CN104818289B - High temperature resistant cabbage type rape aldoketonutase gene and albumen and its application - Google Patents

Abstract

The invention discloses a kind of high temperature resistant cabbage type rape aldoketonutase gene and albumen and its application, it is related to and belongs to mutation and genetic engineering technology field.The nucleotide sequence of high temperature resistant cabbage type rape aldoketonutase gene BnGLYI genes, its nucleotide sequence is as shown in SEQ ID NO1；The albumen of high temperature resistant cabbage type rape aldoketonutase gene BnGLYI genes, its amino acid sequence is as shown in SEQ ID NO2.The present invention is promotes the exploitation of cabbage type rape fine genes, and raising cabbage type rape is heat-resisting, cold resistance has established experiment basis；Heat-resisting, cold tolerance gene resource is further expanded；By BnGLYI genetic transformation to yeast cells, the heat-resisting and cold resistance of yeast cells is significantly improved, be expected to further improve the heat-resisting and cold resistance of other microorganisms and plant.

Description

High temperature resistant cabbage type rape aldoketonutase gene and albumen and its application

Technical field

The present invention relates to mutation and genetic engineering technology field；More particularly to a kind of high temperature resistant cabbage type rape aldoketonutase Gene and albumen and its application；Specifically related to a kind of high temperature resistant cabbage type rape aldoketonutase gene (Glyoxalase I, BnGLYI nucleotide sequence), is related to the amino acid sequence of high temperature resistant BnGLYI protein, BnGLYI genes are in Pichia pastoris The recombinant bacterial strain of middle expression, is directed to use with the gene constructed expression Yeast expression carrier, further relates to enter using said gene sequence The method of row yeast conversion.

Background technology

Seed supplies animal and the raw material of industry of most of foods and important sources for us, and high-quality seed is to society Meeting economy has great importance^[1].Many biological and abiotic factor influence seed vitalities are sprouted and storage property, such as environment Temperature is higher or seed may reduce seed vitality because of the high temperature that autoxidation is produced in storage, slows down or presses down completely System germination^[2].But, although there are part research and inquirement plant heat-resisting quantity molecule aspect, physiology and science of heredity to grind at present Study carefully^[2], but to the research of seed heat resistance also than sparser.

Methyl-glyoxal (Methylglyoxal, MG) is a kind of cytotoxic metabolin, and MG results from glycolysis, ammonia Base acid catabolism, the eubolism of acetone and environmental stress stress, in microorganism, yeast, animal and higher plant all In the presence of^[3].What is completed in aldoketonutase (glyoxalase, GLY) approach is mainly to MG metabolism in organism, the approach bag Two enzymes, i.e. glyoxalase I (GLYI) and glyoxalase I I (GLYII) are included, are a kind of endocellular enzymes, are prevalent in various thin In born of the same parents' device.MG and glutathione (glutathione, GSH) are condensed into generation S-D- lactic acid paddy Guangs under GLYI effects first Sweet peptide, reduces MG physiological active concentration, S-D- lactic acid glutathione then is hydrolyzed into nontoxic GSH in the presence of GLYI And D-ALPHA-Hydroxypropionic acid^[4].In recent years, research finds that GLYI genes are one and contain polygenic gene family^[5], it is not only involved in plant growth Growth course, also tackles biotic in plant, and the responsing reaction that produces of the abiotic stress such as heavy metal, high salt, arid and Alleviate and play an important role in terms of the damage that it causes^[5][6][7], but in higher plant, it is resistance to whether relevant GLYI genes have Hot function is also fresh for report.2002, separation acquisition Tengchong bit the high temperature methylglyoxal synthease base in hot anaerobic bacteria Cause^[8].2010, research found the ability of the brewing yeast cell resistance extreme temperature of conversion banana aldoketonutase gene significantly Degree is improved^[9]。

Bibliography

[1] Janmohammadi M, Fal lahnezhad Y, Golsha M, Mohammadi H, 2008, Controlled ageing for storabi lity assessment and predicting seedling early Growth of canola cultivars (Brassica napus L.) .J Agric Biol Sci 3,22-26.

[2] Wahid A, Gelani S, Ashraf M, Foolad MR, 2007, Heat tolerance in plants： An overview.Environ Exp Bot 61,199-223.

[3] Singla-Pareek SL, Reddy MK, Sopory SK, 2003, Genetic engineering of the glyoxalase pathway in tobacco leads to enhanced salinity tolerance. Proc Natl Acad Sci U S A 100,14672-14677.

