CN109929858B

CN109929858B - Banana fruit glycogen initiation synthase gene MaGN12 and encoding protein and application thereof

Info

Publication number: CN109929858B
Application number: CN201910266092.0A
Authority: CN
Inventors: 金志强; 苗红霞; 徐碧玉; 孙佩光; 刘菊华; 贾彩红; 张建斌; 王静毅; 王卓; 苗雨露
Original assignee: Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
Current assignee: Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
Priority date: 2019-04-03
Filing date: 2019-04-03
Publication date: 2020-05-12
Anticipated expiration: 2039-04-03
Also published as: CN109929858A

Abstract

The invention provides a banana fruit glycogen initiation synthase gene MaGN12, the nucleotide sequence of which is shown in SEQ ID NO. 1. The invention also provides a protein coded by the banana fruit glycogen initiation synthase gene MaGN12 and application thereof. The banana fruit glycogen initiation synthase gene MaGN12 can improve the sweet taste quality of fruits and increase the content of glucose and fructose in the fruits, for example, the gene is introduced into tomatoes to obtain 8 independent strains of MaGN12 transgenic tomatoes driven by a 35S promoter, and the overexpression gene MaGN12 improves GN enzyme activity, the expression quantity of MaGN12, the content of glycogen, the content of fructose and glucose, and has important significance for controlling the fruit quality and cultivating high-quality new banana varieties.

Description

Banana fruit glycogen initiation synthase gene MaGN12 and encoding protein and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a banana fruit glycogen initiation synthase gene MaGN12, and a coding protein and application thereof.

Background

Glycogen is a branched macromolecular polysaccharide consisting of glucose and is widely stored in human bodies, animals, fungi, bacteria and plant tissues, wherein the deficiency of glycogen in heart and skeletal muscle of human bodies causes cardiomyopathy and myasthenia, animal glycogen mainly controls the steady state of blood sugar, glycogen in fungi and bacteria mainly exists in the form of basic energy, can be rapidly used in a large amount in a short time when needed and can be rapidly recovered and stored when not needed, and phytoglycogen is soluble α -D-glucan existing in plant deficiency isoamylase mutants (sul mutants), is the most main storage polysaccharide in plant bodies and is a basic substance consisting of carbon frames of all organs of plants.

Glycogen synthesis is a complex biochemical process requiring the extensive involvement of a series of enzymes, such as: glycogen-initiating synthetases (Glycogenin, GN), Glycogen Synthases (GS), Glycogen Branching Enzymes (GBE) and Glycogen Phosphorylases (GP). GN is the first rate-limiting enzyme that initiates glycogen synthesis. At present, most of the research on GN is mainly focused on animals (Bischof et al, 2013; Li et al, 2017), and information on plant GN is very little, especially for important tropical fruit bananas, so far, no relevant report on GN exists. Therefore, the development of related research on banana gene MagN12 is brand new, and has important significance for regulating fruit quality and cultivating new high-quality banana varieties.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a banana fruit glycogen initiation synthase gene MaGN12 which is obtained from Brazilian banana and can improve the sweet taste quality of a plant fruit, and a product and application thereof.

The first aspect of the invention provides a banana fruit glycogen starting synthetase gene MaGN12, the nucleotide sequence of which is shown in SEQ ID NO. 1.

In a second aspect, the invention provides a protein encoded by the banana fruit glycogen initiation synthase gene MagN12 according to the first aspect, and the amino acid sequence of the protein is shown in SEQ ID NO. 2.

The third aspect of the invention provides the application of the banana fruit glycogen synthase initial gene MaGN12 in improving the sweet taste quality of the plant fruit.

Wherein, the expression of the banana fruit glycogen synthesis initiator enzyme gene MaGN12 improves the expression quantity of the MaGN12 gene, GN enzyme activity, glycogen content, glucose content and fructose content.

In a fourth aspect of the invention, an expression vector is provided, which comprises an original vector and the banana fruit glycogen starting synthase gene MaGN12 according to the first aspect of the invention.

As the original vector, there can be used a vector commonly used in the field of gene recombination, such as a virus, a plasmid, etc. The invention is not limited in this regard. In one embodiment of the present invention, the original vector is a pBI121 vector plasmid, but it is understood that other plasmids, viruses, etc. may be used.

