CN112430602B

CN112430602B - Application of Populus tomentosa HDA909 gene in improving antifungal capacity of plants

Info

Publication number: CN112430602B
Application number: CN202011409624.0A
Authority: CN
Inventors: 马旭俊; 李淑娟; 程玉祥
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2022-05-13
Anticipated expiration: 2040-12-04
Also published as: CN112430602A

Abstract

The invention discloses an application of a Populus tomentosa HDA909 gene in improving the antifungal capacity of plants, and relates to the technical field of genetic engineering. According to the invention, an HDA909 overexpression vector of the populus trichocarpa is constructed according to the HDA909 gene sequence of the populus trichocarpa, and is introduced into a receptor plant by adopting an agrobacterium-mediated method, so that the HDA909 gene in the transgenic plant is overexpressed, and an antifungal plant is obtained. The alternaria alternate infects plants, and the quantity and area of bacterial plaques of the transgenic plants are obviously reduced, and the transgenic plants are slightly infected and damaged by the alternaria alternate. The invention proves that the over-expression of the populus tomentosa HDA909 gene can improve the antifungal capability of plants, and provides a new idea for further creating antifungal transgenic plants in the future.

Description

Application of Populus tomentosa HDA909 gene in improving antifungal capacity of plants

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of a populus tomentosa HDA909 gene in improving the antifungal capacity of plants.

Background

With the development of social economy, environmental pollution is becoming more serious, and original forest tree species can meet environmental stress pressure which is greatly different from that under natural selection. In order to ensure that the living environment and the quality of life of human beings do not deteriorate any more, human beings must learn to assist forest trees to better and more quickly adapt to various environmental stress factors which are aggravated along with the modernization of the human society and come up newly. Because the growth period of the forest is long and the genetic heterozygosity is high, the requirement of directionally cultivating new species of trees with different purposes is often difficult to meet by using the conventional breeding means. In recent years, the development of biotechnology and the rise of molecular breeding research lay a foundation for improving forest trees and accelerating the breeding of new forest tree varieties by using genetic engineering means.

Pathogen infestation is an important factor contributing to large losses in plant productivity. There are 299 species of Alternaria alternata, which is not only an allergen of humans to pollens and asthma, but is more a major plant pathogen. The spores of alternaria alternate can produce a large amount of hypha when growing and propagating on the surface of the plant, and destroy the cell tissue of the plant, so that the plant is damaged.

Disclosure of Invention

The invention aims to provide application of a populus tomentosa HDA909 gene in improving the antifungal capability of plants so as to solve the problems in the prior art.

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

the invention provides application of a Populus deltoides HDA909 gene in improving the antifungal capacity of plants, wherein the gene sequence of the Populus deltoides HDA909 gene is shown as SEQ ID No.1, and the amino acid sequence coded by the Populus deltoides HDA909 gene is shown as SEQ ID No. 2.

The invention also provides a method for creating an antifungal plant, wherein an HDA909 overexpression vector is constructed according to the HDA909 gene sequence of the populus trichocarpa as claimed in claim 1, and is introduced into a receptor plant by adopting an agrobacterium-mediated method, so that the HDA909 gene in the transgenic plant is overexpressed, and the antifungal capacity is obtained.

Preferably, the recipient plant is populus trichocarpa.

Preferably, the fungus is alternaria alternata.

Preferably, the construction method of the populus trichocarpa HDA909 overexpression vector comprises the following steps:

1) taking leaf cDNA of wild type Chinese white poplar as a template, and taking HDA909-Primer-F and HDA909-Primer-R as primers to obtain a target fragment of the HDA909 gene of the Chinese white poplar;

2) connecting the obtained poplar HDA909 gene target fragment with a pMD18-T Vector by using a plasmid Mini Kit I, wherein the connection product is pMD18-HDA 909; transforming the ligation product into an escherichia coli competent cell to obtain a pMD18-HDA909 recombinant plasmid;

3) carrying out double enzyme digestion on pMD18-HDA909 recombinant plasmid and pROK II plasmid respectively by using BamHI and Kpn I, recovering the HDA909 gene fragment and pROK II vector linear fragment obtained after double enzyme digestion by using Gel Extraction Kit, connecting the recovered products by using T4 ligase to obtain pROK II-HDA 909 plasmid, namely the Populus deltoides HDA909 overexpression vector.

