CN104450748B - The rough fatty acid desaturase ApD6D gene families of careless △ 6 and its recombinant expression carrier and application - Google Patents

Abstract

The invention discloses the rough careless fatty acid desaturase ApD6D gene families of △ 6 and its recombinant expression carrier and application, ApD6D gene families include two members of ApD6D1 and ApD6D2, with the fatty acid desaturases of △ 6 activity, linoleic acid formation acid and gamma-linolenic (GLA) can be catalyzed, also alpha linolenic acid (ALA) formation parinaric acid (SDA) can be catalyzed, ApD6D1 and ApD6D2 justice is transferred to after rough grass itself, GLA and SDA contents are greatly improved in the rough grass seed of transgenosis；It is transferred to after cabbage type rape, GLA and SDA that non-transgenic rape seed does not have is have accumulated in transgene rape seed；Show that using ApD6D gene families new resources material can be created, and for industrial production GLA and SDA, or it is directly used in health edible oil of the production rich in GLA and SDA.

Description

Rough careless △ 6- fatty acid desaturase ApD6D gene families and its recombinant expression carrier And application

Technical field

The invention belongs to genetic engineering field, and in particular to rough careless △ 6- fatty acid desaturases ApD6D gene families, also It is related to recombinant expression carrier and the application of ApD6D gene families.

Background technology

Polyunsaturated fatty acid (Polyunsaturated Fatty Acids, PUFA) is to contain two between carbon atom The unrighted acid of individual above double bond, its metabolic pathway is with linoleic acid (Linoleic acid, LA；C18:3Δ^9,12,n- 6) it is initial substrate, under the catalysis of a series of fatty acid desaturases (fatty acid dehydrogenase) and fatty acid elongase, successively Generate gamma-Linolenic acid (γ-linolenic acid, GLA；C18:3Δ^6,9,12, n-6), alpha-linolenic acid (α-linolenic acid,ALA；C18:3Δ^9,12,15, n-3), parinaric acid (stearidonic acid, SDA, octadecatetraenoic acid,OTA,C18:4Δ^6,9,12,15, n-3), double high gamma-linolenic acids (DHLG), arachidonic acid (ARA), the long-chain such as eicosapentaenoic acid (EPA, n-3), clupanodonic acid (DPA), DHA (DHA, n-3) Polyunsaturated fatty acid (LC-PUFAs) or very long-chain polyunsaturated fatty acids (VLC-PUFAs).

PUFA can enhancing development, the lipid-metabolism of regulation human body, moreover it is possible to treat and prevent cardiovascular and cerebrovascular disease, simultaneously With the important physiological function such as immunological regulation, anticancer, anti-aging, very important effect is played to human health.Ω3 (n-3) series fatty acid has important physiological function to human body, and wherein ALA is n-3 PUFA (EPA, DHA) synthesis precursors；EPA It is the important polyunsaturated fatty acids chemical messenger thing of a class, serves in immune and inflammatory reaction vital；DPA It is the intermediate product for generating DHA, there is potential inhibitory action to coronary heart disease；DHA is retina normal development and plays it just Necessary to Chang Gongneng, DHA content is significantly larger than body its hetero-organization in brain and nerve fiber, and nervous function is played extremely Close important effect.(n-6) series fatty acids of Ω 6 also have important physiological function to human body, and wherein GLA is composition human body each group The structural material of biomembrane is knitted, it is also the precursor of synthesis of prostaglandins, GLA levels can not causes the disorder of internal function completely, Cause some diseases, such as diabetes, high fat of blood.

△ 6- fatty acid desaturases (Δ 6fatty acid desaturase, D6D) are a kind of multifunctional enzymes, are also PUFA biosynthesis pathways are through first step rate-limiting enzyme, and it can catalyze and synthesize GLA by substrate of LA, also can be using ALA as substrate Catalyze and synthesize SDA, it was reported that the biochemical reaction using 24C-PUFA as substrate can also be catalyzed in its mammal.

Human body only needs to take in enough LA and ALA by approach such as foods in theory, it is possible to utilize human body itself They are respectively synthesized as GLA and EPA, DHA by the enzyme systems such as D6D, but cruel reality is that human body D6D activity is very low and easy quilt A variety of environmental factors (such as the fat in meals) suppress.Even if this results in human body and has taken in enough substrate LA and ALA, but Synthesize GLA, EPA, DHA level still wretched insufficiency, it is therefore desirable to which it is not enough to supplement its directly to take in GLA, EPA, DHA etc..

GLA is as required unrighted acid in human body, and adult's daily requirement amount is about 36mg/kg.Tradition supplement Human body EPA and DHA main path are that the PUFA in deep sea fish oil, fish oil is actually that fish is enriched with by taking food algae thing etc. In vivo, this kind of mode is by the very big limitation of stock of fish amount, and source is extremely limited, and lacks sustainability, and technique is multiple Miscellaneous, high cost is expensive.It has recently been demonstrated that human body can quickly and effectively synthesize EPA using the SDA of intake, DHA is further synthesized, it is much more much better than taking in ALA effect although its effect is slightly poorer than directly intake EPA and DHA, can Effectively to solve the problem of human body EPA and DHA are not enough.Therefore, directly supplement EPA and DHA mode completely can be by supplementing SDA Mode replaced.Research is found, not only can synthesize SDA in some fungies and algae species, and a small number of plants also can SDA is synthesized, Boraginaceae (Boraginaceae) is particularly Echium (Echium) plant, a small number of primulas (Primula) and planted SDA is rich in the seed grease of thing, reason is that their D6D enzymes effectively can generate SDA in SDA, other plants using ALA Situation and D6D enzymes then require study to the utilization ratio problem of ALA substrates.

Commercially supply falls short of demand always by PUFA, and market requirement also increases constantly.Cultivate algae and fungi It is an important study hotspot to produce PUFA, and has been achieved with serious achievement, but still by complex operation, high cost, The puzzlement of price.PUFA is produced using the seed of the especially large oil crops of botanical system, compared to animal, fungi, algae The production ways such as class, have the advantages that easily to go up that scale, plantation be simple, advantage of lower cost, security are good.Although large oil plant GLA is not present in crop such as rape, soybean, peanut, cotton, sunflower, palm, olive, all higher plants are all without conjunction Into EPA and DHA key enzyme, and it is technically extremely difficult by plant metabolic engineering production EPA and DHA, but if logical Cross genetic engineering mode and import and also can then utilize large oil plant using the external source D6D genes that ALA is substrate using LA Crop produces GLA and SDA, meets market demand, and reduce cost.

Rough grass (Asperugo procumbens) is that the rough grass of Boraginaceae belongs to one to biennial sprawling herbs plant, in China The ground such as the Tibet, Xinjiang, Qinghai, Gansu, Shaanxi, Inner Mongol of China are distributed mainly on, are adapted to Qinghai-Tibet Platean, to cold, drought, Qiang Zi Outside line has very strong adaptability, seed kind oil content up to 26.65%, unrighted acid up to 90% or so, ALA, SDA, GLA content respectively reaches 36.46%, 11.75%, 5.35%, be can while provide a variety of PUFA rare new oil resource plant, Paid close attention to by industry and start to be planted by domestication.But, GLA and SDA content is not still high in natural rough seed oil, Itself GLA and SDA content is greatly improved by metabolic engineering to have important practical significance.

