CN101993483B

CN101993483B - Protein related to plant stress tolerance, coding gene and application thereof

Info

Publication number: CN101993483B
Application number: CN 200910091729
Authority: CN
Inventors: 夏新界; 徐孟亮
Original assignee: Individual
Current assignee: CHANGSHA ZHONGKEJINGBO BIOTECHNOLOGY Ltd
Priority date: 2009-08-24
Filing date: 2009-08-24
Publication date: 2013-08-14
Anticipated expiration: 2029-08-24
Also published as: CN101993483A

Abstract

The invention discloses protein which is named as OsMSr1 and comes from rice. The protein is (1) or (2) as follows: (1) protein consisting of an amino acid sequence shown as a sequence 2 in a sequence table; and (2) protein which is formed by substituting and/or deleting the amino acid sequence in the sequence table 2 and/or adding one or more amino acids to the amino acid sequence, derived from the protein (1) and related to plant stress tolerance. The invention also can protect the coding gene of the protein, and the cold resistance of the transgenic rice obtained by introducing the gene into the rice is obvious enhanced.

Description

Albumen and encoding gene and application with the anti-retrocorrelation of plant

Technical field

The present invention relates to biological technical field, particularly with albumen and encoding gene and the application of the anti-retrocorrelation of plant.

Background technology

Paddy rice is one of most important food crop, it is staple food with rice that the whole world has the population of half approximately, increase along with population, demand to rice is more and more, meanwhile, along with industrialization, fast development of urbanization, the rice field is the trend of reduction, and how to produce more grain at limited even littler rice field area is a global significant problem that must solve at present.Theoretical calculation and Rice Production facts have proved that the yield potential of modern high-yield rice can reach 18-22.5t/ha, the average per unit area yield of actual global paddy rice has only about 3.8t/ha (China is 6.3t/ha), mainly be the performance that adverse circumstance has restricted its genetic potential, comprising abiotic stress factors such as arid, saline and alkaline, low temperature, high temperature, nutrient deficiencies.Therefore, the resistance of reverse that improves modern high-yield rice kind is further to excavate rice yield potentiality, guarantee quality, widen one of important channel of cultivated area, and the essential method of the resistance of reverse that will improve modern high-yield rice kind is cultivated anti-contrary high-yield rice new variety exactly, traditional breeding practice proves, resistance of reverse is the quantitative character of a controlled by multiple genes, is difficult to be used by traditional breeding method.Along with development of molecular biology, then be to be proved to be valid approach by genetically modified molecular breeding approach.Carry out molecule by the important anti-contrary gene to paddy rice itself and control the resistance of reverse that (crossing expression etc.) is expected to significantly improve paddy rice.For this reason, at first must know anti-reversed reaction and the adaptive process that has those important gene to participate in paddy rice on earth.Yet, the anti-contrary gene of cloning from paddy rice at present is very limited, therefore, clone the anti-contrary gene of how new paddy rice, be very necessary, the cultivation that not only can be the anti-rice against the current of China's transgenosis new germ plasm, new variety provides the goal gene with independent intellectual property right, and can deepen that paddy rice, the anti-contrary understanding of molecular mechanism of grass are had important realistic price and scientific meaning.

Summary of the invention

The object of the present invention is to provide a kind of albumen, called after OsMSr1 derives from paddy rice, is following 1) or 2) albumen:

1) protein that the aminoacid sequence shown in the sequence 2 is formed in the sequence table;

2) in sequence table the aminoacid sequence of sequence 2 through replacement and/or disappearance and/or add one or several amino acid and with the anti-retrocorrelation of plant by 1) protein of deriving.

Sequence table sequence 2 is the aminoacid sequence of OsMSr1, comprises 89 amino acid, in this protein sequence, hydrophobic amino acid accounts for 39, and hydrophilic amino acid accounts for 50, and basic aminoacids accounts for 13, acidic amino acid accounts for 16, and the molecular weight of this protein is 9.973KD, and iso-electric point is 5.05.This protein is the new protein of not reported in the world.

In order to make 1) in OsMSr1 be convenient to purifying, label as shown in table 1 on N-terminal that can the protein that the aminoacid sequence shown in the sequence 2 is formed in by sequence table or C-terminal connect.