Preferably, the original vector is a pBI121 vector plasmid, and the nucleotide sequence shown in SEQ ID NO. 1 is positioned between Xba I and Sac I restriction endonuclease sites of the pBI121 vector plasmid.

In a fifth aspect of the invention, there is provided the use of an expression vector according to the fourth aspect of the invention for improving the sweetness quality of a plant fruit.

Wherein the expression vector of the fourth aspect of the invention increases the expression level of MaGN12, GN enzyme activity, glycogen content, glucose content and fructose content.

In the sixth aspect of the invention, a method for improving the sweet taste quality of tomato fruits is provided, and the tomato leaf discs are transformed by the expression vector recombinant plasmid in the fourth aspect of the invention.

The seventh aspect of the invention provides a primer pair for amplifying the banana fruit glycogen initiation synthase gene MaGN12, and the nucleotide sequences are shown as SEQ ID NO. 3 and SEQ ID NO. 4.

The banana fruit glycogen initiation synthase gene MaGN12 can improve the sweet taste quality of fruits and increase the content of glucose and fructose in the fruits, for example, the gene is introduced into tomatoes to obtain 8 independent strains of MaGN12 transgenic tomatoes driven by a 35S promoter, and the overexpression gene MaGN12 improves GN enzyme activity, the expression quantity of MaGN12, the content of glycogen, the content of fructose and glucose, and has important significance for controlling the fruit quality and cultivating high-quality new banana varieties.

Drawings

FIG. 1 is a diagram of the result of electrophoresis of amplification of a banana fruit glycogen initiation synthase gene MaGN12, wherein M: DL2000 DNAmarker; lane 1: gene MagN12 PCR product.

FIG. 2 is a diagram of the result of double restriction enzyme validation electrophoresis of pBI121-MaGN12, wherein M: DL2000 DNA Marker, lane 1: the recombinant plasmid pBI121-MaGN12 was double digested.

FIG. 3 is a PCR assay of a MaGN12 transgenic tomato line (A) and the changes in flower and fruit shape (B), GN enzyme activity (C), fructose content (D), MaGN12 gene expression (E) and glucose content (F). Wherein, M: DL2000 DNA Marker, lane 1: positive control, lane 2: wild type (negative control). WT: a wild type; VC: a pBI121 vector; GN-3, GN-14, GN-22: MaGN12 transgenic plants; values representing MaGN12 transgenic plants reached significant levels of difference from wild type (. p < 0.05;. p < 0.01).

Detailed Description

The invention will be better understood from the following description of specific embodiments with reference to the accompanying drawings.

First, obtaining a Gene

Taking Brazil banana fruit cDNA as a template, and

5’-GCTCTAGAGATGATGTACATGGGGAC-3’

5’-CGAGCTCGGTTAATACAGTTTGTTGAACTCAG-3’

is used as a primer, the sequence of the primer is 1407bp (figure 1) containing alkali sequence obtained by PCR amplification, and the sequence is shown as SEQID No. 1; the amino acid sequence of the gene encoding MaGN12 is shown in SEQ ID No. 2.

Second, expression vector construction

And (2) carrying out double enzyme digestion on the target fragment and the pBI121 vector plasmid respectively by using the nucleotide sequence of the banana fruit glycogen initiation synthase gene MagN12 through two restriction endonucleases of Xba I and Sac I, and carrying out recovery, connection, transformation and sequencing verification on the digested target fragment and the plant expression vector pBI121 fragment to obtain the expression vector of the banana fruit glycogen initiation synthase gene MagN12 (figure 2).

Thirdly, transforming the expression vector to tomato leaf disc

The recombinant plasmid is transformed into tomato leaf disc by agrobacterium with the expression vector. The specific experimental steps of the agrobacterium leaf disc transformation method are as follows:

(1) agrobacterium transformation of the pBI121-MaGN12 vector:

taking 200 mu L of Agrobacterium tumefaciens LBA4404 competent cells melted on ice bath, adding 2 mu g of pBI121-MaGN12 recombinant plasmid, gently mixing, and placing in ice bath for 30 min; freezing in liquid nitrogen for 3min, and rapidly incubating in 37 deg.C water bath for 5 min; adding 800 mu L YEP liquid culture medium, pre-culturing at 28 ℃ and 250rpm for 4-5 h; sucking 300 mu L of bacterial liquid to YEP solid selection culture medium containing 50mg/LRif, and uniformly coating the bacterial liquid on the whole plate; and (3) placing the flat plate at 28 ℃ until the liquid is absorbed, inverting the flat plate, culturing for 2-3 days at 28 ℃, selecting a single colony, verifying and detecting, and using the correctly transformed agrobacterium liquid for the next experiment.

