CN108003226B - Chlorella ellipsoidea NF-YB gene and application thereof - Google Patents
Chlorella ellipsoidea NF-YB gene and application thereof Download PDFInfo
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- CN108003226B CN108003226B CN201711397021.1A CN201711397021A CN108003226B CN 108003226 B CN108003226 B CN 108003226B CN 201711397021 A CN201711397021 A CN 201711397021A CN 108003226 B CN108003226 B CN 108003226B
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Abstract
The invention provides a chlorella ellipsoidea NF-YB gene and application thereof, belonging to the technical field of microalgae genetic engineering. The NF-YB gene sequence of the Chlorella ellipsoidea is shown as SEQ ID No.1, and the coding protein sequence thereof is shown as SEQ ID No. 2. The gene is derived from Chlorella ellipsoidea (Chlorella ellipsoidea), and is transformed into Arabidopsis thaliana, and the result shows that the total fatty acid content of the Arabidopsis thaliana transformed with CeNF-YB is improved by 7.1-11.2% compared with that of wild type Col-0, and the thousand seed weight is increased by 24.5-51% compared with that of the wild type Col-0. The NF-YB gene of the chlorella ellipsoidea can greatly improve the total oil content of plant cells, can be applied to the field of biological oil preparation and crop yield improvement, and has good application prospect.
Description
Technical Field
The invention belongs to the field of microalgae genetic engineering, and particularly relates to a chlorella ellipsoidea NF-YB gene, and a coding protein and application thereof.
Background
With the excessive consumption of traditional energy and the increasing energy demand, the energy problem is becoming more serious, and the research of alternative energy becomes a focus of attention. Microalgae are easy to culture due to high oil content, and the oil yield of unit land is far higher than that of other oil crops, so that microalgae are considered as one of energy resources with optimal potential.
The microalgae is a unicellular lower plant, has strong reproductive capacity, low requirement on growth environment, easy culture, high photosynthesis efficiency and high oil content. Triacylglycerols (TAGs) in microalgal cells can reach 20% -50% of dry weight. Triacylglycerols (TAGs) are the main storage forms of plant storage lipids, are widely distributed in organs such as seeds, pollen, fruits, leaves and the like of plants, and mainly participate in numerous vital activities such as cell membrane lipid construction, energy metabolism, stress reaction, seed germination, pollen development and the like. Therefore, microalgae have excellent candidate gene resources related to oil and fat metabolism. The NF-Y (nuclear Factor Y) super gene family is a transcription Factor family which can combine CCAAT-box and is widely existed in eukaryote, and is composed of 3 gene families, namely NF-YA, NF-YB and NF-YC. In higher plants, the members of the gene family have important influence on the synthesis and accumulation of oil and fat, the growth and development of plants, stress resistance and the like. Studies have shown that higher Plant-derived NF-Y genes driven by the CaMV35S promoter or other seed-specific promoters such as the unmodified Napin promoter cause dysplasia or abnormal seed germination (Mu et al, 2008, Plant Physiology,148: 1042-1584; Tan et al, 2011, Plant Physiology, 156: 1577-1588). This limits the application of the gene. At present, no relevant research report exists about NF-YB genes in Chlorella ellipsoidea and functions thereof.
Disclosure of Invention
The invention aims to provide Chlorella ellipsoidea (Chlorella ellipsoidea) NF-YB and application of a coding protein thereof.
The invention firstly provides chlorella ellipsoidea NF-YB protein, which comprises the following components:
1) an amino acid sequence shown as SEQ ID No. 2; or
2) Protein which is derived from the protein 1) and has the same activity and is obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.
The invention provides a gene for coding a chlorella ellipsoidea NF-YB protein, which has the following characteristics:
1) a nucleotide sequence shown as SEQ ID No. 1; or
2) The nucleotide sequence shown in SEQ ID No.1 is substituted, deleted and/or added with one or more nucleotides; or
3) Nucleotide sequences which hybridize under stringent conditions with the DNA sequences defined in 1).
The invention provides a biological material containing the gene for coding the chlorella ellipsoidea NF-YB protein, and the biological material is a vector, a host cell or an expression cassette.
The invention provides application of the chlorella ellipsoidea NF-YB protein or a gene encoding the protein or a biological material containing the gene in improving the total fatty acid content in cells.