Preferably, the HDA909-Primer-F Primer sequence is shown in SEQ ID NO.3, and the HDA909-Primer-R Primer sequence is shown in SEQ ID NO. 4.

The invention discloses the following technical effects:

the present invention utilizes agrobacterium mediating process to transfer the HDA909 gene of Chinese white poplar into Chinese white poplar, so as to make it over-express in transgenic plant. The leaves of the 3 rd and 4 th nodes of HDA909 transgenic and non-transgenic plants are selected to carry out the in vitro fungus inoculation experiment. After 1 week of alternaria inoculation, the number of plaques and the area of plaques in the transgenic plants were significantly reduced compared to the non-transgenic plants. Dyeing affected leaves by a diaminobenzidine method, and the result shows that the number of brown red spots generated by wild plant diseased leaves is obviously more than that of transgenic plant infected leaves, and the color of the diseased spots is darker, which indicates that H accumulated in wild plant leaf cells₂O₂More H accumulated in leaf cells of transgenic plants₂O₂Less; the leaves of the wild plants are seriously damaged by the infection of alternaria alternate, and the leaves of the transgenic plants are slightly damaged by the infection of alternaria alternate. The results show that the overexpression of the HDA909 gene in the populus trichocarpa can obviously improve the resistance of the leaves of the transgenic plants to alternaria alternata. The invention proves that the over-expression of the populus tomentosa HDA909 gene can improve the antifungal capability of plants, and provides a tool for constructing antifungal plant varieties in the next step.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the result of PCR amplification of a HDA909 gene fragment, in which M is DL 2000 Marker; 1 is the result of the amplification of the HDA909 coding region fragment;

FIG. 2 shows the restriction enzyme digestion identification result of pROK II-HDA 909 recombinant plasmid, wherein M is DL 2000 Marker; 1 is a coding region fragment of populus trichocarpa HDA 909; 2 is the restriction enzyme product of pROK II-HDA 909 recombinant plasmid; 3 is pROK II plasmid enzyme digestion product;

FIG. 3 shows the result of PCR electrophoresis of pROK II-HDA 909 recombinant plasmid, in which M is DL 10000 Marker; 1-3 are detected plasmids; negative control (ddH)₂O)；

FIG. 4 is a growth diagram of HDA909 gene genetic transformation Chinese white poplar, wherein A is the growth condition of infected stem section in the differentiation culture medium of Chinese white poplar, B is the growth condition of transgenic Chinese white poplar in the rooting culture medium containing Kan, and C is the rooting condition of transgenic plant on the rooting culture medium containing Kan;

FIG. 5 shows the RT-PCR detection result of transgenic plants, in which FIG. 1 shows non-transgenic populus trichocarpa; 2 is HDA909 transgenic plant Tr-43; 3 is HDA909 transgenic plant Tr-10; 4 is HDA909 transgenic plant Tr-46; negative control (ddH) 5₂O)；

FIG. 6 shows the condition of leaf infection of hairy fruit poplar after inoculation of fungus, wherein A in the figure is leaf infection of Alternaria alternata; b is the infection rate of alternaria alternate inoculated on the hairy fruit poplar leaves; p <0.01, P < 0.0001;

FIG. 7 shows the lesion spots generated by transgenic hairy fruit poplar leaves, wherein A is the lesion spots of the diseased leaves of hairy fruit poplar; b is the lesion area of alternaria alternate inoculated on the hairy fruit poplar leaves; p < 0.0001;

FIG. 8 shows DAB staining of transgenic hairy fruit poplar diseased leaves.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

The materials and reagents used in the embodiments of the invention are commercially available.

Example 1

Cloning of Populus mauritiana HDA909 gene

1. Experimental Material

The plant material is wild type Chinese white poplar (Populus trichocarpa) aseptic tissue culture seedling, the strains and the carriers are shown in table 1, and the main reagents are shown in table 2.

TABLE 1 strains and vectors

TABLE 2 major reagents

2. Solution preparation

(1) 0.1% aqueous DEPC: mixing ultrapure water and DEPC stock solution at a ratio of 9:1, stirring for 2 hr, and sterilizing for 20 min.

(2)5M NaCl solution: 14.625g of NaCl was dissolved in 45mL of 0.1% DEPC water, the volume was adjusted to 50mL, the mixture was incubated at 37 ℃ for 12 hours, and the mixture was sterilized for 20 min.

