CN102634497B - Protein and gene for adjusting and controlling low temperature saccharification of potato as well as application thereof - Google Patents
Protein and gene for adjusting and controlling low temperature saccharification of potato as well as application thereof Download PDFInfo
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- CN102634497B CN102634497B CN 201210077910 CN201210077910A CN102634497B CN 102634497 B CN102634497 B CN 102634497B CN 201210077910 CN201210077910 CN 201210077910 CN 201210077910 A CN201210077910 A CN 201210077910A CN 102634497 B CN102634497 B CN 102634497B
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Abstract
The invention belongs to the technical field of the plant gene engineering, and relates to a key gene StSnSnRK1a for adjusting and controlling low temperature saccharification of potatoes and application thereof. A gene StSnSnRK1, which is obviously relative to the low temperature saccharification of potatoes, is cloned, and the nucleotide sequence of the gene is shown as the sequence table SEQIDNO:1, and an amino acid sequence is shown as the sequence table SEQIDNO:2. The biological function tests and transgenic strain phenotype approve that the gene StSnSnRK1a in the invention has obvious effects on preventing the low temperature saccharification of the potatoes, and provides a novel efficient path for the mechanism research in preventing the low temperature saccharification of the potatoes and the improvement of the existing varieties.
Description
Technical field
The invention belongs to the plant gene engineering technology field, be specifically related to the key gene of a regulation and control potato low temperature saccharification
SbSnRK1 α 1And use.This gene has significant function aspect the potato tuber low temperature saccharification degree of low temperature storage regulating, and can explode to use aspect the sheet processing behind storage of potato.
Technical background
The fried processing of potato is very strict to the requirement of stem tuber quality, and except the outward appearance quality, main is that requirement stem tuber high-content of starch and low reducing sugar content are lower than the 0.25%(fresh weight) and low temperature resistant storage.Reason is that in frying course, the reducing sugar in the stem tuber and the a-amino acid of nitrogen compound carry out " Maillard Reaction " causes potato chips or French fries surface color to be deepened, thereby has a strong impact on processing quality (Qu Dongyu etc., 2001).For extension of the work cycle and suppress degradation phenomenon under the stem tuber quality that room temperature storage causes, in the potato processing industry often with the stem tuber Long-term Storage at low temperatures.Yet low temperature accelerates starch cracking conversion reducing sugar (glucose and fructose) in the stem tuber, and this " the stem tuber saccharification of low temperature induction " phenomenon is the major cause that most of kinds are not suitable for processing.Therefore, the molecule mechanism effect in the existing explanation low temperature saccharification of the research of the potato tuber low temperature saccharification metabolic process has the Economic Application of pair potato low temperature storage post-treatment to be worth again.
The potato tuber low temperature saccharification mainly is because low temperature has changed starch in the stem tuber-glycometabolic balance, causes sucrose to be accumulated in stem tuber, and sucrose further is converted into (Isherwood, 1973) due to glucose and the fructose again.Studies show that saccharase in this pathways metabolism (Invertase) is the key enzyme that directly causes the reducing sugar accumulation.As far back as Uppal and Verma(1990) research in find that invertase activity and reducing sugar content are utmost point significant correlation (r=0.821) during the low temperature storage.Follow-up a large amount of research shows equally, invertase activity and reducing sugar content significant correlation (Richardson et al. 1990 in the low temperature storage stem tuber; Cheng et al. 2004; McKenzie et al. 2005).
SnRK1(sucrose non-fermenting-1-related protein kinase plant sucrose non-fermentation-1 related protein kinase) as important family in the vegetable-protein kinases family, SNF1(sucrose non-fermenting-1 in itself and the yeast, SNF1) and the AMPK(AMP-activated protein kinase in the Mammals, AMPK) homology and have function with they phase Sihes self uniqueness, three subfamilies of ubiquity in plant materials.