Cabbage type rape (Brassica napus L., 2n=38, AACC) is economy first of the big oil crops of China five Character is good, and yield is high, and oil content is high, strong stress resistance, is important edible oil and protein feeds source, is also important industry Raw material.The important content of rape seed oil quality-improving is to improve the composition and content of wherein aliphatic acid, for common edible-type vegetable seed For oil, it is concentrated mainly on reduction or eliminates erucic acid, increase the ratio of oleic acid, reduce the ratio for the ALA for being easily caused oily corruption, carry Floorboard with high oil content.By importing external source D6D genes, by the use of rape, this traditional large oil crops is used as bioreactor, next life The PUFA such as GLA, SDA are produced, are another importances of rapeseed quality improvement and molecular breeding, in the medicines and health protection field of the mankind Have great importance.

The content of the invention

In view of this, an object of the present invention is to provide rough careless △ 6- fatty acid desaturases ApD6D gene families, The second object of the present invention is to provide the recombination expression containing rough careless △ 6- fatty acid desaturases ApD6D gene families to carry Body；The third object of the present invention is to provide the transformant containing recombinant expression carrier described above；The fourth object of the present invention It is to provide rough careless △ 6- fatty acid desaturase ApD6D gene families to improve rough careless platymiscium gamma-Linolenic acid and 18 carbon Application in tetraene acid content；The fifth object of the present invention is to provide rough careless △ 6- fatty acid desaturase ApD6D genes man The application that race is rebuild in gamma-Linolenic acid and parinaric acid route of synthesis in large oil crops.

For achieving the above object, the present invention provides following technical scheme：

1st, rough careless △ 6- fatty acid desaturases ApD6D gene families, the ApD6D gene families include ApD6D1 and Two members of ApD6D2；The amino acid sequence of ApD6D1 amino acid as shown in SEQ ID NO.5 or shown in SEQ ID NO.5 Sequence substitution, lack or add at least one amino acid, and careless there is △ 6- fatty acid desaturases activity from rough Amino acid sequence；The amino acid sequence of ApD6D2 amino acid sequence as shown in SEQ ID NO.6 or shown in SEQ ID NO.6 Replace, lack or add at least one amino acid, and from the rough careless amino with △ 6- fatty acid desaturases activity Acid sequence.

It is preferred that, the amino acid sequence of the ApD6D1 is as shown in SEQ ID NO.5；The amino acid sequence of the ApD6D2 As shown in SEQ ID NO.6.

It is preferred that, the gene order of ApD6D1 nucleotides sequence as shown in SEQ ID NO.3 or shown in SEQ ID NO.3 Row are substituted, lack or added at least one nucleotides, and have the work of △ 6- fatty acid desaturases from rough careless coding The nucleotide sequence of property；The gene order of ApD6D2 nucleotides sequence as shown in SEQ ID NO.4 or shown in SEQ ID NO.4 Row are substituted, lack or added at least one nucleotides, and have the work of △ 6- fatty acid desaturases from rough careless coding The nucleotide sequence of property.

It is preferred that, the gene order of the ApD6D1 is as shown in SEQ ID NO.3, and the gene order of the ApD6D2 is such as Shown in SEQ ID NO.4.

2nd, the recombinant expression carrier containing the rough careless △ 6- fatty acid desaturases ApD6D gene families.

It is preferred that, the recombinant vector contains the sequence of ApD6D1 gene coding regions or ApD6D2 gene coding regions, described The sequence of ApD6D1 gene coding regions corresponds to shown in the bit bases of SEQ ID No.3 the 162nd~1511, and the ApD6D2 genes are compiled The sequence in code area corresponds to shown in SEQ ID No.4 the 390th~1739 bit base.

It is furthermore preferred that the recombinant expression carrier is the sequence by ApD6D1 gene coding regions or ApD6D2 gene coding regions Insert between the NapA promoters and Nos terminators of pC2301M1NPB carriers and obtain；

Or the recombinant expression carrier is the sequence insertion pYES2 of ApD6D1 gene coding regions or ApD6D2 gene coding regions XbaI the and BamHI restriction enzyme sites of plasmid and obtain；

The pC2301M1NPB carriers are prepared by following methods：The GUS bases on pBI121 are cut with HindIII and EcoRI Because expression cassette is inserted at HindIII the and EcoRI restriction enzyme sites of pCAMBIA2301 carriers, then cut with SacI and XmaI Go after gus gene to use the sequence with SacI and XmaI cohesive ends to connect, obtain pC2301M1 carriers, then carried in pC2301M1 It is connected at body HindIII restriction enzyme sites and drives bar gene expressions, MAS terminators to terminate the bar genes of expression by MAS promoters Expression cassette, obtains pC2301M1B carriers；Finally by the CaMV35S promoter seed-specific expression promoters on pC2301M1B carriers NapA is replaced and is produced pC2301M1NPB carriers.

3rd, the transformant containing the recombinant expression carrier.It is preferred that, the transformant is Agrobacterium, preferred described Agrobacterium is LBA4404.

4th, the rough careless △ 6- fatty acid desaturase ApD6D gene families improve rough careless platymiscium gamma-Linolenic acid and Application in stearidonic acid content.

5th, the rough careless △ 6- fatty acid desaturase ApD6D gene families rebuild gamma-Linolenic acid in large oil crops With the application in parinaric acid route of synthesis.It is preferred that, large oil crops be rape, soybean, peanut, cotton, Sunflower, palm or olive, it is furthermore preferred that large oil crops are rape；Most preferably, the rape is cabbage type rape.

The beneficial effects of the present invention are：The invention provides rough careless 2 member ApD6D1 of ApD6D gene families and ApD6D2 full length cDNA sequence and gDNA sequences, coding protein sequence and architectural feature, evolutionary relationship, the organ-tissue of expression Specificity etc., and confirm that ApD6D gene families member encodes the △ 6- fatty acid desaturases of double activated, using turning base , will because technology will can increase substantially the content of GLA and SDA in seed after the rough grass of ApD6D1 and ApD6D2 genes justice conversion Accumulation GLA and SDA in seed can be caused after ApD6D1 and ApD6D2 justice conversion cabbage type rapes, it was demonstrated that ApD6D genes Family has good application prospect in terms of the molecular breeding of plant GLA and SDA character, and its genetically modified plants can be applied to work Industry extracts GLA and SDA, or directly applies to health edible oil of the production rich in GLA and SDA.

Brief description of the drawings

In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below：

Fig. 1 is clone's electrophoretogram of ApD6D gene families, and wherein a is the expansion of ApD6D gene families conserved region sequence label Increase (A is ApD6D1, and B is ApD6D2), (E is ApD6D1 to the nested amplification that b is ApD6D1 and ApD6D2 5 '-RACE, and F is ApD6D2), the nested amplification (G is ApD6D1) that c is ApD6D1 3 '-RACE, d is ApD6D2 3 '-RACE nested amplification (H is ApD6D2), the full-length cDNA amplification (E and F are ApD6D1, and G, H are ApD6D2) that e is ApD6D1 and ApD6D2, M is Trans2K plus DNA marker。

(A is ApD6D1 to the nucleotide sequence and its amino acid sequence figure of coding that Fig. 2 is ApD6D1 and ApD6D2, and B is ApD6D2, initiation codon and terminator codon add square frame, and transcription initiation site and poly (A) tailings site underline (main It is runic to want site).

Fig. 3 is the network analysis figure [D6D of ApD6D1 and ApD6D2 albumen：△ 6- fatty acid desaturases；sD8D：△8- Sphingolipid desaturase (also referred to as SLD)；Angle tooth moss (Ceratodon purpureus, CAB94993.1)；Marchantia (Marchantia polymorpha, AAT85661.1)；Pasqueflower (Anemone leveillei, AAQ10731.1)；Yin Lian Flower (Anemone leveillei, AAQ10732.1)；Blackcurrant (Ribes nigrum, ADA60230.1)；Blackcurrant (Ribes Nigrum, ADA60228.1)；Arabidopsis (Arabidopsis thaliana, AAM648951)；Common Borage (Borago Officinalis, AAG43277.1)；Common Borage (Borago officinalis, AAD01410.1)；Blueweed (Echium Gentianoides, AAL23580.1)；Tobacco (Nicotiana tabacum, ABO31111.1)；Oil tea (Camellia Oleifera, ADD10720.1)；Khuskhus (Stylosanthes hamata, ABU98945.1)；Primula malacoides (Primula Vialii, AAP23036.1)；Primula malacoides (Primula vialii, AAP23035.1)].

