Silencing vector for silencing cupula mori G protein alpha subunit coding gene CsGPA3 and application and method thereof

文档序号：1884826 发布日期：2021-11-26 浏览：20次中文

阅读说明：本技术 沉默桑实杯盘菌G蛋白α亚基编码基因CsGPA3的沉默载体及其应用和方法 (Silencing vector for silencing cupula mori G protein alpha subunit coding gene CsGPA3 and application and method thereof ) 是由赵爱春朱攀攀李若兰张帅刘长英范伟周玉平向仲怀于 2021-08-31 设计创作，主要内容包括：本发明公开了沉默桑实杯盘菌G蛋白α亚基编码基因CsGPA3的沉默载体及其应用和方法,桑实杯盘菌G蛋白α亚基编码基因CsGPA3的核苷酸序列如SEQ ID NO.1所示；通过利用宿主诱导的基因沉默技术,将含有桑实杯盘菌G蛋白α亚基编码基因CsGPA3的沉默片段的载体在植物中表达,通过对桑实杯盘菌致病力鉴定,转基因植物能够显著提高对桑实杯盘菌的抗性,因此可以利用此方法提高植物对桑实杯盘菌的抗性,防治桑实杯盘菌的感染。(The invention discloses a silencing vector for silencing a cupula mori G protein alpha subunit encoding gene CsGPA3, and application and a method thereof, wherein the nucleotide sequence of the cupula mori G protein alpha subunit encoding gene CsGPA3 is shown as SEQ ID No. 1; by utilizing a host induced gene silencing technology, a vector containing a silencing fragment of the Morusia casserole G protein alpha subunit coding gene CsGPA3 is expressed in a plant, and through the pathogenicity identification of the Morusia casserole, the resistance of a transgenic plant to the Morusia casserole can be obviously improved, so that the resistance of the plant to the Morusia casserole can be improved by utilizing the method, and the infection of the Morusia casserole can be prevented and treated.)

1. A silencing vector for silencing a cupula mori G protein alpha subunit coding gene CsGPA3 comprises an expression vector and an expression frame which is inserted into the expression vector and contains a forward silencing fragment and a reverse silencing fragment, and is characterized in that: the expression vectors are pBin19 and pCAMBIA1300, and the forward silencing fragment is shown as SEQ ID NO. 2; the reverse silent fragment is shown as a reverse complementary sequence of SEQ ID NO. 2.

2. The silencing vector for silencing cupula mori G protein alpha subunit coding gene CsGPA3 according to claim 1, wherein the silencing vector comprises: the expression vector is pBin19, and the construction method of the expression frame is as follows: the sequence shown in SEQ ID NO.2 is linked into pHANNIBAL plasmid through Kpn I and Xho I to obtain an intermediate vector pHANNIBAL-SiCSGPA3-F, then the sequence shown in SEQ ID NO.2 is reversely linked into the intermediate vector pHANNIBAL-SiCSGPA3-F through Xba I and Hind III to obtain a vector pHANNIBAL-SiCSGPA3-FR, and the expression frame is obtained by digestion with Sac I and Spe I.

3. The silencing vector for silencing cupula mori G protein alpha subunit coding gene CsGPA3 according to claim 1, wherein the silencing vector comprises: the expression vector is pCAMBIA 1300; the expression frame construction method comprises the following steps: the sequence shown in SEQ ID NO.2 is connected into a pSilent-1 plasmid through Xho I and Hind III to obtain a pSilent-SiCSGPA3-U plasmid, and the reverse-phase fragment shown in SEQ ID NO.2 is connected into the pSilent-SiCSGPA3-U plasmid through Kpn I and Bgl II to obtain a vector pSilent-SiCSGPA 3-UD.

4. Use of the silencing vector of claim 1 or 2 for reducing the virulence of cupule sorosis in mulberry fruits.

5. Use of the silencing vector of claim 1 or 3 for increasing the resistance of a plant to cupule sorangium.

6. A method for improving the resistance of plants to the cupule fungi of mulberry fruits is characterized by comprising the following steps: transforming the silencing vector for silencing cupula mori G protein alpha subunit coding gene CsGPA3 of claim 1 or 3 into a plant, and expressing a silencing fragment in the transgenic plant to obtain the transgenic plant resistant to cupula mori, wherein the nucleotide sequence of the cupula mori G protein alpha subunit coding gene CsGPA3 is shown in SEQ ID No. 1.

