CN112159816A

CN112159816A - Application of tomato hydroxyproline-rich systemin precursor protein gene SlHypSys in improving plant resistance to Verticillium wilt

Info

Publication number: CN112159816A
Application number: CN202011073407.9A
Authority: CN
Inventors: 李先碧; 裴炎; 金丹; 范艳华; 于晓涵; 侯磊; 赵娟; 李美华; 郑雪丽; 唐梦
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2020-10-09
Filing date: 2020-10-09
Publication date: 2021-01-01
Anticipated expiration: 2040-10-09
Also published as: CN112159816B; WO2022073260A1

Abstract

The invention discloses the application of tomato hydroxyproline-rich systemin precursor protein gene SlHypSys in improving the resistance of plants to Verticillium wilt. The hydroxyproline-rich systemin precursor protein gene SlHypSys is cloned from tomato, The plant expression vector for constitutive expression of SlHypSys was constructed by the method of molecular biology, and then the SlHypSys gene was introduced into the plant by genetic engineering method, and the transgenic Arabidopsis thaliana, tobacco and cotton lines with normal transcription and expression were obtained; The disease index of the plant was significantly lower than that of the wild-type control, and the resistance to Verticillium wilt was significantly improved, indicating that the SlHypSys gene can be used to improve the resistance of plants to Verticillium wilt, which is of great significance for plant disease resistance genetic engineering.

Description

Application of tomato hydroxyproline-rich systemic precursor protein gene SlHypSys in improvement of verticillium wilt resistance of plants

Technical Field

The invention relates to the technical field of plant biology, in particular to application of a tomato hydroxyproline-rich systemic precursor protein gene SlHypSys in improving verticillium wilt resistance of plants.

Background

Plant diseases are one of natural disasters which endanger agricultural production for a long time, and more than 10% of the plant diseases are lost in the world due to the year of the diseases (Von Zhan et al, 2013). Plant diseases not only cause crop reduction, but also seriously threaten the quality safety and international trade of agricultural products, and when serious, also cause food shortage and a series of social problems (Punja, 2004). Pathogenic bacteria harm not only causes direct economic loss, but also can generate toxin, thereby bringing serious food safety problems. Therefore, improving the disease resistance of crops is not only a key to solving the food problem, but also an important measure for improving the food safety.

Verticillium wilt is a worldwide disease, and the onset of verticillium wilt is reported from temperate zone, subtropical zone to tropical zone (Pegg GF, 2002). Verticillium wilt is a pathogenic bacterium of the soil-borne vascular bundle, with facultative nutritional properties (Steven j. klosterman et al, 2009). The pathogenic bacteria have fast mutation frequency and multiple physiological species, can survive in soil for 20 years, and can infect more than 200 plant varieties, all plants except monocotyledons, including various vegetables, fruit trees, crops, forest trees, flowers and plants, and the like are hosts of verticillium wilt bacteria, and the crop yield loss caused by the verticillium wilt disease per year reaches billions of dollars, wherein the loss of the potato infected with the verticillium wilt bacteria can reach more than 50 percent, the lettuce is very easy to reach 100 percent, the cotton is easy to cause no grain harvest in the years with serious verticillium wilt disease, and the disease loss caused by the verticillium wilt bacteria is the most serious in all vascular bundle diseases (Pegg GF, 2002; Steven J. osteerman et al, 2009; Klrios G, 2005). At present, the only gene clonally obtained for resistance to verticillium wilt is Ve1 from tomato, which is also present in lettuce but resistance is lost after several years, and more importantly, most crops lack antigens for resistance to verticillium wilt and it is difficult to obtain resistant antigens (Steven j. klosterman et al, 2009).

In the face of rapid variation of pathogenic bacteria and specificity of physiological race, it is the fundamental to solve the problem to create a material with broad-spectrum resistance. The genetic engineering can overcome the defects of many traditional breeding, and the usable genes are more and the sources are wider. In addition, genetic engineering can also achieve a broader spectrum of disease resistance against a wider range of pathogenic bacteria, with minimal impact on soil-beneficial microorganisms (Owen Wally et al, 2010). However, the success of transgenic plants with increased resistance to fungal and bacterial diseases has been very limited compared to herbicide and insect resistant transgenic plants that have been grown extensively around the world for more than 10 years. There are also many successful reports of improving the resistance of transgenic crops to certain diseases, for example, overexpression of the chitinase gene CHIT36 from Trichoderma in carrot improves the resistance of transgenic plants to fungal diseases (Baranski R et al, 2008). Overexpression of defensin genes RsAFP2 and DmAMP from different sources in tomato and rice improves their disease resistance (Jha S et al, 2009), expression of peak toxin genes in rice improves resistance to bacterial blight in rice (Wei Shi, 2016), and so on. Although these exogenous genes have been successful in improving plant disease resistance, there is no report on their application to production, and the main problem is that the obtained resistance is only strong against a certain pathogen or a certain physiological race (or strain), and is difficult to persist and has no broad-spectrum resistance (Yan-Jun Chen, 2012).

Improving the self-defense ability of plants is an important means for improving the broad-spectrum and durable resistance of the plants. Plant hormones play an important role in plant defense response, SA, JA and ET signal pathways for regulating plant disease resistance are always hot points of research, and with the deepening and expanding of research, a new class of defense signal peptides capable of activating plant resistance to pathogenic bacteria and insects attracts attention of researchers in recent years, and particularly, the role of the plant-derived defense signal peptides in regulating plant immunity to insects and pathogenic bacteria is more important (Yube Yamaguchi ET al, 2011).