(2) Agrobacterium tumefaciens mediated genetic transformation of tomatoes:

soaking tomato seeds in 5mL 75% ethanol sterile centrifuge tube on a clean bench for 1min, shaking thoroughly during soaking, and usingWashing with sterile water for three times; soaking in 20% sodium hypochlorite solution for 15min, shaking, and washing with sterile water for three times; placing tomato seeds on sterile filter paper, airing water, sowing the tomato seeds in an MS solid culture medium, and culturing for 4-5 days at 25 ℃ under a dark condition; after the seeds begin to germinate, transferring the seeds to the conditions of 25 ℃, 1800LUX illumination intensity, 16h illumination and 8h dark for culture; cutting with sterile blade to obtain about 0.5 × 0.5cm cotyledon²Placing the large and small leaves on a tomato differentiation medium (MS solid medium +2.0 mg/L6-BA/ZT +0.2mg/L IAA), culturing at 25 deg.C in the dark for about 2 days until the cut of the leaves just begins to expand; 20 mu L of the bacterial liquid of the Agrobacterium tumefaciens LBA4404 transformed with the pBI121-MaGN12 recombinant vector is taken out to 10mL of YEP liquid culture medium containing 50mg/L Kan and 50mg/L Rif for activation culture overnight; sucking 1mL of activated and cultured bacterial liquid into a new 50mL of YEP liquid culture medium containing 50mg/L Kan and 50mg/L Rif to culture until the OD600 is about 0.5; transferring the bacterial liquid with the required concentration to a 50mL sterile centrifuge tube on an ultra-clean workbench, centrifuging at 4 ℃ and 6000rpm for 5min, removing supernatant, and adding an MS liquid culture medium with the same volume (the volume of the bacterial liquid before centrifugation) to resuspend the thalli; transferring the bacterial liquid into a 100mL sterile triangular flask, adding 0.1% of Acetosyringone (AS) in volume (the volume of the resuspended bacterial liquid), fully mixing uniformly, transferring the pre-cultured leaf explant into the agrobacterium liquid, soaking for 15min, and shaking the bacterial liquid to make the explant fully contact with the bacterial liquid; taking out the explant, placing the explant on sterile filter paper, sucking off redundant bacterial liquid on the surface of the explant, transferring the explant to an MS differentiation culture medium containing acetosyringone, and culturing for 2 days in the dark at 25 ℃; transferring the co-cultured leaf discs to an MS differentiation medium containing 200mg/L timentin, and culturing for a week under the conditions of 25 ℃, 2000LUX illumination intensity, 16h illumination and 8h dark; the leaf discs were transferred to MS differentiation medium containing 200mg/L timentin and 15mg/L hygromycin and cultured at 25 ℃ under 2000LUX light intensity, 16h light, 8h dark conditions. Subcultured every two weeks. Cutting and culturing on MS rooting culture medium (MS solid culture medium +0.2mg/L IAA) containing 200mg/L timentin and 20mg/L hygromycin when adventitious bud grows to about 2cm from leaf disc callus, culturing at 25 deg.C,the culture was carried out under the conditions of 2000LUX illumination intensity, 16h illumination and 8h dark. After the tomato seedlings are cultured for two weeks and roots are differentiated, the tomato seedlings capable of normally growing are selected for hardening and adaptive culture, and then are transplanted.

Fourth, detection of transformed strains

(1) Tomato genome DNA extraction and positive transformation plant detection

The plant genome DNA extraction kit (TIANGEN, Tiangen Biochemical technology (Beijing) Co., Ltd.) is used for extracting the transformed tomato genome DNA, and the specific experimental method is shown in the specification.

