CN104450743A - Application of eggplant anthocyanidin synthesis associated gene SmMYB1 in cultivation of solanum aethiopicum group gilo variety - Google Patents
Application of eggplant anthocyanidin synthesis associated gene SmMYB1 in cultivation of solanum aethiopicum group gilo variety Download PDFInfo
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- CN104450743A CN104450743A CN201410797154.8A CN201410797154A CN104450743A CN 104450743 A CN104450743 A CN 104450743A CN 201410797154 A CN201410797154 A CN 201410797154A CN 104450743 A CN104450743 A CN 104450743A
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Abstract
The invention discloses application of eggplant anthocyanidin synthesis associated gene SmMYB1 in cultivation of solanum aethiopicum group gilo variety. Solanum aethiopicum group gilo explants are converted by using agrobacterium containing recombinant plasmid pBI121:: SmMYB1 to obtain transgenosis solanum aethiopicum group gilo plants; petals, anthers, stems, fruits and leaves of the transgenosis solanum aethiopicum group gilo are purple; the solanum aethiopicum group gilo can be cultivated by being taken as ornamental plants; in addition, the pulp of the transgenosis eggplants contains a certain quantity of components such as anthocyanin beneficial to a human body; the risk of suffering from cancer and cardiovascular diseases can be reduced; the solanum aethiopicum group gilo variety has good market prospects and economic values.
Description
Technical field
The invention belongs to genetically engineered field, be specifically related to eggplant anthocyanidin synthesis related gene SmMYB1 and cultivating the application in purple African red eggplant kind.
Background technology
Cultivation eggplant, as the farm crop of extensively cultivation in a kind of world wide, is extensively by popular with vegetables always, in recent years along with the development of flower gardening, and the African red eggplant of the same race with cultivation eggplant, the also attention being subject to breeder gradually.African red eggplant not only has certain sight, has also been called the important germ plasm resource of plant breeding because having high disease-resistant characteristic.Anthocyanogen is as a kind of water-soluble flavonoid class pigment be extensively present in plant materials, increasing research proves that anthocyanogen is not only played a positive role in plant reply poor environment (uv irradiating, low temperature, arid and pathogenic bacteria invasion) time, while anthocyanogen also have certain help to the treatment of a large amount of chronic disease (cardiovascular diseases and tumour) of the mankind.Great mass of data confirms, the higher food of regular edible anthocyanin content has important value for the health care of human body.Eggplant is as a kind of fruit that the frequency of occurrences is higher on people's dining table or vegetables, and the eggplant of cultivation naturally only has in epidermis and have accumulated a small amount of anthocyanogen, and the fruit of African red eggplant is not then containing anthocyanogen.By engineered method, the transcription factor process LAN of regulation and control eggplant fruit anthocyanogen synthesis in African red eggplant, is obtained African red eggplant plant and the fruit of purple by us.The color and luster of eggplant fruit is the major issue that breeder pays close attention to always, and we are by engineered method, not only changes the color of whole eggplant plant, also changes the color of pulp simultaneously to a certain extent.Traditional breeding method cycle is long, cost is many and effect unstable.In recent years, to develop rapidly with transgenic technology day by day perfect along with engineered, and genetic engineering technique shows great potentiality in cultivation elite crop variety.In the middle of the improvement that genetic engineering technique will certainly more and more be widely used in Eggplant Varieties and breeding.
Summary of the invention
In view of this, an object of the present invention is to provide eggplant anthocyanidin synthesis related gene SmMYB1 cultivating the application in purple African red eggplant kind; Two of object of the present invention is to provide the application in eggplant anthocyanidin synthesis related gene SmMYB1 raising African red solanberry meat in anthocyanin content; Three of object of the present invention is the method for cultivating purple African red eggplant kind.
