CN113493792B - Method for improving biosynthesis of plant proanthocyanidins and application thereof - Google Patents

Abstract

The invention provides a method for improving biosynthesis of proanthocyanidins in plants and application thereof, and relates to the field of genetic engineering application, wherein the proanthocyanidins content in plants is increased by over-expressing CsANR2 gene, PAP1 gene and CsF3'5' H gene, or over-expressing CsANR2 gene and PAP1 gene, or over-expressing CsANR2 gene in plants, so that the problem of too low proanthocyanidins content in plants is solved.

Description

A kind of method and application of improving plant proanthocyanidin biosynthesis

technical field

The invention relates to the application fields of biotechnology and genetic engineering, in particular to a method for improving the biosynthesis of plant proanthocyanidin and its application.

Background technique

Proanthocyanidins are flavonoid secondary metabolites with important activity in plants, which widely exist in various plants and have strong interactions with proteins. Previous research results have shown that they not only play an important role in the regulation of seed dormancy, lifespan and germination, but also participate in the regulation of biotic and abiotic stresses in plants (Debeaujon et al., 2003). For human health, it also has antioxidant, anti-inflammatory and anti-cancer effects (Dixon et al., 2005). Currently the most widely used brown cotton - natural colored cotton, the brown pigment in its fiber is proanthocyanidin (Xiao et al., 2014; Yan et al., 2018). In the animal husbandry industry, although high-quality protein-rich pastures will provide ruminants with rich protein, minerals and vitamins and other important nutrients, their rich protein will also cause bloat in ruminant cattle and sheep. However, studies have shown that when the content of proanthocyanidins in alfalfa is higher than 2% of the dry weight, it can effectively prevent the occurrence of bloat in ruminants such as cattle and sheep (Verdier et al., 2012). However, proanthocyanidins in alfalfa The pigment content has not yet reached 0.2% of the dry weight, so it is of great significance to increase the proanthocyanidin content in plants.

According to the research of Proanthocyanidin Biosynthesis in Plants:Purification of Legume Leucoanthocyanidin Reductase And Molecular Cloning OfIts cDNA disclosed by Gregory J.Tanner etc., the steps and components of the proanthocyanidin synthesis pathway are more complicated, and there are many influencing factors. There are still some difficulties in the content of proanthocyanidins. Even in the Arabidopsis mutants that can accumulate a large amount of anthocyanins, the total content of proanthocyanidins per gram of leaf is only 2.76mg/g, which is far from the actual production. need.

Contents of the invention

In order to solve the existing problems in the prior art, the present invention provides a method for improving the biosynthesis of plant proanthocyanidins and its application in plant breeding and ruminant anti-bloat disease. The present invention changes the gene of plant flavonoid secondary metabolism The expression increases the content of proanthocyanidins in plants, especially the content of proanthocyanidins in plants can reach more than 4 times that of wild-type plants.

In order to realize the technical purpose of the present invention, the present invention provides a method for improving the biosynthesis of plant proanthocyanidins, which makes the proanthocyanidins in plants overexpress CsANR2 gene, PAP1 gene and CsF3'5'H gene at the same time. Anthocyanin content increased.

In particular, the simultaneous overexpression of CsANR2 gene, PAP1 gene and CsF3'5'H gene in plants can be achieved by inserting CsANR2 gene and CsF3'5'H gene into plants capable of overexpressing PAP1 gene by genetic engineering methods.

Wherein, the method for increasing the biosynthesis of plant proanthocyanidins can also increase the content of proanthocyanidins in plants by overexpressing CsANR2 gene and PAP1 gene in plants.

Wherein, the method for increasing the biosynthesis of plant proanthocyanidins can also increase the content of proanthocyanidins in plants by overexpressing CsANR2 gene in plants.

Wherein, the plant is a high-protein forage grass.

In particular, the plants include, but are not limited to, Medicago, Arabidopsis, and the like.

To achieve the technical purpose of the present invention, the present invention further provides a plant with a high content of proanthocyanidins, which is obtained by the above method.

To achieve the technical purpose of the present invention, the present invention also provides a use of the above-mentioned method for increasing the biosynthesis of plant proanthocyanidins to prevent the occurrence of bloat disease in ruminants such as cattle and sheep.

Wherein, the purpose is to increase the content of proanthocyanidins in high-protein pasture plants by using the method for increasing the biosynthesis of plant proanthocyanidins, and then eat the pasture plants with high proanthocyanidin content as feed for ruminants such as cattle and sheep, Then prevent the occurrence of bloat disease in ruminants such as cattle and sheep.

