CN116064566A

CN116064566A - Poplar bark rot-resistant bacterium gene PtoCXE06 and application thereof

Info

Publication number: CN116064566A
Application number: CN202210837131.XA
Authority: CN
Inventors: 韩学敏; 曾庆银; 刘妍婧; 金宇霆; 王丹
Original assignee: CHINESE ACADEMY OF FORESTRY
Current assignee: CHINESE ACADEMY OF FORESTRY
Priority date: 2022-07-15
Filing date: 2022-07-15
Publication date: 2023-05-05

Abstract

The invention provides a gene PtoCXE06 of poplar anti-skin rot germ and application thereof, and relates to the technical field of molecular breeding. The invention provides a gene PtoCXE06 of poplar bark rot bacteria, which can obviously improve the resistance of poplar bark rot bacteria by over-expressing PtoCXE06 gene in poplar. Experiments prove that the PtoCXE06 gene is excessively expressed in the populus tomentosa, a novel poplar disease-resistant germplasm material with obviously enhanced resistance to the poplar bark rot germ can be created, and a foundation is laid for the cultivation of Yang Shuxin varieties.

Description

Poplar bark rot-resistant bacterium gene PtoCXE06 and application thereof

Technical Field

The invention relates to the technical field of molecular breeding, in particular to a gene PtoCXE06 of poplar anti-skin rot germ and application thereof.

Background

Poplar (Populus spp.) is an important ecological and economic tree species, playing an important role in national economic construction and ecological environment construction. However, the occurrence and hazard of poplar diseases, especially fungal diseases, are increasingly serious, which greatly threatens the further development of poplar artificial forests in China and also has great influence on forest resources and environmental safety in the world. Therefore, the creation of new poplar disease-resistant germplasm materials has important significance for cultivating new varieties of forest trees which meet the development requirements of national economy.

Based on traditional methods such as crossbreeding, mutation breeding and the like, new germplasm materials for poplar disease resistance are created, the defects of low efficiency, difficulty in controlling mutation inducing direction and the like are overcome, and the new germplasm materials with obviously changed characters are difficult to obtain in the perennial woody plant poplar.

Disclosure of Invention

Therefore, the invention aims to provide the gene PtoCXE06 of the poplar skin rot resistance bacteria, and the PtoCXE06 gene is overexpressed in the poplar, so that the resistance of the poplar to the poplar skin rot resistance bacteria can be obviously improved.

In order to solve the technical problems, the invention provides the following technical scheme:

the invention provides a gene PtoCXE06 of poplar anti-skin rot germ, wherein the nucleotide sequence of the gene PtoCXE06 is shown as SEQ ID NO. 1.

The invention provides a poplar skin rot resistance germ protein, the amino acid sequence of which is shown as SEQ ID NO. 2.

The invention provides an over-expression vector comprising the gene PtoCXE06, wherein the basic vector of the over-expression vector is a plant binary expression vector delta pCAMBIA1302.

The invention also provides application of the gene PtoCXE06, the protein or the over-expression vector in creating new poplar disease-resistant germplasm materials.

The invention also provides application of the gene PtoCXE06, the protein or the over-expression vector in enhancing the resistance of poplar to the poplar skin rot germ.

Preferably, the resistance of poplar to the species Populus rotten skin bacteria is enhanced by overexpressing the gene PtoCXE06 in the poplar.

The invention also provides a method for improving the resistance of poplar to the poplar skin rot germ, which comprises the following steps:

constructing a gene PtoCXE06 into a plant binary expression vector delta pCAMBIA1302 to obtain an excessive expression vector PtoCXE06-OE; the over-expression vector PtoCXE06-OE is transformed into poplar, so that the gene PtoCXE06 is over-expressed, and the resistance of poplar to the poplar skin rot germ can be improved.

Preferably, the gene PtoCXE06 is extracted and amplified from root, stem and leaf tissues of populus tomentosa.

Preferably, the poplar of the transgenic PtoCXE06 can be screened and cultivated to obtain the new poplar disease-resistant germplasm material with enhanced resistance to the poplar skin rot germ.

Preferably, the screen is a hygromycin screen.

The invention has the beneficial effects that:

the invention provides a gene PtoCXE06 of poplar bark rot bacteria, which can obviously improve the resistance of poplar bark rot bacteria by over-expressing PtoCXE06 gene in poplar. Experiments prove that the PtoCXE06 gene is excessively expressed in the populus tomentosa, a novel poplar disease-resistant germplasm material with obviously enhanced resistance to the poplar bark rot germ can be created, and a foundation is laid for the cultivation of Yang Shuxin varieties.