For achieving the above object, the invention provides following technical scheme:
1, eggplant anthocyanidin synthesis related gene SmMYB1 is cultivating the application in purple African red eggplant (Solanum aethiopicum groupGilo) kind, and the nucleotide sequence of described eggplant anthocyanidin synthesis related gene SmMYB1 is as shown in SEQ ID NO.7.
2, eggplant anthocyanidin synthesis related gene SmMYB1 improves the application in African red solanberry meat in anthocyanin content, and the nucleotide sequence of described eggplant anthocyanidin synthesis related gene SmMYB1 is as shown in SEQ ID NO.7.
3, the method for purple African red eggplant kind is cultivated, comprise the steps: to utilize containing the Agrobacterium-mediated Transformation African red eggplant explant of recombinant plasmid pBI121::SmMYB1, then containing the sucrose of 30g/L, 1mg/L indolylacetic acid, 1.75mg/L zeatin, pH be in the MS solid medium of 5.8 in 28 ± 2 DEG C, dark under Dual culture 48 hours; Proceed to the sucrose containing 30g/L, 1.0mg/L indolylacetic acid, 1.75mg/L zeatin, 500mg/L Pyocianil and 50mg/L kantlex again, pH be in the MS solid medium of 5.9,28 ± 2 DEG C, photoperiod 16h/d, be cultured under intensity of illumination 1000-2000lx condition and grow regeneration bud; Regeneration bud is cut when bud grows to 3-5cm, proceed to the sucrose containing 30g/L, 250mg/L Pyocianil and 50mg/L kantlex, pH is in the MS solid medium of 5.9,28 ± 2 DEG C, photoperiod 16h/d, be cultured under intensity of illumination 1000-2000lx condition and take root, obtain purple African red eggplant kind.
Preferably, described Agrobacterium is LBA4404.
Beneficial effect of the present invention is: the recombinant expression vector containing eggplant anthocyanidin synthesis related gene SmMYB1 is passed through Agrobacterium-medialed transformation by the present invention, after proceeding to African red eggplant in gained transgenosis African red eggplant positive plant SmMYB1 gene comparatively nontransgenic plants have significant expression, and whole African red eggplant plant presents the eggplant fruit of purple and purple; Compared with traditional breeding way, the present invention adopts genetic engineering technique, efficient and Absorbable organic halogens adds the anthocyanin content of African red eggplant plant hereditarily, the pattern breeding that positive exploration and ornamental plant have been made in breed improvement for African red eggplant provides reference and reference, has good market outlook and economic worth.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:
Fig. 1 is the phenotype comparison diagram of transgenosis African red eggplant and wild-type African red eggplant.
Fig. 2 is that PCR detects the expression of NPTII reporter gene in transgenosis African red eggplant plant (M is Maker, CK-is negative control, and CK+ is positive control, and CK-is blank, and N is negative control, and 1-3 is transgenosis eggplant plant).
Fig. 3 is the expression amount of SmMYB1 gene in transgenosis African red eggplant positive plant (WT is non-transgenic eggplant, T#1, and 2,3,4 is the different positive strains of transgenosis eggplant).
Fig. 4 is transgenosis African red solanberry skin and flesh genotype.
Fig. 5 is the measurement result of transgenosis African red solanberry meat anthocyanin content.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, the such as condition described in Molecular Cloning: A Laboratory guide (third edition, the work such as J. Pehanorm Brooker), or according to the condition that manufacturer advises.
The African red eggplant kind used in the embodiment of the present invention is " Solanum aethiopicum group Gilo ", for academy of agricultural sciences of Chongqing City vegetables and flowers institute give.PGEM-T Easy carrier is Promega Products, and pBI121 carrier, bacillus coli DH 5 alpha, intestinal bacteria " assistance " bacterium (being called for short helper) containing travelling plasmid pRK2013 and agrobacterium tumefaciens lba4404 are preserved by this laboratory; RNA extraction test kit, RNA PCR Kit (AMV) Ver.3.0 test kit, DNA extraction kit Universal Genomic DNA Extraction Kit Ver.3.0, connection test kit DNA Ligation Kit Ver.2.0 (T4DNA ligase enzyme/SolutionI), restriction enzyme, Taq archaeal dna polymerase, PrimeSTAR HS archaeal dna polymerase are Dalian TaKaRa Products; DNA purification kit (centrifugal column type) is TIANGEN Products; All the other reagent are analytical reagent.