Fig. 4 is that (A is for the fluorescence quantitative PCR detection result of ApD6D1 and the ApD6D2 transcriptional expression in rough careless each organ ApD6D1 and its allele ApD6D1 ', B are ApD6D2 and its allele ApD6D2 ').

Fig. 5 is the fat that yeast strain INVScI turns pYES2 (control), pYES2-ApD6D1 and pYES2-ApD6D2 respectively Sour GC detections figure (A：Turn pYES2 plasmid yeast strain INVScI aliphatic acid GC testing results；B：Turn pYES2-ApD6D1 plasmid ferment Mother strains INVScI aliphatic acid GC testing results；C：Turn pYES2-ApD6D2 plasmid yeast strain INVScI aliphatic acid GC detection knots Really).

Embodiment

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.It is unreceipted specific in embodiment The experimental method of condition, generally according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brookers etc. write) Described in condition, or according to the condition proposed by manufacturer.

The vegetable material that the embodiment of the present invention is used picks up from Qinghai Province Xining for rough careless (Asperugo procumbens) City Huangzhong County Shang Xinzhuan towns Di Guang villages (height above sea level about 3150m), while the rough careless mature seed of harvest is used for transgenic research.Wild cabbage type Double tenth-seededs are studied by Inst. of Oil Crops, Chinese Academy of Agriculture Zhang Xuekun in rape (Brassica napus) kind Member give, and oily other kind materials such as 821 preserve for oneself in rape variety, and planting conditions are normal test conditions.

Reagent and kit that the embodiment of the present invention is used：SMARTer^TMRACE cDNA Amplification Kit are U.S.'s Clontech Products；PrimeScript^TM RT reagent Kit with gDNA Eraser(Perfect Real Time)、Premix Ex Taq^TM II(Tli RNaseH Plus)ROX plus、DNA Ligation Kit, pMD18-T, pMD19-T, Taq archaeal dna polymerase, DNase I (RNase-free) and buffer, RNase Inhibitor, DL-2000 and λ-HindIII DNA Marker are purchased from the limited public affairs of precious biological (TaKaRa) bioengineering in Dalian Department；A small amount of plant tissue RNA extraction agents boxes, glue reclaim kit, a small amount of method plasmid extraction kits are purchased from Shanghai Hua Shunsheng Thing Technology Co., Ltd.；Restriction enzyme is purchased from MBI Fermentas companies of Lithuania；MS(Murashige&Skoog Medium, including vitamins) culture medium be Holland's Duchefa Products；DL-2000plus, Easy-Taq enzyme, The reagents such as dNTPs are purchased from golden (Transgen) Bioisystech Co., Ltd of the full formula in Beijing；X-Gluc(5-bromo-4-chloro- 3-indolyl- β-D-glucuronic acid), rifampin (Rif), streptomysin (Str), kanamycins (Kan), ammonia benzyl mould Plain (Amp), agarose, Tris, CTAB, Tris saturated phenol (pH=8.0), Tryptone, Yeast Extract, X-gal, Other biochemistry and molecular biology reagents such as IPTG, CTAB are purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd；Plant Hormone sows the companies such as rich garden supplies purchased from Shanghai.

The key instrument that the embodiment of the present invention is used：Veriti^TMMultiple temperature control PCR instrument is purchased from U.S. Applied Biosystems companies；CFX96 quantitative real time PCR Instruments are purchased from Bio-Rad companies of the U.S.；Gas chromatograph (Gas Chromatography, GC) it is Japanese Shimadzu GC-7A types；And the Other Instruments equipment of molecular biology and genetic engineering.

PCR primer used by the embodiment of the present invention and linking subsequence by Shanghai English fine horse/Invitrogen Corp., Shanghai give birth to work or Beijing six directions Hua Da company completes.

The clone of embodiment 1, rough careless D6D (ApD6D) gene family

(1) extraction of rough careless genome DNA and total serum IgE

The tender leaf of rough careless plant is taken, genome DNA is extracted using CTAB (CTAB) method, is used 1.0% agarose gel electrophoresis method and AAS evaluate the quality and concentration of nucleic acid samples.As a result show, the rough grass of extraction The integrality of genome DNA is good, and mean molecule quantity is slightly larger than λ-HindIII DNA Marker 23kb bands, RNA digestion Completely, its purity of spectrophotometry is also higher, available for PCR amplifications.

Meanwhile, with rough careless root (Ro), stem (St), leaf (Le), flower bud (Bu), flower (Fl), early stage seed (ES), mid-term seed (MS), later stage seed (LS) is material, and a small amount of plant tissue RNA extraction agents boxes are respectively adopted and extract total serum IgE, DNase I are used Remove the DNA impurity that contains in total serum IgE, the quality of electrophoresis detection total serum IgE, ultraviolet specrophotometer determine total serum IgE concentration and Purity.Electrophoresis detection shows that the total serum IgE characteristic bands obtained are clear, without obvious RNA degradeds and DNA pollution, AAS inspection The quality of test and appraisal valency also preferably, disclosure satisfy that the requirement of aftermentioned experiment, be then stored in -80 DEG C of refrigerators standby.

(2) acquisition of the total chains of cDNA first of rough grass seed RACE

Each 1 μ g mixing of total serum IgE of rough careless each stage of development is taken, using SMARTer^TM RACE cDNA Amplification Kit is operated according to its specification, respectively obtains 5'-RACE-Ready cDNA and 3'-RACE-Ready the first chains of cDNA standby.

(3) amplification of ApD6D gene families conserved region

By Vector NTI Advance 9.0 to Common Borage (Borago officinalis), blueweed (Echium Gentianoides), anemone rivularis (Anemone leveillei), high Honoka are heralded spring the D6D of plants such as (Primula vialii) Gene carries out multiple alignment, designs degenerate primer according to conservative point, sense primer is FBD6DO, and anti-sense primer is RBD6DO, tool Body sequence is as shown in table 1.Then using the μ l of 3'-RACE-Ready cDNA 1 as template, FBD6DO and RBD6DO are primer, amplification The conserved region sequence of ApD6D gene families.PCR reaction program be：94 DEG C of pre-degeneration 2min；94 DEG C of denaturation 1min, 56 DEG C are moved back Fiery 1min, 72 DEG C of extension 2min, 35 circulations；72 DEG C of extension 10min.Pcr amplification product is subjected to electrophoresis detection, as a result as schemed In 1 shown in a.As a result show, amplified 1 nearly 1.4kb specific band, then reclaimed specific band, and carry out TA clones After convert Escherichia coli, send the sequencing of 7 positive clone molecules, sequencing result carried out finding to obtain 2 independences after multiple alignment The sequence label of gene (unigene), clear length is respectively 1350bp and 1353bp (disregarding the artificial restriction enzyme site on primer), Its nucleotide sequence is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.Through nucleotide blast (http:// blast.ncbi.nlm.nih.gov/) find that they are most homologous with plant particularly Boraginaceae D6D genes after comparison, show The sequence is the sequence label of 2 members of ApD6D gene families, is respectively designated as ApD6D1 and ApD6D2.