The research of SnRK1 in plant materials develops very fast this year, and SnRK is at carbohydrate metabolism and the multiple environment-stress of plant, and the ABA approach is disease-resistant degeneration-resistant, even yield and quality all is critical regulatory enzyme (Halford and Hey 2009; Hey et al. 2010; Coello et al. 2011; Halford et al. 2011).In potato, be cloned into α, the β subgroup is respectively StubSNF1 and Gal83, the sprouting of potato SnRK1 and stem tuber (Halford et al. 2003), starch accumulation, (McKibbin et al. 2006) is closely related and affect for example expression of ADP-glucose pyrophosphorylase of plurality of enzymes (Tiessen et al. 2003) in the carbohydrate metabolism approach under the content of glucose.And about the interact protein of SnRK1 also (Beczner et al. 2010 in further investigation gradually; Shen et al. 2011).But the report of pertinent literature is not but seen in the influence in the low temperature saccharification metabolic process, so saccharase, and the regulation relationship of doing mutually that saccharase suppresses between son and each protein subunit of SnRK1 has good biology and economic implications to the influence of low temperature saccharification.
Summary of the invention
Of the present invention
SbSnRK1 α 1Gene is the wild species strain with anti-low temperature saccharification
S.berthaultiiHigh-quality full-length cDNA yeast two-hybrid library be the basis, be one of potential interact protein candidate gene of obtaining of this library screening of erbium protein hybridization with the saccharase.Subsequent experimental proof SnRK1 protein kinase α and β subgroup (SbSnRK1 α 1, StSnRK1 β 1) and saccharase, saccharase suppresses to have man-to-man specific selectivity interaction between the son, and 1 pair of low temperature saccharification of SbSnRK1 α has played extremely significant regulating effect simultaneously.The influence of this interaction and 1 pair of low temperature saccharification of SbSnRK1 α does not still all have the report of pertinent literature at home and abroad.
Given this, the object of the present invention is to provide a kind of potato low temperature saccharification of regulating and control
SbSnRK1α
1Gene and albumen and application thereof.
To achieve these goals, first aspect of the present invention provides a kind of SbSnRK1 α 1 albumen, and its aminoacid sequence is shown in the sequence table SEQ ID NO:2.
Second aspect of the present invention provides the polynucleotide sequence of this SbSnRK1 α 1 albumen of encoding; Wherein preferred polynucleotide are SEQ ID NO:1.
The 3rd aspect of the present invention provides the expression vector that comprises above-mentioned polynucleotide sequence; Wherein preferred expression vector is pMD18-T carrier or pBIC carrier.
The 4th aspect of the present invention provides the cell that comprises above-mentioned expression vector; Wherein preferred cell is bacillus coli DH 5 alpha or Agrobacterium LBA4404.
The present invention finds by experimental study, overexpression in the potato strain of fried sheet processing after being not suitable for low temperature storage
SbSnRK1 α 1Gene can significantly improve the ability of the anti-low temperature saccharification of this transfer-gen plant, reduces the fried sheet color and luster degree after the low temperature storage, makes its 4 ℃ storages still can reach the standard of commercial processing after one month substantially.Potato strain behind improvement of genes, more low temperature resistant storage, but prolonged from adopting the back to the low temperature storage time of fried sheet processing.Be specially adapted to the fried processing of potato, thereby be conducive to the improvement of processing variety, the reduction of cost and the raising of utilization rate of raw materials are commercially produced in the establishment of new processing variety.
Therefore, the 5th aspect of the present invention provides above-mentioned aminoacid sequence SEQ ID NO:2 or the application of polynucleotide sequence SEQ ID NO:1 in the anti-low temperature saccharification of potato.
Compared with prior art, the present invention utilizes the clone's
SbSnRK1 α 1Gene is overexpression in potato, overexpression
SbSnRK1 α 1But the utmost point significantly improves the ability of the anti-low temperature saccharification in the potato low temperature storage process behind the gene.The present invention can improve the anti-low temperature saccharification ability of plant by this gene of overexpression in the potato body, can make plant more responsive to low temperature saccharification in this expression of gene of potato body internal interference.
Description of drawings
Fig. 1:
SbSnRK1 α 1Gene overexpression vector construction schema.
Fig. 2: pBIC carrier structure figure.
Fig. 3:
SbSnRK1 α 130 days fried sheet color and luster indexes of 4 ℃ of storages of transgenic line potato piece low temperature.
Fig. 4:
SbSnRK1 α 130 days reducing sugar content of 4 ℃ of storages of transgenic line potato piece low temperature detect.
