CN113493792A

CN113493792A - Method for improving biosynthesis of plant proanthocyanidins and application thereof

Info

Publication number: CN113493792A
Application number: CN202010188984.6A
Authority: CN
Inventors: 江文波; 庞永珍; 魏解冰
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2020-03-18
Filing date: 2020-03-18
Publication date: 2021-10-12
Anticipated expiration: 2040-03-18
Also published as: CN113493792B

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 low proanthocyanidins content in plants is solved.

Description

Method for improving biosynthesis of plant proanthocyanidins and application thereof

Technical Field

The invention relates to the application field of biotechnology and genetic engineering, in particular to a method for improving biosynthesis of plant proanthocyanidins and application thereof.

Background

Proanthocyanidins (Proanthocyanidins) are flavonoid secondary metabolites having important activities in plants, are widely present 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, longevity and germination, but also participate in the regulation of biotic and abiotic stress in plants (Debeaujon et al, 2003). It also has antioxidant, anti-inflammatory and anticancer effects for human health (Dixon et al, 2005). At present, brown cotton, which is the most widely used natural colored cotton, has proanthocyanidin as brown pigment in the fiber (Xiao et al, 2014; Yan et al, 2018). In the animal husbandry industry, although high-quality protein-rich pasture provides important nutritional ingredients such as rich protein, minerals and vitamins for ruminants, the protein-rich pasture can also cause ruminants such as cattle and sheep to suffer from tympany, but researches show that the tympany of the ruminants such as the cattle and the sheep can be effectively prevented when the content of proanthocyanidin in alfalfa is higher than 2% of dry weight (Verdier et al, 2012), but the content of proanthocyanidin in alfalfa does not reach 0.2% of the dry weight, so that the significance of improving the content of proanthocyanidin in plants is great.

According to the research Of the Proanthocyanidin Biosynthesis in Plants disclosed in Gregory J.tanner et al, Purification Of Proanthocyanidin production And Molecular Cloning Of Its cDNA, it is known that the steps And components Of the Proanthocyanidin synthesis pathway are complex, And the influence factors are more, so that the increase Of the Proanthocyanidin content in Plants is difficult, And even in the existing Arabidopsis thaliana mutants capable Of accumulating a large amount Of anthocyanidins, the total Proanthocyanidin content per gram Of leaves is only 2.76mg/g, which is far from the requirement Of actual production.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a method for improving the biosynthesis of plant proanthocyanidins and application of the method in plant breeding and ruminants to resist tympanites.

In order to achieve the technical object of the present invention, the present invention provides a method for increasing the biosynthesis of proanthocyanidins in plants, which comprises overexpressing CsANR2 gene, PAP1 gene, and CsF3 '5' H gene simultaneously in plants, thereby increasing the proanthocyanidins content in the plants.

In particular, the overexpression of the CsANR2 gene, the PAP1 gene and the CsF3 '5' H gene in a plant body at the same time can be achieved by inserting the CsANR2 gene and the CsF3 '5' H gene into a plant capable of overexpressing the PAP1 gene by using a genetic engineering method.

The method for improving the biosynthesis of the plant proanthocyanidins can also increase the proanthocyanidins content in the plant by over-expressing the CsANR2 gene and the PAP1 gene in the plant.

The method for improving the biosynthesis of plant proanthocyanidins can also increase the proanthocyanidins content in plants by overexpressing the CsANR2 gene in plants.

Wherein the plant is high protein pasture.

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

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

In order to achieve the technical purpose of the invention, the invention also provides application of the method for improving the biosynthesis of plant proanthocyanidins in preventing tympanites of ruminants such as cattle, sheep and the like.

The method for improving the biosynthesis of plant proanthocyanidins is used for increasing the proanthocyanidins content in the high-protein pasture plants, and then the pasture plants with the high proanthocyanidins content are eaten as feed of ruminants such as cattle and sheep, so that the ruminants such as the cattle and the sheep are prevented from generating tympany.

In order to achieve the technical purpose of the invention, the invention also provides the application of the plant with high content of proanthocyanidin in preventing the tympanites of the ruminants such as cattle, sheep and the like.

Advantageous effects

1. The method of the invention utilizes a genetic engineering method to improve the proanthocyanidin content in the plant by changing the gene expression of the flavonoid secondary metabolism in the plant, thereby solving the problem of low proanthocyanidin content in the plant.

