CN102517262B - Divinyl reductase from corn and coding gene and application thereof - Google Patents
Divinyl reductase from corn and coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a divinyl reductase (DVR) from corn and coding gene and application thereof. The DVR provided by the invention is a protein shown as 1) or 2): 1) a protein comprising an amino acid sequence shown as a sequence 2 in the sequence table; and 2) a protein derived from 1), with DVR activity and obtained by substituting and / or deleting and / or adding one or more amino acid residues on the sequence 2 in the sequence table. Experiments have proved that the DVR provided by the invention can converse DV chlorophyll a into MV chlorophyll a, converse DV chlide a into MV chlide a, converse DV Pchlide a into MV Pchlide a, converse DV MPE into MV MPE and converse DV Mg-proto into MV Mg-proto.
Description
Technical field
The present invention relates to a kind of divinyl reductase enzyme and encoding gene and application that derives from corn.
Background technology
Chlorophyll is that plant carries out photosynthetic important pigment, and its effect is to catch luminous energy and luminous energy is transferred to reactive center, so growth and the output thereof of plant is played a very important role.According to the difference of ethylene lateral chain number, chlorophyll can be divided into 3,8-divinyl chlorophyll (DV Chls) and 3-ethene chlorophyll (single ethene chlorophyll, MV Chls).Under normal circumstances, it all is MV chls that nearly all plant and bacterium are used for photosynthetic chlorophyll, that only find i.e. " the marine prochlorophyte Prochlorococcus marinus " utilization of class marine organisms at present is DVChls (Chisholm SW, Frankel SL, Goericke R, Olson RJ, Palenik B, Waterbury JB, West-Johnsrud L, Zettler ER (1992) Prochlorococcus marinus nov.gen.nov.sp.:A marine prokaryote containing divinylchlorophyll a andb.Arch.Microbiol 157:297-300.).
The biosynthetic diversity of chlorophyll is that mainly it has two parallel synthetic approach of single ethene chlorophyll (MV Chls) and divinyl chlorophyll (DV Chls), DV Chls and intermediate product thereof are by divinyl base reductase enzyme (DVR, be C-8 vinyl reductase enzyme again) be catalytically converted into MV Chls and intermediate product (Rebeiz CA thereof, Kolossov VL, Briskin D, Gawienowski M (2003) Chloroplast biogenesis:chlorophyll biosynthetic heterogeneity, multiple biosynthetic routes, and biological spin-oVs, in:H.S.Nalwa (Ed.), Handbook of Photochemistry and Photobiology, vol.4, American ScientiWc, Los Angeles:183-248.).Up to now, on 5 kinds of substrate levels, detect the divinyl reductase activity, be respectively divinyl Mg-protoporphyrin IX (DV Mg-proto), divinyl Mg-protoporphyrin IX mono-methyl (DV MPE), (DV Pchlide a) for divinyl protochlorophyllide a, (a) (DV Chl is (Kolossov VL a) with the divinyl chlorophyll a for DV Chlide for divinyl chlorophyllide a, Bohnert HJ, Rebeiz CA (2006) Chloroplast biogenesis 92:In situ screening for divinyl chlorophyll (ide) a reductase mutants by spectroXuorometry.Analytical Biochemistry 348:192-197).Now from paddy rice, corresponding divinyl reductase gene DVR, bciA and slr1923 (Wang PR have been cloned among Arabidopis thaliana, green sulphur bacteria Chlorobium tepidum and the cyanobacteria Synechocystis sp.PCC6803 respectively, Gao JX, Wan CM, Zhang FT, Xu ZJ, Huang XQ, Sun XQ, Deng XJ (2010) Divinyl chlorophyll (ide) a can be converted to monovinyl chlorophyll (ide) a by a divinyl reductase in rice.Plant Physiol 153,994-1003; Nagata N, Tanaka R, Satoh S, Tanaka A (2005) Identification of a Vinyl Reductase Gene for Chlorophyll Synthesis in Arabidopsis thaliana and Implications for the Evolution of Prochlorococcus Species.Plant Cell 17,233-240; Chew AGM, Bryant DA (2007) Characterization of a Plant-like Protochlorophyllide a Divinyl Reductase in Green Sulfur Bacteria.J.Biol.Chem 28:2967-2975; Islam MR, Aikawa S, Midorikawa T, Kashino Y, Satoh K, Koike H (2008) slr1923 of Synechocystis sp.PCC6803 Is Essential for Conversion of 3,8-Divinyl (proto) chlorophyll (ide) to 3-Monovinyl (proto) chlorophyll (ide) .Plant Physiology 148:1068-1081).Existing experimental results show that: use NADPH as reductive agent, green sulphur bacteria (Chlorobium tepidum) the BciA