CN103540602B - A kind of gene and application improving chlorophyll content of plant and rape oleaginousness - Google Patents

Abstract

The invention discloses a kind of gene and the application that improve chlorophyll content of plant and rape oleaginousness.This gene is the BnCHS gene deriving from rape, and its nucleotides sequence is classified as shown in SEQ ID NO.1; The aminoacid sequence of the protein of its coding is for shown in SEQ ID NO.2.Prove that its proteins encoded has activity of long-chain acylcoa synthetase by yeast complementation experiment; By the expression pattern analysis in cole crop, show its main to express in leaf in ripe plant in seedling and then mainly to express spending medium fat to synthesize vigorous position; Improve BnCHS expression amount by genetic engineering technique and find to improve the chlorophyll content of tobacco and swede type rape blade and yeast and swede type rape seed fat content, and add the biomass of swede type rape.Show that the albumen that BnCHS encodes has the function improving swede type rape chlorophyll content, improve swede type rape seed oil content.

Description

A kind of gene and application improving chlorophyll content of plant and rape oleaginousness

Technical field

The invention belongs to plant genetic engineering field.Specifically, relate to a kind of raising swede type rape chlorophyll content, improve the gene of swede type rape biomass and Semen Brassicae campestris oleaginousness.Adopt molecular biology method, clone and isolate from rape can improve chlorophyll content in leaf blades gene BnCHS ( brassica napus chloropyll synthesis), its coding fatty acyl-CoA synthetase.By at yeast, overexpression analysis in tobacco and rape, proves the biochemical function of BnCHS and participates in the biological function of Chlorophyll synthesis and fat metabolic synthesis.In rape, overexpression can improve the chlorophyll content of rape and the oleaginousness of seed.

Background technology

Green plants can carry out luminous energy Synthesis, be exactly under the irradiation of visible ray, through photoresponse and dark reaction, utilize Chlorophyll absorption luminous energy, carbonic acid gas (or hydrogen sulfide) and water are converted into organism, and discharge the biological process of oxygen (or hydrogen).Blade carries out photosynthetic major organs, and chloroplast(id) absorbs luminous energy then to be completed by chlorophyll.The summation of the metabolic reaction of photosynthesis series of complex is the source of plant own growth institute energy requirement, and to be also other with plant be relies on the species organic substance of life and the source of energy.Visible chlorophyll is closely bound up with biological existence.For nearly all biology of organic sphere, Chlorophyll absorption luminous energy thus to carry out photosynthesis at chloroplast(id) be the key that they are depended on for existence.At present by having been found that the research of model plant and having identified all enzymes (Beale etc. 2005) participating in Chlorophyll synthesis process.But chlorophyllous building-up process is a complicated process, except affecting by external environment condition and growth and development process, some gene also has regulating and controlling effect to chlorophyllous building-up process.Thus affect chlorophyllous content.We utilize genetic engineering technique by regulating and controlling a certain gene thus reaching the object increasing chlorophyllous content.We find BnCHS gene under study for action, overexpression in rape and tobacco, can improve the chlorophyll content of blade and the accumulation of biomass.In addition due to the raising of chlorophyll content, add photosynthetic efficiency, the oleaginousness of Semen Brassicae campestris is compared with contrast and is also improved.At present, mankind's edible oil more than 70% is vegetables oil.In China, edible oil needs more than 50% dependence on import, and the content improving vegetables oil is significant.Therefore BnCHS gene can as having the candidate gene improving oil crop seeds by using oleaginousness.Because BnCHS can improve chlorophyll content, increase the photosynthetic efficiency of plant, so it also has important application prospect in raising crop yield.

Summary of the invention

The object of the present invention is to provide a kind of gene BnCHS improving oil crops swede type rape chlorophyll content and oleaginousness.

Derive from a gene BnCHS for rape, its nucleotides sequence is classified as shown in SEQ ID NO.1.

The protein of BnCHS genes encoding, its aminoacid sequence is for shown in SEQ ID NO.2.

Invention further provides described BnCHS gene and improving the application in chlorophyll content of plant, and improve the application in oil crops oleaginousness.The present invention reaches the object improving swede type rape chlorophyll content and oleaginousness by this albumen of overexpression.

The invention also discloses the recombinant vectors pB2WG7.0-BnCHS with BnCHS gene, pK7GW2.0-BnCHS, pYES2-BnCHS.

