CN104131013A - Gene OsSWEET5 for regulating and controlling paddy rice leaf blade senility and application thereof - Google Patents

Abstract

The invention belongs to the field of plant gene engineering, and particularly relates to a gene OsSWEET5 for regulating and controlling paddy rice leaf blade senility and an application thereof. The gene has the nucleic acid sequence shown in 1-1363 sites of SEQ ID NO:1, and has the coded protein sequence shown in SEQ ID NO:2. A binary expression vector is constructed, wherein a CaMV35S promoter drives the gene to express the binary expression vector; a paddy rice host is transformed by an agrobacterium mediated transformation method, and an overexpressed transgenic paddy rice plant is obtained. Testing results show that a leaf blade senility process of a transgenic paddy rice plant is accelerated obviously, and the gene is declared to have the function of regulating and controlling the leaf blade senility in paddy rice. The obtained overexpressed transgenic plant allows the leaf blade senility process to be changed, and thus a new resource is provided for genetic engineering and molecular breeding.

Description

Gene OsSWEET5 and the application of an adjusting and controlling rice leaf senile

Technical field

The invention belongs to plant gene engineering technology field, be specifically related to Senescence of Rice genes involved OsSWEET5 and application.

Background technology

The blade of higher plant is the major organs of carrying out the synthetic photosynthate (being mainly carbohydrate) of photosynthesis.After photosynthate is synthetic in the blade of source, first from mesophyll cell, be transported to phloem, then, after the loading of phloem, operation, unloading, be transported to storehouse organ, for growing of storehouse organ.If increase the stowage capacity in farm crop source or the offloading capacity in storehouse, will have more photosynthate input magazine organ, this will be very helpful to improving the output of farm crop.And leaf senile can cause photosynthetic decline, reduce carbon assimilation, thereby affect the output of farm crop.Therefore, the generation of planned control leaf senile, makes photosynthate transport towards desirable direction, for the output of controlling farm crop, has great importance.

Monose is the main source of carbon source and the energy in most of heterotrophs, also be basic energy and nutritive substance (the B ü ttner etc. of growth and development of plants, Monosaccharide transporters in plants:structure, function and physiology.Biochimica Biophysica Acta, 2000,1465:263-274).Because monose is easy to be hydrolyzed in the process of transportation, and often can cause the variation that Premeabilisation of cells is pressed, thereby the form that conventionally changes sucrose or derivatives thereof into is carried out long-distance transportation; When it arrives after the organ of storehouse, be again hydrolyzed into monose again, by monose translocator, be transported to storehouse organ, thereby be absorbed and utilize (Wang Jungang etc., plant monose translocator, Plant Physiology Communications, 2007,43 (6): 1195-1200; Guo Ting etc., sugarcane monose transporter gene ShPST2a clone and Subcellular Localization, tropical crops journal, 2013,34 (3): 429-432).Therefore, transportation and the distribution of monose in plant materials seems extremely important.Monose translocator affects transhipment, the accumulation and distribution of monose, thus the impact important on growing of plant.

MtN3/saliva/SWEET family protein generally contains 2 MtN3/saliva membrane spaning domains, and its member is extensively present in animals and plants and microorganism.There is recently investigator to analyze the MtN3/saliva/SWEET family in each species, identified that some can transport the MtN3/saliva/SWEET albumen (Yuan etc. of monose, Rice MtN3/Saliva/SWEET family genes and their homologs in cellular organisms.Mol Plant, 2013,6:665-674; Yuan etc., Rice MtN3/saliva/SWEET gene family:Evolution, expression profling, and sugar transport.J Integr Plant Biol, 2014,56:559-570).Research shows that some MtN3/saliva/SWEET albumen can be used as monose translocator and affect growing of plant.AtSWEET16 is positioned vacuole, can transport glucose, fructose and sucrose, in the transfer-gen plant of overexpression AtSWEET16 all there is obvious change in monose and sucrose content, germination rate and winter hardiness all significantly strengthen (Klemens etc. simultaneously, Overexpression of the vacuolar sugar carrier AtSWEET16 modifies germination, growth, and stress tolerance in Arabidopsis.Plant Physiol, 2013,163:1338-1352).AtSWEET17 is a vacuole fructose translocator in Arabidopis thaliana, can be by fructose in vacuole transporte to cells matter, its mutant plant strain growth is slow, blade sugar content generation considerable change, and seed production has been subject to impact, illustrate that AtSWEET17 can regulate and control the content of fructose in Arabidopsis leaf, (Chardon etc. play an important role in the distribution process of plant carbohydrates, Leaf fructose content is controlled by the vacuolar transporter SWEET17 in Arabidopsis.Curr Biol, 2013, 23:697-702).