(3) 75% ethanol solution: mixing 0.1% DEPC water and anhydrous ethanol at a ratio of 1:3, sterilizing at high temperature, cooling to room temperature, and storing at-20 deg.C.

3. Experimental methods

3.1 extraction of Total RNA from Populus tomentosa

(1) The hairy fruit poplar leaves were ground to powder in liquid nitrogen in a pre-cooled mortar.

(2) Approximately 0.1g of the sample powder was mixed well with the pBIOZOL reagent pre-cooled at 4 ℃ and then left for 5 min.

(3) Centrifugation at 12000 Xg for 2min, 400. mu.L of supernatant was added to a new RNase-free centrifuge tube.

(4) Adding 100 μ L of 5M NaCl solution, mixing by inversion, adding 300 μ L of chloroform, mixing, and centrifuging at 12000 Xg for 10min at 4 deg.C.

(5) Adding the supernatant into a new RNase-free centrifuge tube, adding equal volume of isopropanol, mixing well, standing at 25 deg.C for 10min, and centrifuging at 12000 Xg at 4 deg.C for 10 min.

(6) The supernatant was discarded (without losing the white precipitate), 800. mu.L of 75% ethanol was added to the precipitate, centrifuged at 12000 Xg for 8min at 4 ℃ and the supernatant discarded, and the residual liquid was air-dried.

(7) To the RNA pellet, 30. mu.L of sterile 0.1% DEPC water was added, ice-cooled for 0.5h, and stored at-80 ℃.

3.2 Synthesis of Populus tomentosa cDNA

The RT reagent Kit is used for synthesizing cDNA from the total RNA of the hairy fruit poplar leaves, and the reaction system and the conditions are shown in tables 3 and 4.

TABLE 3 removal of genomic DNA reaction

Reaction conditions are as follows: 42 ℃ for 10 min.

TABLE 4 reverse transcription reaction

Reaction conditions are as follows: 60min at 42 ℃; storing at 85 deg.C for 5min and 4 deg.C.

3.3 cloning of Populus tomentosa HDA909 Gene

3.3.1 PCR amplification of fragments of interest

The cDNA of the hairy fruit poplar leaf is used as a template, the full-length coding sequence of the HDA909 gene is amplified by utilizing the specific primer of the HDA909 gene, and the reaction system and the reaction conditions are shown in tables 5 and 6.

TABLE 5PCR amplification reaction System

TABLE 6PCR amplification reaction procedure

3.3.2 recovery and purification of the fragment of interest

And (3) carrying out PCR product electrophoresis, and purifying and recovering the gene fragment by using a gel recovery kit.

3.3.3 ligation of the HDA909 Gene fragment to the pMD18-T Vector

The HDA909 gene fragment was ligated with the pMD18-T Vector in the system shown in Table 7.

TABLE 7pMD18-T Vector ligation System

Connection conditions are as follows: the temperature was kept constant at 16 ℃ overnight. The ligation product was pMD18-HDA 909.

3.3.4 transformation of competent cells with ligation products and characterization

The ligation product was transformed into DH 5. alpha. and applied evenly to solid LB medium containing Amp (25mg/L) and cultured in an inverted state at 37 ℃ for 16 hours. The white single colony was inoculated into liquid LB medium containing Amp (25mg/L) and subjected to scale-up culture at 37 ℃ and 200 rpm/min. PCR identification is carried out by taking the bacterial liquid as a template (the reaction system and conditions are shown in tables 8 and 9), and the bacterial liquid with the correct identification result is sent to Beijing Liuhe Huada Gene science and technology Co., Ltd for sequencing.

TABLE 8 bacterial liquid PCR reaction system

TABLE 9 PCR reaction procedure for bacterial solutions

4. Results of the experiment

The invention utilizes the HDA909 specific primer to clone the coding region fragment of the HDA909 gene from the populus trichocarpa, and the length of the nucleotide sequence is 1323bp after sequencing. The PCR result is shown in figure 1, the ORF sequence length of the populus trichocarpa HDA909 gene is 1323bp, and is shown in SEQ ID NO. 1; the protein coded by the HDA909 gene consists of 440 amino acids and is shown as SEQ ID NO. 2.