(4) the amplification of-cDNA ends of ApD6D gene families 5 ' and 3 '-cDNA ends

After the conserved region sequence label obtained according to clone, the homology for analyzing conserved region sequence, clone is devised 4 reverse primers that ApD6D1 and ApD6D2 5 ' are held, respectively following RBD6D5-1, RBD6D5-2, RApD6D2S, RApD6D2SN, its primer sequence is as shown in table 1.Then using the μ L of 5'-RACE-Ready cDNA 1 as template, combined with primer The first time PCR that UPM and RBD6D5-1 and UPM and RApD6D2S carries out 5 '-RACE of ApD6D1 and ApD6D2 genes respectively expands Increase.PCR response procedures are：94 DEG C of pre-degeneration 2min；94 DEG C of denaturation 30sec, 62 DEG C of annealing 30sec, 72 DEG C extend 1min, 22 Circulation；72 DEG C of extension 10min.Again to expand the μ L of PCR primer 0.1 for the first time as template, with primer combine NUP and RBD6D5-2 and NUP and RApD6D2SN carries out 5 '-RACE of ApD6D1 and ApD6D2 genes nested PCR amplification respectively, and PCR response procedures are same First time PCR is expanded, and is distinguished as annealing temperature for 60 DEG C and amplification 30 is circulated.Nested PCR amplification product is subjected to electrophoresis inspection Survey, as a result as shown in b in Fig. 1.As a result show, 5 '-RACE of ApD6D1 and ApD6D2 genes generate 1 treaty 800bp's Broadband, the similar length predicted with other plant D6D gene orders.Reclaim the 5 '-RACE amplifications of ApD6D1 and ApD6D2 genes Band, cloned through TA after convert Escherichia coli, transformant is detected with PCR, as a result shown, ApD6D2 Insert Fragments exist Length polymorphism, 7 representative clone sequencings are respectively sent according to testing result, as a result show that ApD6D1 5 '-cDNA ends are net Length is 760bp, and ApD6D2 5 '-cDNA ends clear lengths are 931bp, 830bp, 817bp, 789bp, 678bp, 607bp 6 Kind, and short-movie section, in long segment sequence, showing ApD6D2 5 '-cDNA has length polymorphism, and this may be by transcribing Caused by the position difference of initiation site.

According to the conserved region sequence label for above cloning acquisition, after the homology for analyzing conserved region sequence, design clone ApD6D1 and ApD6D2 3 ' holds 4 forward primers, as follows respectively：FBD6D3-1, FBD6D3-2, FApD6D2S, FApD6D2SN, Primer sequence is as shown in table 1.Then using 3'-RACE-Ready cDNA1 μ L as template, with primer combine FBD6D3-1 and UPM and The 3 '-RACE first times PCR that FApD6D2S and UPM carries out ApD6D1 and ApD6D2 genes respectively are expanded, the same 5'- of response procedures RACE.It is template to take the μ L of first time pcr amplification product 0.1, respectively with primer combine FBD6D3-2 and NUP and FApD6D2SN with NUP carries out ApD6D1 and ApD6D2 3 '-RACE nested PCR amplification, and PCR response procedures are expanded with first time PCR, amplification production Thing carries out electrophoresis detection respectively, as a result respectively as shown in c in Fig. 1 and d.As a result show that-the RACE of ApD6D1 3 ' are generated about 600bp the band ,-RACE of ApD6D2 3 ' generate about 400bp band, with being predicted according to other plant D6D gene orders Similar length.Escherichia coli are converted after amplified production is carried out glue reclaim, cloned through TA, transformant is then entered into performing PCR detection, As a result show ,-the RACE of ApD6D2 3 ' clone has the polymorphism of length, 2 the-RACE of ApD6D1 3 ' and 4 is sent respectively The sequencing of-the RACE of ApD6D2 3 ' representational clones, as a result show ApD6D1 3 '-cDNA ends clear lengths for 519bp this 1 Kind, ApD6D2 3 '-cDNA ends clear lengths are 302,300,279, this 4 kinds of 192bp, length polymorphism may be by poly (A) Caused by the difference of tailing position.

(5) ApD6D gene families full-length cDNA and gDNA clone

According to the conserved region of ApD6D gene families, 5 '-RACE, 3 '-RACE sequencing result, can be distinguished using software ApD6D1 and ApD6D2 full length cDNA sequence are spliced into, the amplification for then having separately designed ApD6D1 full-length cDNAs and gDNA is drawn Thing combination FApD6D1 combined with the amplimer of RApD6D1, ApD6D2 full-length cDNA and gDNA FApD6D2u or FApD6D2d and RApD6D2, primer sequence is as shown in table 1.

Using the μ L of 3'-RACE-Ready cDNA 1 as template, respectively with primer combine FApD6D1 and RApD6D1, FApD6D2u is expanded with RApD6D2, FApD6D2d and RApD6D2, and PCR response procedures are：94 DEG C of pre-degeneration 2min；94℃ It is denatured 1min, 56-60 DEG C of annealing 1min, 72 DEG C of extension 2min, 35 circulations；72 DEG C of extension 10min.Amplified production carries out electrophoresis Detection, as a result as shown in e in Fig. 1.As a result the specific band for obtaining about 1.8kb, 1.9kb, 1.8kb respectively is shown, will be expanded Condition reclaimed, cloned through TA after conversion Escherichia coli, send clone to be sequenced.As a result show, ApD6D1 full-length cDNA is 1768bp, nucleotide sequence is as shown in SEQ ID NO.3；ApD6D2 full-length cDNA is respectively 1912bp or 1811bp, nucleosides Acid sequence as shown in SEQ ID NO.4 and shown in SEQ ID NO.4 the 102nd to shown in 1912, expand the total length obtained CDNA sequence is consistent with splicing sequence.

Then using the μ L of genome DNA 1 as template, with primer combine FApD6D1 and RApD6D1, FApD6D2u with RApD6D2, FApD6D2d and RApD6D2 enter performing PCR amplification, and amplified production reclaims, clones and then be sequenced through TA.As a result show, Sequence is obtained by template amplification of DNA and the full length cDNA sequence of acquisition is completely the same, illustrates that the two genes are not included Son.

Table 1, ApD6D gene family cloning primers

The bioinformatic analysis of embodiment 2, ApD6D gene families

Gene and albumen annotation, sequence alignment, ORFs (ORF) are carried out on Vector NTI Advance 9.0 Search and translation, Parameter analysis, in http:BLAST and PROTEIN C DD is carried out on //www.ncbi.nlm.nih.gov/ websites to search Rope, in http:The bioinformatics website that //bip.weizmann.ac.il/ and www.expasy.org etc. provides link is enterprising Row structural analysis of protein, in http://prodes.toulouse.inra.fr/multalin/multalin.html and http:Gene and protein sequence multiple alignment are carried out on the websites such as //www.ebi.ac.uk/clustalw/ and system occurs Credit is analysed.

(1) structure and nucleic acid profile of ApD6D gene family：

ApD6D1 is as shown in Fig. 2 result shows that ApD6D1 genes are 1768bp, and mRNA (disregards poly (A) tail for 1768bp Bar, similarly hereinafter), wherein A1 is transcription initiation site, and 5 ' UTR (non-translational region) are 161bp, there is C₁₇₆₈For poly (A) tailing position Point, 3 ' UTR are 257bp, and ORF is 1350bp.

ApD6D2 is as shown in Fig. 2 ApD6D2 genes are 1912bp, and mRNA is 1912bp, there is A₁、A₁₀₂、A₁₁₆、G₁₄₃、 A₂₅₄、A₃₂₅This 6 kinds of transcription initiation sites, most long 5 ' UTR is 389bp, there is T₁₈₀₀、C₁₈₈₈、G₁₉₀₉And T₁₉₁₂This 4 kinds of poly (A) Tailing site, most long 3 ' UTR is 173bp, and ORF is 1350bp.

