CN111187783B - OPR gene for regulating and controlling rape fatty acid and expression vector and application thereof - Google Patents

Abstract

The invention belongs to the field of crop genetic breeding, and particularly relates to an OPR gene for regulating and controlling rape fatty acid, an expression vector and application thereof. The base sequence of the OPR gene is shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 or SEQ ID NO: 4, respectively. The invention researches the molecular mechanism and molecular biological function of the rape OPR gene for regulating the fatty acid component by promoting different miRNA of the OPR gene to regulate the gene expression quantity in the regulation period and analyzing the final fatty acid component by gas chromatography, and provides a potential new gene resource for cultivating high oleic acid or other high-quality rapes.

Description

OPR gene for regulating and controlling rape fatty acid and expression vector and application thereof

Technical Field

The invention belongs to the field of crop genetic breeding, and particularly relates to an OPR gene for regulating and controlling rape fatty acid, an expression vector and application thereof.

Background

Fatty acid (C)Fatty acid) Is an important substance for maintaining human health, and can be divided into saturated fatty acid and unsaturated fatty acid, and the components of the saturated fatty acid and the unsaturated fatty acid have great influence on the components of the fatty acid. In animal and vegetable oil, the saturated fatty acid content of animal oil is higher, and the unsaturated fatty acid content of vegetable oil is higher. The saturated fatty acids mainly comprise palmitic acid (Palmitic acid) And stearic acid (b), (c), (dStearic acid) Too high intake of saturated fatty acids can easily cause elevation of blood cholesterol, triacylglycerol, and low density lipoprotein cholesterol, leading to stenosis of arterial lumen and increasing the risk of coronary heart disease. The unsaturated fatty acid mainly comprises oleic acidOleic acid) Linoleic acid (linoleic acid) and Linolenic acid (alpha-Linolenic acid) are key factors for regulating various metabolisms in animal bodies, are important nutrient substances in the development process of human bodies, and have the functions of preventing and treating certain diseasesAnd (4) acting. Oleic acid is a monounsaturated fatty acid, is a fatty acid essential to human bodies, and exists in all animal and vegetable fats in the form of glyceride. Oleic acid is mainly concentrated on sn-2 position of triglyceride, is vegetable oil with high nutritive value, can more effectively reduce accumulation of cadmium in liver and kidney tissues of mice, relieve acute cadmium poisoning of mice, and reduce TC and TG contents (p) in serum of white mice<0.01), has good nutrition and health promotion effects. Linoleic acid is essential fatty acid in human and animal nutrition, has the effects of reducing blood fat, softening blood vessels, reducing blood pressure and promoting microcirculation, can prevent or reduce the incidence rate of cardiovascular diseases, is particularly favorable for preventing and treating hypertension, hyperlipidemia, angina pectoris, coronary heart disease, atherosclerosis, senile obesity and the like, can prevent the deposition of serum cholesterol in the blood vessel wall of a human body, has the reputation of 'blood vessel scavenger', and has the health-care effect of preventing and treating atherosclerosis and cardiovascular diseases. Linolenic Acid belongs to omega-3 series polyenoic fatty Acid, is all cis-9, 12 and 15 octadecatrienoic Acid, exists in dark green plants in the form of glyceride, is a main component forming human tissue cells, cannot be synthesized and metabolized in vivo, and is converted into the vital active factors of Docosahexaenoic Acid (DHA) and Eicosapentaenoic Acid (EPA) necessary for the organism. However, it cannot be synthesized in humans and must be taken in vitro. Once the human body is deficient, the lipid metabolism disorder of the body can be induced, and the symptoms of immunity reduction, amnesia, fatigue, hypopsia, atherosclerosis and the like can be caused. Especially if infants and teenagers lack linolenic acid, the normal development of intelligence can be seriously influenced, which is proved by scientists at home and abroad and is generally accepted by the world nutritional science. Since the sum of the ratios of the various fatty acids is 1, the saturated fatty acids in the rape oil severely inhibit the ratio of the unsaturated fatty acids, so that the content of the unsaturated fatty acids can be increased by reducing the saturated fatty acids.