Fig. 5:
SbSnRK1 α 130 days invertase activities of 4 ℃ of storages of transgenic line potato piece low temperature detect.
Embodiment
Below in conjunction with specific embodiment the present invention is made more detailed description.According to following description and these embodiment, those skilled in the art can determine essential characteristic of the present invention, and under the situation that does not depart from spirit and scope of the invention, can make various changes and modification to the present invention, so that its suitable various uses and condition.
Cloned genes of the present invention is named as
SbSnRK1 α 1, can pass through agriculture bacillus mediated genetic transforming method or other transgenic methods in the gene transferred plant, the transfer-gen plant or the strain system that obtain by Screening and Identification all belong to protection scope of the present invention.The present invention can improve the anti-low temperature saccharification ability of plant by this gene of overexpression in the potato body; can make plant more responsive to low temperature saccharification in this expression of gene of potato body internal interference, it all belongs to the scope of protection of the invention in the application aspect degeneration-resistant.
1 the present invention
SbSnRK1 α 1The clone of gene and sequential analysis
Among the present invention
SbSnRK1 α 1Gene is by the anti-low temperature saccharification wild species of the potato strain to making up
S.berthaultiiFull-length cDNA yeast two-hybrid library is that erbium albumen carries out hybrid experiment respectively and obtains with the saccharase, finds that there are protein-protein interaction in this gene and saccharase.Yeast two assorted and evaluation and screening result in library proves,
SbSnRK1 α 1The RD district of gene and saccharase can realize that in the yeast body protein-protein does mutually and present the very high positive.
The design amplification
SbSnRK1 α 1Full length gene cDNA aligning primer:
Forward primer is: 5 '-ATGGACGGAACAGCAGTGCAAGGCA-3 '
Reverse primer is: 5 '-AAGTACTCGAAGCTGAGCAAGAAAA-3 '
By PCR method, from the potato wild species strain of anti-low temperature saccharification
S.berthaultiiStem tuber cDNA in amplify
SbSnRK1 α 1Full length cDNA sequence.
Amplification method is: (give birth to worker company available from Shanghai with test kit earlier, China) the total RNA of extracting potato tuber, detailed process is referring to specification sheets, use the reverse transcription test kit (available from TOYOBO company then, Japan) be total cDNA with the mRNA reverse transcription, detailed process is referring to specification sheets, use the LA enzyme (available from Takara company as template with above forward and reverse primer again, Japan) total length of amplification SbSnRK1 α 1 gene, product is after 1% agarose gel electrophoresis detects to single band, reclaim test kit with glue and (give birth to worker company available from Shanghai, China, detailed process is referring to specification sheets) the recovery target stripe, be cloned into the pMD18-T carrier (available from Takara company, Japan), get 5 μ L connection product and carry out thermal shock transformed into escherichia coli DH5 α competence (with reference to works such as J. Sa nurse Brookers, Huang Peitang etc. translate, molecular cloning experiment guide (third edition), Science Press, 2002 editions), coat on the LB solid plate that contains penbritin/isopropyl-/5-bromo-4-chloro-3-indyl-B-D-semi-lactosi sweet (Amp/IPTG/X-gal) of new configuration, 37 ℃ of overnight incubation, it is some to select hickie, in the liquid LB substratum that contains Amp 50 mg/L, spend the night to muddiness in 37 ℃ of 200 r/min shaking culture, whether positive, send three positive colonies to the big gene biological technology company of China to finish examining order if detecting bacterium liquid with primer PCR.
Sequencing result shows, clones
SbSnRK1 α 1The cDNA total length be that 1545bp(sees in the sequence table shown in the SEQ ID NO:1), 514 amino acid (shown in the SEQ ID NO:2) of encoding.Analyze with the Blastn of GenBank and to show, with known potato StubSNF1(U83797.4) cds regional sequence similarity 99 ﹪ are arranged, the difference of 17 bases is arranged.The amino acid similarity of this protein and potato StubSNF1 albumen (NP_001233965.1) is having 4 amino acid whose differences at the 286th, 334,438 and 462 respectively equally up to 99 ﹪.Predict that this protein molecular weight is 58.881KD, iso-electric point is 8.61, does not find sequence leader sequence or special compartment target protein signal with the SignalP prediction, and Expasy protein structure prediction result shows not have obviously strides the film district.
