CN111534540A

CN111534540A - Application of cucumber CsHMGB gene in reducing pesticide residue and relieving pesticide toxicity

Info

Publication number: CN111534540A
Application number: CN202010429337.XA
Authority: CN
Inventors: 辛明; 秦智伟; 李胜男; 周秀艳; 王蕾
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2020-05-20
Filing date: 2020-05-20
Publication date: 2020-08-14
Anticipated expiration: 2040-05-20
Also published as: CN111534540B

Abstract

An application of a cucumber CsHMGB gene in reducing pesticide residue and relieving pesticide toxicity relates to an application of the CsHMGB gene in the agricultural field. From the genetic aspect, the invention metabolizes pesticides in plants, improves the safety quality of cucumbers, reduces pesticide residues and avoids the problems of environmental pollution and the like. The amount of propamocarb in cucumber lines silent on CsHMGB was increased and that in cucumber lines overexpressing CsHMGB was decreased. After the cucumber is stressed by the propamocarb, the cucumber is interfered by ROS, and the over-expressed CsHMGB can improve the activities of antioxidant enzymes SOD, POD, CAT and the like and improve the circulating capacity of ASA-GSH. The over-expressed CsHMGB induces the up-regulation expression of partial genes of detoxification genes, glutathione family and membrane transporter ABC family. CsHMGB can alleviate the toxicity of propamocarb through an antioxidase system.

Description

Application of cucumber CsHMGB gene in reducing pesticide residue and relieving pesticide toxicity

Technical Field

The invention relates to application of a CsHMGB gene in the field of agriculture.

Background

Cucumber (Cucumis sativus) is one of the main cultivated crops in vegetable production of Chinese facilities, and has a wide planting area. Because of special climatic conditions and continuous planting in the greenhouse, the cucumber is very easy to be attacked by diseases and insect pests, and the cucumber downy mildew is a destructive disease.

Propamocarb is a novel, efficient and broad-spectrum carbamate bactericide, can effectively prevent downy mildew of melons, is one of common pesticides for preventing and treating downy mildew of cucumbers, but is very easy to accumulate in cucumber fruits to form pesticide residues after application due to certain volatility and systemic property, and the rest pesticide residues are deposited in soil or diffused in the atmosphere, so that on one hand, cucumber pesticide residues are caused, the quality is influenced, and the food safety problem is caused; on the other hand, the environment pollution is caused, and the human health is harmed. At present, effective ways for reducing pesticide residues in fruits and vegetables are to reduce pesticide investment and accelerate the metabolism of pesticides in plants, but exogenous substances increase production cost and even cause secondary pollution.

Disclosure of Invention

From the genetic aspect, the invention metabolizes pesticides in plants, improves the safety quality of cucumbers, reduces pesticide residues and avoids the problems of environmental pollution and the like.

The application of the cucumber CsHMGB gene in reducing the pesticide residue and relieving the pesticide toxicity.

The total length of the cucumber CsHMGB gene is 624bp, 207 amino acids are coded, and the gene has the highest homology with the melon and the balsam pear on the same evolutionary branch; the amount of propamocarb in cucumber lines silent on CsHMGB was increased and that in cucumber lines overexpressing CsHMGB was decreased. After the cucumber is stressed by the propamocarb, the cucumber is interfered by ROS, and the over-expressed CsHMGB can improve the activities of antioxidant enzymes SOD, POD, CAT and the like and improve the circulating capacity of ASA-GSH. The over-expressed CsHMGB induces the up-regulation expression of partial genes of detoxification genes, glutathione family and membrane transporter ABC family. CsHMGB can alleviate the toxicity of propamocarb through an antioxidase system.

Drawings

FIG. 1 shows the expression level of CsHMGB at different tissue sites of the D0351 cucumber line induced by the propamocarb signal in example 1.

FIG. 2 shows the expression level of CsHMGB at different tissue sites induced by propamocarb signal in the D9320 cucumber line in example 1.

FIG. 3 is a graph comparing the CsHMGB expression levels of the D0351 cucumber line in example 1 in different time periods under propamocarb stress and a control.