The sequence of table 1. label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tagII	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned 2) but in the OsMSr1 synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.Above-mentioned 2) encoding gene of the OsMSr1 in can be by the codon with sequence in the sequence table 1 one or several amino-acid residue of disappearance in the dna sequence dna shown in 5 ' terminal the 7th to 273 bit base, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

The encoding gene of above-mentioned albumen, called after OsMSr1 also belongs within protection scope of the present invention.

Above-mentioned encoding gene is following 1) or 2) or 3) gene:

1) encoding sequence be in the sequence table sequence 1 from the gene of 5 ' end shown in the 7th the-the 273rd;

2) under the rigorous condition of height with 1) gene recombination that limits and the gene of encoding said proteins;

3) with 1) gene that limits has homology more than 90% and the gene of encoding said proteins.

Above-mentioned encoding gene specifically can be the gene shown in the sequence 1.

Sequence table 1 is 1 global cDNA clone of coding OsMSr1.This sequence is altogether by 328 based compositions, and wherein, the untranslated district of 5 ' end comprises 6 bases, the untranslated district of 3 ' end comprises 52 bases, the coding region is made up of 267 bases (from the 7th to the 273rd), and coding has the OsMSr1 albumen of the aminoacid sequence of sequence 2 in the sequence table, in the coding region, A accounts for 14.6% (48), C accounts for 28.0% (92), and G accounts for 44.8% (147), and T accounts for 12.5% (41), A+T accounts for 27.1% (89), and C+G accounts for 72.9% (239).

Above-mentioned high rigorous hybridization conditions refers to, with Hybond membrane place prehybridization solution (the 0.25mol/L sodium phosphate buffer, pH7.2,7%SDS) in, 65 ℃ of prehybridization 30min; Abandon prehybridization solution, add hybridization solution (0.25mol/L sodium phosphate buffer, pH7.2,7%SDS, isotope-labeled nucleotide fragments), 65 ℃ of hybridization 12hr; Abandon hybridization solution, (20mmol/L sodium phosphate buffer, pH7.2 5%SDS), wash film 2 times for 65 ℃, each 30min to add film washing liquid I; (20mmol/L sodium phosphate buffer, pH7.2 1%SDS), wash film 30min for 65 ℃ to add film washing liquid II.

The primer of above-mentioned OsMSr1 full length gene or its arbitrary fragment of increasing is to also belonging to protection scope of the present invention.

The transgenic cell line that contains said gene also belongs to protection scope of the present invention.

The reorganization bacterium that contains said gene also belongs to protection scope of the present invention.

The recombinant vectors that contains said gene also belongs to protection scope of the present invention.

Available existing plant expression vector construction contains the recombinant expression vector of OsMSr1 gene.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, as pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN, pBY505 or other plant expression vector of deriving.

When using the gene constructed recombinant expression vector of OsMSr1, can before its transcription initiation Nucleotide, add any enhancement type, composing type, organizing specific type or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin (Ubiquitin) gene promoter (pUbi), Actin promotor etc., they can use separately or be used in combination with other plant promoter.

In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.

For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, as be added in the plant to express and to produce the enzyme of colour-change or the gene of luminophor (gus gene, GFP gene, luciferase genes etc.), have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (as anti-weedkiller gene) etc.

The concrete construction process of above-mentioned recombinant vectors is as follows: be the multiple clone site of above-mentioned gene being inserted pJIT163, obtain intermediate carrier pJIT163-OsMsr1;

Described intermediate carrier is cut with EcoR V enzyme and pCAMBIA1300 is connected with the fragment that obtains after Sma I enzyme is cut through Sac I through Sac I, obtained recombinant vectors pCAMBIA1300-2 * CaMV35S-OsMsr1-CaMV35S-Term.

Another object of the present invention is to provide a kind of method of cultivating the gene plant of anti-the reverse, is that above-mentioned gene is imported the purpose plant, obtains the gene plant of anti-the reverse.

The described gene plant of anti-the reverse is cold-resistant transgenic plant.

