CN114517202B

CN114517202B - Application of aquaporin gene SlPIP, 2 in improving resistance of tomatoes to facility continuous cropping soil

Info

Publication number: CN114517202B
Application number: CN202210364402.4A
Authority: CN
Inventors: 张大龙; 魏珉; 陈晓璐; 娄洁; 李晓甜; 赵晗; 杨慧华
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2022-04-08
Filing date: 2022-04-08
Publication date: 2024-04-19
Anticipated expiration: 2042-04-08
Also published as: CN114517202A

Abstract

The invention discloses aquaporin gene SlPIP1;2 in improving the resistance of tomatoes to continuous cropping soil of facilities, belonging to the technical field of facility gardening. The research of the invention finds that the water channel protein gene SlPIP is overexpressed; 2 can improve the resistance of tomatoes to the continuous cropping soil of the facility, improve the yield of tomatoes planted in the continuous cropping soil of the facility, and lay a foundation for the high-yield cultivation of tomatoes in the stress of the facility.

Description

Aquaporin gene SlPIP1;2 application in improving soil resistance of tomatoes to continuous cropping soil of facilities

Technical Field

The invention relates to the technical field of facility gardening, in particular to a aquaporin gene SlPIP1;2 in improving the resistance of tomatoes to continuous cropping soil.

Background

The first country of world facility vegetable production is about 80% of the world, and tomatoes are the vegetable crops with the largest facility cultivation area in China. Continuous cropping obstacles are commonly existed in the production of facility crops, and become the largest limiting factor for restricting the development of facility agriculture in China. The continuous cropping obstacle is a phenomenon that after continuous cropping of the same crop or a nearby crop, yield is reduced, quality is deteriorated, and growth conditions are deteriorated even under the normal cultivation and management conditions. The continuous cropping obstacle is complex in cause, and is generally attributed to the physical and chemical property malignant change of soil, imbalance of diversity of soil microorganisms, root system autotoxicity, salinization, accumulation of pathogenic microorganisms and the like, and is the external appearance of the comprehensive effect result of multiple factors such as soil, plants, microorganisms and the like.

The continuous cropping soil of the facility causes soil nutrient deficiency or unbalance and soil-borne disease and pest accumulation due to a large amount of fertilization and high multiple cropping index; in addition, the facility is a relatively closed cultivation environment, and the lack of rain water leaching causes salt to be accumulated on the surface layer of soil along with the evaporation of water, so that secondary salinization occurs. Meanwhile, the volume weight of the soil is increased, the aggregate structure is deteriorated, the ventilation and water permeability is deteriorated, and the growth of root systems and the absorption and utilization of moisture and nutrients are not facilitated. Thus, as the growth years increase, both the yield and quality of the greenhouse vegetable will be significantly reduced.

Aquaporins are widely present in plant leaves, root systems and other organs, and are of great interest because of their wide involvement in responding to plant stress. The aquaporin is a receptor for cell osmotic pressure and turgor pressure, can sensitively respond to water stress signals, and each monomer of the aquaporin can be used as a functional pore structure to regulate the rapid transmembrane transport of water in a 'gate control' manner, change the permeability of cell membranes to water, and effectively maintain the water balance inside and outside cells in response to circadian rhythm, drought, saline-alkali and other stresses. However, the function of aquaporins in continuous cropping soil stress in vegetable crop response facilities is reported in a few days.

Disclosure of Invention

In view of the above prior art, an object of the present invention is to provide aquaporin gene SlPIP1;2 in improving the resistance of tomatoes to continuous cropping soil. The research of the invention finds that the water channel protein gene SlPIP is overexpressed; 2 can improve the resistance of tomatoes to the facility continuous cropping soil and improve the yield of tomatoes planted in the facility continuous cropping soil.

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

in a first aspect of the present invention, there is provided aquaporin gene SlPIP1;2 in improving the resistance of tomatoes to continuous cropping soil.