Paddy rice is one of main food crop of the mankind, and the means that adopt molecular genetic to regulate and control are controlled the aging of blade, and photosynthate is transported towards desirable direction, and this has great importance for output of controlling farm crop.

Summary of the invention

The object of the present invention is to provide the application of a Senescence of Rice genes involved in adjusting and controlling rice Leaf senescence development.

In research before applicant, one of a MtN3/saliva/SWEET gene OsSWEET5 coding can be transported semi-lactosi, and can not transport the albumen of sucrose, not only Leaf senescence development acceleration of the overexpression transfer-gen plant of OsSWEET5 simultaneously, and in blade all there are obvious change (Zhou etc. in monose and sucrose content, Overexpression of OsSWEET5 in rice causes growth retardation and precocious senescence.PLoS One, 2014,9:e94210).The present invention utilizes relevant molecular biology method exactly, built the binary expression vector of being expressed by CaMV35S promoters driven OsSWEET5, and use agrobacterium-mediated transformation rice transformation, and by it improvement of the genetically engineered for paddy rice, thereby distribution and the senescence process of adjusting and controlling rice blade carbohydrate.

The gene source of above-mentioned regulation and control leaf senile is in paddy rice, and it is one of following Nucleotide:

1) nucleotide sequence shown in the 1-1363 position of SEQ ID NO:1;

2) protein sequence shown in SEQ ID NO:2.

3) in addition, the invention still further relates to the expression vector establishment of Senescence of Rice genes involved OsSWEET5, this carrier includes the gene of nucleotide sequence as described in the 1-1363 position of sequence table SEQ ID NO:1 of overexpression.

The present invention clone's Senescence of Rice genes involved OsSWEET5 has following DEVELOPMENT PROSPECT:

1) for the ease of genetically modified cell or plant are screened, can transform used carrier, such as adding the alternative mark of plant (such as Bar gene, gus gene, GFP gene and Lux gene etc., these genes are all the common tagging genes of report);

2) in order to change the expression amount of goal gene OsSWEET5, can before its transcription initiation Nucleotide, use different promotors, as enhancement type promotor, inducible promoter, constitutive promoter, tissue-specific promoter, developmental stage specificity promoter etc.

3) with the expression vector of OsSWEET5 gene of the present invention except can transforming by Agrobacterium Ti-plasmids mediated method, can also transform different vegetable cells or tissue by methods such as Ri plasmid, plant viral vector, microinjections, and be bred as plant, to obtain the transgenic plant of speeding-up blade aging.

(4) the invention still further relates to the Transgenic Rice Plants of described Senescence of Rice genes involved OsSWEET5 expression vector.

(5) gene of the present invention is having important application aspect regulating plant Leaf senescence development, and wherein, the host who is converted can be monocotyledons, as paddy rice, corn, wheat and turfgrass etc.; Also can be dicotyledons, as Arabidopis thaliana, willow, soybean and cotton etc., but be not limited to the above species.

Concrete technical scheme of the present invention is as follows:

(1) take full length cDNA clone J023023E05 as template, through the full-length cDNA of pcr amplification OsSWEET5, its nucleotide sequence is as shown in SEQ ID NO:1;

(2) DNA fragmentation obtaining in step (1) is connected on carrier pCAMBIA1300s, obtain p1300s-OsSWEET5, then imported the agrobacterium tumefaciens EHA105 (being so kind as to give by CAMBIA) of agropine type, utilize agriculture bacillus mediated genetic transforming method in described carrier p1300s-OsSWEET5 Introduced into Rice kind, to spend 11, obtain transfer-gen plant;

(3) utilize the method for Northern blot to detect the expression amount of OsSWEET5 gene in transgenic rice plant and wild-type (not transgenosis) plant.