FIG. 4 is a graph comparing the CsHMGB expression levels of the D9320 cucumber line in example 1 stressed by propamocarb with that of a control over different periods of time.

FIG. 5 is a confocal laser scanning microscope image of Arabidopsis protoplasts transferred into a vector in example 1.

FIG. 6 is a confocal laser scanning microscope image of epidermal cells of tobacco leaves transfected with the vector in example 1.

FIG. 7 shows the whitening of leaves of cucumber plants inoculated with TRV2-PDS in example 2.

FIG. 8 is a graph comparing the CsHMGB expression levels of 3 groups of cucumber lines inoculated with TRV2-HMGB in example 2 with TRV2 empty control and wild type control.

FIG. 9 is a comparison of the propamocarb residual levels of the 3 groups of cucumber lines inoculated with TRV2-HMGB in example 2 with TRV2 empty control and wild type control.

FIG. 10 is a time chart of the residual amount of propamocarb from T1P-dais fruit sprayed with 400pm propamocarb in example 2.

FIG. 11 is a time chart of the residual amount of propamocarb from T1P-daidzein sprayed with 1mM propamocarb in example 2.

FIG. 12 is O in cucumber bodies after propamocarb stress in example 2^2-The content is compared with the figure.

FIG. 13 shows H in cucumber bodies after Pericarb stress in example 2₂O₂The content is compared with the figure.

FIG. 14 is a graph comparing the MDA content in cucumber bodies after propamocarb stress in example 2.

FIG. 15 is a graph comparing the antioxidant enzyme activity in cucumbers after propamocarb stress in example 2.

FIG. 16 is a comparative set of ASA-GSH cycles in cucumbers after propamocarb stress in example 2.

FIG. 17 is a graph showing comparison of expression levels of detoxification genes in cucumbers after the stress of propamocarb in example 2.

Detailed Description

The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.

The first embodiment is as follows: the application of the cucumber CsHMGB gene in reducing the pesticide residue and relieving the pesticide toxicity is provided.

In the present embodiment, the pesticide is propamocarb.

In this embodiment, specific primers (CsHMGB-F:5 'ATGGAAATTATGGAACACAACAG-3', CsHMGB-R: 5'-TCAAGAGATGAAATCTAGAGAGTCA-3') are designed for PCR amplification.

And (3) PCR system: upstream primer (20. mu. mol. L)^-1) 0.5. mu.L, downstream primer (20. mu. mol. L)^-1) 0.5. mu.L, 10 × PCRbuffer 2. mu.L (containing 20 mmol. multidot.L)^-1Mg²⁺)，dNTPs(10mmol·L^-1)2 μ L of Taq enzyme (2U)0.2 μ L, add ddH₂O to 20. mu.L.

PCR procedure: 3min at 94 ℃; 1min at 94 ℃, 1min at 54 ℃, 1min at 72 ℃ and 30 cycles; 10min at 72 ℃; storing at 4 ℃.

Using the online NCBI (http:// www.ncbi.nlm.nih.gov) database, sequences homologous to CsHMGB were analyzed and obtained by BLAST alignment. Phylogenetic tree analysis was performed using MEGA7 software, phylogenetic tree construction using the neighbor method (NJ) of Poisson model (Poisson model) and 1000 resampling confidence analyses according to Bootstrap, and domain sequence multiple alignment analysis was performed using DNAMAN (http:// www.lynnon.com /) software. Obtaining the cucumber CsHMGB gene (the CDS sequence of which is shown as SEQ ID NO: 1 in the sequence table.)

The full length of the sequence is amplified by PCR by taking fruit cDNA as a template, the sequence of the sequenced CsHMGB is completely consistent with CsAV3_5G021890 in a cucumber genome database, and the full length is 624bp, which indicates that the gene is successfully cloned. A total of 207 amino acids were encoded and sequence analysis showed 363bp 5 'untranslated region (UTR), 188bp 3' UTR, exons and 7 introns. And (3) analyzing the genetic relationship between the CsHMGB and HMG family genes of species such as melons, hot peppers, rice, arabidopsis thaliana and the like, and constructing a phylogenetic tree by an adjacent tree construction method. The result shows that the CsHMGB has high homology with melon (CmHMGB7), pepper (Cp HMGB7) and balsam pear (Mn HMGB7) on the same evolutionary branch, and the CsHMGB has highly conserved homologous sequences with homology of more than 80 percent with balsam pear, melon, pepper and the like.