Above-mentioned gene is to import in the purpose plant by above-mentioned recombinant vectors.Carry OsMSr1 gene of the present invention plant expression vector can by Ti-plasmids, Ri plasmid, plant viral vector, as particle bombardment, pollen tube channel, microinjection, electricity lead, conventional biological method such as agriculture bacillus mediated is transformed in vegetable cell or the tissue.

Above-mentioned plant is dicotyledons or monocotyledons; Described monocotyledons is grass; This grass specifically can be paddy rice.

Experimental results show that: the rice shoot survival rate that mistake is expressed after cold the coercing of OsMsr1 trans-genetic hybrid rice 93-11 can reach 71.5%.Rice shoot surviving rate after cold the coercing, two key indexs of the degree of keeping of photosynthetic capacity are weighed, and cross that expression OsMsr1 trans-genetic hybrid rice 93-11 resistance to cold in seedling stage significantly is better than the wild-type contrast and empty carrier contrasts.

Description of drawings

Fig. 1 is the figure that comprises the object tape of OsMsr1 cDNA segment with EcoR I and the acquisition of Hind III double digestion pMD18-T positive colony, and wherein, M is dna molecular amount standard; 1-5 is digested positive colony.

Fig. 2 detects transgenic paddy rice figure for using hygromycin gene PCR primer PCR, wherein, the positive plant of 3-9,2 is wild-type contrast (WT2), 1 is no template contrast, 10 positive contrasts (pCAMBIA1300-OsMsr1), M is dna molecular amount standard.

Fig. 3 is the special primer PCR detection transgenic paddy rice figure with the mosaic gene of 2 * 35S promoter and OsMsr1 composition, wherein, 1 positive contrast, the positive plant of 2-4,5 is empty carrier contrast (WT1), M is the DNA standard.

Fig. 4 is the artificial cold back transgenic plant growth figure that coerces.

Fig. 5 coerces back transgenic plant growth figure for nature is cold.

Fig. 6 is the artificial cold Net Photosynthetic Rate (Fig. 6 A) and electronics transmission speed (Fig. 6 B) of coercing front and back of regenerated transgenic seedling (T0).

Fig. 7 is the artificial cold maximum photochemistry quantum yield of PSII (Fig. 7 A) and actual light chemistry quantum yield (Fig. 7 B) of coercing front and back of regenerated transgenic seedling (T0).

Embodiment

The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.

Among the following embodiment, if no special instructions, be ordinary method.

Embodiment 1, the anti-reverse trans-genetic hybrid rice of cultivation

One, the structure of recombinant expression vector

1, the clone of OsMsr1 gene

The a pair of primer of synthetic, and add XbaI and Pmac I restriction enzyme site respectively at its 5 ' end.Upstream primer and downstream primer are as follows:

OsMsr1-F：5′- TCT AGA GAG GCA ATG GAG GTG GA-3′，

OsMsr1-R：5′- CAC GTG CAG CAA CCA ACA CAC TG-3′。

Extract paddy rice and train short 64S (by National Engineering and Technological Research Center of Interbreed Paddy's sieve filial piety with provide, the public can and obtain from Hunan Academy of Agricultural Sciences germplasm resource bank or Pei ' ai64 S sieve's breeder filial piety), document: Luo Xiaohe, Qiu's toe loyalty, Li Renhua. cause the low dual purpose genic male sterile line of critical male sterile temperature to train short 64S. hybrid rice, 1992, (1): RNA 27-29) obtains article one chain of cDNA through reverse transcription.

Being primer with above-mentioned OsMsr1-F and OsMsr1-R, is that template is carried out pcr amplification to train short 64ScDNA the 1st chain; Amplification system is: 2 * GC buffer I, 10.0 μ L, dNTPmix (2.5mM) 1.6 μ L, F primer (10 μ M) 0.4 μ L, R primer (10 μ M) 0.4 μ L, TaKaRa LA Taq enzyme 0.6 μ L, template 4 μ L (containing the short 64S cDNA 0.2 μ g of training approximately) complement to 20.0 μ L with the sterilization distilled water; Amplification program is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 30s, 58 ℃ of annealing 45s, 72 ℃ are extended 1-min, and totally 35 circulations are extended 10min down at 72 ℃ at last.