(2) architectural feature of ApD6D family proteins

The ApD6D1 albumen of derivation is 449 amino acid, and amino acid sequence is as shown in SEQ ID NO.5, and molecular weight is 51.61kD, isoelectric point is 8.85, in alkalescence, and electrically charged, acid, alkaline, polarity, hydrophobic amino acid number are accounted for respectively 26.28%th, 6.90%, 8.69%, 27.62%, 38.75%, in the composition of amino acid number with leucine (11.14%) most Height, is secondly serine (8.46%), valine (8.24%), phenylalanine (7.35%), glycine (6.24%), lysine (6.01%), with cysteine content minimum (2.23%) (Fig. 2).

The ApD6D2 albumen of derivation is 449 amino acid, and amino acid sequence is as shown in SEQ ID NO.6, and molecular weight is 51.47kD, isoelectric point is 8.75, in alkalescence, and electrically charged, acid, alkaline, polarity, hydrophobic amino acid number are accounted for respectively 26.50%th, 7.13%, 8.69%, 26.50%, 40.09%, in the composition of amino acid number with leucine (11.36%) most Height, is secondly valine (8.46%), serine (8.02%), phenylalanine (7.57%), glycine (6.90%), lysine (5.35%), with cysteine content minimum (2.45%) (Fig. 2).

SignalP3.0(http://www.cbs.dtu.dk/services/SignalP/) prediction ApD6D1 and ApD6D2 Albumen does not have a signal peptide, PSORT (http://psort.hgc.jp/form.html) predict that they can be located at cytoplasma membrane Or on plastid film, TMpred (http://www.ch.embnet.org/software/TMPRED_form.html) predict them There are 4 and 5 membrane-spanning domains respectively, REP predicts that they do not have a repetitive structure, NetPhos2.0 (http:// www.cbs.dtu.dk/services/NetPhos/) predict that they have 13 and 15 potential phosphorylation sites, silk respectively Propylhomoserin phosphorylation site is slightly more, next to that threonine and Tyr phosphorylation site.

NCBI protein blast guard domain search (NCBI Conserved Domain Search) and shown, ApD6D1 The L of albumen₁₄₂-L₄₀₄With the L of ApD6D2 albumen₁₄₁-L₄₀₃Domain (cd03506) is guarded in the presence of a Delta6-FADS-like, should Conservative domain is present in fatty acid desaturases of △ 4 in vertebrate, higher plant, fungi, bacterium, the aliphatic acid of △ 5 Desaturase, the fatty acid desaturases of △ 6, the fatty acid desaturases of △ 8, the sphingolipid desaturases (SLD) of △ 8, the aliphatic acid of △ 11 In desaturase, it is to be integrated in the key enzyme that PUFA is synthesized on plasma membrane, keeps membrane fluidity, influence various biological function. The I of ApD6D1 albumen₁₀-Y₈₁With the I of ApD6D2 albumen₉-Y₈₀Domain is guarded in the presence of a Cyt-b5 [pfam00173], it is cell Pigment b5 erythrocruorins/steroids binding domain, are also a feature of typical D6D protein structures.

SOPMA(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.plPage=npsa_ Sopma.html alpha-helix accounts for 45.43% He respectively in) software prediction, the secondary structure of ApD6D1 and ApD6D2 albumen 47.44%, random coils account for 35.63% and 33.18% respectively, and extended chain accounts for 15.37% and 15.37%, β-corner point respectively 3.56% and 4.01% are not accounted for.

With SWISS-MODEL (http://swissmodel.expasy.org/interactive) can only predict The tertiary structure of ApD6D1 and ApD6D2 protein cytochrome b5 domains, respectively with 3 d structure model 1do9.1 (rabbit cells Pigment B5) there is 33.71% and 31.17% uniformity, the tertiary structure at the other positions of albumen is predicted not due to lacking model Out.

(3) homology analysis of ApD6D gene families

Comparison two-by-two on Vector NTI Advance 9.0 shows, the tetraploid rice between ApD6D1 and ApD6D2 Height, the concordance rate in full-length gene/mRNA level in-site is 78.6%, and the uniformity and similitude of encoding proteins level are respectively 79.3% and 88.0%.Uniformity between them in code area is 83.8%, and 5 ' UTR concordance rate is 55.6%, 3 ' UTR's Concordance rate is 61.3%, and code area is substantially more conservative than noncoding region, meets the feature of functional gene.

BLASTn analysis shows, the nucleotide sequence of two members of ApD6D gene families is particularly with a variety of other plants The D6D genes of Boraginaceae have significant homology.BLASTp and Phylogenetic analysis show, two members of ApD6D gene families Derive protein sequence with from plant kingdom, the D6D of mycota or sD8D/SLD (Δ 8-sphingolipid desaturase, Δ 8- sphingolipids desaturase) there is extensive homology, the D6D albumen homologies highest (Fig. 3) with Boraginaceae.

Embodiment 3, ApD6D gene families express the fluorescence quantitative PCR detection of organ specificity

With rough careless root (Ro), stem (St), leaf (Le), flower bud (Bu), flower (Fl), early stage seed (ES), mid-term seed (MS), The total serum IgE of later stage seed (LS), then is handled to eliminate DNA pollution with RNase-free DNase I by its specification.Take every Each 1 μ g of total serum IgE of individual organ, 20 μ l systems use PrimeScript^TMRT reagent Kit with gDNA Eraser enter Row reverse transcription, which obtains total chains of cDNA first, is used for gene expression detection.Fluorescence quantitative PCR detection is on CFX96 type quantitative PCR apparatus Carry out, kit isPremix Ex Taq^TMII (Tli RNaseH Plus) ROX plus, F25S is combined with primer Carry out amplification of internal standard with R25S, the combination of ApD6D1 and ApD6D2 primers be respectively FApD6D1S and RApD6D1S, fApD6D2S with RApD6D2S, primer sequence is as shown in table 2.

Fluorescence quantitative RT-RCR testing result is as shown in Figure 4.As a result show, ApD6D1 genes mesoderm growing early stage, mid-term and Predominant expression in the seed in later stage, leaf, root, flower bud, spend in also have considerable expression, have very weak expression in stem；ApD6D2 bases Because having stronger organ specificity, mainly the predominant expression in root, has weak expression in other nutrition organs such as stem, leaf, The expression of various reproductive organs is very weak.

Table 2, fluorogenic quantitative detection primer

The Yeast expression detection of embodiment 4, ApD6D gene families encoding proteins activity

According to the coding region sequence of ApD6D1 and ApD6D2 genes design Yeast expression primers F ApD6D1Y, RBD6D1Y, FApD6D2Y, RBD6D2Y, for convenience of follow-up connection, introduced respectively at 5 ' ends of sense primer and anti-sense primer BamH I and XbaI enzyme cutting site, specific primer sequence is as shown in table 3.With primer combination FApD6D1Y and RBD6D1Y, FApD6D2Y with RBD6D2Y expands 1370bp the and 1370bp coding region sequences of ApD6D1 and ApD6D2 genes respectively, is then carried out with pMD19-T TA clones obtain pMD19-T-ApD6D1Y and pMD19-T-ApD6D2Y respectively, after proving that sequence is not mutated through sequencing, under progress One step yeast vector is built.

