CN111303257B - A positive regulator gene of plant seed oil content AtMIF1 and its application - Google Patents

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

A positive regulator gene of plant seed oil content AtMIF1 and its application

technical field

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

Background technique

Vegetable oil is a renewable resource, of which 86% is used as the main oil source for mankind and 14% is used as biofuel and oleochemicals. Global demand for vegetable oils for food is expected to increase by 219.8 tonnes by 2026, equivalent to an increase of 16% (OECD/FAO, 2017). In the past, more arable land was devoted to the cultivation of oil crops to meet the increasing demand. However, arable land has been destroyed and ecologically polluted, and the current rate of increase in production cannot meet future demand. There are nearly 1.4 billion people in my country, and most of them use vegetable oil as their main source of oil. How to ensure the safety of oil crop production in China has become a hot and difficult issue in current oil crop production. Using modern molecular biology techniques, breeding high-yielding varieties is the main way to solve this problem.

The yield of oil crops directly depends on the accumulation of oil in seeds, and the study of the regulatory genes that regulate the oil content of seeds can help to solve the problem of oil crop yield. Embryos are the main components of oil crop seeds and the main place for oil formation and storage. Functional studies of genes regulating lipid accumulation in embryos are critical. But in oil crops, the genes that promote increased seed oil content are poorly understood. F-box proteins have been widely studied in both animals and plants. F-box proteins are a member of the SKP1-Cullin1-F-box (SCF) complex in the 26S protein degradation pathway. The N-terminal F-box of the protein is linked Into the complex, the C-terminus directly interacts with the degraded protein. The F-box protein in Arabidopsis has nearly 700 members, AtMIF1 is highly conserved in angiosperms, and the similarity of MIF1 in different species of cruciferous is very high. This would suggest that the AtMIF1 gene has the potential for broad application.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the present invention aims to provide the positive regulation gene AtMIF1 of the oil content of plant seeds and its application.

In order to realize the above-mentioned purpose of the invention, the technical scheme adopted in the present invention is:

A kind of positive regulation plant seed oil content gene AtMIF1, its nucleotide sequence is as shown in 1) or 2) as follows:

1) 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 positive regulation plant seed oil content gene AtMIF1, the amino acid sequence is shown in 1) or 2) below:

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

2) a fusion protein obtained by linking the N-segment and/or C-terminal of the protein with the amino acid sequence shown in SEQ ID NO.2 to a tag;

3) A protein with the same function obtained by substitution and/or deletion and/or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO. 2.

In the above scheme, the protein with the same function obtained by the substitution and/or deletion and/or addition of one or several amino acid residues as the amino acid sequence shown in SEQ ID NO.2 in 2) refers to: The amino acid sequence shown in ID NO.2 has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology, and has a positive regulation of plant seed oil content functional protein.

The overexpression vector containing the positive regulation plant seed oil content gene AtMIF1 includes the GCD1 promoter sequence-AtMIF1CDS sequence-transcription termination sequence.

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

In the above scheme, the C-terminus of the AtMIF1CDS sequence in the overexpression vector is fused with a small peptide tag 1×FLAG.

The above-mentioned construction method of the overexpression vector containing the positive regulation plant seed oil content gene AtMIF1, comprising the following steps: 1) using the Arabidopsis thaliana genome as a template, amplifying the AtMIF1 CDS sequence and the GCD1 promoter sequence; 2) adding the GCD1 promoter, NOS The transcription termination sequence and AtMIF1 CDS sequence were respectively connected to the pCAMBIA 1300 vector by double digestion method, and the pCAMBIA 1300 overexpression vector of AtMIF1 CDS driven by the GCD1 promoter was constructed.

In the above scheme, the construction method further comprises: connecting the small peptide tag 1×FLAG to the C-terminus of the AtMIF1 CDS, and connecting the LAT52-RFP-NOS sequence to the pCAMBIA 1300 vector by double digestion after the NOS transcription termination sequence. Above, the pCAMBIA 1300 overexpression vector containing AtMIF1 CDS driven by GCD1 promoter and RFP driven by LAT52 promoter was constructed.