Pcr amplification obtains the fragment of 340bp, the PCR product that obtains is reclaimed, connect pMD18-T cloning vector (available from precious biotechnology (Dalian) company limited), obtain recombinant vectors pMD18-T-OsMsr1, then with this recombinant vectors transformed into escherichia coli, screening positive clone, then positive colony is identified (as Fig. 1) with EcoR I and Hind III double digestion, send the order-checking of Invitrogen company after identifying correctly, sequencing result shows that the dna sequence dna of purpose fragment has the nucleotide sequence from the 1st the-the 328th at 5 ' end shown in the sequence 1 in the sequence table, wherein the coding region is from the 7th the-the 273rd at 5 ' end, the albumen shown in can encoding sequence 2.

2, make up recombinant expression vector

1) structure of intermediate carrier pJIT163-OsMsr1

The pMD18-T-OsMsr1 that above-mentioned steps 1 is obtained is with BamH I and Hind III double digestion, pJIT163 (the pGreen of the fragment that contains the OsMsr1 gene that obtains and the same double digestion of warp, http://www.pgreen.ac.uk/) is connected, obtains pJIT163-OsMsr1.

2) structure of pCAMBIA1300-2 * CaMV35S-OsMsr1-CaMV35S-Term

The intermediate carrier pJIT163-OsMsr1 that step 1) is obtained cuts with Sac I and EcoRV enzyme, obtain containing the fragment of OsMsr1,2 * CaMV35S promotor and CaMV35S terminator, with this fragment and the pCAMBIA1300 (CambiaLabs behind Sac I and SmaI double digestion, http://www.cambia.org/daisy/bioforge legacy/3725.html) is connected, obtains pCAMBIA1300-2 * CaMV35S-OsMsr1-CaMV35S-Term.Wherein, the reason of structure intermediate carrier pJIT163-OsMsr1 is 2 * CaMV35S promotor and the CaMV35S terminator that needs to obtain from its carrier mosaic gene.

Two, cultivate anti-reverse trans-genetic hybrid rice and detection thereof

1, the acquisition of anti-reverse trans-genetic hybrid rice

1) with freeze-thaw method the recombinant expression vector pCAMBIA1300-2 * CaMV35S-OsMsr1-CaMV35S-Term of the step 2 of step 1 is transformed Agrobacterium EHA105 (purchasing in TIANGEN Biotech (Beijing) Co., Ltd.).

Wherein the step of freeze-thaw method is as follows: take out the Agrobacterium EHA105 competent cell of-70 ℃ of preservations, ice bath melts; Get 10-20 μ l pCAMBIA1300-2 * CaMV35S-OsMsr1-CaMV35S-Term plasmid DNA (about 1-2 μ g), join in the Agrobacterium competent cell of 200ml thawing, stir evenly with sterilization rifle head, left standstill several minutes; Place liquid nitrogen 1min, 37 ℃ of water-bath 5min add 700-800 μ l LB liquid nutrient medium, 28 ℃, 200rpm, concussion 4h; 1000g 30sec removes the part supernatant, stays 100-150 μ l, inhales to beat again with the rifle head to suspend, and coats on the LB flat board that contains 50mg/L kantlex, 50mg/L Rifampin, 34mg/L paraxin, is inverted for 28 ℃ and cultivates 2d.

Transformant is identified: the mono-clonal bacterium colony of picking 5-8 tool kantlex, Rifampin, 3 kinds of resistances of paraxin from the LB flat board, be inoculated in the 3ml LB liquid nutrient medium that contains 50mg/L kantlex, 50mg/L Rifampin, 34mg/L paraxin 28 ℃ of shaking culture 40h of 220rpm.Getting 1-2 μ l bacterium liquid is template, be primer with the OsMsr1-F in the step 1 and OsMsr1-R, carry out pcr amplification, identify in the Agrobacterium and whether contain target gene fragment, pcr amplification reaction condition and thermal cycling arrange same step 1, the result shows that pCAMBIA1300-2 * CaMV35S-OsMsr1-CaMV35S-Term successfully transforms Agrobacterium.

