CN111303257B

CN111303257B - Positive regulation gene AtMIF1 for oil content of plant seeds and application thereof

Info

Publication number: CN111303257B
Application number: CN201911265532.7A
Authority: CN
Inventors: 孙蒙祥; 程天河; 赵鹏
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2019-12-11
Filing date: 2019-12-11
Publication date: 2022-04-15
Anticipated expiration: 2039-12-11
Also published as: CN111303257A

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to a positive regulation gene AtMIF1 for the oil content of plant seeds and application thereof. The CDS sequence of the gene AtMIF1 is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. The AtMIF1 gene is separated from Arabidopsis thaliana, and is expressed in early embryo and endosperm of Arabidopsis thaliana and stops being expressed when the embryo reaches heart-shaped embryo. Through overexpression vector construction and transgenic technology, researches prove that AtMIF1 has an important regulation and control effect on oil accumulation in seeds, the overexpression of AtMIF1 gene promotes the oil content of the seeds to be remarkably improved, the expression regulation and control of AtMIF1 has no influence on the vegetative growth and the reproductive process of plants, and the yield and the growth and development characteristics of the original variety before the gene expression regulation and control can be well maintained.

Description

Positive regulation gene AtMIF1 for oil content of plant seeds and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a positive regulation gene AtMIF1 for the oil content of plant seeds and application thereof.

Background

Vegetable oil is a renewable resource with 86% as the main oil source for humans and 14% as biofuels and oleochemicals. By 2026, global food vegetable oil demand is expected to increase 219.8 tons, corresponding to a 16% increase (symposium/grain and agriculture organization, 2017). In the past, more cultivated land has been used for the cultivation of oil crops to meet increasing demands. However, cultivated lands are ecologically damaged and polluted, and future demands cannot be met at the current rate of yield increase. In our country, nearly 14 billion people have vegetable oils as the main source of oil. How to ensure the safety of the oil crop yield in China has become a hot point problem in the current oil crop production. By utilizing modern molecular biology technology, the cultivation of high-yield varieties is a main approach for solving the problem.

The yield of the oil crops is directly determined by the accumulation of oil in seeds, and the research on regulating genes for regulating the oil content of the seeds is helpful to solve the yield problem of the oil crops. The embryo is the main component of oil crop seeds and is the main place for forming and storing oil. The functional research of the regulatory gene of the accumulated oil in the embryo is very important. However, in oil crops, the genes that promote the increase in seed oil content are poorly understood. F-box protein is widely studied in animals and plants, and is a member of SKP1-Cullin1-F-box (SCF) complex in 26S protein degradation pathway, wherein the N terminal F-box of the protein is connected into the complex, and the C terminal directly interacts with the degraded protein. The F-box protein has nearly 700 members in Arabidopsis, AtMIF1 is highly conserved among angiosperms, and MIF1 similarity is very high among different species of the Brassicaceae family. This would indicate that the AtMIF1 gene has the potential for broad application.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a positive control gene AtMIF1 for the oil content of plant seeds and application thereof.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

a gene AtMIF1 for positively regulating the oil content of plant seeds, the nucleotide sequence of which is shown in the following 1) or 2):

1) a CDS sequence as shown in SEQ ID No. 1;

2) a DNA sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the CDS sequence defined in 1).

A protein encoded by a gene AtMIF1 for positively regulating the oil content of plant seeds, the amino acid sequence of which is shown in 1) or 2) below:

1) an amino acid sequence shown as SEQ ID NO. 2;

2) the fusion protein is obtained by connecting a label to the N section and/or the C end of the protein of the amino acid sequence shown as SEQ ID NO. 2;

3) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2 and has the same function.

In the above scheme, the protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2) described in 2) and having the same function refers to: a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequence shown in SEQ ID No.2, and having a function of positively regulating the oil content of plant seeds.

An overexpression vector containing a gene AtMIF1 for positively regulating the oil content of plant seeds comprises a GCD1 promoter sequence-AtMIF 1CDS sequence-transcription termination sequence.

In the above embodiment, the overexpression vector further comprises LAT52 promoter-RFP sequence-transcription termination sequence.

In the scheme, a small peptide tag 1 XFLAG is fused at the C terminal of the AtMIF1CDS sequence in the overexpression vector.

The construction method of the overexpression vector containing the oil content gene AtMIF1 of the positive control plant seed comprises the following steps: 1) amplifying an AtMIF1CDS sequence and a GCD1 promoter sequence by taking an arabidopsis genome as a template; 2) the GCD1 promoter, the NOS transcription termination sequence and the AtMIF1CDS sequence are respectively connected to a pCAMBIA 1300 vector by a double-enzyme digestion method, and the pCAMBIA 1300 overexpression vector of the AtMIF1CDS driven by the GCD1 promoter is constructed.