Plant defense signal peptides are a large class of peptide hormones, one of which is derived from a precursor protein peptide with an N-terminal secretion signal. Such peptides include hydroxyproline-rich systemic substances Hypsys of tobacco, tomato, petunia, etc. The tomato hydroxyproline-rich systemin (SlHypSys) comprises 3 glycopeptides rich in hydroxyproline, namely SlHypSysI, II and III, and the length of the glycopeptides is 20, 18 and 15 amino acids respectively, and the three mature peptides come from the same precursor protein, have the same function and have the function of defense signals. Upon tomato plant injury, induction by systemin and methyl jasmonate, slpohhpsys is synthesized in the vascular bundle parenchyma of the leaves and localized in the cell wall matrix (Javier, 2005).

Plant defense signal peptides have an important feature, their precursor proteins or mature peptides accumulate in vascular tissues and amplify defense signals within the vascular bundle, while activating the JA signaling pathway (Yube Yamaguchi et al, 2013). Verticillium dahliae is a typical vascular bundle disease, is continuously infected and expanded in vascular tissues after entering a plant body, and a JA signal path plays an important role in defense reaction of verticillium dahliae (Wei Gao et al, 2013). The two characteristics of the accumulation of the defense signal peptide and the spatial overlapping of the infection of the verticillium wilt bacteria, the signal path activated by the defense signal peptide and the overlapping of the defense of the verticillium wilt bacteria on the signal path are combined, and the obtained defense signal peptide capable of resisting the verticillium wilt bacteria has important significance for improving the verticillium wilt resistance of plants.

Disclosure of Invention

In view of the above, the invention aims to provide an application of a tomato hydroxyproline-rich systemic precursor protein gene SlHypSys in improving the resistance of plants to verticillium wilt, and provides a strategy of the invention by utilizing two characteristics of spatial overlapping of the accumulation of SlHypSys and the infection of verticillium wilt and overlapping of a signal path activated by SlHypSys and the defense of verticillium wilt on the signal path, and improving the resistance of plants to verticillium wilt by utilizing the SlHypSys gene.

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

the application of the tomato hydroxyproline-rich systemic precursor protein gene SlHypSys in improving the resistance of plants to verticillium wilt is disclosed, wherein the nucleotide sequence of the tomato hydroxyproline-rich systemic precursor protein gene SlHypSys is shown in SEQ ID No.1, or the nucleotide shown in SEQ ID No.1 is subjected to substitution and/or deletion and/or addition of one or more bases and has the nucleotide sequence with the same function.

In the present invention, the plant may be other plants which can be infected with verticillium wilt, and preferably, the plant is arabidopsis thaliana, tobacco or cotton.

The second purpose of the invention is to provide a method for improving the verticillium wilt resistance of plants by using a tomato hydroxyproline-rich systemic precursor protein gene SlHypSys.

the method for improving the verticillium wilt resistance of plants by using the tomato hydroxyproline-rich systemic precursor protein gene SlHypSys expresses the tomato hydroxyproline-rich systemic precursor protein gene SlHypSys constitutively in the plants to obtain the verticillium wilt resistance plants;

the nucleotide sequence of the tomato hydroxyproline-rich precursor protein gene SlHypSys is shown in SEQ ID No.1, or the nucleotide sequence which is shown in SEQ ID No.1 and has the same function after one or more base substitutions and/or deletions and/or additions.

Preferably, the constitutive expression method of the tomato hydroxyproline-rich precursor protein gene SlHypSys in plants is to construct a constitutive plant expression vector containing the tomato hydroxyproline-rich precursor protein gene SlHypSys and then obtain transgenic plants through agrobacterium mediation.

In the invention, the constitutive plant expression vector is used for regulating and controlling the expression of a tomato hydroxyproline-rich systemic precursor protein gene SlHypSys by a constitutive promoter.

In the present invention, the constitutive promoter may be any constitutive promoter capable of being expressed in plants, and preferably, the constitutive promoter is CaMV35S promoter.

In the invention, the constitutive plant expression vector is a plant expression vector pLGN-35S-SlHypSys obtained by passing a sequence shown in SEQ ID NO.1 through BamHI and KpnI plant expression vector pLGN (CN 105671076A).

In the invention, the plants can be other plants which can be infected with verticillium wilt, such as vegetables like eggplant, tomato, pepper, lettuce and the like, oil plants like rape and olive and ornamental plants like red leaves; preferably, the plant is arabidopsis, tobacco or cotton.

The specific operation is as follows: the method comprises the steps of obtaining SlHypSys genes (SEQ ID NO.1) from tomatoes, operably connecting the SlHypSys genes with a promoter, constructing a plant expression vector for constitutive expression of the tomato hydroxyproline-rich phylogenin precursor protein genes SlHypSys, transforming the vector into a host to obtain a transformant, then transferring the transformant into plants (such as arabidopsis thaliana, tobacco, cotton and the like), and obtaining transgenic plants after resistance screening and a series of molecular identifications.

In the invention, the method for obtaining the SlHypSys gene and constructing the expression vector for constitutive expression of the SlHypSys gene by fusing the promoter and the SlHypSys gene is a conventional method in the field, and the used vector is a conventional vector used in the field of plant genetic engineering.

The method used for transferring the transformant into the plant is an agrobacterium tumefaciens mediated method, which is a commonly used plant transgenic method.

It is a further object of the present invention to provide a plant expression vector having improved resistance to verticillium wilt in plants.

a plant expression vector capable of improving the resistance of a plant to verticillium wilt comprises an expression frame of a constitutive promoter for regulating and controlling the expression of a tomato hydroxyproline-rich precursor protein gene SlHypSys, wherein the nucleotide sequence of the tomato hydroxyproline-rich precursor protein gene SlHypSys is shown in SEQ ID No.1, or the nucleotide shown in SEQ ID No.1 is subjected to substitution and/or deletion and/or addition of one or more bases and has a nucleotide sequence with the same function.