Wherein recombinant vectors pYES2-BnCHS inserts gene BnCHS at the BamHI/KpnI restriction enzyme site of pYES2.

Recombinant vectors pB2WG7.0-35S-BnCHS and recombinant vectors pK7GW2.0-BnCHS, with BnCHS-F (SEQ ID NO.3), goal gene, for primer amplification goes out goal gene BnCHS, is then connected to by BnCHS-R (SEQ ID NO.4) (Invitrogen Corporation), so according to gateway test kit ( lR Clonase ^tMiI Enzyme Mix, InvitrogenCorporation) goal gene is cloned into pB2WG7.0 by operation respectively, on pK7GW2.0 (from plantsystems biology http://gateway.psb.ugent.be/search), obtain pB2WG7.0-35S-BnCHS and pK7GW2.0-BnCHS.

The present invention also comprises the recombinant vectors pB2WG7.0-35S-BnCHS containing BnCHS gene, and pK7GW2.0-BnCHS, pYES2-BnCHS transgenic cell line and Host Strains, and Semen Brassicae campestris, all belong to protection scope of the present invention.

An object of the present invention is to provide the method that is improved content of chlorophyll in plant.

Another object of the present invention is to provide and a kind of improves in plant, particularly the method for fat content in plant seed.

The method of raising chlorophyll content provided by the invention and oil content in plants BnCHS is imported plant tissue viable cell thus improves chlorophyll content and oil content in plants.

The present invention also comprises the direct flower-dipping method of method-Agrobacterium BnCHS being imported plant tissue viable cell.Step is:

(1), in the test tube containing corresponding antibiotic LB liquid nutrient medium, cultivate the Agrobacterium GV3101 containing recombinant plasmid, then make Agrobacterium nutrient solution final concentration be that OD600 is about about 2.0 in the triangular flask of ratio access containing corresponding antibiotic LB liquid nutrient medium of 10%.

(2), in substratum, transfer buffer is added:

0.1% Silwet-77

2ng/L 6-BA

8mg/L Syringylethanone

(3), the whole bud part do not opened of rape be immersed in 3-5min in Agrobacterium solution, shake makes bud fully contact agrobacterium liquid gently simultaneously.The bud soaked is inserted in sheepskin paper bag 24h, to keep bud to have higher humidity, avoids bud to be exposed to the sun in excessive sunlight, prevent Agrobacterium from losing activity.

(4), every one day repeating step (3), and whole bud is enclosed within 48h in sheepskin paper bag.

(5) after, soaking bow structure bundle, remove the sheepskin paper bag on inflorescence, shear the side shoot inflorescence newly born and the bud newly sprouted through its top of inflorescence of leaching flower, and utilize colored silk thread to carry out anchor line (string) mark to the inflorescence contaminating Agrobacterium, the structure of different recombinant plasmid carries out label.

(6), normal pouring is cultivated plant, and is regularly wiped out newborn side shoot inflorescence and newborn bud, until stop during seed maturity irrigating.Results dry seeds.The screening of transformant is carried out by the selective marker that recombinant plasmid carries.By this method, we have filtered out a large amount of transfer-gen plants.

The present invention isolates a cDNA, called after BnCHS from swede type rape flower cDNA.Sequential analysis display BnCHS belongs to AMP-and connects albumen (AMP) superfamily.Yeast complementation experiment proves that BnCHS has LACS activity.RT-PCR analyzes and is presented at the tender leaf of children and spends BnCHS in tissue highly to express.This gene is proceeded to overexpression in tobacco, TAG, CoA, fatty acid content significantly improves, and proceeds in rape by this gene, and Semen Brassicae campestris oleaginousness significantly improves, and this gene causes the raising of chlorophyll content.These results show that BnCHS has and significantly improve oil crops oleaginousness, and improve the function of chlorophyll content.

Advantage of the present invention:

1. from cDNA, clone gene reliability is high on a molecular scale, speed is fast, efficiency is high.

2. the BnCHS gene of the present invention clone can significantly improve Oil Content In B. Napus L, and this is significant to the biological function and application aborning of illustrating BnCHS gene.

The transfer-gen plant of 3.BnCHS significantly improves oleaginousness, and this has important application prospect to the production of vegetables oil.

The transfer-gen plant chlorophyll content of 4.BnCHS significantly improves, and this is to the raising photosynthetic ability of plant thus raising biomass is significant.