protein of reorganization is reduced into MV Pchlide a (Chew AGM, Bryant DA (2007) Characterization of a Plant-like Protochlorophyllide a Divinyl Reductase in Green Sulfur Bacteria.J.Biol.Chem 28:2967-2975) with DV Pchlide a; The Arabidopis thaliana DVR protein of reorganization is reduced into MV Chlide a with DVChlide a, but 4 kinds of DV materials such as DV Pchlide a, DV Chlide b, DV Chl a and DV Chl b can not be converted into corresponding M V material (Nagata N, Tanaka R, Tanaka A (2007) The Major Route for Chlorophyll Synthesis Includes[3,8-divinyl]-chlorophyllide a Reduction in Arabidopsis thaliana.Plant Cell Physiol 48:1803-1808).We can not only be reduced into DV Chlide a MV Chlide a by the paddy rice DVR protein of zymetology experiment confirm reorganization, and to reduce DV Chl a be MV Chl a (Wang PR, Gao JX, Wan CM, Zhang FT, Xu ZJ, Huang XQ, Sun XQ, Deng XJ (2010) Divinyl chlorophyll (ide) a can be converted to monovinyl chlorophyll (ide) a by a divinyl reductase in rice.Plant Physiol 153,994-1003).At present, this 2 kinds of divinyls (DV type) material of divinyl Mg-protoporphyrin IX (DV Mg-proto) and divinyl Mg-protoporphyrin IX mono-methyl (DV MPE) is converted into corresponding single ethene (MV type) material, also do not obtain utilizing the confirmation of the external zymetology experiment that purifying DVR zymoprotein done, simultaneously, not clear is, these divinyl intermediate materials are to have the catalysis of extensive specific divinyl base reductase enzyme by one, still generate corresponding single ethene material by a plurality of divinyl base reductase enzymes (each enzyme all has specific substrate) catalysis.
Summary of the invention
The purpose of this invention is to provide a kind of albumen with divinyl reductase activity, this dietary protein origin is in corn (Zea mays L.).
Albumen with divinyl reductase activity provided by the present invention is following 1) or 2) protein:
1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;
2) with replacement and/or disappearance and/or the interpolation of the amino acid residue sequence of sequence in the sequence table 2 through one or several amino-acid residue, and have the divinyl reductase activity by 1) deutero-protein.
The above-mentioned proteic gene (ZmDVR) of encoding also belongs to protection scope of the present invention.
Described gene is following 1)-3) in any:
1) its encoding sequence is the dna molecular of sequence 1 in the sequence table;
2) under stringent condition with 1) or 2) the dna molecule hybridize and the above-mentioned proteic dna molecular of divinyl reductase activity that has of encoding;
3) with 1) or 2) dna molecular have the homology more than 90% and encode the above-mentioned proteic dna molecular of divinyl reductase activity that has.
The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain said gene also belong to protection scope of the present invention.
Described recombinant expression vector specifically can be and insert the recombinant expression vector that said gene obtains between the multiple clone site of pET-30a (+).In an embodiment of the present invention, described multiple clone site is specially BamH I and EcoR I.
Described expression cassette is served as reasons and can be started the promotor of ZmDVR genetic expression, the ZmDVR gene, and transcription termination sequence is formed.
In one embodiment of the invention, described reorganization bacterium is specially the intestinal bacteria that carry the ZmDVR gene; Described intestinal bacteria are specially BL21.
Albumen provided by the present invention is also belonging to protection scope of the present invention as the application in the divinyl reductase enzyme.
Above-mentioned albumen with divinyl reductase activity or ZmDVR gene are following 1)-5) application in arbitrary also belongs to protection scope of the present invention:
1) transforming the divinyl chlorophyll a is single ethene chlorophyll a;
2) transforming divinyl chlorophyllide a is single ethene chlorophyllide a;
3) transforming divinyl protochlorophyllide a is single ethylogen chlorophyllide a;
4) transforming divinyl Mg-protoporphyrin IX mono-methyl is single ethene Mg-protoporphyrin IX mono-methyl;
5) transforming divinyl Mg-protoporphysin is single ethene Mg-protoporphysin.