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

Example 1

In this example, a cucumber low propamocarb residual strain 'D0351' (0.014mg/kg) and a cucumber high propamocarb residual strain 'D9320' (0.171mg/kg) were selected as test materials ('D0351' and 'D9320' provided by the cucumber subject group of northeast university of agriculture). In the autumn of 2017, the tested materials are cultured at a gardening test station of northeast agricultural university, and a conventional ridge culture rack type cultivation method is adopted for unified management. The plant is sprayed with 400 times of propamocarb hydrochloride solution in the maturity stage of the tenth section melon (initial harvest stage, 34 days after planting), and the sprayed water is used as a control and is sprayed to the leaf margin and the fruit dropping degree. And respectively taking the peels of the upper part, the middle part and the lower part of the fruit and a sample with the thickness of 2mm for 3h, 6h, 9h, 12h, 24h, 36h, 48h, 72h, 96h and 120h, mixing, quickly freezing by using liquid nitrogen, and storing at-80 ℃ for later use.

Propamocarb residue measurement method: 12.5g of the sample is weighed, 25ml of acetonitrile is added to the sample, homogenized for 2min, and kept stand for 0.5 h. The shaken acetonitrile was extracted into a centrifuge tube containing 3g NaCl, vortexed for 1min, and centrifuged directly. Taking 5ml of supernatant, putting the supernatant in a water bath at 60 ℃, evaporating acetonitrile to dryness, dissolving the acetonitrile by using 2.5ml of acetone after evaporation to dryness, filtering the solution by using an organic phase filter membrane (0.22 for 2 cases), and then putting the filter membrane into a small bottle (500-1000 mu l) for later use. After each solution in the small bottle is diluted by 50 times, a batch of small bottles are arranged for testing. The solution after the filtration of the filter membrane needs to be clear and has no particles, whether the solution after the filtration of the filter membrane is turbid or not can be observed after the solution after the filtration of the filter membrane is static for a plurality of minutes, and if the solution has the particles, the solution needs to be filtered again until the solution is clear. The detection was carried out with a gas chromatograph-Agilent 7890B-5977A.

Total RNA of tissues such as root, stem, leaf, female flower, male flower and the like of the cucumber D0351 and D9320 is extracted, and the RNA is reversely transcribed into cDNA according to the instruction of a Dining reverse transcription Kit ReverTra Ace qPCR RT-Kit. The PCR reaction system is SYBRGreen PCR Master Mix 10. mu.L, upstream primer (10. mu. mol. L)^-1) 0.5. mu.L, downstream primer (10. mu. mol. L)^-1) 0.5. mu.L, cDNA template 2. mu.L, plus ddH₂Performing PCR amplification on a BIO-RAD iQ5 fluorescent quantitative PCR instrument under the reaction conditions of 95 ℃ for 3min, 95 ℃ for 10s, 60 ℃ for 30s and 72 ℃ for 30s for 40 cycles, collecting fluorescent signals in a 72 ℃ extension step, setting 4 technical repeats and 3 biological repeats for each sample, wherein the reference gene is CsEF1 α^[The relative expression level of the target gene was defined as 2^-ΔΔCTCalculated relative to the quantitative analysis method. Adopting a Turkey test method to carry out single-factor analysis of variance, and carrying out difference significance analysis by taking the level of 0.05 as a standard; the mean and standard error data of the triplicates were plotted.

