting solution includes buffer: 100 mM Tris-HCl (pH 7.6), 1.0 mM EDTA, 1.0 mM MgCl₂-6H₂O, 10 MM beta -mercaptoethanol), after sufficiently oscillation mixes, 15,000g is centrifuged 20min at 4 DEG C, and supernatant is crude enzyme liquid.GS enzyme activity Property measurement using glutamine synthelase testing cassete (Bioengineering Research Institute is built up in Nanjing, article No. A047) and it is measured. Control and three transgenosis systems take 20 repetitions respectively.

As a result as shown in Fig. 3, no matter in low nitrogen level, or normally under nitrogen level, three transgenosis system GS are active It is significantly higher than control, shows target gene high level expression in transgenic wheat offspring tissue.

The root system development of embodiment 5, transgenic wheat under Different nitrogen levels

In order to observeTaGS2-A1The influence that overexpression develops wheat root, by control and number obtained above The T4 seed for turning TaGS2-A1 wheat for the T4 generation of OEGS2-53, OEGS2-57 and OEGS2-94, through 1% sodium hypochlorite room Temperature sterilizing 10 minutes, after sterile distilled water rinses 5 times, is sprouted with distilled water, waits for plumule in 24 DEG C of illumination boxs after germination After length to 3-4 cm, chooses the almost the same plant of growth and carry out low nitrogen (0.1 mM NH respectively₄NO₃) and normal nitrogen (1.44 mM NH₄NO₃) under water planting, it is every processing three repeat, processing 7 days and 12 days after observation transgenic plant and nontransgenic plants root system hair Situation is educated, to turn empty carrier wheat as control, each each repetition of strain takes 16 single plants to carry out root growth analysis.

5.1, the root system phenotype of transgenic wheat, lateral root number and the not measurement of fixed number under two kinds of nitrogen levels

After handling 15 d under Different nitrogen levels, entire root system is taken out from nutrient solution, after cleaning, uses desktop scanners Two-dimentional root system is scanned, calculates separately the morphological parameters such as total root long, root surface area through WinRhizo software；The results are shown in attached figure 4,5, 6。

5.2, the measurement of total root long and root surface area, as a result see the table below 1.

In table 1 T4 generation, stablizes the root growth performance results that transgenic wheat ties up under Different nitrogen levels

By data in chart as it can be seen that in low nitrogen and normally for nitrogen level under cultivate 7 d, three independent transgenosis system lateral roots Number and indefinite root long relatively control wheat averagely increase 71.27%, 34.43% and 74.63%, 40.61% respectively, in low nitrogen and normally For cultivating 12 d under nitrogen level, three independent transgenosis system lateral root numbers and indefinite root long relatively control wheat averagely increase respectively 70.36%, 43.05% and 27.11%, 21.09%；In low nitrogen and normally for nitrogen level under, three independent transgenic line roots Biomass relatively control wheat averagely improves 38.55% and 28.95% respectively, and total root long averagely improves 46.3% He respectively 32.72%, root surface area averagely improves 69.81% and 40.34% respectively.Show to overexpressTaGS2-A1bTransgenic wheat is obvious The growth and development for promoting lateral root and adventitious root increases total root long, root surface area and root biomass, may advantageously facilitate and turn base Absorption and utilization because of wheat to soil nutrient and moisture.

Embodiment 6, transgenic wheat are in grouting later period Delaying Leaf-Senescence

Transgenic wheat boot leaf chlorophyll content and pustulation period photosynthetic measurement

Chlorophyll content: accurately weighing the boot leaf sample that 0.2g is shredded, set in 50 mL plug test tube, each pipe difference The acetone of 30 mL 80% is added, is protected from light condition in room temperature dark and extracts 16 h, until leaf sample bleaches completely, then with 80% Acetone returns to zero as blank, measures optical density at 645 nm and 663 nm wavelength；The results are shown in attached figure 7,8.

Photosynthesis: at portable photosynthetic instrument (U.S., genome company) the measurement field Different nitrogen levels of Li-Cor6400X Manage the photosynthetic rate of lower Post flowering wild type control and transgenosis system boot leaf, every 10 leaves of processing replication；As a result see attached Fig. 9.

