Silencing vector for silencing cupula mori G protein gamma subunit coding gene CsG gamma, and application and method thereof

文档序号：1884827 发布日期：2021-11-26 浏览：28次中文

阅读说明：本技术 沉默桑实杯盘菌G蛋白γ亚基编码基因CsGγ的沉默载体及其应用和方法 (Silencing vector for silencing cupula mori G protein gamma subunit coding gene CsG gamma, and application and method thereof ) 是由赵爱春朱攀攀张帅李若兰刘长英范伟马舒煜夏中强向仲怀于 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γ亚基编码基因CsGγ的沉默载体及其应用和方法,桑实杯盘菌G蛋白γ亚基编码基因CsGγ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,将含有靶向桑实杯盘菌G蛋白γ亚基编码基因CsGγ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 cupula mori G protein gamma subunit coding gene CsG gamma, and application and a method thereof, wherein the nucleotide sequence of the cupula mori G protein gamma subunit coding gene CsG gamma is shown as SEQ ID No. 1; by utilizing a host induced gene silencing technology, a vector containing a silencing fragment of a target cuprum mori G protein gamma subunit coding gene CsG gamma is expressed in a plant, and by identifying the pathogenicity of the cuprum mori, the resistance of a transgenic plant to the cuprum mori can be obviously improved, so that the resistance of the plant to the cuprum mori can be improved by utilizing the method, and the infection of the cuprum mori can be prevented and treated.)

1. A silencing vector for silencing a G protein gamma subunit coding gene CsG gamma of cuprum mori 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 gamma subunit encoding gene CsG gamma 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-SiCSG gamma-F, then the sequence shown in SEQ ID NO.2 is reversely linked into the intermediate vector pHANNIBAL-SiCSG gamma-F through Xba I and Hind III to obtain a vector pHANNIBAL-SiCSG gamma-FR, and the vector pHANNIBAL-SiCSG gamma-FR is obtained by digestion with SacI and speI to obtain an expression frame.

3. The silencing vector for silencing cupula mori gamma subunit encoding gene CsG gamma 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 pSilent-1 plasmid through Xho I and Hind III to obtain pSilent-SiCSG gamma-U plasmid, and the reverse fragment shown in SEQ ID NO.2 is connected into pSilent-SiCSG gamma-U plasmid through Kpn I and Bgl II to obtain vector pSilent-SiCSG gamma-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 of the cupula mori L.G protein gamma subunit coding gene CsG gamma in the claim 1 or 3 into a plant to enable the transgenic plant to express a silencing fragment so as to obtain the transgenic plant resistant to the cupula mori L.G protein gamma subunit coding gene CsG gamma, wherein the nucleotide sequence of the cupula mori L.G protein gamma subunit coding gene CsG gamma 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 gamma subunit coding gene CsG gamma comprises the following steps: and (3) transforming the silencing vector for silencing cupule mulberry G protein gamma subunit coding gene CsG gamma 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 G protein gamma subunit encoding gene CsG gamma of calix sanguinea, and also relates to application and a method of the silencing vector for silencing the G protein gamma subunit encoding gene CsG gamma of the calix sanguinea.

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 glumae, magnaporthe grisea and the like, the pathogenicity of the fungi is obviously reduced after the G gamma subunit genes are knocked out, which indicates that the G gamma 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 for improving the resistance of the cupule fungi of the mulberry fruits, which has short period and good tolerance to pathogenic bacteria, 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 G protein gamma subunit encoding gene CsG gamma of cuprum mori; 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 cupula mori G protein gamma subunit coding gene CsG gamma 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-SiCSG gamma-F, then the sequence shown in SEQ ID NO.2 is reversely linked into the intermediate vector pHANNIBAL-SiCSG gamma-F through Xba I and Hind III to obtain a vector pHANNIBAL-SiCSG gamma-FR, and the expression frame is obtained by digestion with SacI and speI.

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 pSilent-1 plasmid through Xho I and Hind III to obtain pSilent-SiCSG gamma-U plasmid, and the reverse phase fragment shown in SEQ ID NO.2 is connected into pSilent-SiCSG gamma-U plasmid through Kpn I and Bgl II to obtain vector pSilent-SiCSG gamma-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 cupule mulberry fungus comprises the steps of transforming a silencing vector of a cupule mulberry fungus G protein gamma subunit coding gene CsG gamma in the plant to enable a silencing fragment to be expressed in a transgenic plant to obtain the transgenic plant resistant to the cupule mulberry fungus, wherein the nucleotide sequence of the cupule mulberry fungus G protein gamma subunit coding gene CsG gamma is shown in SEQ ID NO. 1.

Preferably, the method for transforming and silencing the cupula mori G protein gamma subunit coding gene CsG gamma comprises the following steps: and (3) transforming the silencing vector for silencing cupule mulberry G protein gamma subunit coding gene CsG gamma 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 cupula mori G protein gamma subunit coding gene CsG gamma, a plant with high resistance to cupula mori is obtained by taking a section of specific sequence of the cupula mori G protein gamma subunit coding gene CsG gamma 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 transgenic plants aim at the gene sequence of the cupula mori G protein gamma subunit coding gene CsG gamma and specifically silence the expression of the cupula mori G protein gamma, so that the influence on the plant per se is small; because a 35S strong promoter is used in the vector, the sRNA of the gene CsG gamma for encoding the G protein gamma subunit of the cupule mulberry fungus can be expressed in a large quantity, and the silencing effect is better; 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. 1 shows the effect of CsG gamma interfering strains on the virulence of fungi.