2
SbSnRK1 α 1The vector construction of gene
What increase
SbSnRK1 α 1The primer both sides of gene add respectively
BamHI and
SacThe I restriction enzyme site is that template is carried out pcr amplification with the pMD18-T carrier that has made up subsequently, and with extension amplification outcome again to the pMD18-T carrier, concrete steps are seen of the present invention
SbSnRK1 α 1The clone of gene and sequential analysis part.Subsequently with
BamHI and
SacThe I double digestion has the pMD18-T carrier of goal gene total length, simultaneously with
BamHI and
SacThe I double digestion has been replaced the PBI121 carrier pBIC of 35S promoter with the promotor CIPP of stem tuber predominant expression, enzyme is separately cut product and reclaim test kit (available from the living worker in Shanghai company with glue behind 1% agarose gel electrophoresis, China) reclaim target stripe, and with target gene fragment and the big fragment of the pBIC carrier mixed with 0.8:1, add T
4Dna ligase (available from Takara company) 1U, 1 * reaction buffer, sterilized water is supplemented to 10 μ L systems, 16 ℃ linked 10 hours, get 5 μ L and connect product and carry out thermal shock transformed into escherichia coli DH5 α competence, coat on that (Km) of card 50 mg/L resistance plates of new configuration screening positive clone and enzyme and cut detection.The carrier figure of pBIC and overexpression vector construction flow process are seen Fig. 1 and Fig. 2.
Utilize electric conversion instrument under the voltage of 1800V, to transform Agrobacterium LBA4404 to the recombinant plasmid that obtains, with containing Rifampin (Rif) 100mg/L, the YEB solid resistance plate screening that blocks those (Km) 50 mg/L transforms positive spot, several positive spots of picking are in containing Rifampin (Rif) 100mg/L, block in the YEB liquid nutrient medium of that (Km) 50 mg/L 28 ℃, 150r/min cultivated 4 hours, get 1 μ L and do the PCR positive detection that template is carried out recombinant plasmid, confirm as positive bacterial strain and preserve for follow-up genetic transformation.
3 genetic transformations
Test tube potato about growth 7-9 week, diameter 0.5 cm is cut into the thick thin slice of 1-2 mm, in containing the Agrobacterium bacterium liquid of corresponding plasmid, soaks 10 min.Contaminate and finish back taking-up potato chips, blot surperficial bacterium liquid with aseptic filter paper.Change over to irritate common culture medium A1(MS minimum medium+3% sucrose+1 mg/L IAA+0.2 mg/L GA3+0.5 mg/L BA+2 mg/L ZT arranged) culture dish in, in 24 ℃ of dark 2 d that cultivate.After dark the cultivation, potato chips forward to irritate again regeneration culture medium A2(A1+ 75 mg/L Kam+400 mg/L Cef) culture dish in, place intensity of illumination 2000 lux, photoperiods 16 h/d cultivates under the condition that temperature is 24 ℃ and regenerates up to resistant buds.Treat that resistant buds grows when reaching 0.5-1 cm, change its cutting-out over to root media A3(MS minimum medium+3% sucrose+50 mg/L Kam+200 mg/L Cef, pH 5.8), transform successful budlet because the Kam resistance is arranged, can take root at A3.
4
SbSnRK1 α 1The Function Identification of gene
In order to verify
SbSnRK1 α 1The function of gene aspect the anti-low temperature saccharification of potato, the applicant is that (PS strain system) and adjoining tree cultivar Hubei Province potato No. 3 (Ep3) are planted in the same booth in base, vegetables branch center, Central China 2011 second half year simultaneously with 13 excess gene transgenic strains, each strain system plantation 24 basin, plant grows fine, potato piece ripening degree height, transgenic line there is no significant difference with contrast in shape in the field agricultural.It is heavy to get each transgenic line list potato〉50g do not have stem tuber that scab has no mechanical damage respectively in the storage 30 days down of 4 ℃ of 20 ℃ of normal temperature and low temperature, carries out potato chips and reducing sugar, saccharase enzyme mensuration such as live, each time point is got 3 biology and is repeated.