CsHMGB is expressed in different tissue parts of the D0351 and D9320 cucumber lines under the stress of propamocarb, but the expression patterns are different. In the D0351 cucumber line, the expression level of CsHMGB in pericarp, leaf and female flower is obviously increased by the induction of propamocarb signal, and is respectively 6.43 times, 3.01 times and 1.85 times of that of the control; while roots, stems, male flowers and pulp also show a tendency to up-regulate expression, but the difference is not significant; the expression quantity in male flowers and roots is low; the expression level of different tissue sites is peel > leaf > female flower > stem > pulp > root > male flower (as shown in fig. 1). In the D9320 cucumber line, CsHMGB expression was highest in fruits, but was not significantly different from the control, followed by leaves, with a low surface mass in male flowers and stems, and expression at different tissue sites was peel > leaf > female flower > root > stem > pulp > male flowers (as shown in fig. 2). RT-PCR is adopted to analyze different time expression modes of CsHMGB genes in the peels of cucumbers D0351 and D9320 under the stress of propamocarb. In the D0351 cucumber line, the expression level of CsHMGB is rapidly increased after the propamocarb stress, and is significantly higher than that of a control, 3.23 times of that of the control in 3h, the expression level of CsHMGB is also increased in the subsequent 48h, the expression levels of CsHMGB are respectively 1.42 times, 2.06 times, 4.43 times and 2.36 times of that of the control in 6h, 12h, 24h and 48h, and the expression level of CsHMGB is significantly reduced in the subsequent 72-120 h, is reduced by about 48.32% compared with that of the control in 48h, and has no significant difference with the expression level of the control (as shown in figure 3). In D9320, the CsHMGB gene expression patterns are obviously different, the expression quantity does not change greatly within 3-12 h, the difference with a control is not obvious, and the expression quantity gradually decreases within 24-120 h later (as shown in FIG. 4). The above results demonstrate that CsHMGB is specifically up-regulated by peronospora threaten in D0351, whereas the absence of this specificity in D9320 suggests that CsHMGB is expressed mainly in pericarp and leaves, and responds positively to the propamocarb signal in the D0351 line.

Construction of transient 35S: CsHMGB-GFP expression vector: adding enzyme cutting site sequences at two ends of the designed CsHMGB full-length cloning primer, wherein the upstream and downstream enzyme cutting sites are Xba I and Bgl II respectively, and the downstream primer removes a stop codon. PCR amplification is carried out by taking CsHMGB clone full-length plasmid as a template, the plasmid is connected to a T vector, the full-length coding sequence (without a termination codon) plasmid with correct sequencing is cut with the pSuper-1300 vector by enzyme and gel recovery is carried out simultaneously (refer to the instruction of a kit), the gel recovery product is connected by T4-DNA ligase overnight at 25 ℃, and bacteria shaking is carried out after transformation of escherichia coli. And finally sequencing the positive monoclonal bacteria liquid containing the target fragment identified by the PCR so as to obtain a successfully constructed transient 35S: CsHMGB-GFP expression vector. The empty vector pSuper-1300 served as a negative control.

Expression of transient expression vectors: the transient expression vector 35S, CsHMGB-GFP and the empty vector pSuper-1300 are transformed into arabidopsis protoplasts and tobacco leaf epidermal cells (the specific method can refer to methods such as YOO), and the expression condition of the fusion protein is observed by a laser confocal microscope (Leica, Germany), wherein the excitation wavelength is 488nm, and the emission wavelength is 530 nm. The results of the localization of Arabidopsis protoplasts showed that the DNA sequence was modified in the 35S-transferred: bright green fluorescence was seen throughout the cells with GFP empty vector, whereas in the case of 35S: the CsHMGB-GFP fusion expression vector is enriched in the nucleus only for green fluorescence (as shown in FIG. 5). Tobacco leaf epidermal cell localization also showed 35S: the CsHMGB-GFP protein localizes to the nucleus (as shown in FIG. 6).

Example 2

And (3) carrying out PCR by using cloning full-length primers CsHMGBF and CsHMGBR and using a CsHMGB open reading frame plasmid as a template and using Taq enzyme to obtain an overhang with a 3' terminal band A. The CsHMGB was further ligated to the pCXSN-1250 vector by T4 ligase (see description) to construct an overexpression vector pCXSN-CsHMGB (+).