(4) measure the chlorophyll content of rotaring gene plant blade, observe its phenotype and take pictures, analyze the difference of the old and feeble situation of transfer-gen plant and wild-type plant leaf.

(5) content of various sugar in mensuration wild-type in seedling stage and rotaring gene plant blade, the relatively difference of sugared content in transgenosis and wild-type plant leaf.

The invention has the advantages that:

(1) the present invention has identified a leaf senile correlation gene OsSWEET5, can be follow-up investigator reference is provided.

(2) in the present invention, identify the method for leaf senile correlation gene, can be follow-up investigator reference is provided.

(3) the present invention has obtained the gene of a coding HUCEP-8, and its expression amount can be controlled the distribution of sugar in blade, for the distribution research of carbohydrate from now on lays the foundation.

(4) in the overexpression rotaring gene plant blade that the present invention obtains there is considerable change in the content of various sugar, for molecular breeding provides new resource.

(5) the overexpression transfer-gen plant that the present invention obtains has changed Senescence of Rice process, for genetically engineered and molecular breeding provide new resource.

Accompanying drawing explanation

Sequence table SEQ ID NO:1 is the full length cDNA sequence of the gene OsSWEET5 that clones of the present invention.Length is 1363bp; Its CDS region is from 337 bp – 1050 bp, and length is 714 bp.

Sequence table SEQ ID NO:2 is the OsSWEET5 protein sequence that the present invention clones, 237 amino acid of encoding.

Fig. 1: the carrier figure that is original pCAMBIA1300s.

Fig. 2: the structure sketch that is improved recombinant vectors p1300s-OsSWEET5.

Fig. 3: be paddy rice wild-type (non-transgenic) and T ₂the picture of OsSWEET5 overexpression transfer-gen plant 15 d after sprouting in generation (transgenosis).In figure, mark Bar value represents 10 cm.WT-wild-type (non-transgenic) plant successively from left to right in Fig. 3; OX1-transfer-gen plant; OX2-transfer-gen plant; OX3-transfer-gen plant; OX4-transfer-gen plant.

Fig. 4: be the expression amount with OsSWEET5 in the methods analyst overexpression Transgenic Rice Plants of Northern blot.Mark in figure: WT represents wild-type (non-transgenic) plant; OX1 – OX4 represents transfer-gen plant.RNA sample comes from T ₂generation single copy family and second leaf in wild-type plant seedling stage.

Fig. 5: the mensuration that is OsSWEET5 overexpression Transgenic Rice Plants and wild-type Leaf chlorophyll content.Sample comes from second leaf of transfer-gen plant in Fig. 3.Test arranges 3 repetitions." * * " represents that the P value of t test is less than 0.01, and difference is extremely remarkable.

Fig. 6: the measurement result that is OsSWEET5 overexpression Transgenic Rice Plants and wild-type rice plant middle period piece candy content.Test arranges 3 repetitions." * " represents that the P value of t test is less than 0.05, significant difference; " * * " represents that the P value of t test is less than 0.01, and difference is extremely remarkable.

Embodiment

Embodiment 1: the acquisition of candidate segment

OsSWEET5 gene source in the SSH library of the people's such as Liu structure (referring to Liu etc., Identification of early senescence-associated genes in rice flag leaves.Plant Mol Biol, 2008,67:37-55), it is for No. LOC LOC_Os05g51090.Applicant utilizes its No. LOC search in TIGR (http://rice.plantbiology.msu.edu), obtain the CDS sequence of OsSWEET5, in KOME (http://cdna01.dna.affrc.go.jp/cDNA/), compare subsequently, obtain the full-length cDNA of OsSWEET5, clone's number is J023023E05, and its nucleotide sequence is as shown in SEQ ID NO:1.