[4] Phi l lips SA and Thornalley PJ, 1993, The formation of methyglyoxal From triose phosphates.Eur J Biochem 212,101-105.

[5] Mustafiz A, Singh AK, Pareek A, Sopory SK, Singla-Pareek SL, 2011, Genome-wide analysis of rice and Arabidopsis identifies two glyoxalase genes that are highly expressed in abiotic stresses.Funct Integr Genomics.11：293– 305.

[6] Espartero J, Sa nchez-Aguayo I, Pardo JM, 1995, Molecular characterization of glyoxalase I from a higher plant:upregulation by Stress.Plant Mol Biol, 29：1223–1233.

[7] Mustafiz A, Ghosh A, Tripathi AK, Kaur C, Ganguly AK, Bhavesh NS, Tripathi JK, Pareek A, Sopory SK, Singla-Pareek SL, 2014, A unique Ni2+-dependent and methylglyoxal-inducible rice glyoxalase I possesses a single active site and functions in abiotic stress response.Plant J 78： 951-63.

[8] Li Wei, Yu Jun, Zhong Lan, Wang Jun, Sun Jiandong, Hangzhou Genomics R ＆ D Center, high temperature methylglyoxal are closed The polypeptide and preparation method of enzyme gene sequence and coding.Application number 01132107.5, publication number CN1364907.

[9] Deng Chengju, Zhang Jianbin, Jia Caihong, Jin Zhiqiang, Xu Biyu.Banana aldoketonutase genes amplification saccharomyces cerevisiae pair The research of abiotic stress resistivity.Chinese biological engineering magazine, 2010,30 (8)：22-26.

The content of the invention

The purpose of the present invention, which is that, overcomes the problem of prior art is present and not enough oily there is provided a kind of high temperature resistant Wild cabbage type Dish aldoketonutase gene and albumen and its application.

The object of the present invention is achieved like this：

The aldoketonutase gene of our the isolated cabbage type rapes in the heat-resisting germ plasm resource of cabbage type rape BnGLYI, and prove that the gene has heat resistance and cold resistance, and the gene and Tengchong by the heterogenous expression of yeast cells Biting the heat-resisting GLYI genes of hot anaerobic bacteria has very big difference, and the homology of both amino acid is only 26%.Therefore, this discovery Not only be found that the New function of cabbage type rape BnGLYI genes, and for transgenic engineered bacteria and plant provide it is new it is heat-resisting, Cold tolerance gene is originated, and then improves the heat-resisting effect of engineering bacteria and Genetically Modified Plant, with important economic, social and life State benefit.

First, high temperature resistant cabbage type rape aldoketonutase BnGLYI genes and albumen

For current world food crop and oil crops storage problems faced, and to high temperature and adjoint arid with Other adverse circumstances are to plant production and the influence and loss of growth, present invention separation gram from heat-resisting cabbage type rape germ plasm resource Grand gene BnGLYI newly, the gene expression product makes yeast cells have higher resistance to high temperature and low temperature, and this hair There is some difference with known GLYI albumen for the amino acid sequence of the gene expression product of bright offer, and we are first Disclose GLYI heat-resisting and cold resistance.This new gene can apply to microbial and plant, be allowed to performance to temperature Spend the patience of stress.

The present invention provides the contributive BnGLYI gene orders of a kind of heat-resisting to cabbage type rape seed and cold resistance, to answer For microbial and plant, it is allowed to show the resistance to associated biomolecule and abiotic stress.

Cabbage type rape germ plasm resource 3382, by National Nature scientific and technological resources e platforms, (national Germplasm Resources of Farm Crop is put down Platform) provide, its preserving number is 00003382, it is ensured that provide within 20 years shared utilize.

Cabbage type rape heat resistance BnGLYI genes, its nucleotide sequence is as shown in SEQ ID NO1；

Cabbage type rape heat resistance BnGLYI albumen, by above-mentioned BnGLYI coded by said gene, its amino acid sequence such as SEQ Shown in ID NO2.

2nd, the application of high temperature resistant cabbage type rape aldoketonutase BnGLYI genes and albumen

1. application of the BnGLYI genes in transgenic microorganism

The Pichi strain GSGLYI of BnGLYI genes is converted, heat resistance and cold resistance is obtained；

2. application of the BnGLYI genes in stress resistance of plant.