Example 2

HDA909 gene genetic transformation Chinese hairy fruit poplar and functional analysis thereof

1. Experimental Material

Wild type populus tomentosa tissue culture seedlings. Alternaria alternata (Alternaria alternata) belongs to the subdivision Deuteromycotina, conidiophores of the Alternaria alternata belong to brown or brownish inverted-bar-shaped conidiophores, are mostly single-grown or multi-root fasciculate, and are mostly 3-6 intercrossed and have 1-2 longitudinal diaphragms and 3-4 transverse diaphragms. Purchased from the institute of forest ecological environment and protection, China department of forestry. The pROK II vector was purchased commercially. The main reagents are shown in table 10.

TABLE 10 major reagents

Hairy fruit poplar rooting culture medium: WPM culture medium 2.41g/L, sucrose 20g/L, agar powder 5.8g/L, pH 5.75, and IBA 0.1 mg/L.

Hairy fruit poplar differentiation medium: WPM medium 2.41g/L, sucrose 20g/L, agar powder 5.8g/L, pH 5.75, 6-BA 0.3mg/L, IBA 0.02mg/L and TDZ 0.0008 mg/L.

2. Experimental methods

2.1 construction of plant overexpression vectors

2.1.1 cleavage and ligation of pMD18-HDA909 and pROK II vectors

The plasmid pMD18-HDA909 recombinant plasmid and pROK II plasmid were prepared using the plasmid Mini Kit I. The above two plasmids were digested separately with BamH I and Kpn I, and the reaction system is shown in Table 11.

TABLE 11 double restriction reaction System

Reaction conditions are as follows: the enzyme was cleaved at 37 ℃ for 2.5 h.

The HDA909 gene fragment obtained after the double digestion and the pROK II vector linear fragment were cut with a clean blade, recovered with Gel Extraction Kit, and the recovered products were ligated with T4 ligase, and the ligation system is shown in Table 12.

TABLE 12 ligation reaction System

Connection conditions are as follows: the metal bath was thermostatted at 16 ℃ overnight.

2.1.2 transformation of E.coli with ligation products and characterization

The ligation product of pROK II-HDA 909 was transformed into DH5 alpha, cultured on a selection medium (containing Kan 50mg/L) for 16h, and several resistant single colonies were randomly picked and placed in 5mL LB liquid medium (containing Kan 50mg/L) for expansion and shaking. And carrying out PCR amplification by using the bacterial liquid as a template.

2.1.3 plasmid extraction and restriction enzyme identification of pROK II-HDA 909

The recombinant Plasmid was prepared using a Plasmid Mini Kit I Kit. The plasmid pROK II-HDA 909 was identified by double digestion with restriction endonucleases BamHI and KpnI, and the reaction system is shown in Table 13.

TABLE 13 double digestion reaction System

Reaction conditions are as follows: the enzyme was digested at 37 ℃ for 2.5 h.

2.1.4 transformation of Agrobacterium with pROK II-HDA 909 plasmid and identification

The pROK II-HDA 909 plasmid was transformed into Agrobacterium EHA105 and cultured on selection medium for 2 d. Resistant single colonies were randomly picked, placed in LB liquid medium (containing Kan 50mg/L), and cultured overnight at 28 ℃ at 180 rpm/min. PCR was performed using the bacterial solution as a template, and the reaction system and reaction conditions are shown in tables 14 and 15. And extracting pROK II-HDA 909 plasmid from the agrobacterium liquid by using a plasmid extraction kit. The pROK II-HDA 909 plasmid was transformed into DH 5. alpha. and identified by double digestion with BamHI and KpnI, as shown in Table 16. The identified positive agrobacterium liquid is mixed with 40 percent of glycerol in equal proportion, and the mixture is stored at the temperature of minus 80 ℃ and is used for genetic transformation experiments of populus tomentosa.

TABLE 14 PCR reaction system for bacterial liquid

TABLE 15 PCR reaction procedure for bacterial solutions

TABLE 16 double digestion reaction System

Reaction conditions are as follows: the enzyme was cleaved at 37 ℃ for 2.5 h.

2.2 cultivation of Populus tomentosa

Cutting fresh and tender stem segments without bud points of three-week-old hairy fruit poplar in a super-clean workbench, wherein the stem segments are about 0.8cm long, placing on a hairy fruit poplar differentiation culture medium, and carrying out stem segment differentiation in an environment with a photoperiod of 16h illumination/8 h darkness at 25 ℃. And cutting the buds into a hairy fruit poplar rooting culture medium when the green buds are differentiated from the stem segments and grow to be about 0.8-1 cm high, cutting the single plant buds into the hairy fruit poplar rooting culture medium for culture when the buds grow to be about 1.5cm, and using the stems of the grown plants after 3 weeks for genetic transformation of the hairy fruit poplar.