Preferably, the gene sequence of SlHypSys (SEQ ID NO.1) is operably connected with a constitutive promoter CaMV35S by applying a genetic engineering technology to construct a plant expression vector, and after a plant is transformed, the gene of SlHypSys rich in hydroxyproline precursor protein of tomato is expressed under the action of the constitutive promoter. Preferred plant expression vectors have the vector structure shown in FIG. 1. Wherein, the tomato hydroxyproline precursor protein rich gene SlHypSys is positively linked to a CaMV35S promoter to form a plant expression vector pLGN-35S-SlHypSys for constitutive expression of the gene.

The fourth purpose of the invention is to provide the application of the plant expression vector.

the plant expression vector is applied to the preparation of transgenic plants with verticillium wilt resistance.

The invention has the beneficial effects that: the invention firstly clones a hydroxyproline-rich systemin precursor protein gene SlHypSys from tomatoes, adopts a molecular biology method to construct a plant expression vector for constitutive expression of the SlHypSys, and then utilizes a genetic engineering method to introduce the SlHypSys gene into arabidopsis thaliana, tobacco and cotton to obtain transgenic arabidopsis thaliana, tobacco and cotton strains with normal transcriptional expression. The disease index of the wild type arabidopsis is 50.69, and the disease index of the transgenic arabidopsis is only 13.52. When the disease index of the wild tobacco is 50.85, the disease index of the transgenic tobacco is only 9.11; when the disease index of wild cotton is 53.43, the disease index of transgenic cotton is only 17.22. The disease index of the transgenic plant is obviously lower than that of a wild control, and the resistance to verticillium wilt is obviously improved. The invention has important significance for promoting the application of the SlHypSys gene in plant disease-resistant genetic engineering.

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 is a map of a plant expression vector pLGN-35S-SlHypSys.

FIG. 2 shows the transcriptional expression level of the SlHypSys gene in transgenic Arabidopsis thaliana (SlHypSys-2, SlHypSys-3, SlHypSys-6, SlHypSys-12 and SlHypSys-15: independent transformants of transgenic Arabidopsis thaliana; WT: Columbia ecotype wild type Arabidopsis thaliana; values are the average of three technical replicates and error bars are the standard error SD).

FIG. 3 shows that the SlHypSys gene improves the resistance of Arabidopsis to verticillium wilt (A: 14 days of species of verticillium wilt bacteria, disease index of Arabidopsis strain; B: 14 days of inoculation of verticillium wilt bacteria, and disease condition of Arabidopsis plant. the disease resistance of transgenic Arabidopsis is evaluated by the disease index. the data is the average value of three repeated tests, and the error bars are standard errors SD. SlHypSys-3, SlHypSys-6 and SlHypSys-15: independent transformant of transgenic Arabidopsis.

FIG. 4 shows the transcriptional expression level of the SlHypSys gene in transgenic tobacco (SlHypSys-1, SlHypSys-2, SlHypSys-3, SlHypSys-4, SlHypSys-12 and SlHypSys-15: independent transformants of transgenic tobacco; WT: wild-type tobacco. values are the mean of three technical replicates and error bars are the standard error SD of three replicates).

FIG. 5 shows that the SlHypSys gene improves the resistance of tobacco to verticillium wilt (A: the disease index of tobacco strains after inoculation of 7 days of verticillium wilt, B: the disease of tobacco leaves after inoculation of 7 days of verticillium wilt, and the disease resistance of transgenic tobacco is evaluated by the disease index, the numerical value is the average value of three repetitions, error bars are standard errors SD. SlHypSys-1 and SlHypSys-4: independent transformants of transgenic tobacco, WT: wild type tobacco: the difference level of the disease index reaches the utmost significance compared with wild type tobacco (p < 0.01)).

FIG. 6 shows the transcriptional expression level of the SlHypSys gene in transgenic cotton (SlHypSys-1, SlHypSys-2 and SlHypSys-5: independent transformants of transgenic cotton; WT: wild-type cotton of transgenic recipient; values are the mean of three technical replicates and error bars are the standard error of three replicates).

FIG. 7 shows that the resistance of SlHypSys transgenic cotton to verticillium wilt is improved (A: disease index of transgenic cotton leaves inoculated with verticillium wilt for 5 days; B: disease of cotton leaves inoculated with verticillium wilt for 5 days; disease resistance of transgenic cotton is evaluated by using disease index. data are average values of three repeated tests, error bars are standard errors SD. SlHypSys-1, SlHypSys-2 and SlHypSys-5: independent transformants of transgenic cotton. WT: wild-type cotton of gene receptor: difference level of disease index reaches very significant (p <0.01)) compared with wild-type cotton.

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 cloning of the tomato SlHypSys Gene

(1) Extraction of total RNA of tomato genome and synthesis of cDNA

And (3) taking tomato seedlings of 4 weeks old, slightly clamping the base parts of the leaf stalks by using forceps, and inducing the expression of the SlHypsys gene. After 6 hours, the damaged leaves are cut and immediately frozen by liquid nitrogen, and ground into powder, then the total RNA of the leaves is extracted by using a plant rapid RNA extraction kit of Promoga, and then cDNA is synthesized by using a one-strand cDNA synthesis kit of TaKaRa, wherein the RNA extraction and the cDNA synthesis are carried out according to the kit instructions. The detailed operation flow is as follows:

quickly freezing fresh tomato leaves with liquid nitrogen, grinding into powder, taking about 100mg of the powder, filling into a 1.5mL centrifuge tube without RNase and DNase, immediately adding 500 mu L of RNA lysate, repeatedly blowing and beating with a pipette until no obvious blocky tissue exists in the lysate, then adding 300 mu L of diluent, reversing the centrifuge tube for 3-4 times, uniformly mixing, and standing at room temperature for 3-5 min. 12000rpm, centrifuging for 5min, taking 500 μ L of supernatant into a new centrifugal tube without nuclease of 1.5mL, adding 250 μ L of absolute ethyl alcohol, immediately blowing and uniformly mixing by a pipette, then transferring the mixed solution into an RNA adsorption column, 10000rpm, centrifuging for 1min, discarding filtrate, adding 600 μ L of rinsing solution into the adsorption column, repeatedly rinsing for 2 times, transferring the adsorption column to an elution tube, adding 100 μ L of water without RNase and DNA enzyme into the adsorption column, standing for about 3min, 10000rpm, centrifuging for 1min, collecting eluted RNA solution, and storing RNA at-80 ℃.