1, 2-O-plant dienoic acid reductase: (1，2-oxo-phytodienoic acid reductase，OPR) Is a key enzyme in jasmonic acid biosynthesis, namely the metabolic pathway of octadecenoic acid, and controls jasmonic acidThe final step of the synthesis is carried out,OPRparticipating in the beta oxidation of fatty acid and reducing alpha-linolenic acid, playing a role in the metabolism of fatty acid, and jasmonic acid formed by the alpha-linolenic acid can widely mediate the transmission of stress signals and the like, and plays an important role in the stress resistance of plants.

Disclosure of Invention

The invention provides an OPR gene for regulating and controlling rape fatty acid, an expression vector and application thereof. Specifically, the OPR gene is overexpressed, and a new rape variety with higher unsaturated fatty acid ratio is created through overexpression.

The technical scheme of the invention is realized as follows:

an OPR gene for regulating and controlling rape fatty acid, which is characterized in that: the base sequence of the OPR gene is shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 or SEQ ID NO: 4, respectively.

The expression vector containing the OPR gene comprises an overexpression vector and an RNAi vector.

The construction method of the expression vector comprises the following steps: OPR gene primers were designed based on the A, C genomic sequence of the OPR gene and amplified. And amplifying by PCR to obtain an amplification product. And connecting the product with a PMD18-T vector after enzyme digestion and recovery. The expression vector carrying the target gene is obtained through screening and identification. The content of fatty acid can be regulated and controlled by introducing the expression vector with the OPR gene into rape. The expression vector is applied to culturing new varieties of high-content unsaturated fatty acid rape.

The invention has the following beneficial effects:

1. the invention aims to provide a gene OPR for regulating and controlling the fatty acid proportion of rape, which has the amino acid sequence shown in SEQ ID NO: 1. The accumulation of the gene is increased along with the change of the growth period of the rape, so that the accumulation of the rape unsaturated fatty acid is increased, and the over-expression of the gene is helpful for understanding the molecular mechanism of the accumulation of the rape fatty acid. By pairsOPRGene is in sweetThe expression rule of the blue high oleic rape is researched, the correlation analysis is carried out by combining the fatty acid composition of related materials, and the blue high oleic rape is further cloned, so that the effect of the blue high oleic rape in the fatty acid metabolism of the high oleic rape is researched, and the improvement and the upgrade of the quality of the rape in China are promoted.

2. The invention uses an overexpression technology to promote the expression of endogenous OPR genes of rape, so as to cause the change of fatty acid components, reduce the proportion of saturated fatty acid, increase the proportion of unsaturated fatty acid and promote the improvement of the quality of edible rapeseed oil. Specifically, the OPR gene is overexpressed, and a new rape variety with higher unsaturated fatty acid ratio is created through overexpression.

3. The invention researches the molecular mechanism and molecular biological function of the rape OPR gene for regulating the fatty acid component by promoting different miRNA of the OPR gene to regulate the gene expression quantity in the regulation period and analyzing the final fatty acid component by gas chromatography, and provides a potential new gene resource for cultivating high oleic acid or other high-quality rapes.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the length distribution of clean reads.

FIG. 2 shows the results of sample clustering analysis.

FIG. 3 is GO enrichment analysis top10 (screen GO entries in top10 of the number of genes in three categories).

FIG. 4 shows the qRT-PCR results of 21 differential miRNAs (low oleic acid rape gene expression is control).

FIG. 5 shows that miR156 & gtc & gtg and its target gene are expressed in different materials of high oleic acid rape near isogenic line.

FIG. 6 shows the expression of the OPR gene in 20 high oleic rape material.

FIG. 7 is the PCR electrophoresis diagram of the RNAi fragment of the rape target gene.

FIG. 8 shows 124 and 829 overexpression vectors.

