CN110241124A - Arabidopsis At4g36920 gene is in regulation plant proanthocyanidin biosynthesis and the application of the anti-hoove of ruminant - Google Patents
Arabidopsis At4g36920 gene is in regulation plant proanthocyanidin biosynthesis and the application of the anti-hoove of ruminant Download PDFInfo
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Abstract
Arabidopsis At4g36920 gene disclosed by the invention is in regulation plant proanthocyanidin biosynthesis and the application of the anti-hoove of ruminant, present invention discover that the biosynthesis of arabidopsis At4g36920 gene negative regulation plant proanthocyanidin, by the expression for inhibiting the gene, increase the accumulation of plant proanthocyanidin, is applied the gene in the purposes for improving the anti-hoove of ruminant with this.The advantage of the invention is that, it was found that purposes of the arabidopsis At4g36920 gene in regulation plant proanthocyanidin biosynthesis, important theory support is provided for improvement plant proanthocyanidin biosynthesis and metabolism, to obtain the high plant variety of proanthocyanidin content, the ability of the anti-hoove of ruminant is further increased.
Description
Technical field
The invention belongs to the technical fields of genetic engineering application, and specially arabidopsis At4g36920 gene is in regulation plant
Proanthocyanidin biosynthesis and the application of the anti-hoove of ruminant.
Background technique
Proanthocyanidin (Proanthocyanidins) is the flavonoids secondary metabolite in plant with important activity.
It not only plays an important role in seed dormancy, service life and the regulation of sprouting, but also participates in the biological and non-life of regulation plant
Object environment stress (Debeaujon et al., 2003);Brown cotton is current most widely used natural color cotton, in fiber
Brown pigmentation be exactly proanthocyanidin (Xiao et al., 2014;Yan et al.,2018);Alfalfa Procyanidins contain
Amount be higher than dry weight 2% when, can be effectively prevented the ruminants clover sick such as ox, sheep generation (Verdier et al.,
2012);In addition to this, also have effects that anti-oxidant, anti-inflammatory and anticancer etc. be beneficial to man health (Dixon et al.,
2005).Therefore, the research of proanthocyanidin biosynthesis and regulation aspect, one become in Secondary Metabolism of Plant field are important
Research direction.
Proanthocyanidin mainly the positions such as kind skin, leaf, flower, fruit and the bark of plant accumulate, content by it is various because
The influence (Dixon et al., 2005) of element.Arabidopsis Procyanidins mainly accumulate in kind of skin, and biosynthesis originates in
1 to 2 days hole of bead regions, then gradually accumulated in endotesta towards the direction of chalaza after double fertilization, until general 5 to 6 days
When biosynthesis in chalaza region terminate (Debeaujon et al., 2003;Lepiniec et al.,2006).Therefore,
More precisely, proanthocyanidin accumulates at the endotesta of arabidopsis, the hole of bead and chalaza position.Currently, the biology of proanthocyanidin closes
It is clearer at approach.But the regulation of proanthocyanidin biosynthesis is a complicated process, the molecule machine of regulation
There are also many problems demands to answer for system.
It is current studies have shown that the biosynthesis of proanthocyanidin is mainly by MBW ternary complex (R2R3-MYB (TT2/
MYB5), bHLH (TT8/GL3/EGL3) and WD40 (TTG1)) regulation.TT2-TT8-TTG1 ternary complex is proanthocyanidin
The main positive regulator protein complex of biosynthesis, and plant the specifically expressed TT2 gene of skin be the key that proanthocyanidin accumulation because
Son.When the overexpression TT2 gene in arabidopsis, the content of proanthocyanidin does not increase (Nesi et al., 2001), into
One step simultaneously overexpression TT2 and PAP1 when also only detect proanthocyanidin (Sharma et in some special cells
al.,2005).Some researchs attempt to increase alfalfa etc. proanthocyanidin content (Verdier et al., 2012;
Escaray et al.,2014;Yuan and Grotewold,2015;Li et al., 2016), it is as a result undesirable, much reach
Less than the level of dry weight 2%, therefore the generation for the ruminants clover sick such as can not be effectively prevented ox, sheep.And it is formed therewith
Sharp contrast shares the biosynthesis of the anthocyanidin of route of synthesis also mainly by MBW ternary complex with proanthocyanidin
The regulation of (R2R3-MYB (PAP1/PAP2/MYB113/MYB114), bHLH (TT8/GL3/EGL3) and WD40 (TTG1)), quasi-
In southern mustard when overexpression PAP1, almost whole plants are all purples, accumulation lot of anthocyanin (Borevitz et al.,
2000) tomato, M. truncatula and the alfalfa etc. of enrichment anthocyanidin, are had been obtained for by genetic engineering at present
(Butelli et al.,2008;Peel et al.,2009).Zhao et al. and Li et al. people think that relatively good original can not be obtained
The biggest obstacle of the improved effect of anthocyanidin is that proanthocyanidin biosynthesis and regulation are extremely complex, still there is many problems demands
Answer (Zhao et al., 2010;Li et al.,2016).Result of study according to prior art, it is presumed that there may be
The function of unknown key gene negative regulation TT2 or associated ternary complex, to inhibit overexpression TT2 gene
The function of promoting proanthocyanidin accumulation, leads to that proanthocyanidin can not be enriched with when overexpression TT2 gene.