Beneficial effects of the present invention: the present invention isolates the AtMIF1 gene from Arabidopsis thaliana, which is expressed in the early embryo and endosperm of Arabidopsis thaliana, and ceases to be expressed when the heart-shaped embryo is reached. Through the construction of overexpression vector and transgenic technology, the study found that AtMIF1 gene is involved in the specific pathway of controlling the final accumulation of oil in Arabidopsis seeds, confirming that AtMIF1 plays an important role in regulating the accumulation of oil in seeds, and the overexpression of AtMIF1 gene promotes oily seeds in seeds The amount of AtMIF1 was significantly increased, and the expression regulation of AtMIF1 had no effect on the vegetative growth and reproductive process of plants, and the yield and growth and development characteristics of the original varieties before the gene expression regulation could be kept intact. development.

Description of drawings

Figure 1 shows the structure of the AtMIF1 gene. In Figure 1, the gray rectangle is the untranslated region (UTR), the lavender is the exon region, the green rectangle represents the F-box domain, and the yellow rectangle represents the FBD domain.

Figure 2 shows the expression pattern of AtMIF1 gene. Figure 2(a) The expression levels of AtMIF1 in roots, stems, leaves, flowers and fruits of Arabidopsis were detected by RT-qPCR. UBQ10 was the internal reference gene polyubiquitinase gene. 10; Figures 2(b) to 2(h) show the activity of the AtMIF1 promoter driven by the AtMIF1 promoter driving β-glucuronidase (GUS) reporter gene, Figure 2b shows no GUS activity at the seedling stage, and Figure 2c shows the zygote and The endosperm has GUS activity. Figure 2d shows that the seeds at the two-cell stage have strong GUS activity, Figure 2e shows that the 16-cell stage seeds have strong GUS activity, and Figure 2f shows that the seeds at the early spherical embryo stage have strong GUS activity, Figure 2g It is shown that the seeds of the late spherical embryo stage have weakened GUS activity compared with the previous stage, and Figure 2h shows that the seeds of the heart-shaped embryo stage have no GUS activity; the length of the scale in a is 1 mm, and the length of the scale in c-h is 20 μm.

Figure 3 shows the construction process of the overexpression vector, in which Figure 3A is the modified pCAMBIA 1300 vector, that is, a transgenic binary vector, in which the SpeI and AvrII restriction sites are added on the basis of the pCAMBIA 1300 vector; Figure 3B is a pseudonym A schematic diagram of the structure of each element of the mustard overexpressing AtMIF1 vector. The GCD1 promoter is connected to the pCAMBIA 1300 vector through SacI and BamHI, the AtMIF1 CDS is connected to the pCAMBIA 1300 vector through KpnI and XbaI, and the NOS transcription termination sequence is connected to the pCAMBIA 1300 vector through SpeI and AvrII. LAT52-RFP-NOS was ligated into pCAMBIA 1300 vector via AvrII and HindIII.

Figure 4 is the molecular biological identification of the overexpression of AtMIF1 gene. Col is the wild-type Arabidopsis thaliana as a control. OE-5 and OE-8 represent the GCD1-driven AtMIF1 overexpression lines 5 and 8, respectively. The mRNA levels of AtMIF1 were up-regulated by nearly 4000-fold and 300-fold in strain No.

Figure 5 is the measurement results of the mucopolysaccharide and oil content phenotype secreted by the transgenic Arabidopsis in the seed after the transformation of the overexpression vector, as well as the seed size, the dry weight of the seed per plant, the fresh weight of the seedling per plant, and the height of the seedling per plant; (a) Ruthenium red staining of mucopolysaccharides on the surface of mature wild-type Col seeds and ruthenium red staining of mucopolysaccharides on the surface of seeds of AtMIF1 overexpression transgenic line 5 and overexpression transgenic line 8; (b) Measurement of wild type and overexpression Thickness of mucopolysaccharides on the surface of seeds in lines 5 and 8; (c) measurement of oil content in seeds in wild type and overexpression lines 5 and 8; (d) measurement of oil content in seeds in wild type and overexpression lines 5 and 8 Size; (e) measurement of dry seed weight per plant in wild type and overexpression lines 5 and 8; (f) measurement of fresh weight per plant seedling in wild type and overexpression lines 5 and 8; (g) measurement Height of individual seedlings in wild type and overexpression lines 5 and 8. The length of the scale bar in a is 500 μm.