2.3 genetic transformation of Populus tomentosa

Taking the stem section of the fresh populus tomentosa tissue culture seedling with good light green state and about 3 weeks old, and utilizing an agrobacterium-mediated method to genetically transform the populus tomentosa.

(1) Under aseptic conditions, the agrobacterium liquid carrying the HDA909 gene is dipped and taken by an inoculating loop which is burned by an alcohol lamp outer flame and cooled to room temperature, and inverted and cultured for 2d on a solid LB culture medium containing Kan (50mg/L) and Rif (250mg/L) by adopting a three-region marking method, wherein the culture condition is constant temperature of 28 ℃.

(2) White circular colonies that grew singly on the wire and had a diameter of about 1-2mm were inoculated in 5mL of liquid LB medium (containing 50mg/L Kan and 250mg/L Rif) and cultured at a constant temperature of 28 ℃ at 220rpm for about 16 hours.

(3) Adding 5mL of cultured bacterial liquid into 30mL of liquid LB culture medium, and culturing at constant temperature of 28 deg.C and 220rpm/min for about 4 hr, at which time the bacterial liquid OD₆₀₀The value is preferably 0.6 to 0.8.

(4) Centrifuging at 4 deg.C and 4000 rpm for 10min, pouring out supernatant, and diluting the residual thallus with sterile water to OD₆₀₀The value was 0.4 and the resuspension was used as the staining solution.

(5) Taking fresh and tender green tissue culture seedlings of populus tomentosa of about 3 weeks old, cutting off leaves of the plants by using scissors which are sterilized by a high-temperature sterilizer and cooled to room temperature, and reserving a part of about 0.8cm of petiole connected with the leaves; the plant stem was cut out, placed in a petri dish with forceps, and cut into stem segments (containing no axillary bud parts) of about 0.8cm in length with an operating blade.

(6) Placing petiole and stem segment with a small part of leaf into prepared staining solution with tweezers for 10min for infection, and gently shaking the staining solution from time to time during the infection process to make it contact with the wound of stem segment more fully. Taking out the stem segments soaked in the infection solution by using tweezers, placing the stem segments on a filter paper which is prepared in advance and sterilized at high temperature for absorbing and removing redundant bacteria liquid on the surface of the plant stem segments, keeping the stem segments moist, placing the stem segments on a differentiation culture medium by using sterile tweezers, and culturing the stem segments in the dark at the temperature of 25 ℃ for 2 days.

(7) After dark culture, placing the infected stem segments on a populus tomentosa differentiation and screening culture medium by using forceps in an aseptic environment, and continuously culturing in an environment with a photoperiod of 16 h/8 h at 25 ℃.

2.4 selection of resistance to transgenic Populus tomentosa

The stem segments with tender resistant buds growing on the hairy poplar differentiation culture medium (containing Kan 40mg/L) are placed into a hairy poplar rooting culture medium (containing Kan 40mg/L) for continuous screening, then fresh, tender and green and single seedlings are cut off by a sterile knife, and the seedlings are inserted into the hairy poplar rooting screening culture medium containing Kan by using sterile tweezers for rooting. When the plant grows to about 5cm, cutting plant leaves in a sterile environment, quickly freezing, and storing at-80 ℃ as an experimental material for subsequent molecular detection.

2.5 molecular detection of transgenic Populus tomentosa

RNA was extracted from leaves of kanamycin-resistant transgenic plants and cDNA was synthesized according to the experimental method of example 1. Quantitative RT-PCR was carried out using the above cDNA as a template, and detection was carried out using HDA909 gene-specific primers, and the reaction system and reaction conditions are shown in tables 17 and 18.