The constructed sense and antisense expression vectors and empty vectors are respectively transferred into agrobacterium tumefaciens LBA4404 by a freeze-thaw method. Adopting optimized agrobacterium-mediated cucumber genetic transformation technology of Wang and the like to infect cucumber cotyledonary nodes of a susceptible cucumber strain L18, and obtaining a resistant single plant through co-culture, adventitious bud induced differentiation, adventitious bud elongation and rooting and regeneration plant domestication. Selecting glufosinate with the concentration of 1mg/L to carry out resistant plant screening to obtain resistant single plants. T for molecular identification of transgenic plant by PCR and qPCR technology with sequence on pCXSN carrier as primer₀And (5) harvesting seeds of the single plant.

The physiological and biochemical index detection method comprises the following steps:

taking 0.5g of fresh cucumber leaves, adding 1ml of phosphate buffer (0.05mol/L, PH ═ 7.8) into the fresh cucumber leaves, grinding in an ice bath, adding 1ml of buffer into the ground cucumber leaves, pouring the ground cucumber leaves into a centrifuge tube, cleaning the mortar by using 2ml of buffer, pouring the ground cucumber leaves into the centrifuge tube, centrifuging the cucumber leaves at a low temperature (0-4 ℃) for 20min (10500rpm) in a balanced manner, and refrigerating and storing the cucumber leaves.

POD (total activity of POD (△ OD 470. multidot.min) is obtained by adding 20. mu.L of supernatant into cuvette (20. mu.L of phosphate buffer solution added as control) and 3ml of reaction solution, immediately reading OD value at 470nm, timing, and reading OD value every 1min (0, 1, 2, 3 min)^-1·g^-1FW (△ OD470 · V)/(a · W · t) { V ═ 4ml, a ═ 0.02ml, W ═ 0.5g, and t ═ 1min }, and POD specific activity (△ OD470 · min) }^-1·g^-1Pr) — total activity/protein concentration. Reaction solution: 0.1mol/L, PH ═ 6.0 phosphorus buffer 200ml, add guaiacol 112. mu.L, dissolve with heating on a magnetic stirrer, add 30% H₂O₂19 μ L was stored in a refrigerator.

SOD (superoxide dismutase): NBT method was used. Taking the same test tube, adding 50 μ L of supernatant (2 control tubes each containing phosphorus)And slowing by 50 mu L), respectively adding 3ml of reaction solution, placing one control tube in the dark, and reacting the other tubes in the sunlight of 4000lx for 20-30 min (requiring that the light receiving conditions of the tubes are consistent, the temperature is high, the time is shortened, and the time is prolonged in the low period). After the reaction, a control tube without illumination is used as a blank, and the absorbance value is measured by colorimetry at 560 nm. The formula: the enzyme quantity required for inhibiting NBT photoreduction by 50 percent is taken as an enzyme activity unit (U), and the total activity of SOD (units g)^-1FW ═ V)/(0.5ACK · W · Vt) [ ACK is the absorbance of the illumination control tube; AE is the absorbance of the sample tube; v total volume of sample solution 4ml, Vt for measuring sample amount 0.05ml, W for measuring sample weight 0.5g, protein content unit mg/gFW]. Specific activity of SOD (units. mg)^-1Pr) — total activity/protein content. Reaction solution: water: phosphorus-containing buffer: met: NBT: EDTA-Na₂: riboflavin-5: 30:6:6:6: 6.

MDA comprises collecting supernatant 1mL (adding water 1mL to control), adding 0.67% TBA (thiobarbituric acid-lucifugal acid) 2mL, sealing in boiling water bath for 15min, rapidly cooling (soaking with cold water), pouring into finger tube, centrifuging at 4000rpm for 20min, collecting supernatant, and performing colorimetry at 600nm, 532nm, and 450nm to obtain MDA content (μmol/g FW) ═ C × V/W0.1548 (OD)₅₃₂-OD₆₀₀)-0.01344OD₄₅₀(W ═ 0.5 g; V ═ 4ml) 0.67% TBA: 0.67g of TBA, a small amount of 1mol/L NaOH solution (100 mL-3.99971 g of NaOH) was added, and 10% trichloroacetic acid was added to make 100 mL.

GST: GST activity was measured in Wangzhou, and the kit was purchased from Nanjing, a institute of bioengineering.