5. oleaginousness genetic research is all significant to instructing breeding practice, breed improvement and variety popularization, therefore excavate and identify and improve oleaginousness gene, and the molecular mechanisms of action of further investigated fat metabolic, there is important theory significance and practical application value.

Accompanying drawing explanation

The amino acid alignment of Fig. 1 .BnCHS and other species LACS.

Fig. 2 .BnCHS in tobacco with the Subcellular Localization in rape.A: tobacco leaf BnCHS and GFP merges transient expression B: Chloroplast auto-fluorescence C:A and B stacking diagram; D: rape cotyledon BnCHS and GFP merges transient expression E: Chloroplast auto-fluorescence F:D and E stacking diagram

The content of each material in the test of Fig. 3 .BnCHS yeast complementation and tobacco transient expression assay fat metabolic approach.A: transformed yeast cell is cultivating logarithmic phase mid-term (84h) containing 16:0 lipid acid as on the liquid nutrient medium of sole carbon source.A: the yeast cell transforming pYES2 empty carrier; B: the yeast cell transforming pYES2-BnCHS.B, C: CoA content in instantaneous overexpression BnCHS blade in tobacco.

Fig. 4. tobacco overexpression BnCHS significantly improves tobacco leaf lipid acid and galactolipid content.

A, B: in tobacco, instantaneous process LAN BnCHS makes fatty acid total amount increase.C: the instantaneous process LAN BnCHS of tobacco makes glycolipid total amount in blade increase

Fig. 5 .BnCHS instantaneous process LAN in tobacco makes tobacco leaf chlorophyll content improve

X-coordinate is chlorophyll concentration, and ordinate zou is sample time.To be respectively after transformation of tobacco the 4th, 5,6,7,8,9 days

Fig. 6. the expression amount of BnCHS in the qualification of transgene rape and the transfer-gen plant of acquisition, and the express spectra of BnCHS in swede type rape.A: the qualification of transgene rape plant, identifies the express spectra of expression amount C:BnCHS in swede type rape of 5 B:5 transfer-gen plants, the expression amount in the different tissues in ripe plant altogether.D: the overexpression transgenosis young plant phase, BnCHS improved at expression amount.

Fig. 7 .BnCHS rotaring gene plant blade chlorophyll content is analyzed.A: cotyledon period; C: cotyledon period chlorophyll content; B: five leaf phase blades; D: five leaf phase chlorophyll contents.Transfer-gen plant offspring chlorophyll content improves.

Fig. 8 .BnCHS transgene rape seed oil content and seedling fresh weight are analyzed.

A: Semen Brassicae campestris oleaginousness analyze B: seedling stage plant fresh weight.Visible transgene rape seed oil content significantly improves, and the biomass of plant increases.

Embodiment

The clone of embodiment one: BnCHS gene and sequential analysis

The extraction of 1.RNA

Get napus lines peaceful oil 16 flower tissue, utilize its total serum IgE of plant Trizol reagent extracting, and reverse transcription becomes cDNA.

2. the acquisition of goal gene BnCHS

With BnCHS-F:5 '-ATGATTCCTTACGCT-3 ' (SEQ ID NO.3)

BnCHS-R:5'-TTAGGAAGCATATAGCTT-3’(SEQ ID NO.4)

For primer, be template with cDNA, utilize LA-Taq enzyme (TaKaRa Biotechnology Co.) to carry out PCR, according to following condition: 94 DEG C of 4min, then 25cycles(94 DEG C of 40s, 54 DEG C of 40s, 72 DEG C of 2min, 72 DEG C of 10min).From sepharose, reclaim PCR primer, then connect pMD18-T carrier (TaKaRa Biotechnology Co.) and check order.Intestinal bacteria containing BnCHS-pMD18-T are stored in life science institute of Jiangsu University at present.The sequence of gene BnCHS is as shown in SEQ ID NO.1.

3.BnCHS gene sequencing

Aminoacid sequence (SEQ ID NO.2) and other species LACS of BnCHS comprise: Arabidopsisthaliana:AtLACS1, AtLACS2, AtLACS3, AtLACS4, AtLACS5, AtLACS6, AtLACS7, AtLACS8, AtLACS9; Saccharomyces cerevisiae:ScFAA1, ScFAA2, ScFAA3; Brassica napus:BnLACS1 and BnLACS2 amino acid sequence analysis, finding that BnCHS has total three motifs (Fig. 1 .A) of LACS family, is the member of LACS family.BnCHS and other species LACS aminoacid sequence Genetic relationship find, BnCHS and Arabidopis thaliana LACS Gene A tLACS9 sibship are recently (Fig. 1 .B).