The application is heat-resisting and resistance to by the expression vector microbial containing BnGLYI genes or plant, to be allowed to produce Cold property.

3rd, concrete technical scheme is realized by following steps：

1. by the screening in heat resistance cabbage type rape, it is found that the heat resistance of cabbage type rape 3382 is significantly larger than other Rape variety；The analysis of dielectrophoresis is carried out to the heat-resisting germplasm that screening is obtained, it is found that about 21kD small molecular protein exists It is 2 times of before processing expression quantity after heat treatment；The protein site carries out mass spectral analysis, finds amino acid identity and the mustard of the albumen Dish type rape BjGLYI homology is high, and score values have reached 117.

2. the RNA of cabbage type rape seed is extracted, and reverse transcription is masterplate into cDNA, and BnGLYI volumes are carried out with primer pair The amplification in code region and clone：

BnGLYI F：5^/-ATGGCGTCGGAAGCGAAGG-3^/, as shown in SEQ ID NO3；

BnGLY R：5^/-TCAAGCTGCGTTTCCGGCTG-3^/, as shown in SEQ ID NO4.

3. KOD-PLUS archaeal dna polymerases are used, enter performing PCR amplification, display amplifies about 558bp band, amplified fragments end End adds after A, is connected to pEASY-T carriers, converts bacillus coli DH 5 alpha, obtains recombinant plasmid GLY-T；Insertion is surveyed Sequence is analyzed, and it contains ORFs to analysis shows, and ORF1 position is 1-558, the albumen of 185 amino acid compositions of coding, Homogeneous assays show that the albumen has compared with high homology with others GLYI albumen；Due to known mustard type rape GLYI Albuminoid amino acid identity is up to 99%, with it has been reported that Tengchong bite the gene of hot anaerobic bacteria and have very big difference, The homology of both amino acid is only 26%, is shown by Blast results, the conservative function knot with GLYI albumen of the gene Structure domain.

4. BnGLYI turns the structure of yeast expression vector, utilizes the primer designed by expression vector establishment：

BnGLYI JF：5^/-GGAATTCCACATAATGGCGTCGGAAGCGAAGG-3^/, as shown in SEQ ID NO5；

BnGLYI JR：5^/-TTGCGGCCGCTCAAGCTGCGTTTCCGGCTG-3^/, as shown in SEQ ID NO6.

5. primer BnGLYI JF introduce EcoRI sites, and BnGLYI JF introduce NotI sites and expand flat end by KOD-PLUS Hold (underscore show restriction enzyme site), using bacterial strain GLYI-T DNAs template, amplification obtains full-length gene and carried out In EcoRI/NotI double digestions, the yeast expression vector Ppic3.5K for inserting EcoRI/NotI double digestions, obtain recombinating matter Grain, converts bacillus coli DH 5 alpha, extracts in plasmid, electroporated Pichia pastoris recipient bacterium GS115, obtains engineering bacteria GSGLYI.

6. respectively using engineered strain GSGLYI and the GS115 of conversion parent vector as control, it is seeded to BMGY shaking flask Middle culture collects cell progress SDS-PAGE electrophoretic analysis after methanol induction, as a result shows engineering bacteria to OD600=2-6 BnGLY genes in GSGLYI are expressed, and the molecular weight of representation is 21kDa or so, and the expression quantity when inducing 2-3 days It is maximum.

7. GSGLYI bacterial strains are with YPD culture mediums (1% yeast extract, 2% peptone, 2% glucose), if solid processed is trained Base is supported, 2% agar powder is added) cultivate and arrive 0D₆₀₀Bacterium solution is diluted 10 by=1.5-2, then dilution with YPD culture mediums^-4, high temperature 42 DEG C and the processing of -20 DEG C of low temperature, it is found that the Pichia yeast engineering GSGLYI of conversion BnGLYI genes is more unloaded than conversion pPIC3.5K The yeast cells of body and the bacterial plaque survival rate of yeast cells are significantly improved, that is, are converted BnGLYI and significantly improved yeast cells Heat resistance and cold resistance.