In the above scheme, the construction method further comprises: a small peptide tag 1 XFLAG is connected to the C end of AtMIF1CDS, a LAT52-RFP-NOS sequence is connected to a pCAMBIA 1300 vector by a double enzyme digestion method behind an NOS transcription termination sequence, and the pCAMBIA 1300 overexpression vector containing AtMIF1CDS driven by a GCD1 promoter and RFP driven by a LAT52 promoter is constructed.

The invention has the beneficial effects that: the AtMIF1 gene is separated from Arabidopsis thaliana, and is expressed in early embryo and endosperm of Arabidopsis thaliana and stops being expressed when the embryo reaches heart-shaped embryo. Through overexpression vector construction and transgenic technology, researches discover that the AtMIF1 gene participates in a specific way of controlling the final accumulation of grease in an Arabidopsis seed, and prove that AtMIF1 has an important regulation and control effect on the accumulation of grease in the seed, the overexpression of the AtMIF1 gene promotes the oil content of the seed to be remarkably improved, the expression regulation and control of the AtMIF1 have no influence on the vegetative growth and reproductive process of a plant, and the yield and growth and development characteristics of an original variety before the gene expression regulation and control can be well maintained, so that the AtMIF1 gene can be applied to the development of yield improvement strains.

Drawings

FIG. 1 shows the structure of AtMIF1 gene, wherein the gray rectangle in FIG. 1 is untranslated region (UTR), the light purple is exon region, the green rectangle represents F-box domain, and the yellow rectangle represents FBD domain.

FIG. 2 shows the expression pattern of AtMIF1 gene, FIG. 2(a) shows the expression levels of AtMIF1 in root, stem, leaf, flower and fruit of Arabidopsis thaliana by RT-qPCR, and UBQ10 is reference gene polyubiquitin gene 10; FIGS. 2(b) -2 (h) show that the AtMIF1 promoter drives beta-Glucuronidase (GUS) reporter gene to show the activity of AtMIF1 promoter, FIG. 2b shows that there is no GUS activity in the seedling stage, FIG. 2c shows that the zygote and endosperm of the seed in the zygote stage have GUS activity, FIG. 2d shows that the seed in the two-cell stage has stronger GUS activity, FIG. 2e shows that the seed in the 16-cell stage has stronger GUS activity, FIG. 2f shows that the seed in the early globular embryo stage has stronger GUS activity, FIG. 2g shows that the seed in the late globular embryo stage has reduced GUS activity compared with the previous stage, and FIG. 2h shows that the seed in the heart-shaped embryo stage has no GUS activity; the length of the scale in a is 1mm, and the length of the scale in c-h is 20 mu m.

FIG. 3 is a process of constructing an overexpression vector, wherein FIG. 3A is a modified pCAMBIA 1300 vector, i.e., a transgenic binary vector, in which SpeI and AvrII cleavage sites are added on the basis of the pCAMBIA 1300 vector; FIG. 3B is a schematic diagram showing the construction of the elements of the Arabidopsis thaliana overexpression AtMIF1 vector, in which the GCD1 promoter was ligated into the pCAMBIA 1300 vector via SacI and BamHI, AtMIF1CDS was ligated into the pCAMBIA 1300 vector via KpnI and XbaI, the NOS transcription termination sequence was ligated into the pCAMBIA 1300 vector via SpeI and AvrII, and LAT52-RFP-NOS was ligated into the pCAMBIA 1300 vector via AvrII and HindIII.

FIG. 4 shows the molecular biological identification of the overexpression of AtMIF1 gene, Col is wild type Arabidopsis thaliana as control, OE-5 and OE-8 represent

overexpression lines

5 and 8 of AtMIF1 driven by GCD1, respectively, and the mRNA levels of AtMIF1 in

lines

5 and 8 are up-regulated by approximately 4000-fold and 300-fold, respectively.

FIG. 5 shows the measurement results of mucopolysaccharide and oil content phenotype secreted from seeds by transgenic Arabidopsis thaliana after transformation of overexpression vector, as well as seed size, dry weight of individual seeds, fresh weight of individual seedlings, and height of individual seedlings; (a) ruthenium red staining of mucopolysaccharide on the surface of mature wild type Col seeds and ruthenium red staining of mucopolysaccharide on the surfaces of seeds of AtMIF1 overexpression

transgenic strains

5 and 8; (b) measuring the thickness of the mucopolysaccharides on the surface of the seeds in the wild type and

overexpression lines

5 and 8; (c) measuring the oil content of the seeds in wild type and

overexpression lines

5 and 8; (d) measuring the size of seeds in wild type and

overexpression lines

5 and 8; (e) measuring the weight of individual dry seeds in wild type and over-expression

lines

5 and 8; (f) measuring the fresh weight of the single seedling in wild type and over-expression

strains

5 and 8; (g) the height of individual seedlings in wild type and over-expression

lines

5 and 8 was measured. The length of the scale in a is 500 mu m.