Table 3, ApD6D gene family Yeast expression primers

Then with complete double digestion plasmid pYES2, the pMD19-T-ApD6D1Y of restriction enzyme XbaI and BamHI and PMD19-T-ApD6D2Y, reclaims pYES2 wire skeleton carrier and ApD6D1, ApD6D2 target gene fragment, then by gene Fragment respectively with carrier framework T₄DNA ligase is attached, and converts Escherichia coli, obtains ApD6D1 and ApD6D2 genes Yeast expression carrier pYES2-ApD6D1 and pYES2-ApD6D2, then extracting the plasmid of recombinant vector is used for saccharomyces cerevisiae Conversion and its induced expression.

Prepared by Wine brewing yeast strain INVScI competent cell, plasmid conversion is entered using the method for Invitrogen companies OK.After three kinds of plasmid transformed yeast INVScI of pYES2-ApD6D1, pYES2-ApD6D2, pYES2 (control, CK), chosen training Support base SD-Ura screening, respectively obtain transformant bacterium solution, with primer combination FApD6D1Y and RBD6D1Y, FApD6D2Y with RBD6D2Y enters performing PCR detection to pYES2-ApD6D1 transformed bacteria solutions and pYES2-ApD6D2 transformed bacteria solutions respectively, and electrophoresis is obtained greatly About 1.4kb specific amplified band, it is consistent with predicting the outcome, and control plasmid pYES2 is turned with this two groups of primer combinations simultaneously Change bacterium solution and enter performing PCR detection, do not find the specific amplified band of purposeful clip size.

Using LiAc- galactolipins abductive approach come to carrying pYES2-ApD6D1 plasmids, pYES2-ApD6D2 plasmids, control Plasmid pYES2 Yeast engineering bacterium strain is induced, and is cultivated after addition LA substrates, then collects the bacterium of three kinds of engineered strains Body carries out methyl esterification of fatty acid, and fatty acid component and content are analyzed with gas chromatograph.As a result show, turn pYES2's A kind of this PUFA of ALA is only detected in control yeast, and the yeast for turning pYES2-ApD6D1 and pYES2-ApD6D2 is detected Three kinds of PUFA, have more two kinds of PUFA than control --- GLA and SDA, it was demonstrated that the ApD6D gene families cloned of the present invention two into Member encodes double activated Δ 6- fatty acid desaturases, that is, is catalyzed LA formation GLA and catalysis ALA formation SDA (Fig. 5).

Embodiment 5, overexpression ApD6D1 and ApD6D2 improve the GLA contents of rough grass seed

(1) transformation pCAMBIA2301 carriers obtain the plant expression vector pC2301M1NPB of seed specific expression

PCAMBIA2301, pBI121, pFGC5941 are wide variety of business plant expression vector, and the present invention is at it On the basis of, build and obtain the plant expression vector pC2301M1NPB of seed specific expression：

A, gus gene expression cassette (3038bp, the CaMV35S cut using HindIII and EcoRI double digestions on pBI121 Promoter driving gus gene code area, is followed by Nos terminators), the HindIII of restructuring to pCAMBIA2301 multiple cloning sites area Between EcoRI, 14602bp plant expression vector pC2301G is obtained.

B, synthetic DNA single-stranded 2301M1F and 2301M1R, particular sequence are shown in Table 4, and 2301M1F and 2301M1R is become Property is simultaneously annealed into complementary double-strand adapter 2301M1, and the end of this adapter is respectively provided with SacI and XmaI cohesive end.Adopt With SacI and XmaI double digestion pC2301G, carrier framework is reclaimed after the gus gene for cutting 1.9kb, then with 2301M1 adapters Restructuring is attached, 12747bp plant expression vector pC2301M1 is obtained.

C, design primers F PbarT and RPbarT, particular sequence are shown in Table 4.Then using pFGC5941 plasmids as template, PCR amplifications 1224bp bar expression casettes (MAS promoters driving bar gene coding regions, be followed by MAS terminators), TA clones And be sequenced determination it is errorless after, the expression cassette is subcloned with HindIII single endonuclease digestions and is inserted into pC2301M1 HindIII point of contacts Place, obtains 13965bp plant expression platform carrier pC2301M1B.

D, design primers F NAP and RNAP, particular sequence are shown in Table 4.Then with oil 821 in cabbage type rape variety Blade genome DNA is material, and PCR amplifications obtain 1146bp seed-specific expression promoter NapA (GenBank accession number：J02798), after TA clones and sequence verification, using PstI and XbaI double digestions, replaced with NapA promoters CaMV35S promoters in pC2301M1B, obtain 14252bp plant expression vector pC2301M1NPB.

Table 4, the primer for building overexpression ApD6D1 and ApD6D2 expression vector and linking subsequence

(2) acquisition of the structure and engineered strain of ApD6D gene families plant expression vector

Plant expression vector construction is carried out using with Yeast expression carrier identical ApD6D1 and ApD6D2 genetic fragment.

PC2301M1NPB plasmids are extracted, double digestion open loop are carried out with BamHI and SpeI, glue reclaim obtains the carrier of wire Skeleton, restructuring is then attached with the ApD6D1 and ApD6D2 of XbaI and BamHI digestions respectively, and (XbaI and SpeI is same tail Enzyme), respectively obtain seed specific type plant expression vector pC2301M1NPB-ApD6D1 (the abbreviation pCN- of ApD6D1 genes ApD6D1,15605bp) and ApD6D2 genes seed specific type plant expression vector pC2301M1NPB-ApD6D2 (referred to as PCN-ApD6D2,15605bp), bacillus coli DH 5 alpha competent cell is converted, to the transformant list of Kan resistance LB plate screenings Clone is respectively adopted primer combination FNAP and RBD6D1Y and FNAP and RBD6D2Y and enters performing PCR detection, and positive clone molecule is using freezing Melt method conversion agrobacterium tumefaciens lba4404, the transformant monoclonal of triple resistances (Kan+Str+Rif) LB plate screenings is carried out PCR detects that positive clone molecule is preserved as engineered strain, for Plant Transformation.

(3) agriculture bacillus mediated ApD6D gene families convert rough grass

All tissue cultures operations are carried out under the conditions of the Plant Tissue Breeding of standard, between superclean bench, culture, are tamed and dociled Clean rank between change is respectively 100 grades, 10000 grades and 100000 grades, and corresponding reagent, material, vessel carry out nothing by code Bacterium is handled.Rough careless mature seed volume fraction is uses aseptic water washing 3 times after 75% ethanol surface sterilization 1min, then With mercury chloride 5~10min of soaking disinfection that mass fraction is 0.1%, sterilized water is repeatedly rinsed well, is then inoculated in MS solids Culture medium [MS powder 2.21g/L+Phytagel 2.6g/L+ sucrose 30.0g/L, pH5.8, autoclave moist heat sterilization；It is not added with Phytagel is fluid nutrient medium], 25 DEG C, 2000Lux illumination, 14h/d photoperiods cultivate (condition of culture between tissue culture below It is identical with this in addition to especially person is indicated).The hypocotyl for cutting seedling age 8d or so aseptic seedling is cut into and is about the small of 0.5~1.0cm Section, is inoculated into pre- training culture medium MSp [MS culture medium+0.5mg/L methyl α-naphthyl acetates (NAA)+0.2mg/L 2,4- dichlorophenoxyacetic acids (2,4-D)+0.8mg/L 6- benzylaminopurine (KT)+1.0mg/L 6- benzylaminopurines (6-BA)] preculture 3d.