By the data in Fig. 7-9 as it can be seen that in low nitrogen and normally for nitrogen level under, transgenic wheat obviously prolongs in late growth stage The aging for having delayed functional leaf, in the grouting middle and later periods, the chlorophyll content decrease speeds of three transgenosis system boot leaves and aging into Journey will be significantly slower than control wheat.It is about 3-5 days late in field under the conditions of group.Under low nitrogen level, three transgenosis systems exist Its boot leaf chlorophyll content is higher by 31.85%, 42.43% and 28.40% respectively than control when 28 d after spending；Under the conditions of normally for nitrogen, Three transgenosis systems its boot leaf chlorophyll content in 28 d after spending is higher by 12.98%, 23.40% and 20.79% respectively than control, and two Kind is all remarkably higher than control for the chlorophyll content of transgenic wheat boot leaf under nitrogen level.Further performance is functional period of leaf Duration is long, and the functional leaf for spending rear transgenic wheat is made to capture the ability enhancing of luminous energy, so that higher photosynthetic rate is formed, Finally obtain higher grain yield, in low nitrogen and normally for nitrogen level under, the grain yield of three transgenosis systems compares respectively Attached drawing 10 is seen according to 10.62% and 6.22%(is averagely improved).In conclusion overexpressionTaGS2-A1Transgenic wheat obviously delays The aging of late growth stage plant, extends the photosynthetic function duration of functional leaf, significantly increases grain yield.

Foregoing description is only proposed as the enforceable technical solution of the present invention, not as to the single of its technical solution itself Restrictive condition.

<110>grain and oil crop research institute, Hebei agricultural and forest science institute；Inst. of Genetics and Development Biology, CAS

<120>a kind of method for cultivating plant root system development enhancing and the genetically modified plants of Delaying Leaf-Senescence

<160> 9

<210> 1

<211> 1284

<212> cDNA

<213>grass family Triticum wheat (Triticum aestivumL.)

<400> 1

ATGGCGCAGG CAGTGGTGCC GGCGATGCAG TGCCAGGTGG GCGTGCGGGG CAGGTCGGCC 60

GTCCCGGCGA GGCAGCCCGC GGGCAGGGTG TGGGGCGTCA GGAGGACCGC CCGTGCCACC 120

TCCGGCTTCA AGGTGCTGGC CCTCGGCCCG GAGACCACCG GCGTCATCCA GAGGATGCAG 180

CAGCTGCTCG ACATGGACAC CACGCCCTTC ACCGACAAGA TCATCGCCGA GTACATCTGG 240

GTTGGAGGAT CTGGAATTGA CCTCAGAAGC AAATCAAGGA CGATTTCGAA GCCAGTGGAG 300

GACCCGTCAG AGCTACCCAA ATGGAATTAT GACGGATCGA GCACCGGGCA GGCTCCTGGA 360

GAAGACAGTG AAGTCATTCT ATACCCACAG GCCATATTCA AGGACCCATT CCGAGGAGGC 420

AACAACATCC TGGTTATCTG TGACACCTAC ACGCCACAAG GGGAACCCAT CCCTACTAAC 480

AAGCGACACA TGGCTGCACA AATCTTCAGT GACCCCAAGG TCACTGCACA AGTGCCATGG 540

TTTGGAATCG AACAGGAGTA CACTCTGATG CAGAGGGATG TGAACTGGCC TCTTGGCTGG 600

CCTGTTGGAG GGTACCCTGG CCCCCAGGGT CCATACTACT GCGCCGTAGG ATCAGACAAG 660

TCATTTGGCC GTGACATATC CGATGCTCAC TACAAGGCCT GCCTTTACGC GGGAATTGAA 720

ATCAGTGGAA CAAACGGGGA GGTCATGCCT GGTCAGTGGG AGTACCAGGT TGGACCTAGC 780

GTTGGTATTG ATGCGGGAGA CCACATATGG GCTTCAAGGT ACATTCTCGA GAGAATCACG 840

GAGCAAGCTG GTGTGGTGCT CACCCTTGAC CCAAAACCAA TCCAGGGTGA CTGGAATGGA 900

GCTGGCTGCC ACACAAATTA CAGTACACTG AGCATGCGCG AAGATGGAGG TTTCGACGTG 960

ATCAAGAAGG CAATCCTGAA CCTTTCACTT CGCCATGACT TGCACATAGC CGCATATGGT 1020

GAAGGGAACG AGCGGAGGTT GACAGGGCTA CACGAGACAG CTAGCATTTC AGACTTCTCA 1080

TGGGGCGTCG CGAACCGTGG CTGCTCTATT CGTGTGGGCC GAGAGACCGA GGCAAAGGGC 1140

AAAGGATACC TGGAGGACCG TCGCCCGGCG TCGAACATGG ACCCGTACAC AGTGACGGCG 1200

CTGCTGGCCG AGACCACGAT CCTGTGGGAG CCGACCCTCG AGGCGGAGGC CCTCGCTGCC 1260

AAGAAGCTGG CGCTGAAGGT ATGA 1284

<210> 2

<211> 427

<212> PRT

<213>grass family Triticum wheat (Triticum aestivumL.)