The present invention has following advantages and good effect：

1. succeeded from heat-resisting cabbage type rape seed, separation obtains heat-resisting, cold-resistant aldoketonutase gene BnGLYI, and the sequence of the gene and albumen is obtained, to promote the exploitation of cabbage type rape fine genes, improve Wild cabbage type oil Dish is heat-resisting, cold resistance has established experiment basis；

2. the homology of heat-resisting aldoketonutase gene of the aldoketonutase gene in the present invention with having reported is very low, is only 26%, the present invention has further expanded heat-resisting, cold tolerance gene resource；

3. by BnGLYI genetic transformation to yeast cells, heat-resisting, the cold resistance of yeast cells is significantly improved, is expected into one Step improves heat-resisting, the cold resistance of other microorganisms and plant.

Brief description of the drawings

Fig. 1 is germination percentage coordinate diagram of the heat resistance cabbage type rape 3382 after high-temperature process,

CK：37 DEG C of processing 2h；

TR-1：37 DEG C, after 2h, 65 DEG C, 4h；

TR-2：37 DEG C, after 2h, 65 DEG C, 6h；

TR-3：37 DEG C, after 2h, 65 DEG C, 8h.

Fig. 2 is that the protein site of the thermally treated front and rear differential expression of Two-dimensional Electrophoresis Analysis (utilizes ImageMaster softwares point Analysis) picture.

Fig. 3 is the conserved domain picture of BnGLYI albumen.

Fig. 4 is heterogenous expression (the methanol induction 2 days) pictures of BnGLYI in Pichia pastoris.

Fig. 5 is positive yeast transformant GSGLYI heat resistance coordinate diagram,

GS115：Recipient cell；

GS3.5K：Empty carrier pPIC3.5K cell is converted；

GSGLYI：The cell for carrying p3.5K-GLY expression vectors is converted；

* represent GSGLYI and 0.05 level of signifiance is reached compared with GS115 and GS3.5K；* represents 0.01 level of signifiance.

Embodiment

Describe in detail with reference to the accompanying drawings and examples：

1st, embodiment 1：The screening of heat resistance cabbage type rape

We obtain different sweet of heat resistance by cabbage type rape mature seed after the screening of high temperature different disposal first Blue type Rape Germplasm Resources, as shown in figure 1, heat resistance cabbage type rape 3382 and thermo-responsive cabbage type rape 2682, at 65 DEG C After high-temperature process there is significant difference in the germination percentage of its seed.

To ensure that seed has the germination percentage of more than half, germ plasm resource 3382 heat-resisting to cabbage type rape after treatment Mature seed, by following processing：

Control：37 DEG C (2h) → stand 2h (3382CK), germination percentage 100%；

Processing：37 DEG C (2h) → 2h → 65 DEG C (2h) (3382TR) is stood, germination percentage is up to more than 80%；

The analysis of follow-up protein extraction and dielectrophoresis is carried out, expects to find the GAP-associated protein GAP and base of response heat treatment Cause.

2nd, embodiment 2：Extraction, dielectrophoresis and the mass spectral analysis of Seed Storage Protein

1) extraction of Seed Storage Protein

Seed before and after 3382 seed high-temperature process, extracting albumen using TCA- acetone methods is used for the analysis of dielectrophoresis (sweet dew, Li Dianrong, Zang Xin etc., 2010, the foundation of cabbage type rape Two-Dimensional Gel Electrophoresis system, Acta Agronomica Sinica, 36 (4)： 612-619), comprise the following steps that：

1. liquid nitrogen grinds sample 1g；

2. in 10ml 10%TCA-0.07%DTT (g/v)-acetone solvent, it is vortexed, high vibration 15s, -20 DEG C of placements 1h, during which interval 15min vibrations 1 time；

3. 15000 × g (revolution), 30min, 4 DEG C of centrifugations, remove supernatant；

4. 15mL 0.07%DTT (g/v)-acetone is added, -20 DEG C of precipitation 1h vibrate 2 times every 1h；

5. 15000 × g, 30min, 4 DEG C centrifugation, remove supernatant, add 80% cold acetone (or add 15mL 0.07% DTT- acetone), -20 DEG C of cleaning 1h；

6. 15000 × g, 30min are centrifuged again, supernatant is removed, and add 15mL 0.07%DTT- acetone；

7. repeat step 3. -4., 2-3 times；

8. dry powder protein frozen is made and dries -70 DEG C of preservations.