And (3) adding 1 mu L of RNase-free DNase and 2 mu L of RNase-free DNase buffer solution into 7 mu L of the extracted RNA solution, uniformly mixing, reacting for 2min at 42 ℃ in a PCR instrument, then adding 4 mu L of reverse transcriptase buffer solution, 1 mu L of reverse transcriptase, 1 mu L of reverse transcription primer mixture and 4 mu L of double distilled water without RNase and DNase, uniformly mixing, reacting for 15min at 37 ℃ to synthesize cDNA, reacting for 5S at 85 ℃, and stopping the reaction. The synthesized cDNA was stored at-20 ℃.

(2) Cloning of the tomato SlHypSys Gene

The NCBI website searches for an SlHypSys sequence (shown in SEQ ID NO.1), and designs and synthesizes an upstream primer and a downstream primer of a complete coding frame according to a nucleotide sequence shown in SEQ ID NO.1, which comprises the following specific steps:

an upstream primer F-SlHypSys: 5'-cgcggatccgcg atgatcagcttcttcagagc-3' (SEQ ID NO. 2);

the downstream primer R-SlHypSys: 5'-cggggtaccccg ttaataggaagcttgaagaggc-3' (SEQ ID NO. 3);

PCR amplification was performed using PrimesSTAR MAX DNA Polymerase using the above synthesized cDNA as a template and SEQ ID NO.2 and SEQ ID NO.3 as a primer pair. And (3) amplification procedure: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 57 ℃ for 10s, extension at 72 ℃ for 20s, and amplification for 30 cycles.

mu.L of the obtained SlHypSys fragment was amplified, and 3. mu.L of pTOPO vector plasmid, 1. mu.L of DNA ligase and 1. mu.L of buffer were added thereto, and the mixture was mixed well and allowed to stand at room temperature for 15 min. The reaction product is mixed and evenly mixed with escherichia coli DH5 alpha competent cells, then heat shock is carried out for 1min and 30s at 42 ℃ to be transferred into escherichia coli DH5 alpha, the transformed engineering bacteria are added into 500 mu L LB culture medium and cultured for 1h at 37 ℃ and 200rpm, then the engineering bacteria are coated on an LB plate containing kanamycin and cultured overnight at 37 ℃, single colony is selected and inoculated into a test tube containing about 3mL of LB culture medium, shaking culture is carried out at 37 ℃ and 200rpm overnight, a proper amount of bacterial liquid is taken and sent to a sequencing company for sequencing, and the engineering bacteria containing the positive clone of the target fragment are stored at-80 ℃.

Example 2 construction of plant expression vector for constitutive expression of SlHypSys Gene and obtaining of engineering bacteria

Extracting the cloning vector engineering bacteria plasmid which is verified to be correct by sequencing, carrying out double enzyme digestion by using BamHI and KpnI, carrying out agarose gel electrophoresis on the product after enzyme digestion, and recovering a target fragment SlHypSys. And carrying out double enzyme digestion on the plant expression vector pLGN by using BamHI and KpnI, carrying out agarose gel electrophoresis after enzyme digestion is finished, and recovering a large fragment. The recovered gene and vector fragment were ligated by T4 DNA ligase, and the ligation product was transformed into E.coli DH 5. alpha. by heat shock. Extracting plasmid of the engineering bacteria of the escherichia coli, carrying out double enzyme digestion verification by using BamHI and KpnI, obtaining a transformation bacteria of the enzyme digestion fragment of the target gene SlHypSyss, namely the plant expression vector pLGN-35S-SlHypSys engineering bacteria, storing at-80 ℃, and showing a vector map in figure 1.

Extracting the plasmid of engineering bacteria of colibacillus, transforming Agrobacterium LBA4404 and GV3101 by electric transformation method, screening and culturing the transformed bacteria on YEB plate with additional Km, Sm, Km and Rif, inoculating resistant single colony into YEB liquid culture medium with additional Km, Sm, Km and Rif, collecting bacteria and extracting Agrobacterium plasmid, double enzyme digestion with BamHI and KpnI, and storing correct engineering bacteria at-80 deg.C.

Example 3 genetic transformation of Arabidopsis, screening of transgenic Arabidopsis and analysis of transcription expression level

(1) Genetic transformation of Arabidopsis thaliana

Genetic transformation was carried out using wild type Arabidopsis thaliana in Columbia as material, according to the floral dip transformation method of Steven J. Clough and Andrew F. bent (1998), and seeds after Agrobacterium maceration were harvested after seed maturation.

(2) Screening of transgenic Arabidopsis plants

The seeds harvested after genetic transformation by a flower soaking transformation method are sterilized by 75% alcohol for 15min, then are evenly inoculated on a screening plate with 100mg/L Km (kanamycin) added for sprouting, if the grown seedlings are green, the seedlings are transgenic plants, the plants are transplanted into special soil for culturing arabidopsis thaliana (grass carbon soil: vermiculite: perlite: 3:1:1) when the plants have more than 2 leaves, and the seeds are harvested after maturation. Each plant is a transformant.

Arabidopsis thaliana screening medium: MS inorganic + MS organic + Km 100mg/L +2.5g/L Gelrite (causticizer), pH6.0

(3) Extraction of transgenic arabidopsis plant RNA and synthesis of cDNA

The method of example 1 was followed to extract the RNA of transgenic Arabidopsis thaliana and synthesize cDNA using young leaves of transgenic plants as material.