2. The CsANR2 gene and the CsF3 '5' H gene are introduced into the arabidopsis mutant, so that the proanthocyanidin content in arabidopsis is increased by more than 4 times, the method has a remarkable technical effect, another feasible way is provided for improving the proanthocyanidin content in plants, the method is simple, and the method has great significance for animal husbandry.

Drawings

FIG. 1 is a graph showing the results of the expression levels of the corresponding genes in each homozygous transgenic material provided in example 1 of the present invention;

FIG. 2 is a graph showing the results of the relative content of anthocyanins in 30-day leaves of each homozygous transgenic material provided in example 1 of the present invention;

FIG. 3 is a graph showing the results of proanthocyanidin content in 30-day leaves of each homozygous transgenic material provided in example 1 of the present invention.

Detailed Description

The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. Unless otherwise indicated, the techniques employed in the examples are conventional and well known to those skilled in the art, and the reagents and products employed are also commercially available. Various procedures and methods not described in detail are conventional methods well known in the art, and the sources, trade names, and components of the reagents used are indicated at the time of first appearance, and the same reagents used thereafter are the same as those indicated at the first appearance, unless otherwise specified.

Example 1 method for increasing the biosynthesis of Arabidopsis thaliana proanthocyanidins

The embodiment of the invention adopts arabidopsis thaliana as a target culture plant, thereby obtaining an arabidopsis thaliana plant with high content, and the specific steps are as follows:

1. selection of expression Material

Because the expression level of PAP1 in the arabidopsis PAP1-D mutant is more than 300 times of that of a wild type, the invention takes the PAP1-D mutant as an overexpression material, and does not need to perform molecular cloning treatment on the arabidopsis to overexpress PAP 1-D.

Of course, one skilled in the art can also overexpress PAP1 in Arabidopsis by molecular cloning using conventional Arabidopsis plants as expression material.

2. Molecular cloning process

The CsANR2 and CsF3 '5' H are cloned by a conventional method, pB2GW7-CsANR2 and pK2GW7-CsF3 '5' H vectors are constructed, plasmids of the two vectors are transformed into agrobacterium GV3101, and a strain capable of expressing CsANR2 and CsF3 '5' H genes is obtained.

3. Plant transformation

Transforming an arabidopsis plant by using a flower infection method to obtain a high-expression transgenic plant, which specifically comprises the following steps:

representative high-expressing transgenic lines were selected and artificially pollinated after artificial emasculation to produce materials of Cs 3 '5' H and PAP1 (Fxp), CsANR2 and PAP1(A xp), and CsF3 '5' H, CsANR2 and PAP1 (Fxa xp), while Arabidopsis expressing only CsANR2, Arabidopsis thaliana of CsANR2, and Arabidopsis thaliana PAP1-D mutants were used as controls. After the homozygote of each material was obtained by identification, the gene expression level was confirmed by the method of qRT-PCR, and the results are shown in FIG. 1.

According to the electrophoretogram of fig. 1A, arabidopsis thaliana (F × p) can express CsF3 '5' H and PAP1 genes, arabidopsis thaliana (a × p) can express CsF3 '5' H and PAP1 genes, arabidopsis thaliana (F × a × p) material can express CsF3 '5' H, CsANR2 and PAP1 genes, and according to the bar chart of fig. 1B, each target plant can express the corresponding target gene at a higher level, so that the target plant which can over-express the corresponding target gene and is required by the invention is obtained by the method.

It should be noted that the unexplained test steps are all conventional steps, and reference may be made specifically to the contents in "transforming arabidopsis thaliana by inflorescence dip dyeing", and the details of the present invention are not repeated.

4. Detection and analysis of flavonoid secondary metabolite content

4.1 detection and analysis of anthocyanin content

Since anthocyanins and proanthocyanidins share a common upstream intermediate anthocyanidins in their synthesis pathways, the biosynthesis of these two metabolites will compete, creating this state of equilibrium. Therefore, we first analyzed the anthocyanin content changes in the 30-day leaves of these materials, and the results are shown in fig. 2.

According to the results of fig. 2, the CsANR2 gene was overexpressed alone, so that the leaf anthocyanin content was reduced to half of that of the wild type; when only CsF3 '5' H is overexpressed, the content of anthocyanin in the CsF3 '5' H is not obviously different from that of the wild type; and the over-expression of PAP1 greatly increases anthocyanin to more than 30 times of wild type. When CsF3 '5' H and PAP1 are simultaneously over-expressed, the content of anthocyanin is reduced to about 25 times of that of wild type; when CsANR2 and PAP1 were simultaneously overexpressed, the content of anthocyanins was further reduced; when these three genes are simultaneously over-expressed, the anthocyanin content is greatly reduced, which is only slightly higher than that of the wild type and is 1.4 times higher than that of the wild type.