Detailed Description

In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.

EXAMPLE 1 cloning and characterization of the Gene AtMIF1

The inventors designed primers using The coding region (CDS) of The Arabidopsis positive control oil content synthetic gene AtMIF1 according to The annotation of The Arabidopsis Information Resource (TAIR). Forward and reverse primers for the coding region of AtMIF1 were designed, the CDS of AtMIF1 was amplified and sequenced using the high fidelity enzyme Phanta of Nanjing Novowed Biotechnology Ltd, and the CDS of AtMIF1 was compared in the nucleic acid sequence of NCBI megablast database, indicating that the CDS of AtMIF1 had a total of 1386bp, and that the transcribed mRNA consisted of 1 exon and encoded a protein of 461 amino acids. The AtMIF1 protein contains an N-terminal F-box domain and a C-terminal FBD domain.

Example 2 expression of AtMIF1 Gene in Arabidopsis thaliana

Taking arabidopsis thaliana as a material, and taking roots, stems, leaves, flowers and fruits of the arabidopsis thaliana. Total RNA was extracted with Invitrogen TRIzol, and first strand cDNA was synthesized by inversion with Invitrogen M-MLV Reverse Transcriptase Kit. RT-qPCR assays were performed on AtMIF1 from different materials. The PCR reaction conditions were: 5min at 95 ℃; 95 ℃ 10sec, 60 ℃ 10sec, 72 ℃ 30sec, 40 cycles. Using Ubiquitin 10 as reference gene, the PCR primers of each gene in the detection process are shown in the following table 1:

TABLE 1 PCR primers

Relative quantification results as shown in figure 2a, AtMIF1 was not expressed in all tissues of vegetative growth and was only expressed during reproductive growth, mainly in flowers and siliques (figure 2 a).

GUS activity detection in transgenic plants with the β -Glucuronidase (GUS) reporter gene driven by the promoter of AtMIF1 was performed to understand the in-situ expression activity of the AtMIF1 promoter in Arabidopsis thaliana (FIGS. 2 b-2 h). Specifically, an AtMIF1 promoter is successfully cloned by taking an arabidopsis genome as a template, the AtMIF1 promoter is inserted in front of GUS of pART27 vector containing GUS-NOS by a double-enzyme cutting method, and the constructed plasmid is electrically stimulated to transform agrobacterium GV 3101. The GUS reporter gene expression vector driven by the AtMIF1 promoter is transferred into the Arabidopsis Col by adopting an agrobacterium GV3101 mediated genetic transformation method. The results of GUS staining are shown in FIGS. 2 b-2 h, wherein FIG. 2b shows that there is no GUS activity in the seedling stage, FIG. 2c shows that there is GUS activity in the zygote stage seed zygote and endosperm, FIG. 2d shows that there is strong GUS activity in the two-cell stage seed, FIG. 2e shows that there is strong GUS activity in the 16-cell stage seed, FIG. 2f shows that there is strong GUS activity in the early stage globular embryo stage seed, FIG. 2g shows that there is reduced GUS activity in the late stage globular embryo stage seed compared with the previous stage, and FIG. 2h shows that there is no GUS activity in the cardioid embryo stage seed. Therefore, the AtMIF1 gene had expression activity in early embryo and endosperm, and gradually decreased to stop expression in heart-shaped embryo.

Example 3 AtMIF1 functional analysis

In this example, an AtMIF1 gene overexpression vector is constructed and transformed into arabidopsis thaliana to achieve the purpose of improving the gene expression in a plant body, and finally, the function of AtMIF1 in arabidopsis thaliana is revealed. The specific operation is as follows:

firstly, a promoter GCD1 which is massively expressed in seeds is selected, a GCD1 promoter is successfully cloned by taking an arabidopsis genome as a template, SacI and BamHI are connected into a pCAMBIA 1300 vector by a double enzyme digestion method, and an Nos termination region is inserted into the pCAMBIA 1300 vector (figure 3A-figure 3B) by the double enzyme digestion method to prepare a GCD1 promoter-Nos-pCAMBIA 1300 intermediate vector. Then, the amplified AtMIF1CDS fragment was inserted into the GCD1 promoter-Nos-pCAMBIA 1300 intermediate vector via KpnI and XbaI. Finally, a pCAMBIA 1300 vector of MIF1CDS driven by GCD1 promoter is successfully constructed, in order to detect the expression of AtMIF1 in plants, a small peptide tag 1 XFLAG is fused to the C end of AtMIF1CDS, and in order to detect the acquisition of transgenic homozygous lines, an RFP element driven by LAT52 promoter is also integrated into the final vector. The schematic diagram of the construction is shown in FIG. 3B, and after the correctness is verified by sequencing, agrobacterium GV3101 is electrically transformed. The AtMIF1 overexpression vector was introduced into Arabidopsis thaliana (Arabidopsis thaliana) using Agrobacterium GV 3101-mediated genetic transformation.