<400> 2

Met Ala Gln Ala Val Val Pro Ala Met Gln Cys Gln Val Gly Val

1 5 10 15

Arg Gly Arg Ser Ala Val Pro Ala Arg Gln Pro Ala Gly Arg Val

20 25 30

Trp Gly Val Arg Arg Thr Ala Arg Ala Thr Ser Gly Phe Lys Val

35 40 45

Leu Ala Leu Gly Pro Glu Thr Thr Gly Val Ile Gln Arg Met Gln

50 55 60

Gln Leu Leu Asp Met Asp Thr Thr Pro Phe Thr Asp Lys Ile Ile

65 70 75

Ala Glu Tyr Ile Trp Val Gly Gly Ser Gly Ile Asp Leu Arg Ser

80 85 90

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

95 100 105

Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro Gly

110 115 120

Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp

125 130 135

Pro Phe Arg Gly Gly Asn Asn Ile Leu Val Ile Cys Asp Thr Tyr

140 145 150

Thr Pro Gln Gly Glu Pro Ile Pro Thr Asn Lys Arg His Met Ala

155 160 165

Ala Gln Ile Phe Ser Asp Pro Lys Val Thr Ala Gln Val Pro Trp

170 175 180

Phe Gly Ile Glu Gln Glu Tyr Thr Leu Met Gln Arg Asp Val Asn

185 190 195

Trp Pro Leu Gly Trp Pro Val Gly Gly Tyr Pro Gly Pro Gln Gly

200 205 210

Pro Tyr Tyr Cys Ala Val Gly Ser Asp Lys Ser Phe Gly Arg Asp

215 220 225

Ile Ser Asp Ala His Tyr Lys Ala Cys Leu Tyr Ala Gly Ile Glu

230 235 240

Ile Ser Gly Thr Asn Gly Glu Val Met Pro Gly Gln Trp Glu Tyr

245 250 255

Gln Val Gly Pro Arg Cys Gly Ile Asp Ala Gly Asp His Ile Trp

260 265 270

Ala Ser Arg Tyr Ile Leu Glu Arg Ile Thr Glu Val Ala Gly Val

275 280 285

Val Leu Thr Leu Asp Gly Tyr Pro Ile Gln Pro Asp Trp Asn Gly

290 295 300

Ala Gly Cys Val Thr Ser Tyr Ser Thr Leu Thr Met Pro Glu Val

305 310 315

Pro Arg Ile Asp Gln Phe Tyr Met Pro Leu Val Phe Leu Thr Leu

320 325 330

Arg Thr Asp Asn Gln Tyr Ala Arg Tyr Pro Glu Gly Phe Glu Ala

335 340 345

Arg Tyr Ser Ala Val His Val Ser Ala Ser Ile Ser Glu Lys Ser

350 355 360

Trp Gly Val Arg Leu Ser Ala Cys Ser Ile Arg Val Gly Arg Glu

365 370 375

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

380 385 390

Ser Asn Met Asp Pro Tyr Ile Ala Thr Ala Thr Leu Gly His Trp

395 400 405

Thr Met Val Cys His Gly Thr Glu Glu Pro His Ala Val Arg Ala

410 415 420

Lys Lys Leu Ala Leu Lys Val

425 427

<210> 3

<211> 1072

<212> promoter DNA

<213>grass family Triticum wheat (Triticum aestivumL.)