Take full length cDNA clone J023023E05 as template, through the full-length cDNA of pcr amplification OsSWEET5.The DNA sequence dna of the primer of this fragment that increases is: F:5 '-ggtaccGGTGCTCTGCCTCTCCATTA-3 '; R:5 '-tctagaCCCGTCACAGTTACATGCAC-3 '.Reclaim after PCR product, be connected into pGEM-T carrier (purchased from Promega company); To connect product and transform bacillus coli DH 5 alpha competent cell, by resistance screening, enzyme, cut and the detection of checking order (for ordinary method), obtain the positive TA clone of OsSWEET5 gene.Positive TA clone is checked order, and result shows, this positive TA clone is containing just like the nucleotide sequence shown in SEQ ID NO:1.

The structure of embodiment 2:OsSWEET5 gene overexpression carrier

In order to illustrate better the function of this gene, applicant, by this gene OsSWEET5 overexpression in paddy rice, verifies its function from the phenotype of transfer-gen plant.

Concrete operations are as follows:

(1) the positive TA clone who contains OsSWEET5 gene in embodiment 1 is used after Kpn I and Xba I double digestion, be connected with carrier pCAMBIA1300s (being so kind as to give by Australian CAMBIA) the skeleton fragment through Kpn I and Xba I double digestion.The transformation of pCAMBIA1300s carrier is from pCAMBIA1300 (http://www.cnki.net/, Ye Shuifeng, cultivation and the functional analysis of the expression pattern analysis of paddy rice CDPKs gene and black algae Hvppc2 transgenic paddy rice.[doctorate paper].Wuhan: Hua Zhong Agriculture University Library, 2009), its carrier figure as shown in Figure 1.

(2) the connection product in step (1) is transformed to bacillus coli DH 5 alpha competent cell.

(3) by resistance screening and enzyme, cut and detect confirmation, between the Kpn I that the recombinant vectors p1300s-OsSWEET5 of take is carrier pCAMBIA1300s and Xba I site, inserted the nucleotide sequence as shown in SEQ ID NO:1, the T-DNA plot structure schematic diagram of restructuring overexpression carrier p1300s-OsSWEET5 as shown in Figure 2.

Embodiment 3: the conversion of restructuring overexpression carrier p1300s-OsSWEET5

The p1300s-OsSWEET5 recombinant vectors that embodiment 2 is built imports the agrobacterium tumefaciens EHA105 of agropine type, forms and transforms bacterial strain.Method shown in " agriculture bacillus mediated genetic transformation operational manual " that agriculture bacillus mediated genetic transforming method is mainly delivered with reference to the applicant's State Key Laboratory of Crop Genetic Improvent (support the army etc. by woods, the foundation of the agriculture bacillus mediated No. 8 high-efficient transgenic systems in Mudanjiang, Acta Agronomica Sinica, 2002,28:294-300).Concrete steps are as follows:

(1) reagent and solution abbreviation

In the present invention, the abbreviation of substratum plant hormone used is expressed as follows: 6-BA (6-BenzylaminoPurine, 6-benzyladenine); CN (Carbenicillin, Pyocianil); KT (Kinetin, kinetin); NAA (Napthalene acetic acid, naphthylacetic acid); IAA (Indole-3-acetic acid, indolylacetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4 dichlorophenoxyacetic acid); AS (Acetosringone, Syringylethanone); CH (Casein Enzymatic Hydrolysate, caseinhydrolysate); HN (Hygromycin B, Totomycin); DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO)); N6max (N6 macroelement composition solution); N6mix (N6 Trace Elements solution); MSmax (MS macroelement composition solution); MSmix (MS Trace Elements solution).

(2) main solution formula

1) N6 substratum macroelement mother liquor (according to the preparation of 10 * concentrated solution):

Mentioned reagent is dissolved one by one, then under room temperature, with distilled water, be settled to 1000 ml.

2) N6 substratum trace element mother liquor (according to the preparation of 100 * concentrated solution)

Mentioned reagent is at room temperature dissolved and be settled to 1000 ml with distilled water.

3) molysite (Fe ²⁺eDTA) stock solution (according to the preparation of 100 * concentrated solution)

By 3.73 g b diammonium disodium edta (Na ₂eDTA2H ₂o) and 2.78 g FeSO ₄7H ₂o dissolves respectively, mixes and is settled to 1000 ml with distilled water, to 70 ℃ of temperature, bathes 2 h, and 4 ℃ save backup.

4) VITAMIN stock solution (according to the preparation of 100 * concentrated solution)

Adding distil water is settled to 1000 ml, and 4 ℃ save backup.