One, containing the agrobacterium mediation converted African red eggplant of recombinant plasmid pBI121::SmMYB1
In the present embodiment, the preparation of recombinant plasmid pBI121::SmMYB1 is the Chinese patent of 103966235A see publication number, and wherein the nucleotide sequence of eggplant anthocyanidin synthesis related gene SmMYB1 is as shown in SEQ ID NO.7.The Agrobacterium tumefaciens attachment containing pBI121::SmMYB1 is obtained, called after pBI121::SmMYB1-LBA4404 after recombinant plasmid pBI121::SmMYB1 being transformed LBA4404.Then being inoculated in by pBI121::SmMYB1-LBA4404 containing massfraction is the agar of 1.2%, 50mg/L Rifampin, 500mg/L Streptomycin sulphate and 50mg/L kantlex, pH is on the YEB solid medium of 7.2, 2-3 days are activated to growing single bacterium colony under 28 ± 2 DEG C of dark conditions, picking list bacterium colony, with containing 50mg/L Rifampin, 500mg/L Streptomycin sulphate and 50mg/L kantlex, pH is the YEB liquid nutrient medium 20mL of 7.2, at 28 ± 2 DEG C, enlarged culturing 1.5 days under 200rpm condition, again gained bacterium liquid is contained 50mg/L Rifampin for 1:100 accesses by volume, 500mg/L Streptomycin sulphate and 50mg/L kantlex, pH is in the YEB liquid nutrient medium of 7.2, at 28 ± 2 DEG C, under 200rpm condition, enlarged culturing is to OD
600be 1.8 ~ 2.0, then centrifugally abandon supernatant for 28 ± 2 DEG C, thalline fresh YEB liquid nutrient medium washing, then with the sucrose, the pH that containing massfraction are 3% be 5.9 MS substratum 100mL resuspended, obtain Agrobacterium engineering bacteria liquid.
With the alcohol-pickled African red eggplant seed 2min that volume fraction is 75%, aseptic water washing 3 times; Sterilized saturated Na
3pO
4soak seed 20min, and thermal agitation frequently, aseptic water washing 3 times; The NaClO aqueous solution soaking seed 10min of 1% (V/V), aseptic water washing 7 times; Sterilized water soaks seed 4h, sows on MS solid medium, and illumination box 28 DEG C (16h illumination)/18 DEG C (8h is dark) is cultivated until cotyledon flattens, and cuts the cotyledon flattened and namely obtains eggplant explant.Cut by the cotyledon of eggplant explant, in MS liquid nutrient medium, soak 1h, filter paper blots residual liquid substratum and is placed on the sucrose that massfraction is 3%, massfraction is the agar of 0.8%, 1mg/L indolylacetic acid, 1.75mg/L zeatin, pH is in the MS solid medium of 5.9, and 28 DEG C (16h illumination)/18 DEG C of (8h is dark) preculture 1d are placed in Agrobacterium engineering bacteria liquid and contaminate after 15 minutes, put back to the sucrose that massfraction is 3%, massfraction is the agar of 0.8%, 1mg/L indolylacetic acid, 1.75mg/L zeatin, pH is in the MS solid medium of 5.9, Dual culture 48 hours under 25 ± 2 DEG C of dark conditions, then to proceed to containing massfraction be the sucrose of 3%, massfraction is the agar of 0.8%, 1.0mg/L indolylacetic acid, 1.75mg/L zeatin, 500mg/L Pyocianil and 50mg/L kantlex, pH is in the MS solid medium of 5.9, at 25 ± 2 DEG C, photoperiod 16h/d, be cultured under intensity of illumination 1000-2000lx condition and grow callus and resistant buds, the resistant buds of long 2-3cm is cut, sucrose, massfraction that to proceed to containing massfraction be 3% be 0.8% agar, 250mg/L Pyocianil and 50mg/L kantlex, pH be in the MS solid medium of 5.9,25 ± 2 DEG C, photoperiod 16h/d, be cultured under intensity of illumination 1000-2000lx condition and take root, obtain transgenosis African red eggplant plant, as shown in Figure 1.