2) analysis of dielectrophoresis

500 μ L lysates (7mol/L urea, 2mol/L thiocarbamides, 4%CHAPS, 1mmol/L are each added in albumen precipitation PMSF, 50mmol/LDTT, 0.5%Triton X-100,0.5% ampholytes), vibration is mixed, and room temperature is placed after 1h 4 Under the conditions of DEG C 20000 × g, 20min is centrifuged, supernatant is taken, repeated centrifugation twice, takes supernatant, referring next to 2-D quant kit eggs White quantification kit, is carried out by operational manual, surveys protein concentration；With 1mg albumen through Two-dimensional Electrophoresis Analysis (sweet dew, Li Dianrong, Zang Xin etc., 2010, the foundation of cabbage type rape Two-Dimensional Gel Electrophoresis system, Acta Agronomica Sinica, 36 (4)：612-619), using point (Bio-Rad Laboratories, Hercules, the CA) softwares of analysis software PDQuest 8.01 are analyzed scan image； Main glue is chosen, maximum point and the weakest point on original image is manually selected, Gauss model is selected, background is removed, automatic spot is run Point detection, then matches the spot differential expression between spot, standardization institute spottiness, analysis gel, draws final report manually Accuse result.

3) mass spectral analysis of protein site

Differential expression is reached twice and the protein site of the above digs glue progress mass spectral analysis, analysis result is as follows：Obtain 3 Agnoprotein, 2 known albumen, wherein the 3rd protein site is Glyoxalase I, confidence level highest, Score value highests are high Up to 117, there are 9 polypeptides to match with the Glyoxalase I proteins of prediction, and the albumen is by various abiotic stress Induction, therefore we select the protein site to carry out follow-up research work.

3rd, embodiment 3：The clone of BnGLYI coding regions

The RNA of seed before and after 3382 heat treatments, the reverse transcription reagent box spun using Japan are extracted using LiCl method FSK100 reverse transcription cDNA, the primer for expanding the full length gene is designed according to the sequence (Y13239) of mustard type rape GLYI genes (table 1).

Table 1 is used for expanding BnGLYI primer

Note：Veena, Reddy VS, Sopory SK, 1999, Glyoxalase I from Brassica juncea： molecular cloning,regulation and its over-expression confer tolerance in transgenic tobacco under stress.Plant J 17：385–395.

Using the cDNA of reversion as template, with KOD-PLUS archaeal dna polymerases, (Japanese TOYOBO products are purchased from the life of Beijing ancient cooking vessel state Thing technology Co., Ltd), enter performing PCR with following system and expand：

Ultra-pure water is mended to 50 μ L, mixes centrifugation, plus the μ L of paraffin oil 30；Amplification cycles：95 DEG C of denaturation 5min, 56 DEG C of annealing 1min, 68 DEG C of extension 1min, 35 circulations, last 68 DEG C of extensions 10min, as a result (see accompanying drawing 2), display amplifies an about 0.5kb left sides Right band, flush end (the golden biotechnology of the full formula in Beijing is limited) connection pEASY-T conversion bacillus coli DH 5 alphas after adding A, is obtained To positive transformant GLY-T；Transformant GLY-T is expanded by PCR and sequence verification, sequencing analysis is carried out to insertion, Obtain BnGLYI sequence, SEQ ID NO 1, sequence 558bps, analysis shows its contain ORFs, ORF1 position It is 1-558 to put, and G/C content is 47%, the albumen of 185 amino acid compositions of coding；After measured, its amino acid sequence is SEQ ID Shown in NO 2.

Homogeneous assays show that the albumen and others GLYI albumen have compared with high homology, and table 2 is its Homology data；By In being up to 99%, the difference of only 2 amino acid sites with known mustard type rape GLYI albuminoid amino acid identities It is different.With it has been reported that Tengchong bite the gene of hot anaerobic bacteria and have very big difference, the homology of both amino acid is only 26% (table 2), we name the gene to be BnGLYI.

Table 2BnGLYI is compared with other GLYI protein similarities

Note：

AtGLYI：Arabidopsis thal iana(Accession no.AT1G08110)；

BjGLYI：B.juncea(Accession no.Y13239)；

AhGLYI：Arachis hypogaea(Accession no.DQ989209.2)；

BnGLYI：B.napus 3382；

CsGLYI：Caldanaerobacter subterraneus(Accession no.NP_622558).

The present invention further analyze BnGLYI albumen learn its molecular weight be 20.8kDa, isoelectric point be pH 5.42 (see Table 3), analyze the biochemical indicator (being shown in Table 3) of albumen, its amino acid composition (being shown in Table 4).