The functional analysis of embodiment two: BnCHS

The structure of 1.BnCHS positioning carrier

In order to study the Subcellular Localization of BnCHS in tobacco and rape, first the goal gene of acquisition is cloned into pENTR(Invitrogene company by us), then gateway technology is utilized, goal gene is cloned into positioning carrier pK7WG2.0(Plant systems biology) on, obtain the positioning carrier pK7WG2.0-BnCHS-GFP expressed with GFP fused in tandem.

2. tobacco, the Subcellular Localization experiment of BnCHS in rape cotyledon

The positioning carrier of structure is proceeded to tobacco and rape cotyledon transient expression, the method transformed adopts micro-syringe injection, take blade at fluorescence microscopy Microscopic observation when transforming the 7th day, find that BnCHS is all positioned at (Fig. 2) on chloroplast(id) in rape and tobacco leaf.Chloroplast(id) is that photosynthesis and lipid metabolism synthesize important organoid.

3. the structure of Yeast expression carrier

In order to express in yeast, design pair of primers BnCHS-F1 and BnCHS-R1 increases BnCHS from cDNA, as follows

BnCHS-F1:5’-ggatccATGATTCCTTACGCT-3’(BamHI)(SEQ ID NO.5)

BnCHS-R1:5'-ggtaccTTAGGAAGCATATAGCTTG-3’(KpnI)(SEQ ID NO.6)

LA-Taq enzyme (TaKaRa Biotechnology Co.) is utilized to carry out PCR.From sepharose, reclaim PCR primer, then connect pMD18-T carrier (TaKaRa Biotechnology Co.).BamHI/KpnI enzyme is utilized to cut the later positive colony of order-checking, cut Yeast expression carrier pYES2 (Invitrogen Corporation) with BamHI/KpnI enzyme simultaneously, BnCHS is cloned into the corresponding site of Yeast expression carrier pYES2, obtains pYES2-BnCHS.

4. yeast complementation test

In order to determine whether BnCHS possesses LACS activity, have employed yeast complementation expression system.BnCHS is cloned in Yeast expression carrier pYES2, then verifies whether BnCHS has the ability remedying defective type in yeast (S.cerevisiae) the bacterial strain YB525 (purchased from ATCC http://www.atcc.org/) of conversion shortage LACS activity.Using pYES2 empty carrier transformed yeast YB525 as negative contrast.Transformant is cultivated containing 18:0 lipid acid, as the default uridylic liquid nutrient medium of sole carbon source, (deaminize sour yeast culture yeast nitrogen base (0.67%), save uridylic mixture (0.077%)) in, cultivate 84h to logarithmic phase mid-term for 30 DEG C.Result shows, and the transformant transforming pYES2-BnCHS can normal growth (Fig. 3 A.b), and the yeast cell transforming pYES2 empty carrier can not grow (Fig. 3 A.a), illustrates that BnCHS can have LACS activity by complementary LACS yeast defective type.

5. tobacco, the structure of rape expression vector

Utilize the method described in 1 that BnCHS is cloned into expression vector pB2GW7.0, obtain the plant expression vector pB2GW7.0-BnCHS with BnCHS.

6. the functional analysis of BnCHS in tobacco

PB2GW7.0-BnCHS is proceeded in tobacco, when transforming 7 days, get blade.Participate in the CoA of oil synthesis with liquid-phase chromatographic analysis, the content of fat, with the content of gas chromatographic analysis lipid acid.Result shows the plant leaf CoA proceeding to BnCHS, lipid acid, and galactolipid content all increases (Fig. 3 .B, C; Fig. 4 .A, B, C).Illustrate that BnCHS can improve the oleaginousness of plant.Get injection pB2GW7.0-BnCHS after 4,5,6,7, within 8,9 days, tobacco leaf analyzes chlorophyll content, gets 0.2g blade and is settled to 5ml, find that the tobacco leaf chlorophyll content compared with only injecting the tobacco leaf of empty carrier injecting pB2GW7.0-BnCHS from the 7th day increases (Fig. 5), illustrate that BnCHS can improve chlorophyllous content.