FIG. 9 shows the overexpression of OPR copies in Arabidopsis thaliana and the change in fatty acid composition due to RNAi.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

miRNA sequencing and fatty acid metabolism related functional gene screening

The high oleic rape near isogenic lines (oleic acid content 81.4%, 56.2% respectively) are provided by national oil improvement center of Hunan agriculture university. Mixing seeds 20-35 days after self-cross pollination, placing in a sterilized mortar, quickly grinding into fine powder, placing the whole operation in a liquid nitrogen environment, and referring to the extraction instruction of the total RNA extraction kit in other steps.

Constructing and sequencing an sRNA library, analyzing miRNA data, identifying differential miRNA and predicting a target gene, verifying differentially expressed miRNA and target gene thereof by qRT-PCR, and analyzing expression rules of differential miRNA related to fatty acid metabolism and the target gene thereof.

The statistical finding of the length of clean reads shows that the peak value of the length distribution in the sample is 21-25bp, which accords with the miRNA characteristics (figure 1), and shows that the sequencing result of the sRNA is high in quality and reliable. By utilizing a cluster analysis method, the similarity between samples is researched by calculating the difference miRNA distance between the high-oleic-acid rape and the low-oleic-acid rape, and three high-oleic-acid or low-oleic-acid samples are respectively found in one cluster, which indicates that the small RNAs may have similar biological functions (figure 2). After identifying differentially expressed small RNAs with p <0.05 and log2 (test/control) absolute values greater than 1.5, 21 differentially expressed small RNAs (8.39% of the total) were detected. Of these, 9 genes (42.86%) were up-regulated and 12 genes (57.14%) were down-regulated.

Predicting miRNA target genes by using Targetfinder software, performing GO annotation and KEGG functional enrichment, and finding that 359 target genes are enriched and expressed in 20 metabolic pathways, and particularly annotating. Wherein GO annotation analysis found that 133 putative target genes were associated with 21 differentially expressed mirnas. Among them, 62 distributed in "biological process" (46.62%), 32 in "cellular component" (24.06%) and 39 in "molecular function" (29.32%) (FIG. 3).

Under "biological processes", most target genes are associated with "transcription" (9.68%) and "transcriptional regulation" (4.84%). In the "cellular component" category, "nucleus" (28.13%) and "cytosol" (9.38%) are the same as "cytosol". In the "molecular function" class of genes, most of the potential functions are associated with "transcription factor activity" (10.26%), "DNA binding" and "catalytic activity" (5.13%). The distribution of these target genes indicates that rapeseed is very metabolically active.

qRT-PCR validation (FIG. 4) was performed on differential miRNAs and found that the relative expression levels of the remaining genes were the same as the results obtained by sequencing analysis, except NC-027772.1-2143, bna-miR156a, bna-miR156b > c > g, bna-miR160a > b > c > d and bna-miR166 f. Wherein, the qRT-PCR results of NC _027760.1_5272, NC _027760.1_5272, NW _013650328.1_26640, bna-miR156b > c > g, bna-miR166f, bna-miR167a > b, bna-miR169m, bna-miR396a and bna-miR824 are very different from the control in the level of P <0.01 (0.77, 0.72, 0.50, 0.74, 1.56, 0.59, 0.54, 0.20 and 1.90 times of the control respectively); the qRT-PCR results for NC _027761.1_6665, NC _027772.1_21433, bna-miR156a, bna-miR160a > b > c > d were significantly different from the control at P <0.05 level (0.87, 0.81, 0.83 times of the control, respectively), with the remainder being not significantly different from the control. However, only the qRT-PCR results of bna-miR167a > b, bna-miR396a and bna-miR824 are more obvious than the sequencing results. In addition, 9 of 13 significant and more miRNAs have target genes, namely NC-027760.1 _5272, NW-013650328.1 _26640, bna-miR156b > c > g, bna-miR166f, bna-miR169m, bna-miR396a, bna-miR824, bna-miR156a and bna-160 miR160a > b > c > d, which are probably novel miRNAs related to rape high oleic acid.