Drawings

FIG. 1 shows the expression pattern of PtoCXE06 under normal conditions and stress treatment conditions.

FIG. 2 shows the construction scheme of PtoCXE06 over-expression vector.

FIG. 3 shows the result of PCR detection of the DNA level of transgenic plants, wherein M is marker (DL 2000), WT is wild 741A. Rubra, PC is pCAMBIA1302-PtoCXE6 plasmid, BC is ddH ₂ O,1-43 are PtoCXE6-OE and 44/45 are the non-loaded groups.

FIG. 4 shows the RT-qPCR detection of mRNA levels of transgenic plants.

FIG. 5 is a comparison of infection rates of control plants and PtoCXE06 overexpressing plants.

Detailed Description

In the invention, the plant binary expression vector delta pCAMBIA1302 is a binary plant expression vector transformed from the traditional plant expression vector pCAMBIA1302. The invention selects the plant binary expression vector delta pCAMBIA1302 as a basic vector to avoid the influence of GFP labels contained in the pCAMBIA1302 vector on the activity of target gene coding proteins.

The invention also provides application of the gene PtoCXE06, the protein or the over-expression vector in enhancing the resistance of poplar to the poplar skin rot germ. In the invention, the resistance of poplar to the poplar skin rot germ is enhanced by over-expressing a gene PtoCXE06 in poplar.

In the invention, the gene PtoCXE06 is preferably extracted and amplified from root, stem and leaf tissues of populus tomentosa; the extraction and amplification preferably comprise the following steps: extracting total RNA of root, stem and leaf tissues of populus tomentosa respectively, mixing the total RNA with the same amount, and performing reverse transcription to obtain cDNA; the PtoCXE06 gene can be obtained by using the cDNA as a template and using a primer for amplification. In the present invention, the transformation is preferably an Agrobacterium tumefaciens-mediated transformation method. In the invention, the poplar of the transgenic PtoCXE06 can be screened and cultivated to obtain a new poplar disease-resistant germplasm material with enhanced resistance to the poplar skin rot germ; the selection is preferably a hygromycin selection, the purpose of which is to obtain transgenic positive plants.

The present invention will be described in detail below with reference to examples for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, but they should not be construed as limiting the scope of the present invention.

In the following examples, conventional methods are used unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1

Analysis of PtoCXE06 expression Pattern

Transcriptome data for wild-type and biological and non-biological treatments of populus tomentosa were downloaded from the GEO database (Gene Expression Omnibus, https:// www.ncbi.nlm.nih.gov/GEO /) under accession GSE109609. Through data analysis, TPM values of the PtCXE06 genes of the populus tomentosa under normal conditions (WT) and different processing conditions are obtained through screening. Wherein the different processes include: salicylic Acid (SA), methyl jasmonate (MeJA), poplar black spot germ (Marssonina brunnea, MB), cold (cold), salt (SN), and mechanical damage (Mechanical wounding, WD). A PtoCXE06 gene expression pattern heat map was generated using log2 (TPM+1) values, and the results are shown in Table 1 and FIG. 1.

TABLE 1 expression of PtoCXE06 under normal conditions and stress treatment conditions

Group of	WT	SA	MeJA	MB	cold	SN	WD
								PtoCXE06 gene	0.197	1.833	3.308	1.513	0.391	1.947	0.837

It can be seen that PtoCXE06 has lower expression under normal growth conditions, and the expression level of PtoCXE06 is obviously increased after stress treatment, especially the highest expression level under the condition of methyl jasmonate treatment, which indicates that PtoCXE06 may play an important role in poplar stress response.