5) MS substratum macroelement mother liquor (according to the preparation of 10 * concentrated solution)

Mentioned reagent is at room temperature dissolved, and be settled to 1000 ml with distilled water.

6) MS substratum trace element mother liquor (according to the preparation of 100 * concentrated solution)

Mentioned reagent is at room temperature dissolved, and be settled to 1000 ml with distilled water.

7) 2, the preparation of 4-D stock solution (1 mg/ml):

Take 2,4-D, 100 mg, with 1 ml 1 N potassium hydroxide, dissolve 5 min, then add after 10 ml distilled water dissolve completely and be settled to 100 ml, under room temperature, preserve.

8) preparation of 6-BA stock solution (1 mg/ml):

Take 6-BA 100 mg, with 1 ml 1 N potassium hydroxide, dissolve 5 min, then add after 10 ml distilled water dissolve completely and be settled to 100 ml, room temperature preservation.

9) preparation of naphthylacetic acid (NAA) stock solution (1 mg/ml):

Take NAA 100 mg, with 1 ml 1 N potassium hydroxide, dissolve 5 min, then add after 10 ml distilled water dissolve completely and be settled to 100 ml, 4 ℃ save backup.

10) preparation of indolylacetic acid (IAA) stock solution (1 mg/ml):

Take IAA 100 mg, with 1 ml 1 N potassium hydroxide, dissolve 5 min, then add after 10 ml distilled water dissolve completely and be settled to 100 ml, 4 ℃ save backup.

11) preparation of glucose stock solution (0.5 g/ml):

Take glucose 125 g, then with distilled water, dissolve and be settled to 250 ml, after sterilizing, 4 ℃ save backup.

12) preparation of AS stock solution:

Take AS 0.392 g, add DMSO 10 ml to dissolve, divide and be filled in 1.5 ml centrifuge tubes, 4 ℃ save backup.

13) 1N potassium hydroxide stock solution

Take potassium hydroxide 5.6 g, with distilled water, dissolve and be settled to 100 ml, room temperature preservation is standby.

(3) for the culture medium prescription of rice transformation

1) inducing culture

Adding distil water to 900 ml, 1N potassium hydroxide regulates pH value to 5.9, boil and be settled to 1000 ml, divide and install to 50 ml triangular flasks (25 ml/ bottle), sterilizing according to a conventional method after sealing (for example sterilizing 25 min at 121 ℃, following medium sterilization method is identical with the sterilising method of basal culture medium).

2) subculture medium

Adding distil water to 900 ml, 1N potassium hydroxide regulates pH value to 5.9, boils and is settled to 1000 ml, divides and installs to 50 ml triangular flasks (25 ml/ bottle), sealing, sterilizing as stated above.

3) pre-culture medium

Adding distil water to 250 ml, 1N potassium hydroxide regulates pH value to 5.6, sealing, sterilizing as stated above.

Use front heating for dissolving substratum and add 5 ml glucose stock solutions and 250 μ l AS stock solutions, (25 ml/ ware) in culture dish poured in packing into.

4) be total to substratum

Adding distil water to 250 ml, 1N potassium hydroxide regulates pH value to 5.6, sealing, sterilizing as stated above.

Use front heating for dissolving substratum and add 5 ml glucose stock solutions and 250 μ l AS stock solutions, (the every ware of 25 ml/) in culture dish poured in packing into.

5) suspension medium

Adding distil water to 100 ml, adjust pH to 5.4, divides and installs in the triangular flask of two 100 ml, sealing, sterilizing as stated above.

Before use, add the aseptic glucose stock solution of 1 ml and 100 μ l AS stock solutions.

6) select substratum

Adding distil water to 250 ml, regulates pH value to 6.0, sealing, sterilizing as stated above.

Before using, dissolve substratum, add 250 μ l HN (50 mg/ml) and 400 μ l CN (250 mg/ml) packing to pour (25 ml/ ware) in culture dish into.(note: selecting for the first time substratum Pyocianil concentration is 400 mg/l, selecting for the second time and later substratum Pyocianil concentration is 250 mg/l).