These results indicate that miRNA sequencing analysis can successfully find new candidate mirnas with high accuracy and efficiency. Screening miRNA with obvious differences in the verification, and selecting bna-miR156b > c > g which is related to fatty acid metabolism. Different materials are taken to carry out qRT-PCR analysis (figure 5) at different development stages of the high oleic rape near-isogenic line, and the expression relationship between bna-miR156b > c > g and the target gene thereof is verified. bna-miR156b > c > g is down-regulated in the whole growth stage and is reduced along with the growth stage, and the OPR of the target gene is opposite to the OPR, and the expression quantity is continuously increased along with the advancing of the growth period.

Second, screening fatty acid metabolism related functional gene

The high oleic rape isogenic line is taken as a material and is provided by the national oil improvement center of Hunan agriculture university. Escherichia coli competent DH5 alpha was purchased from Beijing Okagaku biosciences. pCAMBIA1300-35S-N1 is used as a vector. The kit comprises 1: the plant total RNA extraction Kit TransZol UP, TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix is purchased from Beijing Quanyujin biotechnology, Inc., Pfu high fidelity enzyme, DNA marker is purchased from Tiangen, and restriction enzyme, dNTP, T4 DNA Ligation Kit is purchased from Beijing engine, Inc. The use instrument comprises: PCR instruments (PTC 200), agarose gel imaging system (Syngene), autoclave (TOMY SX-500), thermostated water bath (Amersham Bioscience), bacterial incubator (EYELA LTI-700), sterile ultra clean bench (ThermoElectro Industries, S1-1203), shaker (Sciencific Industries, S1-1203), GelDoc2000 gel imager, and the like. The procedure was performed according to the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit instructions.

The verified OPR gene is searched by using TAIR and Brassica Database, 4 copies of the OPR gene are found, A, C genomes are two respectively, and the subsequent primer design is carried out according to the sequence.

To pairOPRGeneIn thatAnalysis (figure 6A) of the expression levels of 20 different rape line materials in the vegetative growth period shows that the expression levels of the two genes are gradually increased in the leaves at the seedling stage, the leaves at the 5-6 leaf stage and the leaves at the bud stage except 6 and 8. And correlation analysis showed (Table 1) that high oleic material was present throughout the vegetative growth phaseOPRThe gene expression level has no significant correlation with 5 fatty acid components.

TABLE 1 vegetative growth phaseOPRCorrelation analysis between gene expression level and different fatty acid components

To pairOPRGeneIn thatAnalysis of expression levels in the flowering flowers of 20 different rape line materials (fig. 6B), with the highest expression level in the blooming flowers and the lowest expression level in the withered flowers; in addition, correlation analysis showed that (Table 2),OPRthe expression quantity in the flower bud and the full-bloom flower has a very obvious correlation with oleic acid, linoleic acid and linolenic acid, and the expression quantity in the withered flower has no obvious correlation with 5 fatty acid components.

TABLE 2 OPRCorrelation analysis of different materials of genes in flowering phase

To pairOPRGeneIn thatThe expression level of 20 different rape lines was analyzed in the silique stage (FIG. 6C), and the gene showed a decreasing trend throughout the silique stage, but the overall expression level was higher in the silique stage than in the other stages. The correlation analysis showed that (table 3),OPRthe genes have very significant/significant correlations with palmitic acid, oleic acid, linoleic acid and linolenic acid throughout the whole silique stage. The silique stage is the key stage of fatty acid accumulation, while Jiangnan and the like find that the oil tea fruit containsOPRThe gene participates in linolenic acid metabolism, and is expressed in different periods of the oil tea seed maturation process, but the expression abundance is lower in different periods; conjecture ofOPRThe gene is involved in the accumulation process of fatty acid in rapeseed.

TABLE 3OPRCorrelation analysis of different materials of gene in silique stage

For 20 rape materialsOPRThe analysis of the expression condition of the gene of the acetyltransferase shows that,OPR3the expression level of the gene in the seedling stage is gradually increased until flowers bloom in the flowering stage, the silique stage is gradually reduced,OPRthe gene expression level is much lower than that of the reference gene; by synthesizing the gene expression and combining fatty acid component analysis, OPR in 35d seeds, the polypeptide has a significant negative correlation with oleic acid and a significant positive correlation with linoleic acid and linolenic acid, and can be a micro-effect gene for controlling the synthesis of oleic acid.