Experimental results show that: the albumen that derives from corn provided by the invention has the divinyl reductase activity, the divinyl chlorophyll a can be converted into single ethene chlorophyll a; Divinyl chlorophyllide a is converted into single ethene chlorophyllide a; Divinyl protochlorophyllide a is converted into single ethylogen chlorophyllide a; Divinyl Mg-protoporphyrin IX mono-methyl is converted into single ethene Mg-protoporphyrin IX mono-methyl; Divinyl Mg-protoporphysin is converted into single ethene Mg-protoporphysin.
Description of drawings
Fig. 1 be with divinyl chlorophyllide a (DV Chlide a) and the divinyl chlorophyll a (DV Chl a) detects spectrogram as the HPLC of the zymetology reaction product of substrate.Wherein, the left hand view of A and B is respectively to react the product of 10min at 440nm and the detected HPLC color atlas of 410nm, and right part of flg all is the absorption spectrums at each peak.It is that (HPLC of DV Chlide zymetology reaction product a) detects spectrogram to divinyl chlorophyllide a that A represents substrate.It is that (HPLC of DV Chl zymetology reaction product a) detects spectrogram to the divinyl chlorophyll a that B represents substrate.Concrete, A1 and A2 are respectively that (a) (MVChlide is a) with single ethene chlorophyllide a for DV Chlide for divinyl chlorophyllide a.B1 and B2 are respectively that (a) (MV Chl a) with single ethene chlorophyll a for DV Chl for the divinyl chlorophyll a.A3 and B3 are respectively DV Chlide a and the reacted pigment of BL21/pET-30a (+) empty carrier (negative control), and DV Chla and the reacted pigment of BL21/pET-30a (+) empty carrier (negative control); A4 and B4 are respectively DV Chlide a and BL21/pET-30a (+)-reacted pigment of ZmDVR intestinal bacteria lysate, and DV Chla and BL21/pET-30 a (+)-reacted pigment of ZmDVR intestinal bacteria lysate.A0 (blank) is the MV-Chlide a that produces behind incubation with residual MV-Chl a in the acetone powder of crowndaisy chrysanthemum blade preparation; Weak peak among A1 and the A3 (peak 2) promptly is the MV-Chlide a that MV-Chl a residual from acetone powder is transformed.
Fig. 2 is that (DV Pchlide a) detects spectrogram as the HPLC of the zymetology reaction product of substrate with divinyl protochlorophyllide a.Wherein, the product of A representative reaction 10min is at the detected HPLC color atlas of 440nm, the product of B representative reaction 2h is at the detected HPLC color atlas of 440nm, and the product of C representative reaction 10h is at the detected HPLC color atlas of 440nm, and D represents the absorption spectrum at each peak.Concrete, A1, B1, C1 all are that (DV Pchlide is a) for divinyl protochlorophyllide a; A2, B2, C2 all are that (MVPchlide a) for single ethylogen chlorophyllide a; A3, B3, C3 all are DV Pchlide a and the reacted pigment of BL21/pET-30a (+) empty carrier (negative control); A4, B4, C4 all are DV Pchlide a and BL21/pET-30a (+)-reacted pigment of ZmDVR intestinal bacteria lysate.
Fig. 3 is with the HPLC detection spectrogram of divinyl Mg-protoporphyrin IX mono-methyl (DV MPE) as the zymetology reaction product of substrate.Wherein, the product of A representative reaction 10min is at the detected HPLC color atlas of 410nm, the product of B representative reaction 2h is at the detected HPLC color atlas of 410nm, and the product of C representative reaction 10h is at the detected HPLC color atlas of 410nm, and D represents the absorption spectrum at each peak.Concrete, A1, B1, C1 all are divinyl Mg-protoporphyrin IX mono-methyls (DV MPE); A2, B2, C2 all are DV MPE and the reacted pigment of BL21/pET-30a (+) empty carrier (negative control); A3, B3, C3 all are DV MPE and BL21/pET-30a (+)-reacted pigment of ZmDVR intestinal bacteria lysate.