7) division culture medium

Adding distil water to 900 ml, 1 N potassium hydroxide regulates pH value to 6.0.Boil and be settled to 1000 ml with distilled water, dividing and install to 50 ml triangular flasks (50 ml/ bottle), sealing, sterilizing as stated above.

8) root media

Adding distil water to 900 ml, regulates pH value to 5.8 with 1N potassium hydroxide.Boil and be settled to 1000 ml with distilled water, dividing to install to and take root (25 ml/ pipe left and right) in pipe, sealing, sterilizing as stated above.

(4) agriculture bacillus mediated genetic transformation step

1) callus of induce

To in maturation, spend 11 rice paddy seeds (from crop science institute of academy of sciences of the Chinese Academy of Agricultural Sciences) to shell, then successively with 70% concentration ethanolic soln process 1 min, 0.15% mercury chloride (HgCl ₂) solution-treated 15 min, aqua sterilisa is transferred to seed on inducing culture after cleaning 4-5 time.By postvaccinal cultivation based on dark cultivation 4 weeks, 26 ℃ of left and right of culture temperature.

2) callus subculture

Select the embryo callus subculture of glassy yellow, consolidation and relatively dry, be put in dark cultivation 2 weeks on subculture medium, 26 ℃ of left and right of culture temperature.

3) preculture

Select the embryo callus subculture of consolidation and relatively dry, be put in dark cultivation 2 weeks on pre-culture medium, 26 ℃ of left and right of culture temperature.

4) Agrobacterium is cultivated

First at the LA substratum of selecting with corresponding resistance, (preparation of LA substratum is with reference to J. Pehanorm Brooker etc., molecular cloning experiment guide, the third edition, Jin Dongyan etc. (translating), Science Press, 2002, Beijing) upper preculture Agrobacterium EHA105 (agrobacterium strains that this bacterial strain is openly used from CAMBIA company) 2 d, 28 ℃ of culture temperature; Subsequently, Agrobacterium is transferred in suspension medium, on 28 ℃ of shaking tables, cultivates 2-3 h.

5) Agrobacterium is infected

First regulate agrobacterium suspension to OD ₆₀₀value for 0.8-1.0, is transferred to pre-incubated callus subsequently in agrobacterium suspension and soaks 30 min, callus is transferred on the filter paper that sterilizing is good and blots, and is then placed on and on common substratum, cultivates 3 d, culture temperature 19-20 ℃.

6) callus washing and selection are cultivated

Aqua sterilisa washing callus is to cannot see Agrobacterium; Be immersed in containing 30 min in the aqua sterilisa of 400 mg/l CN; Shift callus blots to the good filter paper of sterilizing; Shift callus to selecting selection on substratum to cultivate 2-3 time, each 2 weeks.

7) differentiation

Kanamycin-resistant callus tissue is transferred on pre-division culture medium in dark place cultivation 5-7 d; Shift the callus of pre-differentiation culture to division culture medium, 30-40 d, 26 ℃ of culture temperature are cultivated in illumination (1500-2000 lx).

8) take root

Cut the root that differentiation phase produces, then transfer them in root media and cultivate 2-3 week, 26 ℃ of culture temperature in illumination (1500-2000 lx).

9) transplant

Wash the residual substratum on root off, the seedling with good root system is proceeded to greenhouse.

Embodiment 4:Northern blot analyzes the expression amount of OsSWEET5 in transfer-gen plant

In the present embodiment, the method for Northern blot analysis reference literature report is carried out (Liu etc., Identification of early senescence-associated genes in rice flag leaves.Plant Mol Biol, 2008,67:37-55).Water intaking rice wild-type (non-transgenic) and T ₂the blade of the OsSWEET5 overexpression transfer-gen plant (being numbered OX1 – OX4) in generation (transgenosis), utilizes Trizol reagent (purchased from Invitrogen company) extracted total RNA (extracting method is with reference to the operational manual of Trizol reagent).Get the total RNA of 15 μ g electrophoretic separation in 1% agarose denaturant gel, then RNA fragment is transferred to Hybond-N ⁺on nylon membrane (transferring film is spent the night), UV-crosslinked fixedly secondary (each 30 s), prehybridization spends the night, then with α- ³²the OsSWEET5 gene probe hybridization of P mark, after hybridization, first use the at room temperature rinsing of 2 * SSC/0.1%SDS film washing liquid in a moment, at 65 ℃, with 2 * SSC/0.1%SDS film washing liquid, wash 5 min again, after nylon membrane dries with filter paper, at phosphorus, shield radioautograph 3-10 h in (purchased from Fujifilm company), then use FLA-5000 System (Japanese fuji company product) scanning.As shown in Figure 4, in transformed plant, the expression amount of OsSWEET5 has rising in various degree to detected result.