(2) PCR identification of transformed tomato lines

In order to further detect the integration of the exogenous gene MaGN12 in the tomato genome, a transformed tomato plant is used as a material, a non-transformed WT wild type tomato plant is used as a negative control, a strain plasmid used for transformation is used as a positive control, and the transformed tomato plant is subjected to PCR identification, wherein the specific experimental method comprises the following steps:

a. the following reagents were added to a 0.2mL PCR tube:

flick and mix evenly, centrifuge instantaneously, then carry on PCR amplification according to the following procedure:

the PCR results were checked by 1.0% agarose gel electrophoresis. The gel imaging system observes and records the electrophoresis results.

As shown in FIG. 3, the positive plasmid has a band, the wild type WT has no band, while the transgenic strains GN12 GN-3, GN-6, GN-14, GN-16, GN-20, GN-21, GN-22 and GN-23 can clearly distinguish a single band, which proves that the gene MaGN12 has been successfully transformed into the tomato genome, and the bands of the strains GN-3, GN-14 and GN-22 are brighter.

(3) Detection of tomato fruit GN enzyme activity at different developmental stages

Fruits of different development stages of the transgenic plant line are taken as materials, and are divided into small parts to be added with PBS (PH7.4), and the small parts are frozen by liquid nitrogen for later use. 1g of the sample was taken and sufficient PBS (pH7.4) was added, and the specimen was thoroughly homogenized. The supernatant was carefully collected after approximately 20min centrifugation (2000-. The specific operation steps are as follows:

① sample adding of standard substance, arranging standard substance wells and sample wells, and adding 50 μ L of standard substance with different concentrations in each standard substance well.

② sample holes and blank control holes (no sample and enzyme labeling reagent in the blank holes, the rest steps are the same) are arranged on the enzyme labeling coated plate, 40 muL of sample diluent is added into the sample holes, 10 muL of sample is added (the sample is finally diluted by 5 times), when the sample is added, the sample is added into the bottom of the enzyme holes, the sample does not contact with the hole walls as much as possible, and the sample is shaken gently.

③ except for blank wells, 100. mu.L of enzyme-labeled reagent was added to each well.

④ after sealing the plate, the plate was left to stand at 37 ℃ for 60 min.

⑤ Dilute the 20 Xconcentrated washings with distilled water.

⑥ removing the sealing film slowly, draining off the liquid, drying, filling each hole with washing liquid, draining after 30 s, repeating the above steps for 5 times, and drying.

⑦ adding 50 μ L of color reagent A and B into each well, adding reagent A, mixing slowly, and standing at 37 deg.C in dark condition for 15 min.

⑧ mu.L of stop solution was added to each well to terminate the reaction, and the OD was measured with a microplate reader.

⑨ the measurement was zeroed with a blank tube and set at a wavelength of 450 nm.

As shown in FIG. 3, the GN-3, GN-14 and GN-22 activities of the transgenic lines increased by about 13-15U/g, 28-45U/g, 13-14U/g and 11-15U/g in the young fruit stage, green mature stage, color transition stage and red mature stage, respectively, compared with the wild type.

(4) Determination of fructose and glucose content of tomato fruits in different development stages

The tomato strain transformed with MaGN12 gene, the tomato strain transformed with pBI121 empty vector and the wild type WT tomato strain are used as the material, and the material is color-protected in 0.5% sodium bisulfite for 10, dried at 40 deg.C to constant weight (about 24h), and ground into powder for use. Accurately weighing 300mg of powder into a 2mL centrifuge tube, adding 1.5mL of distilled water, carrying out water bath at 80 ℃ for 30min, centrifuging at 10000r/min for 10min, transferring supernatant to the 50mL centrifuge tube, and extracting residues twice by using 1mL of distilled water; adding 15mL of absolute ethyl alcohol into the supernatant, fully and uniformly mixing, and standing overnight at-4 ℃ to precipitate protein; centrifuging at 10000r/min for 10min, transferring supernatant to a small beaker, and completely drying the solution at 80 ℃; adding 2mL of ultrapure water for dissolving, standing overnight at-20 ℃, and centrifuging until no residue exists; the sample was aspirated with a 5ml syringe and filtered through a 0.25nm pore water system filter needle into a sample vial, which was ready for loading.