Detailed ways

In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments.

Example 1 Cloning and identification of the gene AtMIF1

The inventors used the coding region (CDS) of an Arabidopsis thaliana positive-regulated oil content synthesis gene AtMIF1 to design primers according to the annotations of The Arabidopsis Information Resource (TAIR). The forward and reverse primers of the coding region of AtMIF1 were designed, and the CDS of AtMIF1 was amplified and sequenced with the high-fidelity enzyme Phanta of Nanjing Novizan Biotechnology Co., Ltd., and the CDS of AtMIF1 was compared in the NCBI megablast database. It shows that the CDS of AtMIF1 has a total of 1386 bp, and its transcribed mRNA is composed of one exon and encodes a protein of 461 amino acids. AtMIF1 protein contains N-terminal F-box domain and C-terminal FBD domain.

Example 2 Expression of AtMIF1 gene in Arabidopsis thaliana

Using Arabidopsis thaliana as the material, the roots, stems, leaves, flowers and fruits are taken. Total RNA was extracted with Invitrogen's TRIzol, and the first strand of cDNA was reverse synthesized with Invitrigen M-MLV Reverse Transcriptase Kit. RT-qPCR detection of AtMIF1 in different materials. PCR reaction conditions were: 95°C for 5 min; 95°C for 10sec, 60°C for 10sec, 72°C for 30sec, 40 cycles. Taking Ubiquitin 10 as the internal reference gene, the PCR primers for each gene in the detection process are as follows in Table 1:

Table 1 PCR primers

The relative quantitative results shown in Fig. 2a show that AtMIF1 was not expressed in all tissues of vegetative growth and was only expressed during reproductive growth, mainly in flowers and siliques (Fig. 2a).

The GUS activity was detected in transgenic plants driven by the AtMIF1 promoter to drive the β-glucuronidase (GUS) reporter gene to understand the in situ expression activity of the AtMIF1 promoter in Arabidopsis (Figure 2b-Figure 2h). Specifically, using the Arabidopsis thaliana genome as a template, the AtMIF1 promoter was successfully cloned, and the AtMIF1 promoter was inserted into the front of the GUS of the pART27 vector containing GUS-NOS by the double-enzyme digestion method, and the constructed plasmid was electro-transformed into Agrobacterium GV3101. The GUS reporter gene expression vector driven by the AtMIF1 promoter was transformed into Arabidopsis Col by Agrobacterium GV3101-mediated genetic transformation. Through GUS staining, the results are shown in Figures 2b to 2h, in which Figure 2b shows that there is no GUS activity at the seedling stage, Figure 2c shows that the zygote and endosperm of the zygote stage seeds have GUS activity, and Figure 2d shows that the seeds at the two-cell stage have strong GUS activity, Figure 2e shows that the seeds at the 16-cell stage have strong GUS activity, Figure 2f shows that the seeds of the early globular embryo stage have strong GUS activity, Figure 2g shows that the seeds of the late globular embryo stage have weakened GUS activity compared with the previous stage, and Figure 2h shows that the heart There is no GUS activity in the seeds at the embryonic stage. Therefore, the expression of AtMIF1 gene was active in early embryos and endosperm, and gradually weakened, and ceased to express in heart-shaped embryos.