The engineered strain of -80 DEG C of preservations is taken in the LB liquid added with 100mg/L Kan+20mg/L Str+40mg/L Rif In 28 DEG C, 250r/min 1~2d of shaken cultivation in culture medium, Agrobacterium is set to grow to logarithmic phase, switching is cultivated once, in Thalline is collected by centrifugation in 5000rpm, 10min room temperature, with dip-dye culture medium MSm [MSp+100 μm of ol/L acetosyringone of liquid (AS) bacterial concentration] is adjusted to OD₆₀₀About 0.5 or so, as dip dyeing liquid for shell.

By 3~5min in the hypocotyl section immersion dip dyeing liquid for shell after preculture, intermittence is gently swayed, then by hypocotyl section Unnecessary bacterium solution is blotted on sterilizing paper, is inoculated into common training culture medium MSc [MSp+100 μm of ol/L AS], 23.5 DEG C of light cultures 48h.With sterilizing liquid culture medium MSk [liquid MSp+500mg/L Cef] washing by soaking 3 × 10min of explant, inhaled with sterilizing paper Dry surface liquid, be transferred to induction screening and culturing medium MSi [MSp+500mg/L Cef+50.0mg/L Kan] culture, about 2 weeks after Generation 1 time, to growing macroscopic kanamycin-resistant callus tissue, then it is transferred to the differential medium LSd [salt and micro-element of LS minimal mediums + 0.5mg/L NAA+0.5mg/L 6-BA+1.0mg/L zeatin (ZT)+500mg/L Cef+50.0mg/L Kan+ sucrose 30.0g/L+Phytagel 2.6g/L, pH5.8, autoclave moist heat sterilization] in culture more than 14d, evoked callus differentiates Budlet and grow unrooted seedling, then be transferred to root media MSr [MS solid medium+0.3mg/L heteroauxins (IAA)+ 1.0mg/L KT] culture to growing flourishing root system, the seedling after taking root after domestication, be transplanted to containing the perlite that sterilizes, vermiculite, (mass ratio is 1 to turf earth mixtures:1:1) in basin alms bowl, it is managed by greenhouse pot culture.

Finally, pCN-ApD6D1, pCN-ApD6D2 are converted after rough grass, respectively acquisition 22,27 plants of regeneration plants；Regeneration is planted 200mg/L Basta solution detection resistance is added dropwise in strain blade, and the leaf block for cutting regeneration plant carries out GUS histochemical stains, and Primer combination FNAP and RBD6D1Y and FNAP and RBD6D2Y progress is respectively adopted in the blade genome DNA for extracting regeneration plant PCR detects, as a result shows to obtain 13 respectively, 11 plants of triple positive transgenic plant.

Comprehensive biology and agronomy observation are carried out to transgenic positive plant, the transfer-gen plant of above-mentioned kind of carrier is not There is obvious side effect, biology and agronomy character do not have notable difference with non-transgenic reference plant.Using gas-chromatography Method careless mature seed rough to transgenosis carries out aliphatic acid GC analyses, and relative quantification is carried out using area normalization method, finds and the moon Property control compare, as a result as shown in table 5.As a result show, in pCN-ApD6D1, pCN-ApD6D2 rough careless mature seed of transgenosis The ratio that GLA and SDA account for total fatty acids is improved largely, it was demonstrated that two member genes of ApD6D gene families are in rough grass Middle overexpression, can greatly improve the content of GLA and SDA in seed, reach the purpose of metabolic engineering molecular breeding.

Table 5, pCN-ApD6D1, pCN-ApD6D2 turn rough careless fatty acid constituents (%)

Self propagated is carried out to the contemporary plant of transgenosis, Basta is added dropwise using the blade as the contemporary plant of transgenosis Resistance detecting and GUS dyeing detections, Screening and Identification go out pCN-ApD6D1, pCN-ApD6D2 rough careless homozygous lines of transgenosis, GC Detection shows that GLA and SDA accounts for total fatty acid content respectively more than 10% and 25% in optimal strain seed, illustrates transgene traits Heredity can be stablized and homozygosis offspring strain is more preferable than the transgene traits of contemporary (heterozygosis) individual plant.

Embodiment 6, heterogenous expression ApD6D1 and ApD6D2 accumulate GLA and SDA in rape seed

All tissue cultures operations are carried out under the conditions of the Plant Tissue Breeding of standard, between superclean bench, culture, are tamed and dociled Clean rank between change is respectively 100 grades, 10000 grades and 100000 grades, and corresponding reagent, material, vessel carry out nothing by code Bacterium is handled.In cabbage type rape typical species the seed of double No. 10 with volume fraction to be used after 75% ethanol surface sterilization 1min Aseptic water washing 3 times, then soaks 20min, sterilized water is repeatedly rinsed well, then with mass fraction for 5% sodium hypochlorite Being inoculated in MS solid mediums, [MS powder 4.41g/L+Phytagel 2.6g/L+ sucrose 30.0g/L, pH5.8, autoclave is damp and hot to go out Bacterium；It is not added with Phytagel as fluid nutrient mediums], 25 DEG C, 2000Lux illumination, 16h/d photoperiods cultivated (between tissue culture below Condition of culture is identical with this in addition to especially person is indicated).Cut seedling age 8d or so aseptic seedling hypocotyl be cut into be about 0.5~ 1.0cm segment, is inoculated into pre- training culture medium MSp [MS culture medium+1.0mg/L 6-BA+1.0mg/L 2,4-D] preculture 3d.

The engineered strain of -80 DEG C of preservations is taken in the LB liquid added with 100mg/L Kan+20mg/L Str+40mg/L Rif In 28 DEG C, 250r/min 1~2d of shaken cultivation in culture medium, Agrobacterium is set to grow to logarithmic phase, switching culture is once. Thalline is collected by centrifugation in 5000rpm, 10min room temperature, with dip-dye culture medium MSm [MS fluid nutrient mediums+1.0mg/L 2,4-D+ 1.0mg/L 6-BA+100 μm ol/L AS] bacterial concentration is adjusted to OD₆₀₀About 0.5 or so, as dip dyeing liquid for shell.

By 5~10min in the hypocotyl section immersion dip dyeing liquid for shell after preculture, intermittence is gently swayed, then by hypocotyl Section blots unnecessary bacterium solution on sterilizing paper, is inoculated into common training culture medium MSc [MS solid medium+2.0mg/L 6-BA+0.5mg/ L NAA] in, 23.5 DEG C of light culture 48h.With sterilizing liquid culture medium MSk [MS fluid nutrient medium+1.0mg/L 2,4-D+ 1.0mg/L 6-BA+500mg/L Cef] washing by soaking 3 × 10min of explant, blots surface liquid with sterilizing paper, is transferred to and lures Lead screening and culturing medium MSi [MS solid medium+1.0mg/L 6-BA+1.0mg/L 2,4-D+500mg/L Cef+12.5mg/L Basta+6mg/L AgNO₃] culture, about 2 weeks subcultures 1 time to growing macroscopic kanamycin-resistant callus tissue, then are transferred to differentiation and cultivate Base MSd [MS solid medium+4.0mg/L 6-BA+2.0mg/L ZT+5.0mg/L AgNO₃+500mg/L Cef+12.5mg/L Basta] middle culture more than 14d, evoked callus differentiates budlet, then is transferred to stem differential medium MSs (MS solid cultures Base+3.0mg/L 6-BA+2.0mg/L ZT+500mg/L Cef+12.5mg/L Basta) culture is to growing small stem, then be transferred to Cultivated extremely in long shoot culture medium MSe (MS solid medium+0.05mg/L 6-BA+500mg/L Cef+12.5mg/L Basta) Long complete unrooted seedling, then root media MSr [MS solid medium+2mg/L NAA] cultures are transferred to growing flourishing root System, the seedling after taking root is transplanted to containing sterilizing perlite, vermiculite, (mass ratio is 1 to turf earth mixtures after domestication:1:1) Basin alms bowl in, be managed by greenhouse pot culture.