The biochemical characteristic of table 3BnGLYI albumen

The amino acid composition of table 4BnGLYI albumen

4th, embodiment 4, BnGLYI turn the construction and expression analysis of yeast expression vector

1) structure of expression vector

By sequence analysis, EcoRI, 3- end plus NotI restriction enzyme sites is added to be used for the expression of gene at 5- ends, expression is with drawing As shown in table 1, underscore show restriction enzyme site to thing.By the flat end of KOD-PLUS polymeric enzymatic amplifications of hi-fi energy, PCR expands Increasing condition is 94 DEG C of 1min, 56 DEG C of 1min, 68 DEG C of 1min, and 30 circulate, and 68 DEG C extend 10 minutes, using hi-fi energy KOD-PLUS polymerases are expanded；Obtain 558bp BnGLYI full length coding regions fragment；Through EcoR I and NotI double digestions Completely after reaction, electrophoresis is carried out, the fragment is reclaimed from gel, carried with EcoR I and the Pichia pastoris intracellular expression of NotI digestions Body Ppic3.5k carriers (plasmid comes from Plant Protection institute, Chinese Academy of Agricultral Sciences's Biotechnology Experiment room) are connected Reaction, 4 DEG C, 12 hours；The μ L Transformed E .coli recipient bacterium DH5 α of connection product 5 are taken, it is positive with ammonia benzyl chloramphenicol resistance plate screening Recombinant plasmid；The positive recombinant plasmid obtained is inoculated in liquid LB test tubes respectively, 37 DEG C, 230rpm is cultivated 12 hours, By PCR amplifications and sequence verification, illustrate for positive transformant, as a result show that constructed expression vector is correct, be named as p3.5K-GLY。

2) Pichia yeast engineering GSGLYI construction and expression Protein Detection

Recombination expression clone p3.5K-GLY from DH5 α is converted into Pichia pastoris GS115, electric shock condition with electric shock method For：The μ of 1500V, 200 Ω, 25 F, 80 μ L GS115 competent cells, 1 μ g DNAs；Converted product is coated with to MD flat boards immediately, 30 DEG C are cultivated 3 days, are identified positive transformant using PCR method, are obtained the yeast-positive containing p3.5K-GLY expression plasmids Transformant, engineering bacteria GSGLYI is named as by the transformant.

Engineered strain GSGLYI and the GS115 of conversion parent vector are seeded in BMGY shaking flask as control respectively Culture collects cell afterwards to OD600=2-6, removes supernatant, and cell is resuspended every 24 hours with BMMY culture mediums, methanol is added To final concentration of 0.5% with continue induction；Respectively induction 1 day, 2 days, 3 days, 4 days, take within 5 days 1ml culture mediums to 1-5ml from Heart pipe；These samples are used to analyze expression and determine to collect the Best Times of cell after induction；Room temperature is maximum with centrifuge Turn the sample taken hanging centrifugation, add the 50 ultrapure aqueous suspensions of μ L, adding 25 3 × sample buffers of μ L, (925 μ L loadings are delayed The μ of fliud flushing+75 L β-mercaptoethanol), 100 DEG C are boiled 10min, are centrifuged off precipitation.Loading 10uL carries out SDS-PAGE electrophoresis point Analyse result.As a result show, the BnGLY genes in engineering bacteria GSGLYI are expressed, the molecular weight of representation is left for 21kDa The right side, and when inducing 2-3 days, expression quantity is maximum (Fig. 4).

5th, embodiment 5：Identification containing the BnGLYI Pichi strain GSGLYI heat resistances converted and resistance

GSGLYI and control strain GS-p3.5k, GS115 bacterial strain YPD culture mediums (1% yeast extract, 2% albumen Peptone, 2% glucose, if solid medium processed, adds 2% agar powder) cultivate and arrive 0D₆₀₀=1.5-2, then dilutes and is cultivated with YPD Bacterium solution is diluted 10 by base^-4, bacterium solution is distributed into 100 every part of μ L, totally 6 processing, often handled at 3 repetitions, 42 DEG C of water-baths of high temperature Manage after 2.5h, 3.5h, 5h, low temperature -20 DEG C of processing 48h, 72h, 10 days (D), YPD plate counts are coated with, while will be untreated The direct spread plate of bacterium solution is counted, and is used as control.3 repetitions are done in each processing, and each experiment is repeated 3 times.Made of SPSS softwares Statistical analysis.Analysis result shows that 42 DEG C of water bath processings 2.5h, 3.5h, -20 DEG C of 5h and low temperature are handled after 10D, 13D, conversion The yeast cells GS3.5K and yeast recipient bacterium cell GS115 bacterial plaque survival rates of pPIC3.5K empty carriers are without significant difference；And turn Change the Pichia yeast engineering GSGLYI of heat-resisting BnGLYI genes, compared with GS3.5K and GS115 cells, engineering bacteria bacterial plaque Survival rate is improved, and reaches the notable or pole level of signifiance (see Fig. 5), that is, is converted BnGLYI and significantly improved the resistance to of yeast cells Hot and cold resistance.