Fig. 4 is with the HPLC detection spectrogram of divinyl Mg-protoporphysin (DV Mg-proto) as the zymetology reaction product of substrate.Wherein, the product of left hand view representative reaction 10h is at the detected HPLC color atlas of 410nm, and right part of flg is represented the absorption spectrum at each peak.Concrete, A1 is divinyl Mg-protoporphysin (DV Mg-proto); A2 is DV Mg-proto and the reacted pigment of BL21/pET-30a (+) empty carrier (negative control); A3 is DVMg-proto and BL21/pET-30a (+)-reacted pigment of ZmDVR intestinal bacteria lysate.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The clone of embodiment 1, corn divinyl reductase gene and the functional verification of proteins encoded thereof
One, the clone of corn divinyl reductase gene
Design following primer:
ZmDVR-F:5 '-CC
GGATCCATGGCGACCATCCTCCTATC-3 ' (the underscore place is a BamH I restriction enzyme site)
ZmDVR-R:5 '-ATG
GAATTCGCCTAGAAGATGGTCTGCTC-3 ' (the underscore place is an EcoR I restriction enzyme site)
The DNA that extracts with corn (Zea mays L.) kind " B73 " self-mating system blade is a template, carry out pcr amplification with primer ZmDVR-F and ZmDVR-R, obtain the fragment of 1206bp, this fragment is connected on pMD-18-T (TaKaRa) carrier, be built into pMD-ZmDVR, be transformed into then in the e. coli jm109 bacterial strain, check order behind the screening positive clone.Sequencing result shows: the fragment that amplification obtains has the sequence shown in the sequence 1 in the sequence table, with its called after ZmDVR.Sequence is the encoding sequence of ZmDVR gene shown in the sequence 1, and its coding obtains the protein that aminoacid sequence shown in the sequence 2 is formed in the sequence table, with this protein called after ZmDVR.
Two, the acquisition and the functional verification thereof of corn divinyl reductase gene proteins encoded
1, the structure of recombinant expression vector pET-30a (+)-ZmDVR
PMD-ZmDVR carrier usefulness BamH I and the EcoR I double digestion that to identify through step 1 order-checking, be connected to the expression vector of cutting through same enzyme and be connected to expression vector pET-30a (+) (Novagen that cut through same enzyme, catalog number 69909-3) on, constitutes recombinant expression vector pET-30a (+)-ZmDVR.
2, abduction delivering
Recombinant expression vector pET-30a (+)-ZmDVR of step 1 is transformed into coli strain BL21 (contrast that pET-30a (+) empty carrier is transformed into coli strain BL21 is set simultaneously), at 37 ℃, incubated overnight on the LB/kan substratum, select mono-clonal in 37 ℃, shaking culture in the LB/kan nutrient solution, extract plasmid DNA (OMEGA test kit), with BamH I and EcoR I double digestion, whether agarose gel electrophoresis detects the ZmDVR gene and inserts among the carrier pET-30a (+), the clone who detection is contained the recombinant expression vector of ZmDVR gene serves the order-checking of Hai Yingjun company, will screen clone's called after BL21/pET-30a (+)-ZmDVR of the recombinant expression vector of the ZmDVR gene that correctly inserts pET-30a (+).Change the coli strain BL21 called after BL21/pET-30a (+) of pET-30a (+) empty carrier over to.Then with reference to (Nagata N, Tanaka R, Satoh S, Tanaka A (2005) Identification of a vinyl reductase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evolution of Prochlorococcus Species.Plant Cell 17:233-240) method is carried out abduction delivering with BL21/pET-30a (+)-ZmDVR, concrete grammar is as follows: BL21 (the empty carrier contrast CK) mono-clonal of selecting BL21/pET-30a (+)-ZmDVR respectively and containing empty carrier PET-30A (+) is in 20 ℃, one night of shaking culture in the LB/kan nutrient solution, get 1ml bacterium liquid respectively in 100ml LB/kan nutrient solution, add isopropylthio-beta galactose glycosides (IPTG) behind 30 ℃ of shaking culture 30min, final concentration is 0.5mM, in 30 ℃ of inducing culture 7h, 4 ℃ of centrifugal 10min of 10000rpm, precipitation is with containing 6.7 μ gml
-1N,O-Diacetylmuramidase and 3.3 μ gml
-1The solution of the Tris-HCl of the 50mM of DNaseI suspends, and lysate is put into-20 ℃ of refrigerators and preserved.