TABLE 17 PCR reaction system for bacterial liquid

TABLE 18 PCR reaction procedure for bacterial liquid

2.6 analysis of resistance to transgenic hairy fruit Populus

HDA909 transgenic Populus tomentosa was analyzed for resistance to Alternaria alternata. In the experiment of inoculating alternaria alternata, the 3 rd node of 2.5-month-old hairy fruit poplar seedling with consistent shape, size and growth state of HDA909 transgene and non-transgene is selectedOr the blade of node 4. Before treatment, the front and back surfaces of the blade were sprayed and cleaned 3 times with sterilized ultrapure water. In a sterile environment, 10mL of sterile water was pipetted to wet the double layer sterile filter paper. 20mL of sterile water was added to the cultured Alternaria alternata plates and scraped repeatedly with a sterile glass rod several times to dissolve a large amount of hyphae and spores in the sterile water. And sequentially spreading 300-mesh, 400-mesh and 500-mesh filter screens in a sterile funnel from top to bottom for filtering. When the concentration of spores in the spore suspension was calculated to 2X 10 using a hemocytometer⁷When the strain is one/mL, the strain can be used for infection. The leaves which are washed 3 times by clean water and dried to remove surface moisture are put into a culture dish, wet filter paper which is well paved is arranged below the leaves, 2 drops of 10 mu L spore suspension are respectively dropped on two sides of the main vein, and 2 drops of sterile water are respectively dropped on two sides of the main vein of the control group. And culturing the infected leaves at 25 ℃ in a 16h light/8 h dark environment.

2.7 dyeing with Diaminobenzidine (DAB)

The leaves of the Populus tomentosa inoculated with Alternaria alternata 7d were stained overnight in 1mg/mL (pH3.8) of DAB solution, decolorized with ethanol the next day, permeabilized in 10% chloral hydrate solution, and the leaves were observed under an optical microscope.

3. Results of the experiment

3.1 construction of HDA909 overexpression vector

The specific primer of the populus trichocarpa HDA909 gene is utilized to obtain the coding region segment of the HDA909 gene from the populus trichocarpa by a PCR method. And the pMD18-T vector is connected with the PCR product and sequenced, thereby obtaining the pMD18-HDA909 intermediate vector. The pMD18-HDA909 intermediate vector and the pROK II vector were subjected to double digestion with BamHI and KpnI, respectively, and the two digestion products were ligated to obtain an overexpression vector of pROK II-HDA 909. And transforming the ligation product into escherichia coli, extracting a recombinant plasmid, and performing double enzyme digestion identification on the recombinant plasmid by using BamH I and Kpn I. The digestion result shows that the size of the digestion fragment is consistent with that of the HDA909 gene fragment, and the successful construction of the pROK II-HDA 909 overexpression vector is proved (figure 2).

The constructed pROK II-HDA 909 recombinant plasmid was transformed into EHA105, and the plasmid was prepared. The primers specific to the HDA909 gene were used, and the transformed bacterial suspension was used as a template for PCR. The electrophoresis result shows that a clear amplification band is formed at 1323bp (figure 3), which indicates that pROK II-HDA 909 is successfully transferred into EHA105 and can be used for genetic transformation of plants.

3.2 obtaining and molecular identification of transgenic Chinese hairy poplar

The HDA909 gene was genetically transformed into populus trichocarpa by Agrobacterium-mediated method. The hairy fruit poplar stem segment was infected with agrobacterium carrying HDA909 gene and then placed in differentiation selection medium (containing Kan) where the cut at both ends of the stem segment could differentiate light green shoots at about 3 weeks (fig. 4A). Transplanting the young sprout into rooting selection culture medium (containing Kan) to continue culturing, and inserting young sprout into new rooting culture medium (containing Kan) to continue culturing when the young sprout grows to single young sprout with plant height of about 3 cm. The HDA909 transgenic plants grew well on the rooting medium of Chinese white poplar (containing Kan) and could normally root (FIGS. 4B and 4C), while the non-transgenic plants could not root normally.

Extracting total RNA of wild type and transgenic hairy fruit poplar leaves, and performing reverse transcription to obtain cDNA serving as a template of RT-PCR; RT-PCR identification was performed using HDA 909-specific primers. The results show that the HDA909 gene is capable of being transcribed in transgenic plants (fig. 5).