The silencing of Phytoene Desaturase (PDS) genes results in photobleaching, a method for controlling the efficiency of VIGS, and has been applied to tobacco, tomato and cucurbits. In the embodiment, TRV2, TRV2-PDS, TRV2-HMGB and PTRV1 are mixed and then injected to cotyledons of D0351-line cucumbers in an agrobacterium infiltration mode, the mixture is planted in a climatic incubator, when two true leaves are planted, downy mildew bacteria liquid is injected into the leaves and stems, the whitening phenomenon (shown in figure 7) occurs on the leaves of cucumber plants inoculated with the TRV2-PDS in succession, each group is sprayed with propamocarb, 400ppm and 1mM are treated with the propamocarb, 400 times of propamocarb hydrochloride solution is sprayed on the plants when the second section melons are mature (in the initial harvest period and 34 days after field planting), and water spray is used as a control to the leaf margins and the fruit drops. And respectively taking the peels of the upper part, the middle part and the lower part of the fruit and a sample with the thickness of 2mm for 3h, 6h, 9h, 12h, 24h, 36h, 48h, 72h, 96h and 120h, mixing, quickly freezing by using liquid nitrogen, and storing at-80 ℃ for later use. Spraying propamocarb on wild plants and distilled water as a reference, and sampling at 3h, 6h, 9h, 12h, 24h, 36h, 48h, 72h, 96h and 120h after treatment for propamocarb residual quantity detection and analysis; and sampling for 0, 2, 4, 6, 8 and 10d after treatment, and detecting physiological and biochemical indexes. When the albinism rate reaches 90%, the expression level of CsHMGB gene inoculated with TRV2-HMGB is detected by an RT-PCR method, the expression level of CsHMGB is obviously lower than that of TRV2 no-load control and wild type in 3 groups of cucumber lines inoculated with TRV2-HMGB, and the expression level is only 42.43%, 43.16% and 45.02% of that of the control (as shown in figure 8), which indicates that the CsHMGB gene is transiently silenced. The determination of the residual amount of propamocarb in leaves of experimental and control groups revealed that the average residual amounts of wild type and TRV2 were 3.02mg/kg and 2.85mg/kg, respectively, whereas the residual amounts of CsHMGB gene-silenced strains were significantly higher than those of wild type, 1.67 times, 1.75 times and 1.72 times (residual amounts of 5.06mg/kg, 5.34mg/kg and 5.22mg/kg, respectively, as shown in fig. 9).

Constructing a plant over-expression vector pCXSN-CsHMGB (+), transforming the D9320 cucumber strain by adopting an agrobacterium-mediated method, finally obtaining 15 positive transgenic plants after RT-PCR identification, and harvesting T after selfing and pollination₁The cucumber can be used as substitute. Detection of T for determination of Stable inheritance₁And (3) planting the single plant in a barrel after the 3-leaf one-heart period, and culturing in an artificial climate chamber. The environmental conditions were controlled as follows: the average humidity is 75% in 16h light and 8h dark and in 28/18 deg.C environment. Transgenic T obtained by cucumber genetic transformation₁The method comprises the steps of sowing and raising seedlings of substitute plants OX2, OX3 and OX5, treating propamocarb with the concentration of 400ppm and 1mm, spraying 400 times of propamocarb hydrochloride solution on the plants when the melons at the tenth section are mature (initial harvest period and 34 days after field planting), and spraying the propamocarb hydrochloride solution on the leaf margins and fruit drops with water spray as a control. And taking the upper, middle and lower three of the fruit at 3h, 6h, 9h, 12h, 24h, 36h, 48h, 72h, 96h and 120h respectivelyMixing part of pericarp with 2mm thick sample, quick freezing with liquid nitrogen, and storing at-80 deg.C. The wild plant is sprayed with propamocarb and distilled water as a contrast, 3 hours after treatment,

sampling at 6h, 9h, 12h, 24h, 36h, 48h, 72h, 96h and 120h for propamocarb residual quantity detection and analysis; and sampling for 0, 2, 4, 6, 8 and 10d after treatment, and detecting physiological and biochemical indexes.