The cell is a plant cell.
The invention provides application of the chlorella ellipsoidea NF-YB protein or a gene encoding the protein or a biological material containing the gene in preparation of transgenic plants with high total fatty acid content.
The plant is oil crop. The oil crops are rape, arabidopsis, sunflower, soybean, tomato, castor, palm, sesame or peanut.
The invention provides application of the chlorella ellipsoidea NF-YB protein or a gene encoding the protein or a biological material containing the gene in improving the thousand seed weight of plant seeds. The plant is rape, arabidopsis, sunflower, soybean, tomato, palm, castor-oil plant, sesame, peanut, rice, wheat or corn.
The invention provides application of the chlorella ellipsoidea NF-YB protein or a gene encoding the protein or a biological material containing the gene in improvement of plant germplasm resources.
The invention provides the application of the chlorella ellipsoidea NF-YB protein or the gene coding the protein or the biological material containing the gene in producing edible oil.
The invention provides the application of the chlorella ellipsoidea NF-YB protein or the gene coding the protein or the biological material containing the gene in producing biodiesel.
According to the invention, a cDNA sequence of CeNF-YB is screened according to the annotation of a transcriptome of Chlorella ellipsoidea, mRNA of the Chlorella ellipsoidea is extracted, after reverse transcription, a full-length CDS sequence of the CeNF-YB is cloned and is constructed on an entry vector, then the full-length CDS sequence is constructed on a plant expression vector through recombination reaction, positive clone is screened, and the plant is infected after agrobacterium is transformed (GV 3101). The result shows that the CeNF-YB which has the nucleotide sequence of SEQ ID NO.1 and is derived from the Chlorella ellipsoidea is heterologously expressed in the Arabidopsis thaliana under the drive of the CaMV35S promoter, the transgenic Arabidopsis thaliana can not have the undesirable agronomic characters such as inhibiting the growth and development of the Arabidopsis thaliana, and the oil content and the thousand seed weight of the transgenic Arabidopsis thaliana seed are obviously improved: the total fatty acid content of the Arabidopsis transformed with CeNF-YB is improved by 7.1-11.2 percent compared with that of the wild type Col-0, and the thousand seed weight is increased by 24.5-51 percent compared with that of the wild type Col-0. The arabidopsis thaliana is a model plant, and genes which can play a role in arabidopsis thaliana have similar effects in various crops, so that the chlorella ellipsoidea CeNF-YB gene with the nucleotide sequence of SEQ ID NO.1 can be used for genetic breeding of oilseed rape, soybean, cotton, peanut, palm and other oil plants to improve the oil content and the thousand seed weight, and for breeding of wheat, rice, corn and other crops to improve the thousand seed weight.
Drawings
FIG. 1 is a map of entry vector of CeNF-YB gene.
FIG. 2 is a CeNF-YB gene plant expression vector map.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 acquisition of Chlorella ellipsoidea NF-YB Gene and construction of expression vector
1. Extraction of total RNA of Chlorella ellipsoidea
Collecting sufficient Chlorella ellipsoidea cells, rapidly adding liquid nitrogen, grinding with mortar, collecting 50-100mg powder, adding 1m L Trizol (Invitrogen) extraction buffer solution, mixing, standing for 10min, adding 0.2m L chloroform, mixing, centrifuging at 13,500rpm for 10min, collecting supernatant, adding 0.2m L chloroform, mixing, centrifuging at 13,500rpm for 10min, collecting supernatant, adding half volume of isopropanol, standing at room temperature for 30-50min, centrifuging at 13,500rpm for 10min, discarding supernatant, adding 75% ethanol, suspending and precipitating at 10,000rpm for 5min, discarding supernatant, adding 100% ethanol, suspending and precipitating, centrifuging at 10000rpm for 5min, discarding supernatant, blow-drying in ultra-clean bench (more than 3-5min), adding 50 μ L DEPC water to dissolve at-80 deg.C, and storing at-80 deg.C
2. Synthesis of Chlorella ellipsoidea cDNA
cDNA is produced by mRNA by using a DNA-removing reverse transcription kit of the whole gold company, wherein the system comprises 5 mu g of totalRNA, 50 mMOoligo (dT18), 10 mu L2 × TS Reaction Mix, 1 mu L TransScript RT/RI Enzyme Mix and 1 mu L g of DNA Redmover, the materials are supplemented to 20 mu L by RNase-free water, the materials are gently mixed, incubated at 42 ℃ for 40-50min and inactivated at 85 ℃ for 5 min-20 ℃ and stored for standby.