Summary of the invention
In order to solve the defect of still indefinite proanthocyanidin regulation synthesis in the prior art, the present invention provides arabidopsis
At4g36920 gene regulation the biosynthesis of plant proanthocyanidin and the application of the anti-hoove of ruminant, the purpose of realization be,
It was found that purposes of the arabidopsis At4g36920 gene in regulation plant proanthocyanidin biosynthesis, to improve plant proanthocyanidin
Biosynthesis and metabolism provide important theory support and further increase to obtain the high plant variety of proanthocyanidin content
The ability of the anti-hoove of ruminant.
To achieve the goals above, it is raw in regulation plant proanthocyanidin that the present invention provides arabidopsis At4g36920 genes
Object synthesis and the application of the anti-hoove of ruminant, the present invention using molecular biology, science of heredity and metabolin by quantitatively being divided
The means of analysis determine target gene and key in arabidopsis At4g36920 gene (AP2) regulation proanthocyanidin biosynthesis pathway
Transcription factor, it was found that the biosynthesis of arabidopsis At4g36920 gene (AP2) negative regulation plant proanthocyanidin passes through inhibition
The expression of the gene increases the accumulation of plant proanthocyanidin, applies the gene with this and is improving the anti-hoove of ruminant
Purposes.
By adopting the above technical scheme, beneficial effect includes: that proanthocyanidin is coerced in the growth and development of plant and adverse circumstance to the present invention
Important regulating and controlling effect is played in compeling, there are many benefits to human health.Many prior art researchs imply the presence of unknown pass
The function of key gene negative regulation TT2 or associated MBW ternary complex, and then the accumulation of negative regulation proanthocyanidin, but it is assorted
Gene is participated, and its molecule mechanism of regulation is still unclear.The present invention utilizes transcription group and reverse genetics
Method, discovery ap2 mutant kernel seed coat colour is deeper than wild type, accumulates more proanthocyanidins;AP2m3 (albumen of coding with
AP2's is identical, but its mRNA can not be by miRNA172 combination) the kind skin of overexpression significantly becomes in addition to the hole of bead and chalaza
Shallowly, proanthocyanidin content declines to a great extent;Mutant and overexpression do not influence the accumulation of flavonols and anthocyanidin.These results
Show AP2 specifically biosynthesis of negative regulation proanthocyanidin in the kind skin except the hole of bead and chalaza.The present invention, which will utilize, to be divided
The means of sub- biology, science of heredity and metabolin quantitative analysis determine the target base in AP2 regulation proanthocyanidin biosynthesis pathway
Cause and key transcription factor illustrate the genetic neural network of AP2 regulation proanthocyanidin biosynthesis, for improvement plant proanthocyanidin biology
Synthesis and metabolism provide important theory support.
Detailed description of the invention
Fig. 1 is the picture of ap2-5, ap2-6, wildtype Arabidopsis thaliana mature seed kind skin after DMACA is dyed.
Specific embodiment
The present invention is further illustrated with reference to embodiments.There is no the operating method of specified otherwise in the present invention
It is all the prior art, the operating condition of specified otherwise is not all under normal temperature and pressure yet, and reagent is commercially available buys by oneself.
Embodiment one: arabidopsis At4g36920 gene (AP2) provided by the invention is closed in regulation plant proanthocyanidin biology
At and the anti-hoove of ruminant application, arabidopsis At4g36920 gene (AP2) negative regulation plant proanthocyanidin biology close
At by inhibiting the expression of the gene, which is applied with this and improving ruminant by the accumulation of increase plant proanthocyanidin
The purposes of anti-hoove.
The present invention confirmed life of the arabidopsis At4g36920 gene (AP2) for plant proanthocyanidin with the following method
Object synthesizes played negative regulation effect:
(1) Mutants homozygous of candidate gene is obtained:
According to proanthocyanidin the after fertilization accumulation in 1-6 days the characteristics of, we obtain the gene tables of 0-6 days pods of after fertilization
Up to data, in conjunction with the data in Arabidopsis eFP Browser, we screened more or less a hundred in kind of skin specifically expressing or
Higher candidate gene is expressed in kind of skin.Then, mutant is had purchased from ABRC, identify and obtains Mutants homozygous.