The obtained transgenic positive plants are subjected to expression identification, the molecular biological identification of the AtMIF1 gene overexpression is shown in figure 4, Col is wild type arabidopsis thaliana as a control, OE-5 and OE-8 respectively represent

overexpression strains

5 and 8 of AtMIF1 driven by GCD1, and the mRNA levels of AtMIF1 in

strains

5 and 8 are respectively up-regulated by about 4000 times and 300 times.

After confirming that the target gene is overexpressed (FIG. 4), phenotypical comparison of growth of transgenic plants at various stages was observed. FIG. 5 shows measurement results of mucopolysaccharide and oil content phenotype secreted by transgenic Arabidopsis thaliana in seeds, seed size, dry weight of single plant seeds, fresh weight of single plant seedlings and height of single plant seedlings after transformation of an overexpression vector, and observation results show that the gene does not affect vegetative growth and reproductive growth of plants, and that the transgenic Arabidopsis thaliana after transformation of the overexpression vector is free of difference in wild type in terms of seed size, dry weight of single plant seeds, fresh weight of single plant seedlings and height of single plant seedlings and also participates in regulation of mucopolysaccharide secretion on seed surfaces and oil accumulation of seeds. In the over-expression strain, the expression level of AtMIF1 is obviously up-regulated, the secretion of mucopolysaccharide on the surface of the seed is reduced compared with that of the wild type, and the oil content is obviously increased. Therefore, the AtMIF1 gene and the overexpression technology are combined to obtain the Arabidopsis seed with higher oil content (figure 5c), and the method has a larger application prospect in increasing the oil content of the seed in oil crops.

It is apparent that the above embodiments are only examples for clearly illustrating and do not limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are therefore intended to be included within the scope of the invention as claimed.

Sequence listing

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Val Ala Arg Tyr Tyr Gln Arg Ala Ala Leu Val Ala Tyr Met Val Gly

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Trp Val Asn Asn Phe Thr Gly Gly Val Val Glu Ser Phe Glu Leu Arg

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Leu Ser Asn Ser Tyr Ala Phe Glu Glu Gly Val Thr Thr Leu Ile Glu

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Phe Ala Val Ser Lys Asn Val Lys His Leu Phe Leu Asp Leu Ser Glu

115 120 125

Pro Arg Trp Val Thr Asn Asn Asp Ala Ala Gln Leu Glu Pro Gly Leu

130 135 140

Ile Lys Leu Pro Glu Ser Phe Tyr Lys Ile Thr Ser Leu Val Thr Leu

145 150 155 160

Lys Leu Phe Gly Cys Arg Phe Glu Pro Ser Arg Leu Ala Lys Pro Gly

165 170 175

Met Val Lys Thr Met Phe Phe Arg Trp Ile Arg Leu Glu Met Leu Ser

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Ala Leu Ile Ala Lys Thr Pro Leu Leu Glu Ile Leu Asn Ile Lys Asn

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Cys Trp Glu Ile Gly Leu Asp Ala Ile Thr Gly Tyr Asn Asp Arg Leu

210 215 220

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Arg Phe Glu Phe Ala Ser Ala Asn Lys Leu Met Glu Glu Ala Tyr Leu

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Claims

The application of AtMIF1 gene in the aspect of positively regulating the oil content of plant seeds, wherein the nucleotide sequence of the gene AtMIF1 is the CDS sequence shown in SEQ ID NO. 1.
2. Use of a protein encoded by the AtMIF1 gene of claim 1, having an amino acid sequence as set forth in 1) or 2) below, for positively regulating oil content in plant seeds:

1) an amino acid sequence shown as SEQ ID NO. 2;

2) the fusion protein is obtained by connecting a label to the N section and/or the C end of the protein of the amino acid sequence shown as SEQ ID NO. 2.
3. Use of an overexpression vector comprising the AtMIF1 gene of claim 1 for positively regulating oil content in plant seeds.
4. Use according to claim 3, wherein the over-expression vector comprises the GCD1 promoter sequence-AtMIF 1CDS sequence-transcription termination sequence.
5. The use of claim 3, wherein said overexpression vector further comprises a LAT52 promoter-RFP sequence-transcription termination sequence.
6. The use of claim 3, wherein the C-terminus of the AtMIF1CDS sequence in the over-expression vector is fused with a small peptide tag 1 XFLAG.