4.2 detection and analysis of proanthocyanidin content

The proanthocyanidin content in the obtained leaf of Arabidopsis thaliana was measured and counted for 30 days, and the results are shown in FIG. 3.

As shown in fig. 3A, when soluble proanthocyanidin was detected by DMACA method, accumulation of soluble proanthocyanidin was detected only in leaf discs overexpressing CsANR2 and PAP 1. When the soluble proanthocyanidin is detected by using n-butanol hydrochloric acid, accumulation of the soluble proanthocyanidin is detected in leaves which excessively express CsANR2 and PAP1 and excessively express the 3 genes, wherein the content of the soluble proanthocyanidin in the leaves of arabidopsis thaliana excessively expressing CsANR2 and PAP1 is 2.11mg/g DW, and the content of the soluble proanthocyanidin in the leaves of arabidopsis thaliana excessively expressing CsANR2, PAP1 and CsF3 '5' H is 2.28mg/g DW, which are both about 3 times of that of a wild type (namely an arabidopsis thaliana PAP1-D mutant, wherein the content of the soluble proanthocyanidin in the leaves is 0.74mg/g DW).

Meanwhile, the content of insoluble proanthocyanidin in the obtained arabidopsis 30-day leaves was measured and counted, and the results are shown in fig. 3B, and it can be seen from the results in fig. 3B that the overexpression of CsF3 '5' H and CsANR2 alone did not affect the biosynthesis of insoluble proanthocyanidin, while the overexpression of PAP1 alone resulted in 2.3 times the content of insoluble proanthocyanidin in the leaves of wild type. Meanwhile, the leaf blade of Arabidopsis thaliana with over-expression of CsF3 '5' H and PAP1 has the insoluble proanthocyanidin content of 4.33mg/g DW, which is 2.14 times of that of wild type; meanwhile, the content of insoluble proanthocyanidin in leaves of Arabidopsis thaliana with over-expression of CsANR2 and PAP1 reaches 5.65mg/g DW, which is 2.8 times of that of wild type; and meanwhile, the leaf blade of the arabidopsis thaliana excessively expressing CsANR2, PAP1 and CsF3 '5' H has the content of insoluble proanthocyanidin reaching 9.11mg/g DW which is 4.51 times of that of the wild type (wherein the wild type is an arabidopsis thaliana PAP1-D mutant, and the content of the insoluble proanthocyanidin in the leaf blade is 2.02mg/g DW).

In conclusion, the total content of proanthocyanidins in the target arabidopsis plant capable of simultaneously overexpressing CsANR2, PAP1 and CsF3 '5' H provided by the invention reaches 11.39mg/g DW, which is more than 4 times of that of the arabidopsis PAP1-D mutant; the total content of proanthocyanidins in the target arabidopsis plant capable of simultaneously over-expressing CsANR2 and PAP1 reaches 7.86mg/g DW, which is about 3 times of that of the arabidopsis PAP1-D mutant; and the content of insoluble proanthocyanidin in a target arabidopsis plant capable of simultaneously over-expressing CsF3 '5' H, PAP1 is more than 1.6 times of that of the arabidopsis pap1-D mutant. Therefore, the target plant capable of simultaneously over-expressing CsANR2, PAP1 and CsF3 '5' H provided by the invention can improve the content of proanthocyanidin in the plant body, and has obvious effect.

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.

Claims

1. A method for increasing the biosynthesis of proanthocyanidins in plants, which comprises overexpressing the CsANR2 gene, PAP1 gene and CsF3 '5' H gene in a plant at the same time, thereby increasing the proanthocyanidins content in the plant.

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

3. A method for increasing the biosynthesis of proanthocyanidins in plants, which comprises overexpressing the CsANR2 gene in plants to increase the proanthocyanidins content in the plants.

4. The method for increasing the biosynthesis of a plant proanthocyanidin according to any one of claims 1-3, wherein the plant is high protein pasture grass.

5. A plant having a high content of proanthocyanidins, obtained by the method according to any one of the preceding claims 1 to 3.

6. Use of the method for increasing plant proanthocyanidin biosynthesis according to any one of claims 1 to 3 for preventing the occurrence of hoove in ruminants such as cattle and sheep.

7. A use of plant with high content of proanthocyanidin for preventing tympanites of ruminant such as cattle and sheep.