Probe be take full length cDNA clone J023023E05 as template, reclaims PCR product and obtain after pcr amplification (for ordinary method).The DNA sequence dna of pcr amplification primer is: forward primer F is: 5 '-cgtgacgatcgtgaagaaga-3 '; Reverse primer R: be 5 '-ggatgtcaaagcggatgaac-3 '.

Embodiment 5: the mensuration of chlorophyll content

The mensuration of chlorophyll content adopts acetone spectrophotometer method.Get approximately 0.04 g paddy rice wild-type (non-transgenic) and T ₂at the sample of sprouting second leaf of latter 15 days, in chlorophyll extract, (extract is acetone to the OsSWEET5 overexpression transfer-gen plant (being numbered OX1 – OX4) in generation (transgenosis): ethanol: water volume ratio=4.5:4.5:1), 4 ℃ of extractings are spent the night, after all bleaching, blade use spectrophotometer at the light absorption value of 663 nm and 645 nm place working samples, chlorophyll content calculates (Mao etc. by the method for bibliographical information, Two complementary recessive genes in duplicated segments control etiolation in rice.Theor Appl Genet, 2011, 122:373-383), with mg/g leaf fresh weight, represent.Result shows: OsSWEET5 overexpression transfer-gen plant chlorophyll content is starkly lower than wild-type (Fig. 5).

Embodiment 6: the mensuration of sugared content

By gas chromatograph-mass spectrometer (GC-MS), by the method for bibliographical information, measure the content (Rojas-Escudero etc. of various sugar, Optimization of carbohydrate silylation for gas chromatography.J Chromatogr A, 2004,1027:117-120; Kenyon etc., Maintenance carbon cycle in crassulacean acid metabolism plant leaves:source and compartmentation of carbon for nocturnal malate synthesis.Plant Physiol, 1985,77:183-189).Sample is paddy rice wild-type (non-transgenic) and T ₂second leaf of OsSWEET5 overexpression transfer-gen plant (being numbered OX2) 15 d after sprouting in generation (transgenosis).Result shows: compare with wild-type, in OX2 blade, the content of semi-lactosi, glucose and fructose obviously rises, and the content of sucrose is obviously decline (Fig. 6).These results show that in rotaring gene plant blade of the present invention, obvious variation has occurred sugared content.

Claims (8)

1. OsSWEET5 gene that derives from paddy rice application in regulation and control Leaf senescence development, is characterized in that, the nucleotide sequence of described gene is as shown in sequence table SEQ ID NO:1.

2. the application of the proteins encoded of an OsSWEET5 gene that derives from paddy rice in regulation and control Leaf senescence development, is characterized in that, the sequence of described DNA encoding the protein is as shown in sequence table SEQ ID NO:2.

3. an overexpression carrier p1300s-OsSWEET5, is characterized in that, this carrier contains gene as claimed in claim 1.

4. the application of gene OsSWEET5 as claimed in claim 1, is characterized in that, by containing the expression vector of gene OsSWEET5 described in claim 1, proceeds to paddy rice, thereby with this gene speeding-up blade senescence process of overexpression.

5. a method that changes object plant leaf senescence process, is characterized in that, by improving the expression amount of gene OsSWEET5 described in claim 1, carrys out speeding-up blade senescence process.

6. method as claimed in claim 4, is characterized in that, described object plant is monocotyledons or dicotyledons.

7. the method as described in claim 5 or 6, is characterized in that, described monocotyledons is paddy rice, corn, wheat and turfgrass.

8. the method as described in claim 5 or 6, is characterized in that, described dicotyledons is Arabidopis thaliana, willow, soybean and cotton.