(2) mutation type surface is analyzed:
The method that we use reverse genetics analyzes the phenotype of candidate gene Mutants homozygous.Since proanthocyanidin is raw
Key structure gene in object route of synthesis, such as the mutation of CHS, CHI and F3H and controlling gene such as TT2, TT8 and TT16 are led
Kernel seed coat colour is caused to shoal, we analyze the variation of kernel seed coat colour first.It was found that ap2-5 (weak mutant) and ap2-6 (strong mutation
Body) mutant kernel seed coat colour deepens than wild type.
Therefore the mutation of AP2 may increase the accumulation of proanthocyanidin, imply that AP2 may negative regulation kind skin Procyanidins
Accumulation.It is mostly identical due to the biosynthesis pathway of proanthocyanidin and anthocyanidin, since AP2 may negative regulation
The biosynthesis of proanthocyanidin, whether it also regulates and controls the biosynthesis of anthocyanidin.We observe after seed is sprouted embryo under 4 days seedling
The anthocyanin accumulation situation of axis, discovery ap2-5 and ap2-6 mutant are similar to the phenotype of wild type.Thus it is speculated that AP2 may
The biosynthesis of anthocyanidin is not regulated and controled.
(3) the flavonoids Methanogenesis of AP2 regulation
Since DMACA is specifically reacted with proanthocyanidin, DMACA dyeing after color the depth and proanthocyanidin content at
Direct ratio.We further use DMACA to dye mature mutant seeds, after the seed kind leather dyeing for finding ap2-5 and ap2-6
(Fig. 1) more darker than wild type.As shown in Figure 1, the mature seed kind skin of ap2-5 and ap2-6 is after DMACA is dyed than open country
Raw type color burn.Bar is 0.5mm.Further demonstrate that ap2 mutant kernel seed coat colour deepens, it may be possible to due to AP2 gene mutation
The accumulation of kind of skin Procyanidins is caused to increase, AP2 is likely to the biosynthesis of negative regulation proanthocyanidin.
The prior art inhibits it to translate into albumen studies have shown that miRNA172 is integrated on the mRNA of AP2 gene, and
The albumen of AP2m3 coding is identical as AP2's, but its mRNA can not be combined by miRNA172 and be inhibited its translation.Therefore our structures
The Overexpression vector of AP2, AP2m3 and AP2m3 fusion MYC label is built, arabidopsis thaliana transformation simultaneously obtains homozygous plants.It is first
First, it has been found that the kernel seed coat colour of the overexpression seed of AP2m3 and AP2m3 fusion MYC label significantly shoals, and AP2 is excessive
The kernel seed coat colour for expressing seed is similar to wild type.Then, mature mutant seeds are dyed using DMACA, is found
After dyeing 3 hours, there is the kernel seed coat colour of 22% ap2-6 seed obviously to deepen;After when dyeing 6 is small, there is 21%
The kernel seed coat colour of ap2-6 seed of ap2-5 and 51% obviously deepen, and wild type is still without being colored significantly at this time;
When dye 12 it is small when after, the ratio that the kind skin of ap2-5 and ap2-6 seed significantly dyes is respectively more than 94% and 96%, and at this time
Only 35% wild type is significantly dyed;After when dyeing 24 is small, ap2-5 and ap2-6 are significantly dyed, and wild type
Only 67% significantly dyed.After dyeing 12 hours, the ratio that the seed kind skin of overexpression AP2 gene is colored is unexpectedly than open country
Raw type is also high, but substantially less than mutant;And other dyeing times, it is not significantly different between overexpression and wild type.Always
For body, it is not significantly different between overexpression and wild type.The studies have shown that miRNA172 of the prior art is integrated to AP2
It is inhibited to translate into albumen on the mRNA of gene, so that the protein level of 35S::AP2 does not increase significantly compared with wild type.
Therefore, our protein levels of overexpression AP2 gene may also be similar to wild type, so as to cause in the kind skin of overexpression
The accumulation of proanthocyanidin is similar to wild type.Other than the hole of bead and chalaza, overexpression AP2m3 and AP2m3 merge MYC label
Seed kind skin cannot be dyed significantly in different dyeing times.
Finally, we have further quantitative determined the content of mature seed Procyanidins, mutant Central Plains pattern is found
The accumulation of element increases significantly, and the seed Procyanidins content of overexpression AP2m3 drastically decreases.These results are clear
Show that AP2 gene inhibits the biosynthesis of proanthocyanidin significantly in the kind skin except the hole of bead and chalaza clearly.