III,OPRGene cloning and vector construction

After target gene amplification, PCR amplification product is subjected to gel electrophoresis, photographed and observed, and cut and recovered. Connecting with PMD18-T carrier, and sequencing by prokaryote. The high-fidelity amplification of bna-miR156b > c > g 4 target genes, the lengths of the target genes are 1122bp (949), 1119bp (124), 1119bp (829), 1125bp (135), 2 target genes 114 and 148 of bna-miR396 are 1350bp respectively, and the target band is in line with the expectation. The synthesized cDNA is used as a template, and PCR high-fidelity amplification is respectively carried out on the 6 target gene specific fragments to obtain DNA fragments with the length of the target fragment. The 124 and 829 copies of the OPR gene are shown in FIG. 7, and the 949 and 135 copies are shown in FIG. 7. The four copies 124, 829, 949 of the OPR gene and the 135 RNAi recombinant plasmid are shown in FIG. 8. The vector construction included two steps of an overexpression recombinant plasmid map (left) and an RNAi recombinant plasmid map (right) (fig. 8). The method comprises the following specific steps:

(1) in vitro recombination

And carrying out double enzyme digestion on the PMD18-T vector, recovering glue, and carrying out in-vitro homologous recombination reaction on a product to obtain a recombinant plasmid.

(2) Transformation of

Adding the ligation product into competent cells (50-100 μ L), mixing well, and ice-cooling for 30 min; performing heat shock at the constant temperature of 42 ℃ for 90 s, and immediately placing in an ice bath for 2-3 min after the heat shock is finished; adding 500 μ L LB liquid culture medium without antibiotics, and shake culturing at 37 deg.C and 220 rpm for 45-60 min; centrifuging at 6000 rpm for 1 min, discarding supernatant, leaving about 100 μ L of suspension, uniformly spreading on LB solid agar medium containing kanamycin (50 mg/L), and performing inverted culture at 37 deg.C for 12-16 h.

(3) Extracting plasmid by shaking bacteria and sequencing

And (3) selecting positive colonies, culturing, sending the bacterial liquid to the organism company of the department of engine for sequencing, comparing the sequencing result with DNAMAN through SnapGene, checking the base interpretation result, and reducing the error of a sequencer.

Enzyme digestion of recombinant plasmid and RNAi fragment:

TABLE 4 Forward cleavage System

TABLE 5 Forward connection System

The linker system was reacted at 22 ℃ for 1 h and after completion of the reaction the linker product was placed on ice for the conversion step.

Fourth, analysis of OPR gene overexpression and RNAi results

As can be seen from FIG. 9, the effects are slightly different after different copy regulation, the overall change is that the ratio of oleic acid to linoleic acid is increased, and the ratio of stearic acid is decreased, and the results show that the over-expression of the OPR gene and RNAi can be used for regulating the ratio of fatty acid, thereby providing a method for cultivating high-quality rape.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

<120> OPR gene for regulating and controlling rape fatty acid and expression vector and application thereof

<141> 2020-03-31

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Claims (4)

1. An OPR gene for regulating and controlling rape fatty acid, which is characterized in that: the base sequence of the OPR gene is shown as SEQ ID NO: 1 is shown.

2. An overexpression vector comprising the OPR gene of claim 1.

3. The method for constructing an overexpression vector of claim 2, which comprises the following steps: designing a primer according to the OPR gene, obtaining an amplification product through PCR amplification, connecting the amplification product with a PMD18-T vector after enzyme digestion and recovery, obtaining an overexpression vector carrying a target gene through screening and identification, and regulating the content of fatty acid after introducing the overexpression vector carrying the OPR gene into rape.

4. The use of the overexpression vector of claim 2 in breeding new varieties of high-unsaturated-fatty-acid-content rape.

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