3. Obtaining of CeNF-YB gene and construction of expression vector
And predicting the complete CDS according to the cDNA sequence in the transcriptome, translating the CDS into an amino acid sequence, and comparing the CDS with the NCBI protein database to further determine the integrity of the CeNF-YB gene. cDNA is used as a template, upstream and downstream amplification primers (an upstream primer 5-AGCAGGCTTTGACTTTATGAGCTCCGAGGCGGGGCT-3 and a downstream primer 5-TGGGTCTAGAGACTTTCCGTAGTCATGCTTGTCTTTC-3) are designed, and the CeNF-YB gene is amplified by using high-fidelity Taq enzyme. The amplification procedure was 2min pre-denaturation at 98 ℃, 30s 60 ℃, 30s 72 ℃, for 35 cycles. And purifying the PCR product for later use.
The PCR product is connected to an entry vector (named pGWC-CeNF-YB, figure 1) by utilizing an In-fusion system, specifically comprises pGWCm (100 ng/mu L cut by AhdI) after 1 mu L enzyme cutting, a 1 mu L PCR product (80 ng/mu L), 2 mu L In-fusion Mix at 50 ℃ for 50-60min, escherichia coli DH5 α, positive clones are identified and screened by PCR, and are constructed on a plant expression vector through a gateway system after sequencing identification, and are named as pCeNF-YB (figure 2).
Example 2 genetic transformation of CeNF-YB Gene and screening of Positive transgenic line
Agrobacterium with pCeNF-YB plasmid obtained in example 1 was cultured in L B liquid (containing 50 mg/L kanamycin, 50 mg/L gentamicin and 200 mg/L rifampicin) medium until OD was 0.8, centrifuged at 10000rpm for 5min to collect the cells, and an equal volume of MgCl suspension (10mM MgCl) was used25% sucrose), collecting thallus by centrifugation, suspending the thallus with suspension until OD is 1.0, adding 0.005% Silwet L-77, transforming Arabidopsis thaliana at the early stage of flowering once, dipping flower once again at intervals of 7 days, collecting seeds of T0 generation after the seeds are mature, planting in a culture dish, spraying Barsta (0.3%) for screening 10 days after emergence, spraying again at intervals of 5 days, and spraying again by PCAnd after the R identification, transplanting the T1 generation positive seedlings into a nutrition pot for culture, and harvesting seeds after the seedlings are mature.
Example 3 analysis of fatty acid composition in seeds transformed with CeNF-YB Gene
The transgenic Arabidopsis seeds of example 2 were collected, dried at 37 ℃ and ground thoroughly, 0.05g was weighed, and 3m L7.5.5% KOH-CH was added3OH (C17: 0 standard substance is added as internal reference), water bath is carried out at 70 deg.C for 3-5h, the middle part is reversed and mixed for several times, 2m L HCl-CH is added3OH (V/V, 1:1) solution, 2m L14% BF3-CH3And (3) adding 1m L0.9% NaCl and 4m L n-hexane into the OH solution in a water bath at 70 ℃ for 1.5h, fully oscillating and uniformly mixing, centrifuging at 4,000rpm for 8min, transferring an upper organic phase into a new tube, drying by nitrogen, dissolving by 300 mu L ethyl acetate, and repeating the experiment for 3 times in parallel by making two samples each time.
The GC/MS instrument is started strictly according to the operating specification of TurboMass (PerkinElmer company), GC parameters are set as follows, a chromatographic column is BPX-70, 30m × 0.25.25 mm × 0.25.25 mu m, the temperature of a column box is set as gradient temperature rise (100 ℃, 1min, 15 ℃/min to 190 ℃, 1min, 10 ℃/min to 220 ℃, 4min), carrier gas is helium, the flow is 1m L/min, a 1 mu L sample is taken for GC-MS detection, and according to the result of gas chromatography analysis, peak areas corresponding to different fatty acids are compared with an internal standard of a C17:0 peak area to calculate the content of each fatty acid component and the content of total fatty acids, wherein the content of each fatty acid component and the content of total fatty acids are shown in Table 1.