(4) Analysis of transcriptional expression level of SlHypSys gene in transgenic Arabidopsis thaliana

And detecting the transcription expression level of the SlHypSys gene in the transgenic arabidopsis thaliana by using a Real-time PCR method.

And amplifying a specific fragment of the SlHypSys gene by using the cDNA as a template. The upstream and downstream primers of the SlHypSys gene are SlHypSys UP: 5'-ttaccacctccttctccc-3' (SEQ ID NO.4) and SlHypSys DN: 5'-tacataatcgtgcctccc-3' (SEQ ID NO.5), respectively. The Arabidopsis AtACT2 gene was used as an internal standard. The upstream and downstream primers of AtACT2 gene were AtACT2 UP: 5'-tatcgctgaccgtatgag-3' (SEQ ID NO.6) and AtACT2 DN: 5'-ctgagggaagcaagaatg-3' (SEQ ID NO.7), respectively.

A20. mu.L Real-time PCR reaction system included: 1 mu L of cDNA template, 1 mu L of each of upstream and downstream primers of target gene, 10 mu L of 2 xiQ SYBR Green Supermix, ddH₂O 7μL。

Real-time PCR amplification conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 10s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 30s, for a total of 40 cycles of amplification. After amplification, the relative expression level of SlHypSys Gene was analyzed by using Gene studio software.

The Real-time PCR result shows that (figure 2) SlHypSys genes in transgenic arabidopsis plants can be effectively transcribed and expressed, and the obtained plants are SlHypSys transgenic plants.

Example 4 resistance of transgenic Arabidopsis to Verticillium wilt

(1) Preparation of verticillium wilt bacteria for disease-resistant identification and inoculation of transgenic arabidopsis thaliana

Selecting a small amount of verticillium dahliae V991 strain (Verticillium dahliae) stored in a solid PD medium (potato medium), inoculating the strain into a liquid PD medium, carrying out shaking culture at 180rpm and 26 ℃ for 7d, inoculating the strain into the liquid PD medium according to the proportion of 10% (bacterial liquid/PD medium), carrying out shaking culture at 180rpm and 26 ℃ for 10d, filtering by four layers of sterile gauze to remove hyphae and impurities in the bacterial liquid, and adjusting the spore concentration to 10 by deionized water⁸One/ml was used as inoculum.

(2) Transgenic arabidopsis disease-resistant identification inoculation method

The arabidopsis seedlings cultured for one week are uprooted, then placed in a 150mm culture dish with soil tidily, the evenly mixed inoculation bacterial liquid is poured, the inoculation dose is 10 mL/plant, after the seedlings are soaked and inoculated for 24 hours at room temperature, the seedlings are transplanted into moist soil, the seedlings are irradiated for 16 hours, dark culture is carried out for 8 hours, and the seedlings are cultured in an illumination incubator with the humidity of 70 percent (20 ℃ (dark culture) -24 ℃ (illumination culture). After 2 weeks of inoculation, the disease grade of the plants was counted according to the 0-4 grade standard and the disease index was calculated. Wild type plants transformed with the recipient material were used as controls.

Grading grade standard: level 0: the plant leaves have no diseases; level 1: 0-25% of leaf development; and 2, stage: 25% -50% of the leaves present with symptoms; and 3, level: 50% -75% of the leaves present with symptoms; 4, level: over 75% of leaves present with disease. The calculation formula of the disease index is as follows:

disease index (Σ [ (number of disease stages)/(number of inoculated plants)/(4 × total number of inoculated plants) × 100

(3) SlHypSys gene for improving resistance of arabidopsis thaliana to verticillium wilt

The Arabidopsis plants are inoculated with verticillium wilt bacteria for 14 days, the disease index of wild plants reaches 50.69, and the disease indexes of transgenic lines SlHypSys-3, SlHypSys-6 and SlHypSys-15 are respectively 13.52, 21.48 and 20.99. The T-test results showed a very significant decrease in disease index of the transgenic lines compared to that of the wild-type control (a in fig. 3). Plant disorders show that the leaves of wild plants basically have the disorders after 14 days of inoculation with verticillium wilt bacteria, while the leaves of transgenic plants only have the disorders in the individual leaves at the base (B in figure 3). The result shows that the SlHypSys gene can effectively improve the resistance of arabidopsis to verticillium wilt.

Example 5 genetic transformation of tobacco and obtaining of transgenic tobacco

(1) Tissue culture medium for tobacco genetic transformation:

seed germination culture medium: MSB (MS inorganic salt + B5 organic), 1.0% agar powder and tap water, and natural pH.

Genetic transformation co-culture medium: MSB (MS inorganic salt + B5 organic) +2mg/L NAA +0.5 mg/L6-BA + 200. mu. mol/L AS, pH5.6, solid medium added with 1.0% agar powder for solidification.

Callus induction medium: MSB (MS inorganic salt + B5 organic) +2mg/L NAA +0.5 mg/L6-BA + 1.0% agar powder, pH5.8.

Shoot induction medium: MSB (MS inorganic salt + B5 organic) +2 mg/L6-BA + 1.0% agar powder, pH5.8.

Rooting culture medium: MSB (MS inorganic salt + B5 organic) + 1.0% agar powder, ph 6.0.

(2) Genetic transformation of tobacco

Inoculating the recombinant agrobacterium containing the pLGN-35S-SlHypSys plant expression vector into a liquid YEB culture medium, and carrying out shake culture at 28 ℃ and 200rpm overnight until OD 6001.0-1.2. And (3) centrifuging the bacterial liquid, collecting the bacteria, and suspending the bacteria by using an equal-volume MSB liquid culture medium, wherein the heavy suspension is a staining solution for transformation.