Finally, pCN-ApD6D1, pCN-ApD6D2 are converted in cabbage type rape after double No. 10, obtain 18 respectively, 16 plants again Raw plant；Regeneration plant blade is added dropwise 200mg/L Basta solution detection resistance, the leaf block for cutting regeneration plant carries out GUS Histochemical stain, and extract the blade genome DNA of regeneration plant be respectively adopted primer combination FNAP and RBD6D1Y and FNAP and RBD6D2Y enter performing PCR detection, as a result show to obtain 9 respectively, 11 plants of triple positive transgenic plant.

Comprehensive biology and agronomy observation are carried out to transgenic positive plant, the transfer-gen plant of above-mentioned kind of carrier is not There is obvious side effect, biology and agronomy character do not have notable difference with non-transgenic reference plant.Using gas-chromatography Method carries out aliphatic acid GC analyses to transgene rape mature seed, finds compared with nontransgenic plants (negative control), pCN- ApD6D1, pCN-ApD6D2 transgene rape mature seed have all had more two new PUFA crests-GLA and SDA, using face Product normalization method, which is calculated, learns that the percentage that GLA accounts for total fatty acids is respectively 6.25%~8.75%, 5.83%~8.94%, The percentage that SDA accounts for total fatty acids is respectively 4.12%~5.80%, 4.83%~5.68%, it was demonstrated that started using seed specific Two member gene's heterogenous expressions in rape of son driving ApD6D gene families, can mediate accumulation GLA and SDA in seed, The purpose of metabolic engineering molecular breeding is reached.

Self propagated is carried out to the contemporary plant of transgenosis, Basta is added dropwise using the blade as the contemporary plant of transgenosis Resistance detecting and GUS dyeing detections, Screening and Identification go out pCN-ApD6D1, pCN-ApD6D2 excellent strain of transgene rape homozygosis, GC detections show in optimal strain seed that GLA accounts for total fatty acid content and reach that 14.2%, SDA accounts for total fatty acid content and reached 9.6%, illustrate that transgene traits can stablize the transgene traits of contemporary (heterozygosis) individual plant of hereditary and homozygosis offspring strain ratio more It is good.

Other explanations

Here the following change on especially statement, application form also all necessarily belongs to the spirit and scope of the present invention and covered Lid：

1st, the gene and its fragment in the present invention, in addition to nucleotide sequence listed in sequence table, also including from In the sequence of rough careless other D6D allele, including come from rough careless other subspecies, the ecotype, kind, cenospecies D6D gene orders, also the D6D sequences highly similar gene order in the nearly edge species including coming from rough grass category to the present invention (the horizontal concordance rate in ORF full codings area is more than 97%).

2nd, the gene and its fragment in the present invention, in addition to nucleotide sequence listed in sequence table, also include and it Have any nucleotide sequence of more than 98.00% uniformity in continuous 80bp and the above.

3rd, the gene and its fragment in the present invention, in addition to nucleotide sequence listed in sequence table, also including coding Albumen has the artificial synthesized nucleotide sequence of 100% concordance rate with SEQ ID No.5 or SEQ ID No.6.

4th, the present invention in gene and its fragment, except lifted in preferred embodiment be used for it is rough grass, cabbage type rape with Outside, other species can also be applied to.

5th, the present invention in gene and its fragment, except lifted in preferred embodiment justice conversion carry out overexpression and Outside heterogenous expression, the technologies such as antisense RNA, RNA interference, genome editor (ZFN, TALEN, CRISPR-Cas) can also be used, To mediate the silence of the endogenous D6D genes of rough careless platymiscium or gene family, GLA and SDA synthesis is prevented, rough careless platymiscium is improved LA and ALA content.

6th, the gene and its fragment in the present invention, except the use pC2301M1NPB lifted in preferred embodiment is planted Beyond thing expression vector establishment, plant expression vector construction can also be carried out using other carriers；Carrier structure in the present invention Thing is built, except the improvement leaf disk method for the use Agrobacterium tumefaciens strain LBA4404 mediations lifted in preferred embodiment carries out heredity Beyond conversion, it would however also be possible to employ other methods carry out Genetic Transformation in Higher Plants.

7th, the effect parameter value acquired by application example of the present invention only refers to is carried out using the kind in example as explant The result of transgeneic procedure, if turned using other kinds (such as superelevation oleic acid, superelevation linolenic acid variety) as explant Genetic manipulation, can obtain more excellent operating effect parameter value.

Finally illustrate, above example is only to illustrate technical scheme, but be not limited to this. Although by referring to the preferred embodiments of the present invention, invention has been described, and one of ordinary skill in the art should Work as understanding, various changes can be made to it in the form and details, limited without departing from appended claims Fixed the spirit and scope of the present invention.

Claims (8)

1. rough careless △ 6- fatty acid desaturases ApD6D Gene As pD6D1 and ApD6D2, it is characterised in that：The ammonia of the ApD6D1 Base acid sequence is as shown in SEQ ID NO.5；The amino acid sequence of the ApD6D2 is as shown in SEQ ID NO.6.

2. rough careless △ 6- fatty acid desaturases ApD6D Gene A pD6D1 and ApD6D2 according to claim 1, it is special Levy and be：The gene order of the ApD6D1 is as shown in SEQ ID NO.3, the gene order such as SEQ ID of the ApD6D2 Shown in NO.4.

3. the recombinant expression carrier containing rough careless △ 6- fatty acid desaturases ApD6D genes described in claim 2.

4. recombinant expression carrier according to claim 3, it is characterised in that：The recombinant vector contains ApD6D1 genes volume Code area or the sequence of ApD6D2 gene coding regions, the sequence of the ApD6D1 gene coding regions correspond to SEQ ID No.3 the 162nd Shown in~1511 bit bases, the sequence of the ApD6D2 gene coding regions corresponds to SEQ ID No.4 the 390th~1739 alkali Shown in base.

5. recombinant expression carrier according to claim 4, it is characterised in that：The recombinant expression carrier is by ApD6D1 bases Because the sequence of code area or ApD6D2 gene coding regions insert pC2301M1NPB carriers NapA promoters and Nos terminators it Between and obtain；

Or the recombinant expression carrier is the sequence insertion pYES2 plasmids of ApD6D1 gene coding regions or ApD6D2 gene coding regions XbaI and BamHI restriction enzyme sites and obtain；

The pC2301M1NPB carriers are prepared by following methods：The GUS bases on pBI121 are cut with HindIII and EcoRI Because expression cassette is inserted at HindIII the and EcoRI restriction enzyme sites of pCAMBIA2301 carriers, then with SacI and XmaI cuts the sequence connection used after GUS genes with SacI and XmaI cohesive ends, obtains pC2301M1 carriers, then It is connected at pC2301M1 carrier HindIII restriction enzyme sites by MAS promoters driving bar gene expressions, MAS terminators The bar expression casettes of expression are terminated, pC2301M1B carriers are obtained；Finally the CaMV35S on pC2301M1B carriers is opened Mover seed-specific expression promoter NapA is replaced and is produced pC2301M1NPB carriers.

6. the transformant containing any one of the claim 3-5 recombinant expression carriers.

7. any one of claim 1-2 rough careless △ 6- fatty acid desaturases ApD6D the Gene As pD6D1 and ApD6D2 Application in rough careless platymiscium γ-leukotrienes and stearidonic acid content is improved.

8. any one of claim 1-2 rough careless △ 6- fatty acid desaturases ApD6D the Gene As pD6D1 and ApD6D2 The application rebuild in rape γ-leukotrienes and parinaric acid route of synthesis.