In order to achieve the technical purpose of the present invention, the present invention also provides a use of the above-mentioned plants with high content of proanthocyanidins for preventing the occurrence of bloat in ruminants such as cattle and sheep.

Beneficial effect

1. The method of the present invention improves the content of proanthocyanidins in plants by changing the gene expression of flavonoid secondary metabolism in plants by using genetic engineering methods, and solves the problem of low proanthocyanidin content in plants.

2. The present invention increases the content of proanthocyanidins in Arabidopsis thaliana by more than 4 times by introducing CsANR2 gene and CsF3'5'H gene into Arabidopsis mutants, which has significant technical effects. The content of anthocyanins provides another practical way, which is simple and of great significance to animal husbandry.

Description of drawings

Fig. 1 is the result graph of the expression level of corresponding gene in each homozygous transgenic material provided by Example 1 of the present invention;

Fig. 2 is the result figure of the relative content of anthocyanins in the 30-day leaves of each homozygous transgenic material provided by Example 1 of the present invention;

Fig. 3 is a graph showing the content results of proanthocyanidins in the leaves of each homozygous transgenic material provided in Example 1 of the present invention at day 30.

Detailed ways

The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are only illustrative and should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents and products used are also commercially available. Various processes and methods that are not described in detail are conventional methods well known in the art. The source, trade name and necessary list of components of the reagents used are all indicated when they appear for the first time. Descriptions are the same as those indicated for the first time.

Embodiment 1 improves the method for Arabidopsis proanthocyanidin biosynthesis

In the embodiment of the present invention, Arabidopsis thaliana is used as the target culture plant to obtain a high-content Arabidopsis plant. The specific steps are as follows:

1. Selection of expression materials

Since the expression level of PAP1 in the Arabidopsis pap1-D mutant is more than 300 times that of the wild type, the present invention uses the pap1-D mutant as an overexpression material, and there is no need to overexpress the pap1-D molecule in Arabidopsis Clone processing.

Of course, those skilled in the art can also use conventional Arabidopsis plants as expression materials, and overexpress PAP1 in Arabidopsis by means of molecular cloning.

2. Molecular cloning

CsANR2 and CsF3'5'H were cloned by conventional methods, pB2GW7-CsANR2 and pK2GW7-CsF3'5'H vectors were constructed, and the plasmids of the two vectors were transformed into Agrobacterium GV3101 to obtain the ability to express CsANR2 and CsF3'5' H gene strains.

3. Plant transformation

Use the flower infection method to transform Arabidopsis plants to obtain high-expression transgenic plants, specifically:

Representative high-expression transgenic lines were selected and pollinated after artificial emasculation to produce CsF3'5'H and PAP1(F×p), CsANR2 and PAP1(A×p) and CsF3'5'H, CsANR2 and PAP1 (F×A×p) materials, while Arabidopsis expressing only CsANR2, Arabidopsis CsANR2 and Arabidopsis pap1-D mutant were used as controls. After the homozygosity of each material was obtained through identification, the gene expression level was confirmed by qRT-PCR, and the results are shown in FIG. 1 .

According to the electropherogram in Figure 1A, it can be seen that Arabidopsis (F×p) can express CsF3’5’H and PAP1 genes, Arabidopsis (A×p) can express CsF3’5’H and PAP1 genes, and Arabidopsis ( F×A×p) material can express CsF3'5'H, CsANR2 and PAP1 gene, according to the histogram of Figure 1B, it can be known that each target plant can express the corresponding target gene at a higher level, it can be seen that the present invention adopts the above method The target plants capable of overexpressing the corresponding target genes required by the present invention were all obtained.

It should be noted that the above-mentioned unexplained test steps are all routine steps. For details, please refer to the content in "Transformation of Arabidopsis thaliana by Inflorescence Dip Dyeing Method", which will not be repeated in the present invention.

4. Detection and analysis of flavonoid secondary metabolite content

4.1 Detection and analysis of anthocyanin content

Since anthocyanins and proanthocyanidins have a common upstream intermediate product anthocyanins in the synthetic pathway, the biosynthesis of these two metabolites will compete, resulting in a balanced state of trade-off. Therefore, we first analyzed the changes of anthocyanin content in leaves of these materials for 30 days, and the results are shown in Figure 2.