7. The method of claim 4, wherein: the method for transforming the silent vector of the silent cupula mori G protein alpha subunit coding gene CsGPA3 comprises the following steps: and (3) transforming the silencing vector of the silencing cupula mori G protein alpha subunit coding gene CsGPA3 into a plant through agrobacterium-mediated transformation, and screening a positive plant.

8. The method of claim 5, wherein: the agrobacterium is LBA 4404.

9. The method of claim 5, wherein: the plant is tobacco or mulberry.

Technical Field

The invention relates to the field of genetic engineering, in particular to a silencing vector for silencing a cupula mori G protein alpha subunit encoding gene CsGPA3, and also relates to application and a method of the silencing vector for silencing the cupula mori G protein alpha subunit encoding gene CsGPA 3.

Background

Mulberry (Morus alba L.) is an important economic forest in China, and can be used for producing mulberry leaves, silkworms, silk and mulberries. The mulberry flesh is juicy, has sweet and delicious taste, is rich in amino acid, vitamin, flavone and anthocyanin which are necessary for human bodies, is listed as one of agricultural products with homology of medicine and food by the national ministry of health, and has high food and medicinal value. In mulberry planting, mulberry is very susceptible to outbreak of disastrous diseases, namely mulberry sclerotinia. The mulberry hypertrophic sclerotinia sclerotiorum pathogenic bacteria of mulberry cupertiria is the mulberry pathogenic bacteria with the largest harm and the widest spread range to mulberry. At present, chemical pesticides are still mainly used for prevention and control of the cupule sarmentosum, pesticide residues can adversely affect the environment and food safety, and meanwhile, due to the genetic characteristics of fungi, the fungi have high drug resistance generation speed, so that the economic cost for prevention and control of the cupule sarmentosum is increased; in recent years, biological control has been applied to control of phytopathogens due to its characteristic of no environmental pollution, but biocontrol bacteria have been limited in practical application due to their characteristics of high cost, slow action, difficult preservation and great environmental impact.

The G protein signal path is a signal path which is conserved in eukaryotes and has very important functions of sensing, transmitting, responding to various external signals and stimulating. In fungi, heterotrimeric G protein participates in physiological processes of regulating and controlling vegetative growth, pathogenicity, sporulation, formation of infection structures and the like by regulating and controlling adenylate cyclase, phospholipase activity, ion channels and the like. Under the condition of no external signal stimulation, G protein G alpha subunit is combined with GDP, G alpha and G beta gamma subunit exist in the form of heterotrimer, G protein is in inactive state, when external signal is sensed, GPCR is combined with signal molecule as Guanylate Exchange Factor (GEF) to cause conformational change, and is combined with G alpha subunit of G protein to cause conformational change to release GDP to be combined with GTP, to form G alpha subunit in active state, and is dissociated with G beta gamma heterodimer to be respectively combined with downstream effector molecule to transmit signal. The G alpha subunit has GTP enzyme hydrolysis activity to hydrolyze GTP into GDP, so that G alpha is combined with GDP and recombined with G beta gamma subunit to be in an inactivated heterotrimer state, and GPCR signals are finally turned off. The rate of GTP hydrolysis can be increased by the G-protein regulator RGS. The G protein alpha subunit is used as an important component of a G protein signal and plays an important role in the processes of growth, development, pathogenicity and the like of fungi. In the plant pathogenic bacteria such as septoria nodorum, aspergillus flavus, botrytis cinerea, fusarium oxysporum and the like, the pathogenicity of the fungi is reduced after the G alpha subunit genes are knocked out, and the G alpha subunit is important for the pathogenicity of the fungi.

Sanford and Johnson transferred pathogen gene fragments into plant or animal hosts to gain resistance to the corresponding pathogens as early as 10 years before RNA silencing was approved, thus proposing the concept of parasite/pathogen-derived resistance (PDR). Subsequently, it is found that a pathogen gene fragment transferred into a double strand can obtain a more effective disease-resistant effect, which is the most widely applied strategy at present called HD-RNAi (Host-delayed RNAi) or Host-induced gene silencing (HIGS). The basic principle is to express RNAi carrier of pathogenic gene of target pest or pathogenic bacteria in host to silence the pathogenic target gene, so as to make host obtain resistance to pathogen. Later studies demonstrated that this approach could be used effectively not only to combat viral entry, but also in pest control and to combat bacterial and fungal infections.