Two, the screening of transgenosis African red eggplant positive plant
1.PCR detects the expression of NPTII reporter gene in transgenosis African red eggplant plant
According to the NPTII reporter sequences in carrier pBI121::SmMYB1, design following specific detection primer:
NPTII-F：5'-gacaatcggctgctctga-3'(SEQ ID No.1)；
NPTII-R：5'-aactccagcatgagatcc-3'(SEQ ID No.2)。
Get transgenosis African red eggplant plant respectively and non-ly turn African red eggplant plant, extract genomic dna, again with gained genomic dna be template, sequence shown in SEQ ID No.1 and SEQ ID No.2 carries out pcr amplification for primer, detects the expression of NPTII reporter gene in transgenosis African red eggplant plant.The agarose gel electrophoresis analytical results of PCR primer as shown in Figure 2, target dna band is there is in the PCR primer of positive transgenic African red eggplant plant in 900bp position, prove consistent with NPTII reporter gene fragment sequence through order-checking, confirm that these plant are transgenosis African red eggplant positive plant.
2.qPCR detects the expression of SmMYB1 gene in transgenosis African red eggplant positive plant
According to SmMYB1 gene order and African red eggplant reference gene GAPDH sequence, design following primer:
qSmMYB1-F：5'-caagtgacaagcaaactaccg-3'(SEQ ID No.3)；
qSmMYB1-R：5'-tcttctccttcaacagcgtc-3'(SEQ ID No.4)；
qSmGAPDH-F：5'-gtacgacaacgaatggggtta-3'(SEQ ID No.5)；
qSmGAPDH-R：5'-tcatatcagcagcaccagca-3'(SEQ ID No.6)。
Get the spire of transgenosis African red eggplant positive plant and non-transgenic African red eggplant plant respectively, extract total serum IgE, be cDNA by total serum IgE reverse transcription, again with gained cDNA for template, respectively with qSmMYB1-F and qSmMYB1-R, qSmGAPDH-F and qSmGAPDH-R is that primer carries out qPCR, and detect the expression amount of SmMYB1 gene in transgenosis African red eggplant positive plant, result as shown in Figure 3.In known transgenosis African red eggplant positive plant, SmMYB1 gene has obvious expression.
Three, the phenotype analytical of transgenosis African red eggplant positive plant
The above-mentioned transgenosis African red eggplant positive plant that filters out and non-transgenic African red eggplant plant are cultivated according to a conventional method, the phenotypic characteristic of observation analysis transgenosis African red eggplant positive plant, result as shown in Figure 4.The whole plant of transgenosis African red eggplant comprises petal, flower pesticide, and fruit and blade have obvious anthocyanogen accumulate and present purple; As shown in Figure 4, the fruit rind of transgenosis African red eggplant and pulp inside have obvious anthocyanogen to accumulate.Carry out detection by the method for high performance liquid chromatography in fact to transgenosis and wild-type African red solanberry to analyze, find that transgenosis African red eggplant local pulp anthocyanin content reaches as high as 0.45mg/g fresh weight (Fig. 5).From above result, the method that this transgenosis African red eggplant obtains not only still produces the functional African red solanberry with food therapy value for ornamental plant breeding and cultivation has obvious using value in fact.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (4)
1. eggplant anthocyanidin synthesis related gene SmMYB1 is cultivating the application in purple African red eggplant (Solanum aethiopicum groupGilo) kind, and the nucleotide sequence of described eggplant anthocyanidin synthesis related gene SmMYB1 is as shown in SEQ ID NO.7.