FIG. 2 shows the PCR detection result of kanamycin resistance gene in transgenic tobacco genome DNA (wherein Marker: DL15000 DNA Marker; WT: amplification with wild type total DNA as template; Positive control).

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

FIG. 4 is the statistics of the lesion area of the leaf after inoculation of the cupule of Morus alba.

FIG. 5 is a statistic of relative biomass of hyphae in leaves after inoculation of Stachys mori.

FIG. 6 shows the relative expression level of CsG γ in leaves of Moringa reticulata after inoculation.

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

CsGγ-F：5’-atgcctcaaggttactcatc-3’(SEQ ID NO.3)；

CsGγ-R：5’-ctacatcaccaaacaacatc-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 a CsG gamma nucleotide sequence obtained after sequencing is shown as SEQ NO. 1.

Example 2

Selecting a specific sequence of about 300bp of a coding region and a 3' non-coding region on CsG gamma to construct a silencing vector of the target cuprum sori CsG gamma, wherein the selected silencing fragment SisG gamma is shown as SEQ ID NO.2, the silencing fragment SisG gamma for constructing the host induced gene silencing vector is connected into pHANNIBAL plasmid EcoRI and Xho I enzyme cutting sites to obtain an intermediate vector pHANNIBAL-SisG gamma-F, and then the SisG gamma is connected into BamH I and Hind III enzyme cutting sites of the pHANNIBAL-SisG gamma-F plasmid to obtain the vector pHANNIBAL-SisG gamma-FR.

Forward silencing fragments were as follows:

SiCsGγ-F：5’-ccgctcgagtttctcaagctgccaagagt-3’(SEQ ID NO.5)；

SiCsGγ-R：5’-ccggaattctgtcctcggtggtgatgtac-3’(SEQ ID NO.6)。

forward silencing fragments were as follows:

SiCsGγ-F1：5’-cgcggatcctttctcaagctgccaagagt-3’(SEQ ID NO.7)；

SiCsGγ-R1：5’-cccaagctttgtcctcggtggtgatgtac-3’(SEQ ID NO.8)。

the SiCsG gamma-containing fragment was then excised using SacI and SpeI from the SacI and XbaI ligated plasmid pBin19 to obtain the final silencing expression vector pBin19-SiCsG gamma.

The fungus silencing expression vector is constructed by the following method: the forward fragment shown in SEQ ID NO.2 is connected into pSilent-1 plasmid through Xho I and Hind III to obtain pSilent-SiCysG gamma-U plasmid, and the reverse fragment shown in SEQ ID NO.2 is connected into pSilent-SiCysG gamma-U plasmid through Stu I and Bgl II to obtain vector pSilent-SiCysG gamma-UD.

Amplification was performed using the following primers:

SiCsGγ-U：5’-ccgctcgagtttctcaagctgccaagagt-3’(SEQ ID NO.9)

SiCsGγ-D：5’-cccaagctttgtcctcggtggtgatgtac-3’(SEQ ID NO.10)

SiCsGγ-U1：5’-aaaaggccttttctcaagctgccaagagt-3’(SEQ ID NO.11)

SiCsGγ-D1：5’-ggaagatcttgtcctcggtggtgatgtac-3’(SEQ ID NO.12)

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

Example 3

The silencing expression vector pCAMBIA1300-SiCsG gamma is used for obtaining a corresponding interference strain by a protoplast transformation method. 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 CsG gamma interfering strains) just growing over 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, the lesion area of infected leaves is calculated by using image J software, and the result is shown in figure 1.

Transferring the final silent expression vector pBin19-SiCsG gamma into Agrobacterium tumefaciens LBA4404 by a chemical conversion method, and identifying a positive transformant by bacteria liquid PCR. The detection primers are as follows:

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

Kan-F：5’-ggtagccaacgctatgtcct-3’(SEQ ID NO.14)。

the results of the detection are shown in FIG. 2. The results show that the positive agrobacterium transforms the sterile seedlings of the nicotiana benthamiana through a leaf disc transformation method to obtain seeds of positive strains (Line1, Line2, Line3, Line4, Line5 and Line 6).

Example 4

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; the tobacco actin gene quantitative primer actin-QF/QR and the fungus tubulin gene quantitative primer tubulin-QF/QR are used, quantitative PCR reaction is carried out by taking the genome of an infected material as a template, and the relative biomass of the fungus is calculated, so that the results show that the relative biomass of the fungus of Line1, Line2 and Line3 strains is obviously lower than that of a wild type strain. The quantitative primer CsG gamma-QF/QR of the fungus CsG gamma gene is used, and the fungus tubulin gene is used as an internal reference. And calculating the silencing effect on the target gene CsG gamma by using cDNA inverted from the infected material RNA as a template.

The detection primers are as follows:

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

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

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

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

CsGγ-QF：5’-cgacccatcgcaaatcaaga-3’(SEQ ID NO.19)；

CsGγ-QR：5’-cttggcagcttgagaaaccg-3’(SEQ ID NO.20)；

the results are shown in FIG. 5, and the results show that the expression level of the target gene of the transgenic line is significantly reduced. Then, viral biomass was calculated based on the expression amount, and the results are shown in FIG. 6, which shows that the transgenic line had lower viral biomass.

The results show that the transgenic tobacco obtained by using the cuprum mori as the target point through the host induced gene silencing technology can obviously improve the tolerance capability of the tobacco to the cuprum mori, and meanwhile, the invention also provides the target point for improving the resistance of other species of cuprum mori 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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