<400> 3

TGGAGGGTGA CTGCTCCAGA GTTCCCCCCC ATTTTTGCCG AACAGTTTGT CCTGTACACA 60

TACTCCGAAG GCGGTGACCG TTGCATCACT GGTTGTTAAC TCGGTCGTGC AACCTGGAGT 120

AGTTTGTGGT TTCCACTTTT GCCGTTCTAC GACGGAGGAT AAACTCGCCT GCTAGGCTAC 180

TAGTGTGGTT GCACTTGCAC AAGAAGGCAG GGCCAAAAGT ACAGGCAGCC CATGTGTGCT 240

ACCGCTGCTG CTGGGCGCTG TCACAGTGTC ACTGCTAGTC CGTCCGTCCC CGGCACCGGG 300

CAGCCCGACT GGAAGTGGAC CAAGCTAGGG TGCCCCCATG ACAGGCCACC CGGCGCAAAG 360

ACATGCTGCA CCTGACAAAG GGGTCCATCC ATCCATCATC CTATACGGCT ACACCCCCAA 420

TTATTGGCCC CGGGGTCAAT GAGACACGCA CGCCGCACAG CACATAGTTT GCTTGCGTTG 480

CGGTGCAGTG CAGGTCTTGC TCCGCTCTGA TGCATCTGGC TGGGTTTGGT TGGAGAGGTC 540

GCTTGGGCTG GGGCGGGAGA CGGACGTGAC AAGGAGAGGC AGCCGCATCT TTCATTTCAT 600

ATCCGGAGCC AGAGGGAAGG AACATTGTAA ACTCTATCCG AGATGTGTGT GCCTTCTTTG 660

GGCAGACGGC GCTTGGTTTC TGGACCGGTT TGCCTCCGGT GCGGCTGCAA GGGGCGCCTC 720

AAATCTCGTC CACCCAGAGC CCTATCGCCT AATCAAGAGC CAGATGCCAC TGCCCGCGCC 780

GCGGCCACTA GAATCTCCCC CGCAATCTAA ATTACTTGTA CCACCGGCCT CCGCTCCCAT 840

AAATATAAAT AATATTTCCA GCGCGACCTC GCAAGGTCGC CTCCCCTTCC CTCCATCCTC 900

TCCCCCCTCG TCTCGTCCGC GTGCTTGTCC GTCTCTCTGG TGAGTGCCGC GGACTCCTCG 960

ATGCAATGCT TCTTGGAGCA CCCGGCTTCT TCAATCTCTG TTGCGTTCAT TCGCTGATCA 1020

ACCGCAATGG CGCTGCTTTG GTTTGGTTGA TCAGCTAGCG CTGAAGGGGA AG 1072

<210> 4

<211> 36

<212> DNA

<213>artificial sequence

<400> 4

AGTGGATCCA TGGCGCAGGC AGTGGTGCCG GCGATG 36

<210> 5

<211> 34

<212> DNA

<213>artificial sequence

<400> 5

TCAGGTACCT CATACCTTCA GCGCCAGCTT CTTG 34

<210> 6

<211> 33

<212> DNA

<213>artificial sequence

<400> 6

AGTCTGCAGT GGAGGGTGAC TGCTCCAGAG TTC 33

<210> 7

<211> 33

<212> DNA

<213>artificial sequence

<400> 7

TCAGGATCCC TTCCCCTTCA GCGCTAGCTG ATC 33

<210> 8

<211> 18

<212> DNA

<213>artificial sequence

<400> 8

ATTTAGGTGA CACTATAG 18

<210> 9

<211> 20

<212> DNA

<213>artificial sequence

<400> 9

TAATACGACT CACTATAGGG 20

Claims (3)

1. enhancing the method for plant root growth development and Delaying Leaf-Senescence, it is characterised in that:

The recombinant expression carrier containing gene shown in SEQ ID NO:1 is constructed first, then utilizes gained recombinant expression carrier structure Purpose plant is built, evaluation and screening positive plant obtains the transgenosis that root system development enhancing and aging are slowed down compared with normal plants Plant；

The method for constructing recombinant expression carrier is as follows: TaGS2-A1 gene shown in SEQ ID NO:1 being first connected to expression and is carried Under the Ubiquitin promoter of body pUBI:cas, TaGS2-A1 own promoter is then replaced into Ubiquitin promoter, Finally it is configured to that there is own promoter+gene recombinant expression carrier pTaGS2::TaGS2-A1；

The plant is wheat.

2. according to the method described in claim 1, it is characterized by: the method for building purpose plant is as follows: will correctly connect The method transformed wheat of the recombinant expression carrier of TaGS2-A1 gene and the pAHC25 carrier biolistic bombardment with Bar gene Rataria, by rataria induction, it is hypertonic culture, shoot and renewal cultivation process, the wheat immature embryo callus after conversion is placed in and is contained Differentiation screening is carried out on the screening and culturing medium for there are double third ammonia phosphorus, the young shoot with Herbicid resistant extends, takes root, practices transplantation of seedlings, T0 is obtained for transgenic plant.

3. according to the method described in claim 1, it is characterized by: the method for evaluation and screening positive plant is as follows: according to recombination Sequence design amplimer on plasmid pTaGS2::TaGS2-A1 carrier, in the nucleotide sequence of forward primer such as sequence table SEQ ID NO:8 shown in, the nucleotide sequence of reverse primer is as shown in the SEQ ID NO:9 in sequence table；Reaction condition For 94 DEG C of 2 min, 98 DEG C of 10 s, 53 DEG C of 30 s, 68 DEG C of 3 min, 39 circulations, 68 DEG C of 7 min of extension, by solidifying Gel electrophoresis map identification screens sun plant, and then PCR trace detection per generation is selfed breeding, it is ensured that and target gene stablizes heredity, Obtain T3, T4 generation stablize transgenic line.