Culturing tobacco sterile seedling leaf for 20 days, cutting into 3-5mm leaf disc, dip-dyeing in the dip-dyeing solution for 1hr, removing bacterial liquid, inoculating the leaf disc into co-culture medium, and dark culturing at 24 deg.C for 2 days. After the co-culture is completed, the explants are subcultured into a callus induction culture medium added with 100mg/L kanamycin and 200mg/L cephamycin, the explants are subjected to photoperiod culture at 25 ℃ under 16hr illumination/8 hr dark culture, the explants are subcultured into a bud induction culture medium after 20 days, the explants are subcultured once after 20 days until buds are generated at the edge of a leaf disc, the buds are cut off and then substituted into a rooting culture medium to root and grow into seedlings, and the seedlings grow to 3-4 leaves and are transplanted into flowerpots for further analysis.

(3) Acquisition and molecular identification of SlHypSys transgenic tobacco

a. GUS histochemical staining of transgenic plants

GUS staining solution: 500mg/L X-Gluc, 0.1mol/L K₃Fe(CN)₆，0.1mol/L K₄Fe(CN)₆，1％Triton X-100(V/V)，0.01mol/L Na₂EDTA, 0.1mol/L phosphate buffer (pH 7.0).

The pLGN-35S-SlHypSys plant expression vector contains GUS gene controlled by CaMV35S promoter, so that transgenic plants can be quickly identified by GUS histochemical staining method firstly. Leaf tissue from Km resistant seedlings was excised as described in Jefferson (1987), stained in GUS histochemical staining solution for 5h at 37 ℃ and destained to green by 95% ethanol. Finally, the blue plants are transgenic plants, otherwise, the plants are non-transgenic plants.

SlHypSys transcriptional expression level analysis

The SlHypSys transgenic tobacco plant takes young and tender leaves as materials, respectively extracts the RNA of GUS positive and wild plant leaves, and synthesizes one-strand cDNA of each sample RNA according to the instruction of a cDNA one-strand synthesis kit (the method for extracting RNA and synthesizing cDNA is the same as that in example 1). Then, a specific segment of the SlHypSys gene is amplified by taking the cDNA as a template. The upstream and downstream primers of the SlHypSys gene are SlHypSys UP: 5'-ttaccacctccttctccc-3' (SEQ ID NO.4) and SlHypSys DN: 5'-tacataatcgtgcctccc-3' (SEQ ID NO.5), respectively. The tobacco 18S gene is used as an internal standard. The upstream and downstream primers of the 18S gene were Nt18S UP: 5'-aggaattgacggaagggca-3' (SEQ ID NO.8) and Nt18S DN: 5'-gtgcggcccagaacatctaag-3' (SEQ ID NO.9), respectively.

A20. mu.L Real-time PCR reaction system included: mu.L of cDNA template, 1. mu.L of each of the upstream and downstream primers of the target gene, 10. mu.L of 2 xiQ SYBR Green Supermix, and 7. mu.L of ddH2O 7.

Real-time PCR amplification conditions: denaturation at 95 deg.C for 3 min; denaturation at 94 ℃ for 10s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 30s, for a total of 40 cycles of amplification. After amplification, the relative expression level of SlHypSys Gene was analyzed by using Gene studio software.

The Real-time PCR result shows that (figure 4) SlHypSys genes in transgenic tobacco plants can be effectively transcribed and expressed, and the expression of the genes is not detected in leaves of wild plants.

Example 6 resistance of transgenic tobacco to Verticillium wilt

(1) Preparation of verticillium wilt bacteria for transgenic tobacco disease-resistant identification and inoculation

The preparation method of verticillium wilt bacteria for identifying and inoculating transgenic tobacco disease resistance is consistent with the embodiment 4, the spore concentration is adjusted to 10¹⁰spores/mL.

(2) Transgenic tobacco disease-resistant identification inoculation method

Cutting the third to fifth leaves from the top to the bottom of 7-8 true-leaf tobacco plants, wrapping petioles with moist absorbent paper, uniformly and neatly placing in an inoculation box, slightly squeezing the junction of the main leaf vein and the secondary leaf vein of the leaves by using a pipette tip to achieve the purpose of artificial injury, and immediately inoculating 10 mu L of verticillium wilt germ inoculation liquid at the injured part after injury. Then, the leaf was covered with a film and kept moist, and the leaf was incubated in an incubator of 16 hours light/8 hours dark culture, 20 ℃ (dark culture)/26 ℃ (light), inoculated for 7 days, and the grade of the leaf was counted according to the 5-grade standard of 0-4 and the disease index was calculated. Wild type plants transformed with the recipient material were used as controls.

Grading grade standard: grade 0, no disease of the leaf; level 1: 0-25% of the leaf area presents with symptoms; and 2, stage: symptoms appear in 25% -50% of the leaf area; and 3, level: 50-75% of the leaf area is diseased; 4, level: over 75% of the leaf area develops symptoms. The calculation formula of the disease index is as follows:

disease index (Σ [ (number of disease stages × number of plants)/(4 × total number of inoculated plants) × 100.

(3) SlHypSys gene for improving resistance of tobacco to verticillium wilt

The verticillium dahliae is inoculated on tobacco leaves for 7 days, the disease index of a wild plant reaches 50.74, and the disease indexes of transgenic lines SlHypSys-1 and SlHypSys-4 are respectively 9.11 and 15.43. The T-test results showed a very significant decrease in disease index of the transgenic lines compared to that of the wild-type control (a in fig. 5). After 7 days of inoculation of verticillium wilt, the disease area of wild type leaves exceeds 50 percent, and the disease area of transgenic tobacco leaves is less than 10 percent (B in figure 5). The result shows that the SlHypSys gene can effectively improve the resistance of tobacco to verticillium wilt.