2. eggplant anthocyanidin synthesis related gene SmMYB1 improves the application in African red solanberry meat in anthocyanin content, and the nucleotide sequence of described eggplant anthocyanidin synthesis related gene SmMYB1 is as shown in SEQ ID NO.7.
3. cultivate the method for purple African red eggplant kind, it is characterized in that, comprise the steps: to utilize containing the Agrobacterium-mediated Transformation African red eggplant explant of recombinant plasmid pBI121::SmMYB1, then containing the sucrose of 30g/L, 1mg/L indolylacetic acid, 1.75mg/L zeatin, pH be in the MS solid medium of 5.8 in 28 ± 2 DEG C, dark under Dual culture 48 hours; Proceed to the sucrose containing 30g/L, 1.0mg/L indolylacetic acid, 1.75mg/L zeatin, 500mg/L Pyocianil and 50mg/L kantlex again, pH be in the MS solid medium of 5.9,28 ± 2 DEG C, photoperiod 16h/d, be cultured under intensity of illumination 1000-2000lx condition and grow regeneration bud; Regeneration bud is cut when bud grows to 3-5cm, proceed to the sucrose containing 30g/L, 250mg/L Pyocianil and 50mg/L kantlex, pH is in the MS solid medium of 5.9,28 ± 2 DEG C, photoperiod 16h/d, be cultured under intensity of illumination 1000-2000lx condition and take root, obtain purple African red eggplant kind.
4. method according to claim 3, is characterized in that: described Agrobacterium is LBA4404.
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Cited By (5)
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| CN105505947A (en) * | 2015-12-28 | 2016-04-20 | 重庆大学 | Application of purple cabbage anthocyanin biosynthesis regulatory gene BrTT8 to photoinduction type purple tomatoes cultivation and method |
| CN109810988A (en) * | 2019-02-18 | 2019-05-28 | 广东省农业科学院蔬菜研究所 | A kind of eggplant fruit gene silencing system and its construction method |
| CN110229928A (en) * | 2019-06-27 | 2019-09-13 | 江西省农业科学院蔬菜花卉研究所 | Molecular labeling combination and its application for Eggplant Germplasm Resources identification |
| CN111926098A (en) * | 2020-08-17 | 2020-11-13 | 广东省农业科学院蔬菜研究所 | InDel molecular marker closely linked with epistatic gene Y of eggplant fruit color and application |
| CN111996198A (en) * | 2020-08-26 | 2020-11-27 | 山东农业大学 | Anthocyanin regulatory gene SmbHLH1 in eggplant stem and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505947A (en) * | 2015-12-28 | 2016-04-20 | 重庆大学 | Application of purple cabbage anthocyanin biosynthesis regulatory gene BrTT8 to photoinduction type purple tomatoes cultivation and method |
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| CN110229928A (en) * | 2019-06-27 | 2019-09-13 | 江西省农业科学院蔬菜花卉研究所 | Molecular labeling combination and its application for Eggplant Germplasm Resources identification |
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| CN111926098A (en) * | 2020-08-17 | 2020-11-13 | 广东省农业科学院蔬菜研究所 | InDel molecular marker closely linked with epistatic gene Y of eggplant fruit color and application |
| CN111926098B (en) * | 2020-08-17 | 2021-04-13 | 广东省农业科学院蔬菜研究所 | InDel molecular marker closely linked with epistatic gene Y of eggplant fruit color and application |
| CN111996198A (en) * | 2020-08-26 | 2020-11-27 | 山东农业大学 | Anthocyanin regulatory gene SmbHLH1 in eggplant stem and application thereof |
| CN111996198B (en) * | 2020-08-26 | 2022-06-24 | 山东农业大学 | Anthocyanin regulatory gene SmbHLH1 in eggplant stem and application thereof |
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