3, enzymatic reaction checking protein function
The used substrate of this step relates to following five kinds: the divinyl chlorophyll a (DV Chl a), divinyl chlorophyllide a (DV Chlide a), divinyl protochlorophyllide a (DV Pchlide a), divinyl Mg-protoporphyrin IX mono-methyl (DV MPE) and divinyl Mg-protoporphysin (DV Mg-proto).
The acquisition approach of these five kinds of substrates is as follows: (DV Chl is a) from paddy rice 824ys mutant (Huang Xiaoqun for the divinyl chlorophyll a, Wang Pingrong, Zhao Haixin, Deng Xiaojian. the genetic analysis of a new rice chlorophyll deletion mutantion gene and molecule marker location. the rice in China science, 2007,21 (4): 355-359; Wang PR, Gao JX, Wan CM, Zhang FT, Xu ZJ, Huang XQ, Sun XQ, Deng XJ (2010) Divinyl chlorophyll (ide) a can be converted to monovinyl chlorophyll (ide) a by a divinyl reductase in rice.Plant Physiol 153,994-1003) separate in the chlorophyll, with 100% acetone lixiviate chlorophyll from the fresh leaf tissue of 824ys mutant, the centrifugal 15min of extracting solution 10000r/min (Eppendorf 5804R), supernatant liquor dries up under nitrogen, heavily is dissolved in 100% acetone.Use C18 post (4.6mm i.d. * 150mm long then; 5 μ m, Agilent, U.S.A) carrying out HPLC separates, elution requirement is a moving phase: methyl alcohol: acetonitrile: acetone=1: 3: 1,40 ℃, flow velocity 1.0mL/min, applied sample amount 5 μ L, the pigment of wash-out 660nm wavelength monitoring in the post is collected DV-Chl a in corresponding retention time, specifically referring to Wang etc. at Wang PR, Gao JX, Wan CM, Zhang FT, Xu ZJ, Huang XQ, Sun XQ, Deng XJ (2010) Divinyl chlorophyll (ide) a can be converted to monovinyl chlorophyll (ide) a by a divinyl reductase in rice.Plant Physiol 153, the method for putting down in writing among the 994-1003.
Divinyl chlorophyllide a (DV Chlide a) be with above-mentioned isolating divinyl chlorophyll a (DV Chl a) with chlorphyllase reaction gained from the acetone powder of crowndaisy chrysanthemum blade preparation.
Wherein, the preparation method of acetone powder (chlorphyllase) is as follows: according to (Ito H such as Ito, Takaichi S, Tsuji H, Tanaka A.Properties of synthesis of chlorophyll a from chlorophyll b in cucumber etioplasts.J Biol Chem, 1994, method 269:22034-22038), (kind is " a delicate fragrance crowndaisy chrysanthemum " with crowndaisy chrysanthemum (Garland chrysanthemum), buy from market of farm produce seed street pedlar) blade prepares acetone powder, be specially: get the fresh crowndaisy chrysanthemum blade of 15g, add 300mL refrigerated acetone, put into tissue mashing machine and smash to pieces fast, 4 ℃ of centrifugal 10min of 10000r/min abandon supernatant liquor; Then with the acetone rinsing precipitation, till chlorophyll is cleaned fully; At last precipitation is placed the reduced vacuum whizzer to dry up and make acetone powder.
The preparation method of DV Chlide a is as follows: according to Holden (Holden M.The breakdown of chlorophyll by chlorophyllase.Biochem J, 1961,78:359-364) and (Ito H such as Ito, Ohtsuka T, Tanaka A.Conversion of chlorophyll b to chlorophyll a via 7-hydroxymethyl chlorophyll.J Biol Chem, 1996, method 271:1475-1479), (a) (above-mentioned DV-Chl through the HPLC resulting separation a) is dissolved in 4mL solution (25mM Tris-HCl to DV Chl with 200 μ g divinyl chlorophyll as, pH 7.5 and 40% acetone) in, with the 200mg acetone powder at 28 ℃, incubation 2h under the dark condition, behind the incubation according to (Ito H such as Ito, Tanaka Y, Tsuji H, Tanaka A.Conversion of chlorophyll b to chlorophyll a by isolated cucumber etioplasts.Arch Biochem Biophys, 1993, method purifying DV Chlide a 306:148-151).