Sequence table

<110>Inst. of Oil Crops, Chinese Academy of Agriculture

<120>High temperature resistant cabbage type rape aldoketonutase gene and albumen and its application

<160>6

<210>1

<211>558

<212>DNA

<400>

ATGGCGTCGGAAGCGAAGGAATCAGCAGCAAACAATCCTGGCTTGTCAACGGTTCGGGATGAGGCTACC

AAAGGGTATATCATGCAGCAGACTATGTTTCGGGTGAAGGACCCTAAGGCTAGTCTTGACTTTTACTCA

CGTGTCCTGGGAATGTCATTGCTGAAGAGATTAGATTTCTCTGAGATGAAGTTCAGCTTGTACTTCCTG

GGCTACGAGGATACTTCGACAGCTCCAACAGATCCTACTGAGAGAACTGTTTGGACCTTTGGTCGACCT

GTAACAATTGAGCTGACTCACAACTGGGGCACAGAGAGTGATCCTGAGTTTAAAGGCTATCATAATGGG

AACTCCGAGCCTCGTGGATTTGGGCATATTGGGGTTACAGTTGATGATGTGCACAAGGCATGCGAGAGA

TTTGAACAACTGGGAGTAGAGTTCGTCAAGAAACCGAATGATGGAAAGATGAAGAATATAGCCTTCATC

AAGGATCCTGATGGCTACTGGATCGAGATCTTTGATCTCAAGACTATCGGGACAACAGCCGGAAACGCA

GCTTGA；

<210>2

<211>185

<212>DNA

<400>

MASEAKESAANNPGLSTVRDEATKGYIMQQTMFRVKDPKASLDFYSRVLGMSLLKRLDFSEMKFSLYFL

GYEDTSTAPTDPTERTVWTFGRPVTIELTHNWGTESDPEFKGYHNGNSEPRGFGHIGVTVDDVHKACER

FEQLGVEFVKKPNDGKMKNIAFIKDPDGYWIEIFDLKTIGTTAGNAA；

<210>3

<211>19

<212>DNA

<400>

ATGGCGTCGGAAGCGAAGG；

<210>4

<211>20

<212>DNA

<400>

TCAAGCTGCGTTTCCGGCTG；

<210>5

<211>32

<212>DNA

<400>

GGAATTCCACATAATGGCGTCGGAAGCGAAGG；

<210>6

<211>30

<212>DNA

<400>

TTGCGGCCGCTCAAGCTGCGTTTCCGGCTG。

Claims (4)

1. a kind of high temperature resistant cabbage type rape aldoketonutase gene, it is characterised in that：

The nucleotide sequence of the high temperature resistant cabbage type rape aldoketonutase gene is as shown in SEQ ID NO1.

2. a kind of high temperature resistant cabbage type rape aldoketonutase albumen, it is characterised in that：

The high temperature resistant cabbage type rape aldoketonutase albumen is as the high temperature resistant cabbage type rape aldoketonutase described in claim 1 Coded by said gene, the amino acid sequence of the high temperature resistant cabbage type rape aldoketonutase albumen is as shown in SEQ ID NO2.

3. a kind of application of high temperature resistant cabbage type rape aldoketonutase gene in transgenic microorganism, it is characterised in that：

High temperature resistant cabbage type rape aldoketonutase gene as claimed in claim 1 is converted into Pichi strain GS115, from And Pichi strain GS115 is obtained heat resistance and cold resistance.

4. a kind of application of high temperature resistant cabbage type rape aldoketonutase gene in stress resistance of plant, it is characterised in that：

Expression vector containing the high temperature resistant cabbage type rape aldoketonutase gene is converted into plant, produces the plant resistance to Heat and cold resistance；

The nucleotide sequence of the high temperature resistant cabbage type rape aldoketonutase gene is as shown in SEQ ID NO1.