Example 7 genetic transformation of Cotton

(1) Common culture medium for cotton genetic transformation

Basic culture medium: MSB (MS inorganic salts + B5 organic) (t.murashige, 1962; o.l.gamborg, 1968);

seed germination culture medium: 1/2MSB +20g/L sucrose +6g/L agar, prepared with tap water, natural pH;

co-culture medium: MSB +0.5mg/L IAA (indoleacetic acid) +0.1mg/L KT (6-furfurylaminopurine) +30g/L glucose + 100. mu. mol/L acetosyringone +2.0g/L Gelrite (Sigma), pH 5.4;

screening a degerming culture medium, namely MSB +0.5mg/L IAA +0.1mg/L KT +75mg/L Km (kanamycin) +500mg/L cef (cefamycin) +30g/L glucose +2.0g/L Gelrite, and pH is 5.8;

the callus induction culture medium comprises MSB, 0.5mg/L IAA, 0.1mg/L KT, 75mg/L Km, 200mg/L cef, 30g/L glucose and 2.0g/L Gelrite, and the pH value is 5.8;

the embryogenic callus induction culture medium comprises MSB +0.1mg/L KT +30g/L glucose +2.0g/L Gelrite, and pH5.8;

liquid suspension culture medium: MSB +0.1mg/L KT +30g/L glucose, pH5.8;

somatic embryo maturation medium: MSB +15g/L sucrose +15g/L glucose +0.1mg/L KT +2.5g/L Gelrite, pH6.0;

seedling medium SH +0.4g/L activated carbon +20g/L sucrose, pH6.0(Schenk & Hildebrandt, 1972).

(2) Method for genetic transformation of cotton

Obtaining of transformed explants: removing shell of seeds of Gossypium hirsutum 14, sterilizing seed kernel with 3% hydrogen peroxide for 60min, rinsing with sterile tap water for 5-6 times, inoculating to seed germination culture medium, and dark culturing at 28 deg.C for 5 d. The sterile hypocotyls were cut into 3-5mm long sections as transformed explants.

Transformation ofPreparation of a liquid for agrobacteria staining: obtaining single colony of Agrobacterium integrating plant expression vector pLGN-35S-SlHypSys by using a line drawing method, then selecting the single colony and inoculating 10mL liquid YEB (5g/L sucrose, 1g/L yeast extract for bacteria, 10g/L tryptone for bacteria and 0.5g/L MgSO (magnesium sulfate)) with additional 50mg/L Km and 125mg/L Sm (streptomycin)₄·7H₂O, pH7.0), cultured overnight at 28 ℃ and 200rpm, and then inoculated with 20mL of a liquid YEB containing no antibiotic at a ratio of 5%, cultured at 28 ℃ and 200rpm until the OD600 is about 0.5. And (3) centrifuging 5mL of bacterial liquid at 6000rpm for 5min to collect thalli, and suspending the thalli by using 5mL of co-culture liquid culture medium without adding Gelrite, wherein the suspended bacterial liquid is the agrobacterium tumefaciens staining solution for staining the explant.

(3) Genetic transformation of hypocotyls and induction of embryogenic callus

The explant is impregnated with an agrobacterium impregnation solution for 20min, a bacterial solution is poured out, redundant bacterial solution on the surface of the explant is absorbed by sterile filter paper, the impregnated hypocotyl is cut into sections and inoculated in a co-culture medium, dark culture is carried out at 26 ℃ for 2d, the hypocotyl is inoculated in a screening and degerming medium, 20d later is substituted into a callus induction medium with kanamycin (Km) cef, callus induction is carried out, subculture is carried out once at intervals of 20d, 60d later is substituted into an embryogenic callus induction medium, and liquid suspension culture is carried out after embryogenic callus is obtained, so that a large amount of embryogenic callus with consistent growth is obtained.

(4) Induction and seedling culture of somatic embryos

And (3) filtering the embryogenic callus cultured by liquid suspension, uniformly and dispersedly inoculating the screened embryogenic callus into a somatic embryo maturation culture medium, generating a large amount of somatic embryos in about 15 days, and subculturing the somatic embryos into an SH culture medium to promote further seedling formation of the somatic embryos. Transplanting the 3-4 leaf regenerated seedlings into a greenhouse for propagation.

Example 8 acquisition and molecular validation of SlHypSys transgenic Cotton

The SlHypSys gene was transferred into the cotton genome according to the cotton genetic transformation and regeneration method of example 7. After induction of Km resistant callus, embryogenic callus and somatic embryos, somatic embryos became seedlings and SlHypSys transgenic cotton plants were obtained.

(1) GUS histochemical staining of transgenic plants

The GUS staining solution was formulated as in example 5.

Cutting a small amount of leaf stalk and leaf tissue of Km resistant seedlings, adding the cut leaves and leaf tissue into GUS histochemical staining solution respectively, staining for 2h at 37 ℃, and then decoloring by 95% ethanol until the leaves and leaf tissue are clean. Finally, the blue plants are transgenic plants, otherwise, the plants are non-transgenic plants.

(2) Analysis of transcriptional expression level of SlHypSys Gene

The SlHypSys transgenic cotton plant takes young and tender leaves as materials, respectively extracts RNAs of GUS positive and wild plant leaves, synthesizes one-strand cDNA of each sample RNA according to the instruction of a cDNA one-strand synthesis kit (the RNA extraction and cDNA synthesis methods are the same as the example 1), and then amplifies a specific fragment of the SlHypSys gene by taking the cDNA as a template. The upstream and downstream primers of the SlHypSys gene are SlHypSys UP: 5'-TTACCACCTCCTTCTCCC-3' (SEQ ID NO.4) and SlHypSys DN: 5'-TACATAATCGTGCCTCCC-3' (SEQ ID NO. 5). The cotton histone GhHIS3 gene is used as an internal standard. The upstream and downstream primers of the GhHIS3 gene are GhHIS3 UP: 5'-GAAGCCTCATCGATACCGTC-3' (SEQ ID NO.10) and GhHIS3 DN: 5'-CTACCACTACCATCATGGC-3' (SEQ ID NO.11) (Zhu YQ et al, 2003).