(DV Pchlide is with reference to (Nagata N a) to divinyl protochlorophyllide a, Tanaka R, TanakaA (2007) The major route for chlorophyll synthesis includes[3,8-divinyl]-chlorophyllide a reduction in Arabidopsis thaliana.Plant Cell Physiol 48:1803-1808) method), from 6 days paddy rice 824ys mutant seedling of dark culturing, extract gained.Concrete operations are: after the paddy rice 824ys mutant seedling of cultivating 6 days in the dark is shredded, mix with acetone, homogenate is 1 minute in refiner, add then Tris-HCL (pH 7.6) to final concentration be 200mM, the centrifugal 20min of 10000rpm, supernatant liquor add isopyknic normal hexane to remove Chlorophylls and Carotenoids.Extract DV Pchlide a with ether from acetone soln at last, dry up with nitrogen, (DV Pchlide a) to obtain described divinyl protochlorophyllide a.
Divinyl Mg-protoporphyrin IX mono-methyl (DV MPE) and divinyl Mg-protoporphysin (DV Mg-proto) are to buy from U.S. Frontier Scientific Inc company.
(MV Chl is a) with reference to above-mentioned divinyl chlorophyll a (DV Chl preparation method a) with the single ethene chlorophyll a that compares, from paddy rice wild-type kind 824B (Huang Xiaoqun, Wang Pingrong, Zhao Haixin, Deng Xiaojian. the genetic analysis of a new rice chlorophyll deletion mutantion gene and molecule marker location. the rice in China science, 2007,21 (4): 355-359; Wang PR, Gao JX, Wan CM, Zhang FT, Xu ZJ, Huang XQ, Sun XQ, Deng XJ (2010) Divinyl chlorophyll (ide) a can be converted to monovinyl chlorophyll (ide) a by a divinyl reductase in rice.Plant Physiol 153,994-1003) the middle extraction.
(a) (DVChlide is the preparation method a), and (MV Chl a) carries out the enzyme reaction preparation as substrate with single ethene chlorophyll a with reference to above-mentioned divinyl chlorophyllide a for MV Chlide with the single ethene chlorophyllide a that compares.
(MV Pchlide is that (DV Pchlide preparation method a) extracts gained from 6 days paddy rice wild-type parent 824B seedling of dark culturing with reference to above-mentioned divinyl protochlorophyllide a a) with the single ethylogen chlorophyllide a that compares.
With BL21/pET-30a (+)-ZmDVR expressed proteins (ZmDVR) respectively with above-mentioned DV Chl a and these two kinds of substrates of DV Chlide a at reaction buffer (40mM citric acid, 80mM K
2HPO
40.5mM NADPH, 20% acetone, pH 7.0) in 30 ℃ of reaction 10min because ZmDVR albumen is slower to substrate DV Pchlide a, DV Mg-Proto and DV MPE catalytic activity, therefore, reaction times to DV Pchlide a and DV MPE is provided with 10min, 2h and three processing of 10h, reaction times to DV Mg-proto extends to 10h, and the reaction of BL21/pET-30a (+)-ZmDVR expressed proteins (ZmDVR) and five kinds of substrates simultaneously all is provided with the empty carrier contrast.Use the acetone termination reaction then, change in the ether, dry up through nitrogen, be dissolved in acetone again, detect with HPLC at last and (be provided as corresponding contrast MV Chl a, the MV Chlide a of above-mentioned DV Chl a, DV Chlide a, three kinds of substrates of DV Pchlide a and MV Pchlide simultaneously a).According to (Zapata M such as Zapata, Rodr í guez F and Garrido JL (2000) Separation of chlorophylls and carotenoids from marine phytoplankton:A new HPLC method using a reversed phase C8 column and pyridine containing mobile phases.Mar.Ecol.Prog.Ser.195:29-45) method is carried out HPLC and is analyzed, wash-out adopts gradient moving phase (table 1), 25 ℃, flow velocity 1.0mL/min, applied sample amount 5 μ L, the product of enzyme reaction DV Chl a and DV Chlide a C8 post (4.6mm i.d. * 150mm long; 3.5 μ m Agilent) detects, the pigment of wash-out is used 410nm and 440nm wavelength monitoring respectively in the post; The product of enzyme reaction DV Pchlide a, DV MPE and DV Mg-proto C8 post (4.6mm i.d. * 150mm long; 3.5 μ m Waters) detects, the pigment of wash-out is used 440nm, 410nm and 410nm wavelength monitoring respectively in the post.