Example 2

Cloning of PtoCXE06 coding sequence

Extracting total RNA of root, stem and leaf tissue of populus tomentosa respectively by using RNAprep Pure Plant Kit (Tiangen, beijing), mixing the total RNA of each tissue in equal quantity, and reversely transcribing the RNA mixed in equal quantity into cDNA by using PrimeScriptTM RT reagent Kit (TakaRa);

the PtoCXE06 gene was amplified using the above cDNA as a template and specific primers. Wherein the upstream primer is 5'-CATACTGTTCTTCATCTCCCTTAG-3' (SEQ ID NO. 3) and the downstream primer is 5'-GCCAAAGTTCATTTCAGTCC-3' (SEQ ID NO. 4). The amplified product is recovered by using a DNA rapid recovery/purification kit (Beijing Ding Guo Changsheng biotechnology Co., ltd.); the recovered DNA fragment is connected with pEASY-Blunt vector (full gold, beijing), and the connection system and reaction conditions are as follows: PCR product, 4. Mu.l, pEASY-Blunt vector 1. Mu.l; the reaction was carried out at room temperature for 5min. The ligation product was then transformed into E.coli DH10b. Through resistance screening, selecting monoclonal, sending to Beijing qingke biotechnology Co, sequencing, obtaining PtoCXE06 gene sequence.

Example 3

PtoCXE06 over-expression vector construction

The PtoCXE06 gene sequence obtained in the example 2 is constructed into a plant binary expression vector delta pCAMBIA1302, and the specific flow is shown in figure 2;

the SalI and PmlI are utilized to enzyme-cut the delta pCAMBIA1302 vector and the cloning vector containing PtoCXE06, the enzyme-cut PtoCXE06 gene fragment is connected to the enzyme-cut delta pCAMBIA1302 vector through T4DNA ligase, and finally the over-expression vector PtoCXE06-OE is obtained. All restriction enzymes were purchased from NEB and were operated according to the instructions for use.

Example 4

1. Obtaining a plurality of poplar candidate transgenic plant lines which overexpress PtoCXE06 through an agrobacterium tumefaciens mediated transformation method, detecting candidate plants, and screening transgenic positive plants, wherein the specific screening steps are as follows:

1) Extracting genomic DNA of candidate transgenic plants by adopting DNAsecure Plant Kit (Tiangen, beijing), wherein the extraction steps are carried out according to the instruction of the kit;

2) And (3) taking the extracted genomic DNA of the candidate transgenic plant as a template, and amplifying an HPT marker gene by PCR to detect whether the T-DNA segment carrying the PtoCXE06 gene is integrated on the poplar genome. Wherein the upstream primer is 5'-TGCTCCATACAAGCCAACC-3' (SEQ ID NO. 5) and the downstream primer is 5'-TGTCCTGCGGGTAAATAGC-3' (SEQ ID NO. 6). The amplification system of the PCR is shown in Table 2;

TABLE 2 amplification System

Reagent(s)	Volume (mul)
		10×ExTaq Reaction Buffer	2.0
dNTP(2.5mM)	2.0
		Forward Primer(2.0mM)	2.0
Reverse Primer(2.0mM)	2.0
		Template (genomic DNA or cDNA)	1.0
Ex Taq polymerase(5U/ul)	0.1
		ddH ₂ O	10.9

Amplification procedure: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30sec, annealing at 55-58℃for 30sec, elongation at 72℃for 1.5min,35 cycles; extending at 72 ℃ for 10min;15℃for 30sec.

The results of PCR amplification of the HPT marker gene are shown in FIG. 3. It can be seen that the T-DNA segment carrying the PtoCXE06 gene is successfully integrated into the poplar genome.

2. RNA was extracted from control wild-type plants and transgenic plants, respectively, and reverse transcribed into cDNA using PrimeScriptTM RT reagent Kit (Perfect Real Time) (TakaRa) with the reverse transcription system shown in Table 3 below;

TABLE 3 reverse transcription system

Reagent(s)	Volume of
		5×PrimeScript Buffer(for Real Time)	2μl
PrimeScript RT Enzyme Mix I	0.5μl
		Oligo dT Primer(50μM)	0.5μl
Random 6 mers(100μM)	0.5μl
		Total RNA	400ng
Rnase Free ddH ₂ O	Supplement to 10. Mu.l

Using cDNA synthesized by reverse transcription as a template, using

Premix Ex Taq ^TM II (Tli RNaseH Plus) RT-qPCR detection was performed to detect the transcript level of PtoCXE06 in poplar transgenic plants. The detection primers for RT-qPCR are shown in Table 4; the reaction system of RT-qPCR is shown in Table 5.

Table 4 primer information for PtoCXE06 RT-qPCR detection

TABLE 5 RT-qPCR reaction System

Reagent(s)	Volume (mul)
		SYBR Premix Ex Taq II(Tli RNaseH Plus)(2×)	10
PCR Forward Primer(10μM)	0.8
		PCR Reverse Primer(10μM)	0.8
cDNA template [ ]<100ng)	2
		Sterilizing water	6.4

The RT-qPCR detection flow adopts a two-step method: (1) Pre-denaturation: 95 ℃ for 30sec; (2) PCR reaction: 95℃for 5sec,60℃for 30sec,40 cycles; (3) dissolution profile analysis: 95℃for 5sec and 60℃for 60sec. The results of RT-qPCR detection are shown in FIG. 4.