The residual amounts of propamocarb of wild cucumber strains and over-expression strains OX-HMGB2, OX-HMGB3 and OX-HMGB5 show a trend of rising firstly and then falling, within 3-48 h, the residual amount of propamocarb gradually rises, the residual amount reaches the maximum value within 48h, the residual amount gradually increases within 3-48 h, the residual amount of propamocarb is the maximum within 48h, the wild type is 4.526mg/kg, and the residual amounts of over-expression strains OX-HMGB2, OX-HMGB3 and OX-HMGB5 are 3.7mg/kg, 3.64mg/kg and 3.53mg/kg, the average value of which is 19.8 percent lower than that of the wild type. The amount of propamocarb remaining after 48h started to decrease, probably due to the half-life of the pesticide,

the residual amount of propamocarb is close to steady at 96-120 h, and the average value of the residual amount of propamocarb of an over-expression strain is still 20.95 percent lower than that of a wild type (as shown in figure 10). The change trend of the residual amount of propamocarb in the peel of cucumber scurrow sprayed with 1mM propamocarb is similar to that of 400ppm (as shown in figure 11), and the experimental result shows that the over-expressed CsHMGB can reduce the residual amount of propamocarb in the peel of cucumber.

When plants are stressed by toxic stresses such as pesticides and the like, excessive accumulation of ROS can be caused, the redox balance in cells is changed, oxidative stress is caused, biomacromolecules such as protein, lipid and the like are damaged, even cells die, and the growth and development of the plants are seriously influenced. O in cucumber bodies after Pericarb stress^2-And H₂O₂The contents all increased rapidly, especially 4d after stress, and reached a maximum and then began to decrease, whereas O was overexpressed in CsHMGB cucumber lines^2-And H₂O₂The content of Malondialdehyde (MDA) in cucumber is further determined, and the content of MDA in cucumber lines over-expressing CsHMGB is found to be significantly lower than that of wild type control (shown in figure 14). The experimental results show that the downy mildew threatensThe stress can cause the accumulation of ROS in cucumber bodies, and the over-expression of CsHMGB helps to eliminate ROS and resist ROS stress.

In order to maintain the normal level of ROS, when plants are damaged by ROS, an antioxidant system can be started quickly, and peroxide formed in vivo is converted into substances with low or no toxicity, so as to resist the toxicity of the outside. In this example, the activities of antioxidase such as SOD, POD and CAT were measured. Peroxidases such as SOD, POD, CAT and the like all show a tendency to increase after the cucumber is stressed by propamocarb. SOD is the first substance for eliminating free radicals in plants, after the downy mildew is stressed, the SOD enzyme activity in cucumber fruits is obviously increased, the SOD enzyme activity of an over-expressed strain shows a trend of increasing firstly and then decreasing, but is still obviously higher than a control, the POD enzyme activity shows a trend of continuously increasing within 2-10 days of the downy mildew stress, and the enzyme activity of the over-expressed strain is higher than that of a wild control. APX is the elimination of H in chloroplasts₂O₂The APX activity and SOD enzyme activity of the over-expressed CsHMGB strain show similar change trend after the propamocarb is stressed; CAT widely exists in plant chloroplasts, an antioxidant mechanism is provided for organisms in mitochondria, after propamocarb is stressed, the CAT activity in cucumber fruits of over-expressed CsHMGB strains is obviously higher than that of a control at 2-4 days, and then the CAT activity tends to be stable. GPX is an important catalytic enzyme in a glutathione system, hydroperoxide is reduced by catalyzing Glutathione (GSH), the activity of the GPX of the over-expressed CsHMGB strain is obviously higher than that of a control 2-6 days after the propamocarb is stressed, and then the activity of the GPX is reduced. In a word, the activity of key enzyme enzymes in the CsHMGB strain antioxidant enzyme system overexpressed after the propamocarb is stressed is obviously enhanced in the initial stage, which shows that the CsHMGB can activate the antioxidant enzyme system after the propamocarb is stressed, so that the interference of ROS is resisted. (as shown in FIG. 15)