Table 1HPLC analyzes the gradient moving phase that is adopted
| Time (min) | Solution A (%) | Solution B (%) |
| 0 | 100 | 0 |
| 22 | 60 | 40 |
| 28 | 5 | 95 |
| 38 | 5 | 95 |
| 40 | 100 | 0 |
Annotate: the prescription of solution A is a methyl alcohol: acetonitrile: the volume ratio of the 0.25M pyrimidine aqueous solution is 2: 1: 1; The prescription of solution B is a methyl alcohol: acetonitrile: the volume ratio of acetone 1: 3: 1.(methyl alcohol, acetonitrile, acetone, pyrimidine are chromatographically pure, and methyl alcohol, acetonitrile and acetone are domestic production, and pyrimidine is available from Sigma company)
The result shows that BL21/pET-30a (+)-ZmDVR expressed protein (ZmDVR) is separately converted to corresponding single ethene material with above 5 kinds of substrates, is about to the divinyl chlorophyll a and is converted into single ethene chlorophyll a; Divinyl chlorophyllide a is converted into single ethene chlorophyllide a; Divinyl protochlorophyllide a is converted into single ethylogen chlorophyllide a; Divinyl Mg-protoporphyrin IX mono-methyl is converted into single ethene Mg-protoporphyrin IX mono-methyl; Divinyl Mg-protoporphysin is converted into single ethene Mg-protoporphysin.And the empty carrier contrast can not transform above material.Simultaneously, BL21/pET-30a (+)-ZmDVR expressed protein (ZmDVR) has very big difference to different substrate catalytic activitys, substrate DV Chl a and DV Chlide a are had very high catalytic activity (Fig. 1), and to substrate DV Pchlide a (Fig. 2), DV MPE (Fig. 3) and DV Mg-proto (Fig. 4) catalysis speed more slowly or very slow.
This shows that clone's ZmDVR genes encoding has the divinyl reductase enzyme of function, and the present invention utilizes the divinyl reductase enzyme of purifying to carry out external zymetology experiment confirm divinyl Mg-protoporphyrin IX (DV Mg-proto) first and this two kinds of divinyls (DV type) material of divinyl Mg-protoporphyrin IX mono-methyl (DV MPE) can be converted into corresponding single ethene (MV type) material.Simultaneously, the present invention confirms first that also above-mentioned five kinds of substrates (the biosynthetic divinyl intermediate material of chlorophyll) all can be converted into corresponding single ethene material by corn divinyl reductase enzyme ZmDVR, illustrate divinyl reductase enzyme ZmDVR in the chlorophyll biosynthetic pathway by a genes encoding, have the divinyl reductase enzyme of wide spectrum substrate specificity, thereby further illustrated the chlorophyll biosynthetic pathway.
Claims (10)
1. albumen with divinyl reductase activity, the protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2.
2. coding claim 1 described proteic gene.
3. gene according to claim 2 is characterized in that: the encoding sequence of described gene is the dna molecular of sequence 1 in the sequence table.
4. the recombinant expression vector or the transgenic cell line that contain claim 2 or 3 described genes.
5. the described albumen of claim 1 is as the application in the divinyl reductase enzyme.
6. described albumen of claim 1 or claim 2 or 3 described genes are application in single ethene chlorophyll a transforming the divinyl chlorophyll a.
7. described albumen of claim 1 or claim 2 or 3 described genes are application among single ethene chlorophyllide a transforming divinyl chlorophyllide a.
8. described albumen of claim 1 or claim 2 or 3 described genes are application among single ethylogen chlorophyllide a transforming divinyl protochlorophyllide a.
9. described albumen of claim 1 or claim 2 or 3 described genes are application in single ethene Mg-protoporphyrin IX mono-methyl transforming divinyl Mg-protoporphyrin IX mono-methyl.
10. described albumen of claim 1 or claim 2 or 3 described genes are application in single ethene Mg-protoporphyrin IX transforming divinyl Mg-protoporphyrin IX.
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