2) Agrobacterium of the reorganization that above-mentioned evaluation is correct is infected rice variety 93-11 by agrobacterium tumefaciens-mediated transformation (Hunan Normal University's school of life and health sciences is so kind as to give, and the public can freely obtain from Hunan Normal University school of life and health sciences paddy rice research department.Document: Yuan Longping chief editor. hybrid rice is learned. Beijing: the .2002 of Chinese agriculture press, callus pp153.).Callus after will infecting is then grown in the screening culture medium that contains Totomycin (50 μ g/ml), obtains the hygromycin resistance regeneration plant.

With with the closely linked hygromycin gene of OsMsr1 be detected object, carry out PCR checking.

Genomic dna with hygromycin resistance regeneration plant blade is template, carries out pcr amplification: hpt-F:5 '-ACC TGC CTG AAA CCG AAC TG-3 ' and hpt-R:5 '-CTG CTC CATACA AGC CAA CC-3 ' with following primer.The PCR detected result as shown in Figure 2, with the closely linked hygromycin gene of OsMsr1 be detected object, regeneration plant all expands and expection DNA band (504bp), and the wild-type contrast is not expanded and DNA band (Fig. 2).Illustrate with the closely linked hygromycin gene of OsMsr1 and be incorporated in the rice genome.

The mosaic gene of forming with 35S promoter and OsMsr1 is detected object, carries out the PCR checking.

Genomic dna with hygromycin resistance regeneration plant blade is template, carries out pcr amplification with following primer:

PML1F：5’-AAC CTC CTC GGA TTC CAT TG-3’；PML1R：5’-CCA CCTCCA TTG CCT CTC T-3’。

The PCR detected result as shown in Figure 3, the mosaic gene of forming with 35S promoter and OsMsr1 is detected object, regeneration plant also all expands and expection DNA band (715bp and 376bp), and the empty carrier contrast is not expanded and DNA band (Fig. 3).Illustrate that the mosaic gene that 35S promoter and OsMsr1 form also is incorporated in the rice genome.

2, detect analysis

Above-mentioned commentaries on classics pCAMBIA1300-2 * CaMV35S-OsMsr1-CaMV35S-Term paddy rice is moved to hot-house culture, selfing, the seed of collection transgenic paddy rice is sprouted into seedling, gets two strains at random and crosses the strain system of expressing OsMsr1, called after T13-1 and T17-1.

With pJIT163 through Sac I and fragment after the EcoRV enzyme is cut be connected with the pCAMBIA1300 that Sma I enzyme was cut through Sac I, obtain empty carrier pCAMBIA1300-2 * CaMV35S-CaMV35S-Term.According to preparing the method for changeing pCAMBIA1300-2 * CaMV35S-OsMsr1-CaMV35S-Term paddy rice, prepare commentaries on classics empty carrier pCAMBIA1300-2 * CaMV35S-CaMV35S-Term paddy rice and contrast called after WT1 as empty carrier.

The seedling that conventional seed germination becomes contrasts called after WT2 as wild-type.

1) the cold rice shoot survival rate of coercing is tested

A. artificial cold coercing

Be that 25 days seedling is put into growth cabinet and handles that (the climate box environment was 11 ℃ of photophases, 8h in 3 days for average 7.7 ℃ with above-mentioned WT1, WT2 and T13-1 and T17-1 seedling age; 6 ℃ of dark phases, 16h).21 ℃ are recovered growth and take pictures after 10 days and add up seedling number alive after artificial cold the coercing then.Three repetitions are established in experiment, respectively handle 20 strains in repeating at every turn.

The result as shown in Figure 4, after deepfreeze and recovering growth, T13-1 and T17-1 strain system is most of can survive, and WT1, WT2 can not be survived substantially.

Concrete outcome is as shown in table 1, and the survival rate of the survival rate 8.5% of WT1, the survival rate 11.5% of WT2, T13-1 is 53.5%, the survival rate of T17-1 is 55.0%.

The artificial cold back rice shoot survival rate (2008) of coercing of table 1 transgenosis 93-11

Title material	Strain number before cold the coercing	Cold strain number of coercing the back survival	The cold back survival rate % that coerces
				WT1	20.0	1.7±1.5	8.5
WT2	20.0	2.3±1.2	11.5
				T13-1	20.0	10.7±1.5**	53.5
T17-1	20.0	11.0±2.0**	55.0

Annotate: data are the mean+SD of 3 repeated sample in the table.* be illustrated in 5% level and 1% level and WT1, WT2 with * * significant difference is arranged.