The traditional breeding mode has the defects of long period, large difference of tolerance capability to pathogenic bacteria, limited promotion of plant resistance and the like. Therefore, a method which has short period and tolerance to pathogenic bacteria and can obviously improve the resistance of plants to the cupule fungi of the mulberry fruits is urgently needed.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a silencing vector for silencing cupula mori G protein alpha subunit encoding gene CsGPA 3; the invention also aims to provide the application of the silencing carrier in reducing the pathogenicity of the mulberry cupule bacilli; the third purpose of the invention is to provide the application of the silencing vector in improving the resistance of plants to the cupule mulberry; the fourth purpose of the invention is to provide a method for improving the resistance of plants to the cupule strain of mulberry fruits.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a silencing vector for silencing a cupula mori G protein alpha subunit coding gene CsGPA3 comprises an expression vector and an expression frame which is inserted into the expression vector and contains a forward silencing fragment and a reverse silencing fragment, wherein the expression vector comprises pBin19 and pCAMBIA1300, and the forward silencing fragment is shown as SEQ ID No. 2; the reverse silent fragment is shown as a reverse complementary sequence of SEQ ID NO. 2.

Preferably, the expression vector is pBin19, and the construction method of the expression frame is as follows: the sequence shown in SEQ ID NO.2 is linked into pHANNIBAL plasmid through Kpn I and Xho I to obtain an intermediate vector pHANNIBAL-SiCSGPA3-F, then the sequence shown in SEQ ID NO.2 is reversely linked into the intermediate vector pHANNIBAL-SiCSGPA3-F through Xba I and Hind III to obtain a vector pHANNIBAL-SiCSGPA3-FR, and the expression frame is obtained by digestion with SacI and Spe I.

Preferably, the expression vector is pCAMBIA 1300; the expression frame construction method comprises the following steps: the sequence shown in SEQ ID NO.2 is connected into a pSilent-1 plasmid through Xho I and Hind III to obtain a pSilent-Si CsGPA3-U plasmid, and the reverse-phase fragment shown in SEQ ID NO.2 is connected into the pSilent-Si CsGPA3-U plasmid through Kpn I and Bgl II to obtain a vector pSilent-Si CsGPA 3-UD.

2. The application of the silencing vector in reducing pathogenicity of the cupule sorangium morganii.

3. The application of the silencing vector in improving the resistance of plants to the cupule bacilli of the mulberry fruits.

4. A method for improving the resistance of a plant to the cuprum morbifidus comprises the steps of transforming a silencing vector for silencing a cuprum morbifidus G protein alpha subunit encoding gene CsGPA3 in the plant to enable a silencing fragment to be expressed in a transgenic plant, and obtaining the transgenic plant which is resistant to the cuprum morbifidus, wherein the nucleotide sequence of the cuprum morbifidus G protein alpha subunit encoding gene CsGPA3 is shown in SEQ ID NO. 1.

Preferably, the method for transforming the silencing vector for silencing the cupula mori G protein alpha subunit coding gene CsGPA3 comprises the following steps: and (3) transforming the silencing vector of the silencing cupula mori G protein alpha subunit coding gene CsGPA3 into a plant through agrobacterium-mediated transformation, and screening a positive plant.

Preferably, the agrobacterium is LBA 4404.

Preferably, the plant is tobacco or mulberry.

The invention has the beneficial effects that: the invention discloses a silencing vector for silencing a gene CsGPA3 encoding a G protein alpha subunit of Morus multicincana, a plant with high resistance to Morus multicincana is obtained by taking a section of specific sequence of the gene CsGPA3 encoding the G protein alpha subunit of Morus multicincana as a target spot and inducing gene silencing through a host, compared with the prior art, the method does not need to use chemical pesticides, and silencing fragments in a transgenic plant aim at the gene sequence of the gene CsGPA3 encoding the G protein alpha subunit of Morus multicincana and specifically silence the expression thereof, so that the influence on the plant per se is small; because a 35S strong promoter is used in the vector, the vector can express a large amount of sRNA of the gene CsGPA3 encoding the G protein alpha subunit of the cupula mori and has a good silencing effect; the seed of the transgenic plant can be stably inherited after being screened to be homozygous, and a continuous antibacterial effect is generated.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1CsGPA3 interference strains influence on fungal virulence;