The ASA-GSH cycle is an important antioxidant system in plants and also an important way to scavenge ROS in plants. In this example, key enzyme activities and levels in the ASA-GSH cycle were determined. As shown in FIG. 16, the content of ASA of the cucumber is reduced at 2-6 days after the cucumber is stressed by propamocarb and is lower than that of a wild type control, while the content of DHA is increased, which indicates that the propamocarb can promote the conversion of ASA to DHA, so that the oxidation resistance of the cucumber is reduced, the ASA and DHA of the transgenic cucumber are both significantly higher than that of the wild type control at 2-10 days, the overall change difference of ASA is small, DHA is increased and then reduced, the total amount of ASA and DHA is increased, the conversion of DHA to ASA is accelerated, the oxidation resistance is improved, and the fact that the cucumber oxidation resistance is enhanced by over-expressing CsHMGB strains is indicated. The MDHAR and DHAR have important effects on the regeneration of ascorbic acid, the activity of the MDHAR and DHAR enzymes of the transgenic plants in the embodiment is obviously higher than that of non-transgenic wild type control plants, and the circulation path of the ascorbic acid is enhanced, and the antioxidant capacity of the plants is enhanced. In addition, although the total content of GSSG and GSH is increased after downy mildew stress, the value of GSH/GSSG is decreased, which indicates that cucumber plants are stressed by ROS and the like, the total amount of GSH and GSSG of transgenic over-expression plants in 2-10 days is higher than that of wild type control, and the value of GSH/GSSG is higher than that of wild type control, further indicates that the oxidation-reduction state of a glutathione system is strong, so that the downy mildew stress is resisted. Similarly, the enzyme activities of the transgenic over-expression plants GST and GR in 2-10 days are also obviously higher than those of the wild control. In conclusion, the cucumber strain of over-expressed CsHMGB enhances the ASA-GSH circulation and improves the oxidation resistance of the plant.

Example 3

This example analyzed expression in CsHMGB transgenic cucumber plants and non-transgenic plants using qRT-PCR. Up-regulated expression of CsGPX, CsGSH2 and CsGST1 in glutathione families is shown in transgenic cucumber strains OX2, OX3 and OX5, wherein the expression amount of the CsGPX of an OX2 strain is increased most obviously and is 5.4 times that of a non-transgenic control (as shown in FIG. 17); the expression amounts of CsGST1 and CsGSH2 were 13.45 times and 8.89 times of the wild type, respectively; up-regulated expression of cytochrome CsP450 gene also occurs; the expression of the transportproteins CsABCA19 and CsPDR is also up-regulated, and the expression amounts of CsABCA19 and CsPDR in the OX2 strain are 5.86 times and 15.6 times of that of the wild type respectively; the experimental result shows that the over-expressed CsHMGB protein can increase the expression of detoxification genes.

Sequence listing

<110> northeast university of agriculture

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>624

<212>DNA

<213> cucumber (Cucumis sativus)

<400>1

atggccggcg gaggatcctc caagtcccgg aagagagtgg aggctactcc tgctgacgtc 60

gccgctactg gtccttcgct tgtgagggcc aaagacggca gtgcttttgc tagatgtgac 120

gagtgcggta aaagtgttcc ggtggcctta atcagcatgc acagttgcag cctcgacgcc 180

aaaattagaa tgaatttaga gtctcagact gttgaaaagc aaacacaatc caaaaagcca 240

gctgaaaaga aaagatcagc atcatctgaa cctaagacta agaaatcccg aactgagaag 300

aaagggaaga aggacaaaga tccaaatgcc cccaaacgcc ctcctacagc tttctttatc 360

ttcatggatg acttcagaaa gtcatataaa gaagccaatc ctgattccaa gggcgttaag 420

gaggttgcaa aggagggtgg tgagaaatgg aagtcaatga ctgatgaaga gaagaagcct 480

taccaagata aagctgccga gctaaaagcg gaatatgaga aggcattgga aagtcgaaat 540

gctgagaatg aagatgatga aaaagagaca gaagagacgg aagagattga agaggaggtt 600

gaaggaataa cggaagaaga gtaa 624

Claims

1. The application of the cucumber CsHMGB gene in reducing the pesticide residue and relieving the pesticide toxicity.

2. Use according to claim 1, characterized in that the pesticide is propamocarb.