3.3 function of HDA909 Gene in fungal stress response

Leaf blight seriously threatens the normal growth of poplar, and as a common disease, the pathogen is alternaria alternate. The paper studies the role of populus trichocarpa HDA909 gene in alternaria leaf infection. And (3) selecting the leaves of the 3 rd node or the 4 th node of the HDA909 transgenic and non-transgenic 2.5-month-old hairy fruit poplar seedlings with consistent shapes, sizes and growth states to perform an alternaria alternate inoculation experiment, and analyzing the resistance of the HDA909 transgenic plants to alternaria alternate. At 1 week after inoculation with Alternaria alternata, leaves of transgenic plants and leaves of non-transgenic plants were attacked to different degrees, however, the resistance of HDA909 transgenic plants to Alternaria alternata was significantly higher than that of wild type plants (FIG. 6A). At 7 days after inoculation with Alternaria alternata, the percentage of plaques produced by the leaves of the wild-type plants was 72.5%, whereas the percentage of plaques produced by the leaves of the transgenic lines Tr-43, Tr-10, Tr-46 was 0%, 25% and 17.5%, respectively (FIG. 6B). The lesion area on the non-transgenic plant leaf is larger than that on the transgenic plant leaf (FIG. 7A), and is 69.6 times, 6.5 times and 9.5 times of the lesion area on the transgenic lines Tr-43, Tr-10 and Tr-46 respectively (FIG. 7B).

Hydrogen peroxide, an important active oxygen, widely affects many vital activities. Pathogen infestation can cause intracellular accumulation of large amounts of H₂O₂. DAB can generate H₂O₂Of and H₂O₂The reaction takes place, producing a brownish red spot. Therefore, by using the DAB dyeing method, the damage degree of plants infected by fungi can be detected, and the more damaged the plants are, the more reddish brown spots are generated, and the darker the color is. When DAB staining of diseased leaves was observed under a 4-fold optical microscope, the transgenic plants had significantly fewer brown-red spots and lighter colors than wild-type plants (FIG. 8). Therefore, the leaves of the HDA909 transgenic plants are less susceptible to Alternaria alternata infection than non-transgenic plants, and the infected leaves are also relatively less damaged. In conclusion, the overexpression of the HDA909 gene in populus trichocarpa obviously improves the resistance of transgenic plants to fungal stress.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

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acggggcaca tcaaggacat tggagttggg aaagggaaga actatgccct gaatattcct 720

ttaaaagatg ggatggatga cgaatgtttc cgtgctctgt ttcggccact catccaaaaa 780

gtgatggagg tttatcaacc tgatgcagtt gttctccaat gtggagcaga ttcattatct 840

ggtgataggt tggggtgttt caacctgtct gttaagggcc atgcagactg ccttagattt 900

attagatctt ttaatgttcc tctgatgatc ttgggtgggg gtgggtatac tatcaggaat 960

gttgcccgtt gctggtgcta tgagacagca gttgcagttg gggtggaacc agataacaaa 1020

ttgccttaca atgagtacta cgagtacttt ggccctgaat acacacttca tgctgaccca 1080

tccaatatgg agaacctaaa cacacccaaa gacatggaga gaataaggaa catactgcta 1140

gagcaacttt ctagactgcc taatgctccc agtgtacctt ttcagacaac accagctaca 1200

actgaagttc cggaagagga tgaagagaac atggatcaaa gaccaaagcg tcatgtatgg 1260

aatggtgttg attatgagtc tgatcatgat gaagatgaga aaccggaacc cggatttttc 1320

tga 1323

<210> 2

<211> 440

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Leu Asp Thr Asn Gly Gly Ala Ser Leu Pro Ser Thr Gly Thr Asp