Embodiment three: BnCHS is at the expression analysis of rape

1. the acquisition of transgene rape

PB2GW7.0-BnCHS is proceeded to Agrobacterium GV3101, contaminate rape flower three times, each five minutes.The seed sprouting of results first screens with weedicide, and then choose the plant of antiweed later, and extract its genome, screen with PCR, primers designed is as follows:

pw35s5’-TAAGGAAGTTCATTTCATTTGGAG-3’(SEQ ID NO.7)

BnCHS2R5’-CCTAAAGACGAGTAGATAGTAACCAC-3’(SEQ ID NO.8)

Obtain 5 transfer-gen plants (Fig. 6 .A).

2. transgene rape plant BnCHS expression analysis

In order to determine the biological function of BnCHS in rape, in rape, QRT-PCR expression analysis is carried out to BnCHS.

Utilize primer BnCHSQRT F(5 '-ATTGGGCACAAGTCTGAGGA-3 ', SEQ ID NO.9) and BnCHSQRT(5 '-TGTAACCTCTGTCTCATTAAGCG-3 ', SEQ ID NO.10), according to BnActin sequences Design Auele Specific Primer BnActin F(5 '-ATGGCCGATGGTGAGGACATTC-3 ', SEQ ID NO.11) and BnActin R(5 '-GGTGCGACCACCTTGATCTT C-3 ', SEQ ID NO.12), serve Hai Shenggong synthesis.According to follow procedure: 95 DEG C of denaturation 30S, then through 40 circulations (95 DEG C of 10s, 57 DEG C of 10s, 72 DEG C of 26s).Same cDNA, also as template, uses Actin F and Actin R as primer, and Actin is as internal reference in amplification.Research finds that the expression amount of BnCHS in the transfer-gen plant obtained all improves (Fig. 6 .B).

The express spectra of 3.BnCHS in rape

To utilize in 2 BnCHS expression amount in primer pair rape different tissues to carry out real-time quantitative (Fig. 6 .C) analysis to find, BnCHS is main at leaf expression in plant shoots, and expression amount in the root and leaf of ripe plant is very low, and the expression amount relatively high (Fig. 6 .C) in flower and stem.Flower is the tissue that grease synthesizes in a large number, and in spending, the great expression of BnCHS illustrates that BnCHS may participate in the anabolism of grease.

Embodiment four: the transgene rape chlorophyll content of process LAN BnCHS and seed oil content analysis.

1. transgene rape chlorophyll content is analyzed

The T1 of results is implanted in constant incubator for seed, daylight 5000, humidity 60%, temperature 25 degree, 12h.Illumination 0 at night, humidity 60%, temperature 22 degree, 12 hours, samsara like this.Identify by transfer-gen plant qualification PCR primer when the 7th day.At the 10th day, get foliar analysis chlorophyll content when 20 days and find.Transgene rape chlorophyll content significantly improves (Fig. 7 .A, B, C, D) than nontransgenic plants.

2. transgene rape T2 analyzes for seed oil content analysis and plant fresh weight

Collect 5 T2 being for seed, survey the oleaginousness of seed by the method for nucleus magnetic resonance.Found that the oleaginousness of 5 transgenic lines seeds all significantly improves.Oleaginousness is high compared with the control 15.34% for its Line12.Prove that BnCHS participates in the synthesis of Lipid Research of Rapeseed, the oleaginousness (Fig. 8 .A) of plant can be improved.And we have surveyed transfer-gen plant the 9th, 16,23, the plant fresh weight of 30,37 days, finds the plant fresh weight heavier than nontransgenic plants (Fig. 8 .B) of transfer-gen plant, we infer because chlorophyll content increases, and add photosynthetic rate thus add the biomass of plant.

More than prove BnCHS participation oil lipid metabolism, and chlorophyll content of plant can be improved and improve oil content in plants.

Claims (2)

1. derive from the gene of rape bnCHSimproving the application in Chlorophyll in Rape content, raising rape oleaginousness, described gene bnCHSnucleotides sequence be classified as shown in SEQ ID NO.1.

2. improve a method for rape Determination of Chlorophyll content and oleaginousness, be by the BnCHS channel genes Oil Rape Tissue viable cell of sequence as shown in SEQ ID NO.1 thus improve chlorophyll content and oleaginousness.