Real-time PCR amplification conditions: 3min at 95 ℃; amplifying at 94 ℃ for 10s, 57 ℃ for 30s and 72 ℃ for 30s for 40 cycles. After amplification, the relative expression level of SlHypSys Gene was analyzed by using Gene studio software.

The Real-time PCR result shows (figure 6) that the SlHypSys gene in the transgenic cotton plant can be effectively transcribed and expressed, and the expression of the gene is not detected in the wild plant.

Example 9 resistance of SlHypSys transgenic Cotton to verticillium wilt

(1) Preparation of pathogenic bacteria for disease-resistant identification and inoculation

The preparation method of pathogenic bacteria for disease resistance identification of transgenic cotton is the same as that in example 4.

(2) In vitro leaf inoculation method

Shearing young and tender leaves of a transgenic cotton plant, putting the aligned leafstalks into a culture bottle containing inoculated bacterial liquid, culturing for 24 hours (namely, inoculating for 24 hours) in the bacterial liquid, dumping the bacterial liquid, injecting a proper amount of sterile water into the culture bottle, putting the culture bottle containing the leaves into a 16-hour illumination environment, carrying out 8-hour dark culture light period, carrying out 20 ℃ (dark culture) and 26 ℃ (illumination) temperature change period, continuing moisturizing culture under the condition of 70% humidity, counting the disease grade of the leaves once every two days, and calculating the disease index. Wild type plants transformed with the recipient material were used as controls. Grading grade standard: level 0: the cotton leaves have no disease; level 1: less than 25% of the leaf area presents with symptoms; and 2, stage: symptoms appear in 25% -50% of the leaf area; and 3, level: disease symptoms occur in 50% -75% of the leaf area; 4, level: over 75% of the leaf area develops disease. The calculation formula of the disease index is as follows:

(3) SlHypSys gene for improving resistance of cotton to verticillium wilt

The verticillium wilt resistance identification is carried out on all transgenic plants according to the method, and the result shows that: and 5d of inoculation, the disease index of wild cotton is 65.83, and the disease indexes of transgenic lines SlHypSys-1, SlHypSys-2 and SlHypSys-5 are 23.61, 18.65 and 16.56 respectively. The T-test results showed that the disease index of the transgenic cotton lines was very significantly lower than that of wild-type cotton (a in fig. 7). After 5 days of inoculation, the wild-type cotton leaves showed severe disease, while the leaves of the transgenic cotton lines showed only a few seed lesions (B in FIG. 7). The result shows that the SlHypSys can obviously improve the resistance of cotton to verticillium wilt.

The experiments prove that the tomato hydroxyproline-rich systemic precursor protein gene SlHypSys is introduced into wild arabidopsis thaliana, tobacco and cotton by a transgenic means to obtain transgenic arabidopsis thaliana, tobacco and cotton, and the disease index of the transgenic plant inoculated with verticillium wilt bacteria is obviously lower than that of the wild control, so that the gene can improve the disease resistance of arabidopsis thaliana, tobacco and cotton to verticillium wilt diseases.

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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Claims

1. The application of the tomato hydroxyproline-rich systemic precursor protein gene SlHypSys in improving the resistance of plants to verticillium wilt is characterized in that: the nucleotide sequence of the tomato hydroxyproline-rich precursor protein gene SlHypSys is shown in SEQ ID No.1, or the nucleotide sequence which is shown in SEQ ID No.1 and has the same function after one or more base substitutions and/or deletions and/or additions.

2. Use according to claim 1, characterized in that: the plant is Arabidopsis thaliana, tobacco or cotton.

3. The method for improving the verticillium wilt resistance of plants by using the tomato hydroxyproline-rich systemic precursor protein gene SlHypSys is characterized by comprising the following steps of: constitutive expression of a tomato hydroxyproline-rich systemic precursor protein gene SlHypSys in a plant to obtain a plant resistant to verticillium wilt;

4. The method of claim 3, wherein: the constitutive expression method of the tomato hydroxyproline-rich systemic precursor protein gene SlHypSys in plants is to construct a constitutive plant expression vector containing the tomato hydroxyproline-rich systemic precursor protein gene SlHypSys and obtain transgenic plants through agrobacterium mediation.

5. The method of claim 4, wherein: the constitutive plant expression vector is used for regulating and controlling the expression of a tomato hydroxyproline-rich systemic precursor protein gene SlHypSys by a constitutive promoter.

6. The method of claim 5, wherein: the constitutive promoter, CaMV35S promoter.

7. The method of claim 5, wherein: the constitutive plant expression vector is obtained by passing a sequence shown in SEQ ID NO.1 through a BamHI and KpnI plant expression vector pLGN, and obtaining a plant expression vector pLGN-35S-SlHypSys.

8. The method according to any one of claims 3 to 7, wherein: the plant is Arabidopsis thaliana, tobacco or cotton.

9. A plant expression vector having increased resistance to verticillium wilt in plants, comprising: the plant expression vector contains an expression frame for regulating and controlling the expression of the tomato hydroxyproline-rich systemic precursor protein gene SlHypSys by a constitutive promoter, wherein the nucleotide sequence of the tomato hydroxyproline-rich systemic precursor protein gene SlHypSys is shown in SEQ ID No.1, or the nucleotide shown in SEQ ID No.1 is subjected to substitution and/or deletion and/or addition of one or more bases and has a nucleotide sequence with the same function.

10. Use of the plant expression vector of claim 9 for the preparation of transgenic plants resistant to verticillium wilt.