It can be seen that the expression level of PtoCXE06 in transgenic plants is significantly increased compared to control plants.

Example 5

Overexpression of PtoCXE06 enhances resistance of poplar to Populus sclerotium rolfsii

1. After the overexpression of PtoCXE06 in poplar according to example 4, when plants were grown to a height of 50cm, 3-5 th leaves of plants overexpressing PtoCXE06-OE and wild type plants (WT) were selected from the top, respectively, the leaves were placed face down on a petri dish with moist filter paper laid thereon, and four days of Poplar sclerotium rolfsii cultured on PDA medium was sampled with a sampler to obtain 6 mm-sized agar blocks, and the side with the bacteria was inoculated onto the back of the leaves. After inoculation, the probability of bacterial plaque occurrence in all leaves inoculated with the poplar skin rot bacteria, namely the infection rate of the poplar skin rot bacteria, is counted every other day, and the result is shown in figure 5.

As can be seen from the results of FIG. 5, ptoCXE06-OE plants were infected at a rate lower than that of the wild type for six days of inoculation with the Poplar skin rot bacteria.

2. According to the disease spot length formed on the surface of the infected leaf as a grading standard, a grading standard of the disease severity of the poplar skin rot bacteria is established, as shown in table 6.

Table 6 Classification Standard of Poplar skin rot disease

Wherein, the unobvious disease spots with the disease spot length below 0.7cm are I-level, and the representative value is 0; the length of the disease spots is 0.7-1.4cm and is grade II, and the representative value is 1; the length of the disease spots is grade III, the length of the disease spots is 1.4-2.1cm, and the representative value is 2; the length of the lesion is grade IV from 2.1cm to 2.8cm, and the representative value is 3; the length of the lesion is V grade above 2.8cm, and the representative value is 4. And (3) respectively measuring the length of each bacterial plaque in the leaves inoculated with the poplar skin rot germs according to the sixth day after inoculation in the step (1), counting the number of the bacterial plaques of each severity level according to the disease grading standard, calculating a Disease Index (DI), and constructing a resistance evaluation system based on the Disease Index (DI) according to the number. The Disease Index (DI) represents the severity of plant contamination and is calculated as follows:

wherein: x is the representative value of each grading standard; n is the representative value 4 of the highest ranking criterion; f is the number of lesions in each grading standard.

Calculated according to the above formula, the wild-type DI was 74.84, whereas plants overexpressing PtoCXE06-OE had a DI of 12.97, significantly lower than the wild-type DI (p <0.001, independent sample t-test). Therefore, the over-expression of PtoCXE06 gene greatly enhances the resistance to the poplar skin rot germ.

The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.

Claims

1. The gene PtoCXE06 of the poplar anti-skin rot germ is characterized in that the nucleotide sequence of the gene PtoCXE06 is shown as SEQ ID NO. 1.

2. The poplar skin rot resistance germ protein is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.

3. An over-expression vector comprising the gene PtoCXE06 of claim 1, wherein the basic vector of said over-expression vector is a plant binary expression vector Δpcambia1302.

4. Use of the gene PtoCXE06 of claim 1, the protein of claim 2 or the overexpression vector of claim 3 for the creation of new germplasm material for poplar disease resistance.

5. Use of the gene PtoCXE06 of claim 1, the protein of claim 2 or the overexpression vector of claim 3 for enhancing resistance of poplar to the species pholiota.

6. The use according to claim 5, characterized in that the resistance of poplar to the species dermatophytosis is enhanced by the overexpression of the gene PtoCXE06 in poplar.

7. A method for increasing the resistance of poplar to the species of the genus poplar bark rot, the method comprising the steps of:

8. The method according to claim 7, wherein the gene PtoCXE06 is extracted and amplified from root, stem and leaf tissues of populus tomentosa.

9. The method of claim 7, wherein the poplar transformed with PtoCXE06 is selected and cultivated to obtain a new poplar disease resistant germplasm material with enhanced resistance to the bark rot of poplar.

10. The method of claim 9, wherein the screening is a hygromycin screening.