Example 3 AtMIF1 functional analysis

In this example, an AtMIF1 gene overexpression vector was constructed and transformed into Arabidopsis thaliana to achieve the purpose of increasing the expression of the gene in plants, and finally reveal the function of AtMIF1 in Arabidopsis thaliana. The specific operations are as follows:

First, a promoter, GCD1, which is abundantly expressed in seeds, was selected, and the GCD1 promoter was successfully cloned using the Arabidopsis genome as a template. SacI and BamHI were linked into the pCAMBIA 1300 vector by double digestion method. The pCAMBIA 1300 vector was inserted into the pCAMBIA 1300 vector (Fig. 3A to Fig. 3B) by the above double restriction digestion method to form a GCD1 promoter-Nos-pCAMBIA1300 intermediate vector. Then, the amplified AtMIF1 CDS fragment was inserted into the GCD1 promoter-Nos-pCAMBIA 1300 intermediate vector through KpnI and XbaI. Finally, the pCAMBIA 1300 vector of MIF1 CDS driven by the GCD1 promoter was successfully constructed. In order to facilitate the detection of the expression of AtMIF1 in plants, a small peptide tag 1×FLAG was fused to the C-terminus of the AtMIF1 CDS. In order to facilitate the detection of the acquisition of transgenic homozygous lines , the LAT52 promoter-driven RFP element was also integrated into the final vector. The schematic diagram of the construction is shown in Fig. 3B. After the correctness was verified by sequencing, Agrobacterium GV3101 was transformed by electric stimulation. The AtMIF1 overexpression vector was introduced into Arabidopsis thaliana by Agrobacterium GV3101-mediated genetic transformation.

The obtained transgenic positive plants were subjected to expression identification. Figure 4 shows the molecular biological identification of the overexpression of AtMIF1 gene. Col is the wild-type Arabidopsis as a control. OE-5 and OE-8 represent the overexpression of AtMIF1 driven by GCD1, respectively. In lines 5 and 8, the mRNA levels of AtMIF1 were up-regulated by nearly 4000-fold and 300-fold in lines 5 and 8, respectively.

After confirming that the target gene was overexpressed (Fig. 4), the phenotypes of the growth conditions of the transgenic plants at each stage were compared and observed. Fig. 5 is the mucopolysaccharide and oil content phenotype secreted by the transgenic Arabidopsis in the seed after the transformation of the overexpression vector, as well as the measurement results of the seed size, the dry weight of the seed per plant, the fresh weight of the seedling per plant, and the height of the seedling per plant, The observation results showed that the gene did not affect the vegetative and reproductive growth of plants, and the transgenic Arabidopsis thaliana after the transformation of the overexpression vector had all the same seed size, dry weight of single seed, fresh weight of single seedling, and height of single seedling. Indifferent to the wild type, it also participates in regulating the secretion of mucopolysaccharide on the seed surface and the accumulation of oil in the seed. In the overexpression lines, the expression of AtMIF1 was significantly up-regulated, the secretion of mucopolysaccharide on the seed surface was decreased, and the oil content was significantly increased compared with the wild type. Therefore, combining AtMIF1 gene and overexpression technology, Arabidopsis seeds with higher oil content can be obtained (Fig. 5c), which has great application prospects in oil crops to improve the oil content of seeds.

Obviously, the above-mentioned embodiments are only examples for clear illustration, and are not intended to limit the implementation manner. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. However, the obvious changes or changes derived therefrom still fall within the protection scope of the present invention.

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Claims (6)

1. The application of AtMIF1 gene in positively regulating the oil content of plant seeds, the nucleotide sequence of said gene AtMIF1 is the CDS sequence shown in SEQ ID NO.1. 2. the application of the protein encoded by the AtMIF1 gene of claim 1 in the aspect of positive regulation of plant seed oil content, the amino acid sequence of the protein is shown in 1) or 2) as follows: 1) an amino acid sequence as shown in SEQ ID NO.2; 2) A fusion protein obtained by linking the N-segment and/or C-terminal of the protein with the amino acid sequence shown in SEQ ID NO.2 with a tag. 3. The application of the overexpression vector containing the AtMIF1 gene of claim 1 in positively regulating the oil content of plant seeds. The application according to claim 3, wherein the overexpression vector comprises a GCD1 promoter sequence-AtMIF1CDS sequence-transcription termination sequence. The application according to claim 3, wherein the overexpression vector further comprises a LAT52 promoter-RFP sequence-transcription termination sequence. 6 . The application according to claim 3 , wherein the C-terminus of the AtMIF1CDS sequence in the overexpression vector is fused with a small peptide tag 1×FLAG. 7 .

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