To between each overexpression strain system under the differing temps different storage time and the contrast fried sheet color and luster index compare, visualized result finds,, preserve 30 days fried sheet color and lusters and all do not have significant difference for 20 ℃ between contrast and the transgenic line.But under 4 ℃ of storages, it is Ep3 that the fried sheet color and luster of each transgenic line all is starkly lower than the contrast strain, PS3 wherein, and PS8, PS15 shows particularly evident (Fig. 3).This result shows overexpression
SbSnRK1 α 1Low temperature saccharification has all been played very obvious suppression effect, made that not being suitable for after the low temperature storage cultivar Ep3 of fried sheet processing has originally obtained to preserve and still can reach the fried lower ability of sheet color and luster index after 1 month.
The assay result of reducing sugar shows that the difference of transgenic line and contrast is more remarkable, compared with the control, each strain system of excess strain system compares mostly to throw light on and shows low (Fig. 4) under the low temperature storage, wherein the PS3 performance is the most obvious, reducing sugar content decline degree surpasses 90% during 4 ℃ of storages in 30 days, be under the low temperature in the transgenic line accumulation rate of reducing sugar very slow, this also is the fried very low main immediate cause of sheet color and luster.Same saccharase enzyme running water is flat also to show same trend, in 4 ℃ of storages in excess strain system the enzyme of saccharase live and extremely significantly be lower than contrast (Fig. 5).
Laboratory test results proves, changes
SbSnRK1 α 1The potato of gene effect aspect anti-low temperature saccharification is extremely remarkable, is a very potential functional gene in the anti-low temperature saccharification application of potato.
Claims (8)
1.StSnRK1 α albumen, its aminoacid sequence are shown in the sequence table SEQ ID NO:2.
2. the polynucleotide sequence of coding StSnRK1 α albumen.
3. polynucleotide sequence according to claim 2, it is characterized in that: this sequence is SEQ ID NO:1.
4. the expression vector that comprises claim 2 or 3 described polynucleotide sequences.
5. the cell that comprises the described expression vector of claim 4.
6. cell according to claim 5, it is characterized in that: described cell is bacillus coli DH 5 alpha or Agrobacterium LBA4404.
7. the application of the described aminoacid sequence SEQ of claim 1 ID NO:2 in the anti-low temperature saccharification of potato.
8. the described polynucleotide sequence of claim 2 or the application of the described polynucleotide sequence SEQ of claim 3 ID NO:1 in the anti-low temperature saccharification of potato.
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| CN103044536B (en) * | 2012-12-28 | 2014-05-28 | 中国农业大学 | Protein related to carbon and nitrogen metabolism, as well as encoding gene and application thereof |
| CN105713991A (en) * | 2016-04-29 | 2016-06-29 | 华中农业大学 | Potato low-temperature sweetening resistant molecular marker combination and application thereof in potato low-temperature sweetening resistant breeding |
| CN107937591B (en) * | 2017-12-12 | 2021-04-13 | 华中农业大学 | SNP marker of QTL site related to low-temperature saccharification resistance at tail end of potato chromosome XI and application of SNP marker |
| CN108707594B (en) * | 2018-05-30 | 2019-04-19 | 山东省农业科学院玉米研究所（山东省农业科学院玉米工程技术研究中心） | 1 gene family of -1 related protein kinase of maize sucrose non-fermented and its application |
| CN109207486B (en) * | 2018-10-31 | 2022-03-22 | 西南大学 | Potato CISR2 gene and application thereof in low-temperature saccharification resistance |
| CN110205326B (en) * | 2019-06-03 | 2020-11-20 | 华中农业大学 | Application of potato StMADS10 gene in low-temperature saccharification resistance |
| CN114716522B (en) * | 2020-12-22 | 2023-07-07 | 中国农业大学 | Application of KIN10 protein and related biological materials thereof in saline-alkali tolerance of plants |
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| WO2010118338A2 (en) * | 2009-04-10 | 2010-10-14 | Dow Agrosciences Llc | Plant snf1-related protein kinase gene |
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| WO2010118338A2 (en) * | 2009-04-10 | 2010-10-14 | Dow Agrosciences Llc | Plant snf1-related protein kinase gene |
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