阅读说明：本技术 一种橡胶树八氢番茄红素脱氢酶基因vigs沉默体系及其构建方法与应用 (Rubber tree phytoene dehydrogenase gene VIGS silencing system and construction method and application thereof ) 是由 李辉亮 彭世清 郭冬 王颖 朱家红 于 2020-05-07 设计创作，主要内容包括：本发明实施例公开了一种橡胶树八氢番茄红素脱氢酶基因VIGS沉默体系及其构建方法与应用,属于生物技术领域。所述橡胶树八氢番茄红素脱氢酶基因VIGS沉默体系具有如SEQ ID NO:1所示的核苷酸序列。本发明操作简单,建立了一套快速、有效的病毒诱导沉默体系,有效避免了传统遗传转化需要繁重的劳动和效率低下的缺点。该体系可有效降低橡胶树中HbPDS基因的表达水平,可以获得病毒诱导的基因沉默性状,进而解决有效验证橡胶树基因功能的问题,为挖掘具有重要农艺性状的橡胶树基因,为培育优良的橡胶树品种奠定基础。(The embodiment of the invention discloses a rubber tree phytoene dehydrogenase gene VIGS silencing system and a construction method and application thereof, belonging to the technical field of biology. The rubber tree phytoene dehydrogenase gene VIGS silencing system has a nucleotide sequence shown as SEQ ID NO. 1. The method is simple to operate, a set of rapid and effective virus-induced silencing system is established, and the defects that the traditional genetic transformation needs heavy labor and is low in efficiency are effectively overcome. The system can effectively reduce the expression level of HbPDS gene in the rubber tree, can obtain virus-induced gene silencing property, further solve the problem of effectively verifying the function of the rubber tree gene, and lay a foundation for mining the rubber tree gene with important agronomic property and cultivating excellent rubber tree varieties.)

1. A rubber tree phytoene dehydrogenase gene VIGS silencing system is characterized by having a nucleotide sequence shown as SEQ ID NO. 1.

2. The method for constructing the rubber tree phytoene dehydrogenase gene VIGS silencing system of claim 1, which is characterized by comprising the following steps:

s1, designing specific primers according to sequence information of rubber tree phytoene dehydrogenase HbPDS gene and combined with sequence information of pTRV2 vector:

HbPDS1F：GAATTCGGCGCTTAACTTTATTAACCCTG，

HbPDS1R：CTCGAGCTTTAGTTTCCTGTCGAACCATATG；

s2, carrying out PCR amplification reaction by using the rubber tree cDNA as a template through specific primers to obtain an HbPDS target gene fragment with the size of 409 bp;

s3, connecting the HbPDS target gene fragment obtained in the step S2 to a PMD-18T vector;

s3, connecting the HbPDS target gene fragment connected to the PMD-18T vector to a pTRV2 vector subjected to the same enzyme digestion after enzyme digestion to obtain a pTRV2-HbPDS recombinant plasmid;

s4, transforming the pTRV2-HbPDS recombinant plasmid into agrobacterium to obtain the VIGS silencing system.

3. The use of the rubber tree phytoene dehydrogenase gene VIGS silencing system according to claim 1, wherein the roots, stems and leaves of the rubber tree seedling in the two-fleabane leaf stage are infected at room temperature or infected by vacuuming; and (3) dark culturing the infected rubber tree seedlings at 28 ℃ for 12h, and then culturing the rubber tree seedlings in a greenhouse nursery.

4. The use of the rubber tree phytoene dehydrogenase gene VIGS silencing system according to claim 3, wherein the infection is with 10mM MgCl₂+10mM MES + 150. mu. MAS.

5. The application of the rubber tree phytoene dehydrogenase gene VIGS silencing system according to claim 3, wherein the vacuum degree is 0.08MPa and the infection time is 1 hour when the rubber tree phytoene dehydrogenase gene VIGS silencing system is vacuumized for infection.

Technical Field

The embodiment of the invention relates to the technical field of biology, and particularly relates to a rubber tree phytoene dehydrogenase gene VIGS silencing system, and a construction method and application thereof.

Background

The Hevea brasiliensis is a plant of genus Hevea of family Euphorbiaceae, native to Amazon forest. The Chinese vegetable gelatin area is mainly distributed in the areas of Hainan, Guangdong, Guangxi, Yunnan and the like, wherein Hainan is the main vegetable gelatin area. The rubber tree is a deciduous tree, and the latex in the bark of the tree produces latex of milky juice, which is a main secondary metabolite of the Brazilian rubber tree and also a main source of natural rubber. The economic life of the rubber tree is about 30 years, and the growth life is about 60 years. Through a conventional crossbreeding means, the cultivation of a new variety of the rubber tree needs 20-30 years. The establishment of the genetic transformation system is a necessary way for effectively verifying the gene function of the rubber tree, shortening the breeding period of the rubber tree and accelerating the breeding of new varieties.

At present, the research on related genes of rubber trees starts, researchers obtain a plurality of gene sequences and clone a plurality of genes after genome sequencing, but the research cannot be further deeply researched, wherein the main reasons are that the genetic transformation system is immature and the detection means of genetic transformation is less. At present, receptors used for agrobacterium tumfaciens genetic transformation are mainly embryogenic callus, and no report is made on the utilization of other materials as the receptors. The embryogenic callus as a transformation receptor has the following defects: 1. the material taking is limited by seasons, labor and time are consumed for peeling stamens, the regenerative capability of anthers is poor, and embryogenic callus is difficult to obtain, so that the transformation requirement of all seasons of the year cannot be met; 2. the tissue regeneration capability is poor, transgenic plants are difficult to obtain, and the increasing scientific research and production requirements are difficult to meet; 3. the callus is used as a transformation receptor, and the genetic transformation efficiency is low. The VIGS (virus-induced gene cloning) technology is a high-efficiency genetic transformation system, is applied to various plants for gene function verification, but the system is not applied to rubber trees at present.