B. cold coercing naturally

A) by late sowing method (sowing on November 1st, 2008), above-mentioned WT1, WT2 and T13-1 and the potted plant seedling of T17-1 are placed under the natural condition of Changsha, through 24-November 30 November continuous 7 days natural low temperature coerce, average average daily temperature was 10.5 ℃ in 7 days, an average day lowest temperature is 6.0 ℃, an average day highest temperature is 17.0 ℃, and 21 ℃ (Artificial Control) recovered growth and added up after 10 days.

The result is as shown in table 2, and the survival rate of the survival rate 3.5% of WT1, the survival rate 16.5% of WT2, T13-1 is 33.5%, the survival rate of T17-1 is 71.0%.

The cold naturally back rice shoot survival rate (Changsha, 2008) of coercing of table 2 transgenosis 93-11

Title material	Seedling number before cold the coercing	The cold back seedling number of coercing	The cold back survival rate % that coerces
				WT1	20.0±0.0	0.7±0.6	3.5
WT2	20.0±0.0	3.3±0.6	16.5
				T13-1	20.0±0.0	6.7±1.2*	33.5
T17-1	20.0±0.0	14.3±0.6**	71.5

B) in 6 days-March 8 March in 2009 and 12-March 14 March above-mentioned WT1, WT2 and T13-1 and T17-1 seedling are placed under the natural condition in Changsha, stand 2 times totally 6 days natural low temperature coerce, average average daily temperature was 9.4 ℃ in preceding 3 days, an average day lowest temperature is 6.3 ℃, an average day highest temperature is 13.9 ℃, the 3 days average average daily temperature in back are 8.6 ℃, an average day lowest temperature is 6.0 ℃, an average day highest temperature is 11.7 ℃, and 21 ℃ (Artificial Control) recovers the seedling number that growth is taken pictures after 10 days and added up survival.

Photo as shown in Figure 5, transfer-gen plant (T13-1 and T17-1) growing way is significantly better than wild-type contrast WT2 and empty carrier contrast (WT1).

Concrete outcome is as shown in table 3, and the survival rate of the survival rate 0.0% of WT1, the survival rate 5.0% of WT2, T13-1 is 35.0%, the survival rate of T17-1 is 45.0%.

The cold naturally back rice shoot survival rate (Changsha, 2009) of coercing of table 3 transgenosis 93-11

Title material	Seedling number before cold the coercing	The cold back seedling number of coercing	The cold back survival rate % that coerces
				WT1	20.0±0.0	0.0±0.0	0.0
WT2	20.0±0.0	1.0±1.0	5.0
				T13-1	20.0±0.0	7.0±1.7*	35.0
T17-1	20.0±0.0	9.0±1.0**	45.0

2) degree of keeping of photosynthetic capacity

To the artificial cold trans-genetic hybrid rice of the expression OsMsr1 excessively 93-11 T that coerces processing among the above-mentioned steps A ₀Measure for the cold maximum photochemistry quantum yield of Net Photosynthetic Rate, electronics transfer rate, photosystem II and the actual light chemistry quantum yield of coercing the front and back blade of regrowth (T13-1 and T17-1), WT1 and WT2.Wherein, Net Photosynthetic Rate adopts LI-6400 portable photosynthesis measurement system (production of U.S. LI-COR company) to measure, and 9:00-11:00 carries out in growth cabinet in the morning, and light intensity is controlled at 1500 μ mol m ^-2S ^-1, produce CO by the red blue-light source (6400-02B led light source) of apparatus preparation ₂Concentration is set to 400 μ mol.mol ^-1, by Small-sized C O ₂Steel cylinder is by the CO of apparatus preparation ₂Injected system provides, and the blade face temperature is set to 25 ℃, by instrument accurately control automatically; Chlorophyll fluorescence parameters adopts MINI-PAM (Pulse-Amplitude-Modulation) pulse-amplitude modulation luminoscope (German WALZ company produce) to measure, after abundant dark adatpation, and the fluorescence induction program determination chlorophyll fluorescence parameters that carries by instrument.Each material is measured 5 blades at least, averages as measurement result.