FIG. 2 shows the PCR detection result of kanamycin resistance gene in transgenic tobacco genome DNA (wherein Marker: DL5000 DNA Marker; WT: amplification with wild type total DNA as template);

FIG. 3 shows the identification of disease resistance and phenotypic analysis of transgenic tobacco;

FIG. 4 is a statistics of the lesion area of the leaves after inoculation of the cupule fungus of mulberry;

FIG. 5 is a statistics of relative biomass of hyphae in leaves after inoculation of colletotrichum sanguineum;

FIG. 6 shows the relative expression level of CsGPA3 in leaves after inoculation of Stachys mori.

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

Example 1

The nucleotide sequence of the Morusis multicinctus CsGPA3 is obtained by searching the G protein alpha subunit genes in the sclerotinia sclerotiorum, the Botrytis cinerea and the Saccharomyces cerevisiae through NCBI and comparing the G protein alpha subunit genes with local Morusis multicinctus genome data. Designing a primer CsGPA3-F/R according to the sequence of the genome, wherein the specific primer is as follows:

CsGPA3-F：5’-atgggttgcggaatgagcac-3’(SEQ ID NO.3)；

CsGPA3-R：5’-ttatatcagaccacacagac-3’(SEQ ID NO.4)；

PCR amplification is carried out by taking Morus multicinctus cDNA as a template, a product is connected with a PMD19-T carrier after being recovered, and the nucleotide sequence of CsGPA3 obtained after sequencing is shown as SEQ ID NO. 1.

Example 2

Selecting a specific sequence of about 300bp on CsGPA3 to construct a silencing vector targeting the cuprum sorrel CsGPA3, wherein the selected silencing fragment SisGPA 3 is shown as SEQ ID NO.2, the silencing fragment SisGPA 3 for constructing the host induced gene silencing vector is connected into pHANNIBAL plasmid Kpn I and Xho I enzyme cutting sites to obtain an intermediate vector pHANNIBAL-SicGPA 3-F, and then the SicGPA 3 is connected into the Xba I and Hind III enzyme cutting sites of the pHANNIBAL-SicGPA 3-F plasmid to obtain the vector pHANNIBAL-SicGPA 3-FR.

The forward silent fragment amplification primers were as follows:

SiCsGPA3-F：5’-ccgctcgagcagcatgattatatgccaaa-3’(SEQ ID NO.5)；

SiCsGPA3-R：5’-cggggtacccaagaacagaatgatggaag-3’(SEQ ID NO.6)；

reverse silent fragment amplification primers were as follows:

SiCsGPA3-F1：5’-tgctctagacagcatgattatatgccaaa-3’(SEQ ID NO.7)；

SiCsGPA3-R1：5’-cccaagcttcaagaacagaatgatggaag-3’(SEQ ID NO.8)；

the fragment containing SiCSGPA3 was then excised using SacI and speI for SacI and XbaI ligated into the pBin19 plasmid to obtain the final silencing expression vector pBin19-SiCSGPA 3.

Constructing a fungus silencing expression vector, specifically, connecting a forward fragment shown in SEQ ID NO.2 into a pSilent-1 plasmid through Xho I and Hind III to obtain a pSilent-SiCSGPA3-U plasmid, and connecting a reverse fragment shown in SEQ ID NO.2 into the pSilent-SiCSGPA3-U plasmid through Kpn I and Bgl II to obtain the vector pSilent-SiCSGPA 3-UD.

Forward silent fragments were amplified using the following primers:

SiCsGPA3-U：5’-ccgctcgagcagcatgattatatgccaaa-3’(SEQ ID NO.9)；

SiCsGPA3-D：5’-cccaagcttcaagaacagaatgatggaag-3’(SEQ ID NO.10)；

SiCsGPA3-U1：5’-cggggtacccagcatgattatatgccaaa-3’(SEQ ID NO.11)；

SiCsGPA3-D1：5’-ggaagatctcaagaacagaatgatggaag-3’(SEQ ID NO.12)。

the fragment containing SiCsG.beta.2 was then excised using XbaI into the XbaI cleavage site of the pCAMBIA1300 plasmid to obtain the final silencing expression vector pCAMBIA1300-SiCsGPA 3.