Disclosure of Invention

Therefore, the embodiment of the invention provides a rubber tree phytoene dehydrogenase gene VIGS silencing system and a construction method and application thereof.

The invention utilizes the characteristic that the VIGS system can move in a plant body, takes the roots, stems and leaves of rubber tree seedlings as receptors, and establishes a high-efficiency rubber tree genetic transformation method through a virus-induced gene silencing system which is carried out under different conditions of infecting the plant parts of the receptors. The method is simple, rapid and effective to operate, and overcomes the defects of heavy labor and low efficiency of the traditional genetic transformation. The system can effectively reduce the expression level of HbPDS gene in the rubber tree, can obtain virus-induced gene silencing property, further solve the problem of effectively verifying the function of the rubber tree gene, and lay a foundation for mining the rubber tree gene with important agronomic property and cultivating excellent rubber tree varieties.

In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:

according to the first aspect of the embodiments of the present invention, the embodiments of the present invention provide a rubber tree phytoene dehydrogenase gene VIGS silencing system, which has a nucleotide sequence shown as SEQ ID NO. 1.

According to a second aspect of the embodiments of the present invention, the embodiments of the present invention provide a method for constructing the above-mentioned ruby tree phytoene dehydrogenase gene VIGS silencing system, which includes the following steps:

s1, designing specific primers according to sequence information of rubber tree phytoene dehydrogenase HbPDS gene and combined with sequence information of pTRV2 vector:

HbPDS1F：GAATTCGGCGCTTAACTTTATTAACCCTG，

HbPDS1R：CTCGAGCTTTAGTTTCCTGTCGAACCATATG；

s2, carrying out PCR amplification reaction by using the rubber tree cDNA as a template through specific primers to obtain an HbPDS target gene fragment with the size of 409 bp;

s3, connecting the HbPDS target gene fragment obtained in the step S2 to a PMD-18T vector;

s3, connecting the HbPDS target gene fragment connected to the PMD-18T vector to a pTRV2 vector subjected to the same enzyme digestion after enzyme digestion to obtain a pTRV2-HbPDS recombinant plasmid;

s4, transforming the pTRV2-HbPDS recombinant plasmid into agrobacterium to obtain the VIGS silencing system.

According to a third aspect of the embodiments of the present invention, the embodiments of the present invention provide an application of the above-mentioned rubus hevea phytoene dehydrogenase gene VIGS silencing system, wherein roots, stems and leaves of rubus hevea seedlings in the two-fleabane leaf stage are infected at room temperature, or infected by vacuuming; and (3) dark culturing the infected rubber tree seedlings at 28 ℃ for 12h, and then culturing the rubber tree seedlings in a greenhouse nursery.

Further, the infection was carried out using 10mM MgCl₂+10mM MES + 150. mu. MAS.

Further, when the vacuum is pumped for infection, the vacuum degree is 0.08MPa, and the infection time is 1 hour. Under such conditions, the infection efficiency can be increased.

The embodiment of the invention has the following advantages:

the transformation receptor of the invention is different from the previous reports, and the rubber tree seedling as the transformation receptor has obvious advantages: can be used for both seed seedlings and tissue culture seedlings, has materials all the year round, and can meet the transformation requirement at any time. Specific primers are designed according to the coding gene sequence of rubber tree phytoene dehydrogenase (HbPDS), and a cDNA fragment of 409bp is obtained in rubber trees by PCR amplification. Constructing the obtained HbPDS cDNA fragment on a pTRV2 vector to obtain a pTRV2-HbPDS recombinant plasmid, and inducing a gene silencing system through agrobacterium-mediated viruses infecting the root, stem and leaf parts of a receptor plant. The method is simple to operate, a set of rapid and effective virus-induced silencing system is established, and the defects that the traditional genetic transformation needs heavy labor and is low in efficiency are effectively overcome. The system can effectively reduce the expression level of HbPDS gene in the rubber tree, obtain virus-induced gene silencing property, further solve the problem of effectively verifying the function of the rubber tree gene, and lay a foundation for excavating the rubber tree gene with important agronomic property and cultivating excellent rubber tree varieties.

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 should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 is a flow chart of a virus-induced rubber tree phytoene dehydrogenase (HbPDS) gene silencing system.

FIG. 2 is an amplification electrophoretogram of HbPDS cDNA fragment and identification of pTRV2-HbPDS recombinant plasmid; wherein: 1: DNAmarker; 2: pTRV2-HbPDS recombinant plasmid; 3: double digestion of pTRV2-HbPDS recombinant plasmid; 4: pTRV2-HbPDS recombinant plasmid PCR product; 5: and (5) DNA Marker.

FIG. 3 shows the phenotype of leaf whitening after control treatment and transgenic rubber tree treated with phytoene dehydrogenase (HbPDS) gene.

FIG. 4 shows the relative expression amounts of HbPDS genes of the control plant leaf and the transgenic plant leaf detected by qRT-PCR.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The reagents and methods used in the examples were, unless otherwise specified, those which were conventional and those which were conventional were used.