Detecting sugar components and content thereof by waters2695-3300ELSD HPLC, wherein the chromatographic conditions comprise acetonitrile and ammonia water (0.1%) -75: 25(V: V) as mobile phase, amino column as column, flow rate of 1mL/min, sample amount of 10 μ L, column temperature of 30 deg.C, and calculating fructose and glucose content in fruit.

As shown in FIG. 3, the fructose content of the transgenic lines GN-3, GN-14 and GN-22 was increased by about 90mg/g, 30mg/g and 40mg/g, respectively, in the red-ripening stage, as compared with the wild type. The glucose content of the transgenic lines GN-3, GN-14 and GN-22 increased by about 20mg/g, 30mg/g and 25mg/g, respectively, during the red stage of maturation, compared to the wild type.

(5) Expression analysis of MaGN12 Gene in tomato

The expression of the MAGN12 gene in different developmental stages of tomato is analyzed by taking a MAGN12 transgenic line and fruit cDNA of different developmental stages (Alba et al, 2005) of a wild young fruit stage, a green mature stage, a color conversion stage and a red mature stage as templates, and a reaction system and a specific experimental method are as follows:

the following components were added to a 200. mu.L PCR tube, and the reaction system was as follows:

sucking, mixing uniformly, performing instant centrifugation for several seconds, and performing amplification detection in a real-time fluorescence quantitative PCR instrument (Mx3000P, Stratagene) by taking MaActin as an internal reference gene, wherein each sample is repeated three times, and the operation program of the amplification reaction is as follows:

as shown in FIG. 3, the relative expression levels of the MaGN12 genes of the transgenic lines GN-3, GN-14 and GN-22 in the young fruit stage, green mature stage, color transition stage and red mature stage were increased by about 8-12 times, 26-51 times, 8-10 times and 3-6 times, respectively, as compared with the wild type.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Sequence listing

<110> research institute of tropical biotechnology of Chinese tropical academy of agricultural sciences

<120> banana fruit glycogen initiation synthase gene MaGN12, and coding protein and application thereof

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Val Glu Ala Asp Leu Val Val Ile Ala Ser Val Asp Val Pro Val Arg

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Trp Ala Gln Thr Leu Gln Glu Glu Asp Gly Val Lys Val Ile Arg Val

85 90 95

Glu Asn Leu Lys Asn Pro Tyr Glu Asn Gln Gln Asn Phe Asn Thr Arg

100 105 110

Phe Lys Leu Thr Leu Asn Lys Leu Tyr Ala Trp Ser Leu Ile Ser Tyr

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Claims

1. A banana fruit glycogen initiation synthase gene MaGN12 is characterized in that the nucleotide sequence is shown in SEQ ID NO. 1.

2. A protein encoded by the banana fruit glycogen initiation synthase gene MaGN12 according to claim 1, wherein the amino acid sequence of the protein is represented by SEQ ID NO. 2.

3. Use of the banana fruit glycogen starter synthase gene MagN12 according to claim 1 for improving the sweet taste quality of a plant fruit.

4. Use according to claim 3, wherein overexpression of the banana fruit glycogen synthesis initiator enzyme gene MaGN12 increases the expression of MaGN12, GN enzyme activity, glycogen content, glucose and fructose content.

5. An expression vector comprising an original vector and the banana fruit glycogen starting synthase gene MaGN12 according to claim 1.

6. The expression vector of claim 5, wherein the original vector is pBI121 vector plasmid, and the nucleotide sequence shown in SEQ ID NO. 1 is located between XbaI and Sac I restriction endonuclease sites of the pBI121 vector plasmid.

7. Use of the expression vector of claim 5 or 6 for improving the sweetness quality of a plant fruit.

8. The use of claim 7, wherein the expression vector of claim 5 or 6 increases the amount of expression of MaGN12, GN enzyme activity, glycogen content, glucose and fructose content.

9. A method for improving the sweet taste quality of tomato fruits, which is characterized in that a tomato leaf disc is transformed by the expression vector recombinant plasmid of claim 5 or 6.

10. A primer pair for amplifying the banana fruit glycogen initiation synthase gene MaGN12 as claimed in claim 1, characterized in that the nucleotide sequences are shown as SEQ ID NO. 3 and SEQ ID NO. 4.