According to the results in Figure 2, it can be seen that the overexpression of the CsANR2 gene alone reduces the anthocyanin content of the leaves, which is only half of that of the wild type; when only overexpression of CsF3'5'H, the anthocyanin content of the leaves is not significantly different from that of the wild type ; and the overexpression of PAP1 greatly increased anthocyanins, reaching more than 30 times that of the wild type. When CsF3'5'H and PAP1 were overexpressed at the same time, the content of anthocyanins decreased to about 25 times that of the wild type; when CsANR2 and PAP1 were overexpressed at the same time, the content of anthocyanins further decreased; when these three genes were simultaneously When overexpressed, the content of anthocyanin decreased significantly, only slightly higher than the level of the wild type, which was 1.4 times of the wild type.

4.2 Detection and analysis of proanthocyanidin content

The content of proanthocyanidins in the 30-day-old leaves of Arabidopsis thaliana was detected and counted, and the results are shown in Figure 3.

According to the results in Fig. 3A, when soluble proanthocyanidins were detected by DMACA method, the accumulation of soluble proanthocyanidins was only detected in the leaves overexpressing both CsANR2 and PAP1. However, when soluble proanthocyanidins were detected by n-butanol hydrochloric acid, the accumulation of soluble proanthocyanidins was detected in the leaves that overexpressed CsANR2 and PAP1 and the three genes at the same time. Among them, the leaves that overexpressed CsANR2 and PAP1 The content of soluble proanthocyanidins in the leaves of Arabidopsis thaliana reached 2.11 mg/g DW, and the content of soluble proanthocyanidins in the leaves of Arabidopsis thaliana overexpressing CsANR2, PAP1, and CsF3'5'H reached 2.28 mg/g DW , all reached about 3 times that of the wild type (namely Arabidopsis pap1-D mutant, the content of soluble proanthocyanidin in its leaves was 0.74mg/g DW).

At the same time, the content of insoluble proanthocyanidins in the 30-day leaves of Arabidopsis thaliana was detected and counted. Affects the biosynthesis of insoluble proanthocyanidins, and the content of insoluble proanthocyanidins in leaves overexpressing PAP1 alone is 2.3 times that of wild type. The content of insoluble proanthocyanidin in the leaves of Arabidopsis thaliana overexpressing CsF3'5'H and PAP1 reached 4.33 mg/gDW, which was 2.14 times that of the wild type; in the leaves of Arabidopsis thaliana overexpressing CsANR2 and PAP1 The content of insoluble proanthocyanidins reached 5.65 mg/g DW, which was 2.8 times that of the wild type; while the content of insoluble proanthocyanidins in the leaves of Arabidopsis thaliana overexpressing CsANR2, PAP1, and CsF3'5'H at the same time reached 9.11 mg/g DW is 4.51 times that of the wild type (wherein the wild type is Arabidopsis pap1-D mutant, the content of insoluble proanthocyanidin in its leaves is 2.02 mg/g DW).

In summary, it can be seen that the total content of proanthocyanidins in the target Arabidopsis plants that can overexpress CsANR2, PAP1, and CsF3'5'H provided by the present invention reaches 11.39 mg/g DW, which is higher than that of the Arabidopsis pap1-D mutant. more than 4 times; and the total content of proanthocyanidins in the target Arabidopsis plants that can overexpress CsANR2 and PAP1 at the same time reaches 7.86mg/g DW, which is about 3 times that of the Arabidopsis pap1-D mutant; and can simultaneously overexpress CsF3'5'H, PAP1's target Arabidopsis plants contained insoluble proanthocyanidins which were 1.6 times higher than those in Arabidopsis pap1-D mutants. It can be seen that the target plants that can simultaneously overexpress CsANR2, PAP1, and CsF3'5'H provided by the present invention can increase the content of proanthocyanidins in plants, and have obvious effects.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (2)

1. A method for increasing the biosynthesis of proanthocyanidins in a plant, which comprises overexpressing the CsANR2 gene, PAP1 gene and CsF3'5' H gene simultaneously in the plant to increase the proanthocyanidins content in the plant;

wherein the plant is Arabidopsis thaliana.

2. A method for increasing the biosynthesis of proanthocyanidins in plants, which comprises overexpressing a CsANR2 gene and a PAP1 gene in plants to increase the proanthocyanidins content in the plants;

wherein the plant is Arabidopsis thaliana.

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