Due to the biosynthesis of AP2 negative regulation proanthocyanidin, whether it can also regulate and control other flavonoids (flavonols and cyanine
Element) accumulation? we are not it has been observed that mutant and the anthocyanin accumulation of wild type have significant change in 4 days seedling, calmly
Amount determines the anthocyanidin content in 4 days seedling and 17 days plant, finds ap2-5, ap2-6 mutant and AP2m3 overexpression
Anthocyanidin content be also not significantly different with wild type.Further demonstrate that AP2 gene does not regulate and control the biosynthesis of anthocyanidin.
Since flavonols mainly accumulates in kind of skin and seedling, we analyze flavonols in maturation using the method for HPLC
Content in seed and 6 days seedling, find the flavones alcohol contents of ap2-5, ap2-6 mutant and AP2m3 overexpressing plants with
There were significant differences for wild type.These results indicate that AP2 gene does not regulate and control the biosynthesis of flavonols yet.In conclusion
AP2 gene does not regulate and control the accumulation of flavonols and anthocyanidin, and the specifically negative regulation original flower in the kind skin except the hole of bead and chalaza
The biosynthesis of pigment.
(4) to cultivate the proanthocyanidin with higher amount, the new variety of plant with meteorism resistent disease quality provides can
Energy.
The method of this research and utilization transcription group and reverse genetics, discovery ap2 mutant kernel seed coat colour is more than wild type
It is deep, accumulate more proanthocyanidins, the accumulation of AP2 gene negative regulation proanthocyanidin;AP2m3 (the phase of the albumen of coding and AP2
Together, but its mRNA can not be by miRNA172 combination) the kind skin of overexpression significantly shoals in addition to the hole of bead and chalaza, former pattern
Cellulose content declines to a great extent;Mutant and overexpression do not influence the accumulation of flavonols and anthocyanidin.These results indicate that AP2
The specifically biosynthesis of negative regulation proanthocyanidin in the kind skin except the hole of bead and chalaza.Utilize the means of genetic engineering, suppression
The expression of AP2 gene processed, to increase the accumulation of proanthocyanidin, to cultivate the proanthocyanidin with higher amount and there is anti-drum
The new variety of plant of abdominal distension quality provides possibility.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (1)
1. arabidopsis At4g36920 gene is regulating and controlling the biosynthesis of plant proanthocyanidin and the application of the anti-hoove of ruminant,
It is characterized in that, the biosynthesis of arabidopsis At4g36920 gene negative regulation plant proanthocyanidin, by the table for inhibiting the gene
It reaches, increases the accumulation of plant proanthocyanidin, applied the gene in the purposes for improving the anti-hoove of ruminant with this.
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CN113957079A (en) * | 2021-10-12 | 2022-01-21 | 中国农业科学院北京畜牧兽医研究所 | Application of MtBGLU17 gene in regulation and control of plant flavonoid synthesis |
CN115305251A (en) * | 2022-08-30 | 2022-11-08 | 中国农业科学院北京畜牧兽医研究所 | Application of AP2-MYBL2 molecular module in regulation and control of proanthocyanidin biosynthesis |
CN116640781A (en) * | 2023-07-21 | 2023-08-25 | 中国农业科学院北京畜牧兽医研究所 | Application of MtAHA5 Gene and MtAHA5 Protein in Alfalfa Plants |
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Publication number | Priority date | Publication date | Assignee | Title |
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CN113493792A (en) * | 2020-03-18 | 2021-10-12 | 中国农业科学院北京畜牧兽医研究所 | Method for improving biosynthesis of plant proanthocyanidins and application thereof |
CN113957079A (en) * | 2021-10-12 | 2022-01-21 | 中国农业科学院北京畜牧兽医研究所 | Application of MtBGLU17 gene in regulation and control of plant flavonoid synthesis |
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CN115305251A (en) * | 2022-08-30 | 2022-11-08 | 中国农业科学院北京畜牧兽医研究所 | Application of AP2-MYBL2 molecular module in regulation and control of proanthocyanidin biosynthesis |
CN115305251B (en) * | 2022-08-30 | 2023-06-27 | 中国农业科学院北京畜牧兽医研究所 | Application of AP2-MYBL2 molecular module in regulating and controlling proanthocyanidin biosynthesis |
CN116640781A (en) * | 2023-07-21 | 2023-08-25 | 中国农业科学院北京畜牧兽医研究所 | Application of MtAHA5 Gene and MtAHA5 Protein in Alfalfa Plants |
CN116640781B (en) * | 2023-07-21 | 2023-11-14 | 中国农业科学院北京畜牧兽医研究所 | Application of MtAHA5 gene and MtAHA5 protein in alfalfa plants |
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