Example 3

The expression vectors constructed in the examples were used to obtain the corresponding interfering strains by protoplast transformation. Culturing wild tobacco in a 16h light/22 h 8h dark incubator at 25 deg.C for 40 days, and placing leaves with the same leaf position and size in a culture dish with soaked sterile filter paper; hyphae (wild type, no-load, three CsGPA3 interfering strains) just growing on the whole plate are selected, a yellow gun head is used for punching holes at the edge of the hyphae, hypha blocks with the same size are inoculated on tobacco and placed in an incubator at 25 ℃, photographing and material taking are carried out 48 hours after inoculation, and the lesion area of infected leaves is calculated by using image J software, and the result is shown in figure 1. The results show that the lesion area of the interfering strain is smaller, indicating that the virulence of the interfering strain is reduced.

Example 4

Transferring the final silent expression vector pBin19-SiCSGPA3 into agrobacterium tumefaciens LBA4404 by a chemical conversion method, and identifying a positive transformant by bacteria liquid PCR, wherein identification primers are as follows:

Kan-F：5’-ggtgccctgaatgaactgca-3’(SEQ ID NO.13)；

Kan-R：5’-ggtagccaacgctatgtcct-3’(SEQ ID NO.14)；

the identification results are shown in FIG. 2, and the results show that the positive agrobacterium is transformed into the nicotiana benthamiana aseptic seedlings by the leaf disc transformation method to obtain seeds of positive strains (Line1, Line2, Line3, Line4 and Line 5).

Example 5

After germination, seeds of (Line1, Line2 and Line3) are selected for subsequent infection experiments. Uniformly sowing wild type and transgenic seeds which are vernalized for 24 hours at 4 ℃ in high-pressure nutrient soil, selecting seedlings with consistent growth vigor after 10 days of germination, transplanting the seedlings into an independent flowerpot, culturing the seedlings in a 16h illumination/22 ℃ 8h dark incubator for 40 days at 25 ℃, and then placing leaves with the same leaf position and consistent size in a culture dish with soaked sterile filter paper; hyphae just growing over the whole plate is selected, holes are punched at the edge of the hyphae by using a blue gun head, hypha blocks with the same size are inoculated on tobacco and placed in an incubator at 25 ℃, photographing is carried out 48 hours after inoculation, and the material is obtained, and the result is shown in figure 3.

The lesion area of the infected leaf was calculated using image J software, and the results are shown in FIG. 4. The results show that the lesion area formed after the transgenic tobacco is infected is obviously smaller than that of the wild tobacco; using a tobacco actin gene quantitative primer actin-QF/QR and a fungus tubulin gene quantitative primer tubulin-QF/QR to perform quantitative PCR reaction by taking the genome of an infected material as a template, wherein the primers are as follows:

NbActin-F：5’-tcacagaagctcctcctaatcca-3’(SEQ ID NO.15)；

NbActin-R：5’-gagggaaagaacagcctgaatg-3’(SEQ ID NO.16)；

Tubulin-F：5’-ttggatttgctcctttgaccag-3’(SEQ ID NO.17)；

Tubulin-R：5’-agcggccatcatgttcttagg-3’(SEQ ID NO.18)；

the relative biomass of the fungus was calculated and the results are shown in figure 5. The results show that the relative biomass of the Line1, Line2 and Line3 strains of fungi is significantly lower than that of the wild type. The quantitative primer CsGPA3-QF/QR of the fungus CsGPA3 gene is used, and the fungus tubulin gene is used as an internal reference.

CsGPA3-QF：5’-aagagggaaaggcgaggaac-3’(SEQ ID NO.19)；

CsGPA3-QR：5’-tgattctctctcgtcgcgtg-3’(SEQ ID NO.20)；

The silencing effect on the target gene CsGPA3 was calculated using cDNA inverted from the RNA of the invaded material as a template, and the results are shown in FIG. 6. The result shows that the expression quantity of the target gene of the transgenic line is obviously reduced.

The results show that the cupellational mulberry fruit SiCSGPA3 is taken as the target point, the transgenic tobacco obtained by the host induced gene silencing technology can obviously improve the tolerance capability of the tobacco to the cupellational mulberry fruit, and meanwhile, the invention also provides the target point for improving the resistance of other species of cupellational mulberry fruit through the host induced gene silencing technology.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

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