The result crosses the cold ratio of coercing before back Net Photosynthetic Rate and photosystem II photochemistry quantum yield and cold the coercing of the 93-11 express the OsMsr1 gene and is significantly higher than also that not genetically modified wild-type contrasts and empty carrier contrasts shown in Fig. 6 and Fig. 7 and table 4 and table 5.

Table 4 changes 2 * CaMV 35S-OsMsr1 93-11 regrowth (T ₀) Net Photosynthetic Rate (μ mol CO2m after artificial cold the coercing ^-2s ^-1)

Annotate: growth cabinet was handled 2 days for 7.7 ℃, and 21 ℃ are recovered growth and measure after 3 days; Every material repeats for 3 times.

Table 5 changes the chlorophyll fluorescence parameters after artificial cold the coercing of 2 * CaMV 35S-OsMsr193-11 regrowth (T0)

BCT：Before cold treatment；ACT：After cold treatment.

Rice shoot surviving rate after above cold the coercing, two key indexs of the degree of keeping of photosynthetic capacity are weighed, and cross that expression OsMsr1 trans-genetic hybrid rice 93-11 resistance to cold in seedling stage significantly is better than the wild-type contrast and empty carrier contrasts.

Sequence table

＜110〉Xiaxin circle

＜120〉with albumen and encoding gene and the application of the anti-retrocorrelation of plant

<130>CGGNARL92497

<160>2

<210>1

<211>328

<212>DNA

＜213〉paddy rice (Oryza sativa L.)

<400>1

gaggcaatgg aggtggagat ggggcacggc gaggaggtgg cggctgcgat ggaggaggag 60

gagctgcagg ggtgggagac gccgaggagg gaggagtgcc gcatcccggt ggtgccgccg 120

cagtgcccgg cgccgccgag gaagaggccg gtggcgctgc cggagctggg gaaggagcgg 180

cgggagccgc ccaagggcgg gtacttccag ccgccggacc tcgagtcgct cttcgtgctc 240

gcgccgccgc ggaggcaggc gtccagctgc gcgtagtgct gctgcgccca gcctccgtgc 300

agcagcacgc acagtgtgtt ggttgctg 328

<210>2

<211>89

<212>PRT

＜213〉paddy rice (Oryza sativa L.)

<400>2

Met Glu Val Glu Met Gly His Gly Glu Glu Val Ala Ala Ala Met Glu

1 5 10 15

Glu Glu Glu Leu Gln Gly Trp Glu Thr Pro Arg Arg Glu Glu Cys Arg

20 25 30

Ile Pro Val Val Pro Pro Gln Cys Pro Ala Pro Pro Arg Lys Arg Pro

35 40 45

Val Ala Leu Pro Glu Leu Gly Lys Glu Arg Arg Glu Pro Pro Lys Gly

50 55 60

Gly Tyr Phe Gln Pro Pro Asp Leu Glu Ser Leu Phe Val Leu Ala Pro

65 70 75 80

Pro Arg Arg Gln Ala Ser Ser Cys Ala

85

Claims

1. method of cultivating the gene plant of anti-the reverse is that the gene from shown in the 7th the-the 273rd at the 5 ' end with sequence in the sequence table 1 imports the purpose plant, obtains the gene plant of anti-the reverse; The gene shown in 5 ' holds the 7th the-the 273rd of sequence 1 is to import in the purpose plant by recombinant vectors in the sequence table; The construction process of described recombinant vectors is as follows: be with sequence in the sequence table 1 insert the multiple clone site of pJIT163 from the gene of 5 ' end shown in the 7th the-the 273rd, obtain intermediate carrier pJIT163-OsMsr1;

Described intermediate carrier is connected through the fragment that Sac I cuts with the EcoRV enzyme and pCAMBIA1300 obtains behind Sac I and Sma I, obtains recombinant vectors pCAMBIA1300-2 * CaMV35S-OsMsr1-CaMV35S-Term;

The described gene plant of anti-the reverse is cold-resistant transgenic plant;

Described plant is paddy rice.