TABLE 1 analysis of fatty acid composition of transgenic lines (μ g/mg)
From experimental results, the fatty acid composition of the seeds of the CeNF-YB-transgenic arabidopsis thaliana is not changed, but the content of the fatty acid composition is obviously changed, the total fatty acid content of the CeNF-YB-transgenic arabidopsis thaliana is improved by 7.1-11.2% compared with that of wild Col-0, the C18:0 is improved by 12.2-24.5%, the C18:1 is improved by 10.8-29.9%, the C18:3 is improved by 3.5-6.4%, and the C20:1 is improved by 6.5-20.6%.
Example 4 thousand kernel weight analysis of seeds transformed with CeNF-YB Gene
The transgenic Arabidopsis seeds of example 2 were collected, dried at 37 ℃ and then counted for 1,000 seeds, and each line was repeated 3 times. The thousand seed weights of Col-0, CeNF-YB-2, CeNF-YB-3, CeNF-YB-4 and CeNF-YB-10 are respectively 16.33 +/-2.08 mg, 21.00 +/-1.00 mg, 20.33 +/-1.16 mg, 23.33 +/-0.58 mg and 24.67 +/-0.58 mg.
From experimental results, the thousand seed weight of arabidopsis can be obviously increased by transferring the CeNF-YB gene, and is increased by 24.5% -51% compared with the wild type.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (8)
1. The chlorella ellipsoidea NF-YB protein is characterized in that the amino acid sequence is shown as SEQ ID No. 2.
2. The gene for coding the protein of claim 1, and the nucleotide sequence of the gene is shown as SEQ ID No. 1.
3. Biological material comprising the gene of claim 2, said biological material being a vector, a host cell or an expression cassette, said host cell being incapable of being propagated as a plant or an animal.
4. Use of a chlorella ellipsoidea NF-YB protein according to claim 1 or a gene according to claim 2 or a biological material according to claim 3 for increasing the total fatty acid content in plant cells.
5. Use of a chlorella ellipsoidea NF-YB protein according to claim 1 or a gene according to claim 2 or a biological material according to claim 3 for the preparation of transgenic plants with a high total fatty acid content.
6. Use of the chlorella ellipsoidea NF-YB protein of claim 1 or the gene of claim 2 or the biological material of claim 3 to increase the thousand kernel weight of plant seeds.
7. Use of the chlorella ellipsoidea NF-YB protein of claim 1 or the gene of claim 2 or the biomaterial of claim 3 for improving plant germplasm resources.
8. Use of the chlorella ellipsoidea NF-YB protein of claim 1 or the gene of claim 2 or the biomaterial of claim 3 in the production of edible oil.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005063988A1 (en) * | 2003-12-23 | 2005-07-14 | Pioneer Hi-Bred International, Inc. | Alteration of oil traits in plants |
| CN102260686A (en) * | 2011-06-29 | 2011-11-30 | 中国科学院遗传与发育生物学研究所 | Method for improving content of total grease, linoleic acid or alpha-linolenic acid in chlorella |
| CN105063060A (en) * | 2015-07-15 | 2015-11-18 | 南京农业大学 | Application of genes in oilseed rape NF-YB gene family |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005063988A1 (en) * | 2003-12-23 | 2005-07-14 | Pioneer Hi-Bred International, Inc. | Alteration of oil traits in plants |
| CN102260686A (en) * | 2011-06-29 | 2011-11-30 | 中国科学院遗传与发育生物学研究所 | Method for improving content of total grease, linoleic acid or alpha-linolenic acid in chlorella |
| CN105063060A (en) * | 2015-07-15 | 2015-11-18 | 南京农业大学 | Application of genes in oilseed rape NF-YB gene family |
Non-Patent Citations (2)
| Title |
|---|
| "The Promiscuous Life of Plant NUCLEAR FACTOR Y Transcription Factors";Katia Petroni et al.;《The Plant Cell》;20121228;第24卷;第4777-4792页 * |
| 植物NF-Y转录因子的生物学功能及其研究进展;宋秋明 等;《植物生理学报》;20150531;第51卷(第5期);第623-632页 * |
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