1 5 10 15

Ala Glu Lys Arg Arg Val Ser Tyr Phe Tyr Glu Pro Thr Ile Gly Glu

20 25 30

Tyr Tyr Tyr Gly Gln Gly His Ser Met Lys Pro His Arg Ile Arg Met

35 40 45

Ala His Asn Leu Ile Ile Asn Tyr Ala Leu His Arg Arg Met Glu Ile

50 55 60

Asn Arg Pro Phe Pro Ala Gly Pro Glu Asp Ile Gly Trp Phe His Ser

65 70 75 80

Asp Asp Tyr Val Glu Phe Leu Ser Ser Val Ser Pro Gln Ser Ala Ser

85 90 95

Asp Pro Ala His Gly Arg Gln Leu Lys Arg Phe Asn Ile Gly Glu Asp

100 105 110

Cys Pro Val Phe Tyr Gly Leu Phe Glu Phe Cys Gln Ala Ser Ala Gly

115 120 125

Gly Ser Ile Gly Cys Ala Val Lys Leu Asn Arg Gly Asp Ala Asp Ile

130 135 140

Ala Leu Asn Trp Ala Gly Gly Leu His His Ala Lys Arg Ser Glu Ala

145 150 155 160

Ser Gly Phe Cys Tyr Val Asn Asp Ile Val Leu Gly Ile Leu Glu Leu

165 170 175

Leu Lys Val His Lys Arg Val Leu Tyr Val Asp Ile Asp Val His His

180 185 190

Gly Asp Gly Val Glu Glu Ala Phe Tyr Thr Thr Asp Arg Val Met Thr

195 200 205

Val Ser Phe His Lys Tyr Gly Asp Phe Phe Pro Gly Thr Gly His Ile

210 215 220

Lys Asp Ile Gly Val Gly Lys Gly Lys Asn Tyr Ala Leu Asn Ile Pro

225 230 235 240

Leu Lys Asp Gly Met Asp Asp Glu Cys Phe Arg Ala Leu Phe Arg Pro

245 250 255

Leu Ile Gln Lys Val Met Glu Val Tyr Gln Pro Asp Ala Val Val Leu

260 265 270

Gln Cys Gly Ala Asp Ser Leu Ser Gly Asp Arg Leu Gly Cys Phe Asn

275 280 285

Leu Ser Val Lys Gly His Ala Asp Cys Leu Arg Phe Ile Arg Ser Phe

290 295 300

Asn Val Pro Leu Met Ile Leu Gly Gly Gly Gly Tyr Thr Ile Arg Asn

305 310 315 320

Val Ala Arg Cys Trp Cys Tyr Glu Thr Ala Val Ala Val Gly Val Glu

325 330 335

Pro Asp Asn Lys Leu Pro Tyr Asn Glu Tyr Tyr Glu Tyr Phe Gly Pro

340 345 350

Glu Tyr Thr Leu His Ala Asp Pro Ser Asn Met Glu Asn Leu Asn Thr

355 360 365

Pro Lys Asp Met Glu Arg Ile Arg Asn Ile Leu Leu Glu Gln Leu Ser

370 375 380

Arg Leu Pro Asn Ala Pro Ser Val Pro Phe Gln Thr Thr Pro Ala Thr

385 390 395 400

Thr Glu Val Pro Glu Glu Asp Glu Glu Asn Met Asp Gln Arg Pro Lys

405 410 415

Arg His Val Trp Asn Gly Val Asp Tyr Glu Ser Asp His Asp Glu Asp

420 425 430

Glu Lys Pro Glu Pro Gly Phe Phe

435 440

<210> 3

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgggatccat gctggacact aatggagg 28

<210> 4

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggggtacctc agaaaaatcc gggttccggt 30

Claims

1. Populus mauritiana HDA909 gene for improving alternaria alternata (A) resistance of plantsAlternar alternata) The application of the gene is characterized in that the gene sequence of the populus trichocarpa HDA909 is shown in SEQ ID NO.1, and the amino acid sequence coded by the populus trichocarpa HDA909 gene is shown in SEQ ID NO. 2.

2. A method for creating a plant resistant to Alternaria alternata is characterized in that an Alternaria alternata HDA909 overexpression vector is constructed according to the gene sequence of the Alternaria solanta HDA909 in the claim 1, and the gene sequence is introduced into a receptor plant by adopting an agrobacterium-mediated method, so that the HDA909 gene in the transgenic plant is overexpressed, and the Alternaria alternata resistance is obtained.

3. The method of claim 2, wherein the recipient plant is Populus tomentosa.

4. The method for creating a Alternaria alternata plant according to claim 2, wherein the method for constructing the Alternaria solani HDA909 overexpression vector comprises the following steps:

2) connecting the obtained target fragment of the HDA909 gene of the populus trichocarpa with a pMD18-T Vector, wherein the connection product is pMD18-HDA 909; transforming the ligation product into an escherichia coli competent cell to obtain a pMD18-HDA909 recombinant plasmid;

3) carrying out double enzyme digestion on pMD18-HDA909 recombinant plasmid and pROK II plasmid respectively by using BamHI and Kpn I, recovering the HDA909 gene fragment and pROK II vector linear fragment obtained after double enzyme digestion by using Gel Extraction Kit, and connecting the recovered products by using T4 ligase to obtain pROK II-HDA 909 plasmid, namely the Populus mauritianus HDA909 overexpression vector.

5. The method for creating an Alternaria alternata-resistant plant according to claim 4, wherein the HDA909-Primer-F Primer sequence is shown as SEQ ID No.3, and the HDA909-Primer-R Primer sequence is shown as SEQ ID No. 4.