In the above application, the aquaporin gene SlPIP1;2 is shown as SEQ ID NO. 1; the method comprises the following steps:

ATGGAGGGGAAAGAAGAGGATGTGAAGGTTGGAGCAAACAAGTATTCAGAAAGGCAGCCATTAGGGACTTCAGCACAGAGCAAGGACTACAAGGAGGCACCACCAGCACCATTATTTGAGGCTGGTGAGCTACATTCTTGGTCTTTTTGGAGAGCTGGGATTGCAGAGTTCATGGCTACTTTTTTGTTCCTTTACATAACTGTATTGACTGTTATGGGTTATTCAAGGGCTAACAGCAAATGTAGTACTGTTGGTGTTCAAGGCATTGCTTGGGCTTTTGGGGGTATGATTTTTGCCCTTGTTTACTGCACTGCTGGCATATCAGGTGGACACATTAACCCTGCTGTGACATTTGGTTTATTTCTGGCAAGGAAATTGTCCTTAACCAGGGCAGTGTTCTACATTGTGATGCAGTGCCTTGGTGCAATCTGTGGTGCTGGTGTTGTCAAAGGGTTCCAGCCATCTTTGTTTGAGACTAAGGGTGGAGGTGCCAATGTTGTTGCCCATGGTTACACCAAGGGAGATGGCCTTGGTGCTGAGATTATTGGCACTTTTGTTCTTGTCTACACTGTCTTCTCTGCTACTGATGCCAAGAGAAATGCTAGAGACTCCCATGTCCCTATTCTGGCTCCTCTCCCAATTGGATTTGCAGTGTTCTTGGTTCATTTGGCTACAATCCCTATTACAGGCACCGGCATTAACCCTGCTAGAAGCCTAGGCGCTGCCATTGTCTACAACAAAGAACATGCATGGGATGATCATTGGATTTTCTGGGTGGGACCATTCATTGGAGCTGCACTTGCTGCCTTATACCACCAAGTTATCATAAGAGCCATTCCATTCAAGAGTGGAAACTGA.

In the above application, the improvement of the soil resistance of tomatoes to continuous cropping soil comprises:

(1) Reducing root knot index of tomato root knot nematode infection;

(2) Improving the water absorption capacity of the tomato root system in the facility continuous cropping soil with secondary salinization and hardening;

(3) Improving the moisture condition of plants and promoting the absorption and transportation of air holes and mesophyll tissues to CO ₂.

(4) The photosynthetic rate is improved, the plant growth is promoted, and the yield is improved.

In a second aspect of the present invention, there is provided a polypeptide comprising aquaporin gene SlPIP; 2, the application of the recombinant expression vector, the transgenic cell line or the engineering bacteria in improving the resistance of tomatoes to continuous cropping soil.

The recombinant expression vector can be constructed by using the existing plant expression vector. The plant expression vector comprises binary agrobacterium vectors, vectors which can be used for plant microprojectile bombardment, and the like, such as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative plant expression vectors. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as cauliflower mosaic virus (CaMV) 35S promoter, ubiquitin gene Ubiquitin promoter (pUbi), stress-inducible promoter rd29A and the like, can be added before the transcription initiation nucleotide thereof, and can be used alone or in combination with other plant promoters; in addition, a green fluorescent protein Gene (GFP) sequence may be added when constructing the recombinant expression vector.

Preferably, the recombinant expression vector is constructed by taking pCAMBIA2301 as a starting plasmid, and is added with a 35S promoter and GFP.

In a third aspect of the invention, there is provided a method of increasing soil resistance of tomatoes to continuous cropping soil of a facility comprising: allowing tomato aquaporin gene SlPIP1;2 over-expression step.

In the method, the aquaporin gene SlPIP1 in tomato; 2 can be transferred into aquaporin gene SlPIP through exogenous gene; 2; or up-regulating aquaporin gene SlPIP1 in tomato genome; 2.

In a fourth aspect of the present invention, there is provided a method for cultivating tomatoes having improved resistance to continuous cropping soil stress, comprising the steps of:

The aquaporin gene SlPIP1;2, transferring the tomato into a tomato starting plant to enable the aquaporin gene SlPIP1;2, over-expressing to obtain tomato transgenic plants; the capability of the tomato transgenic plant for resisting the continuous cropping soil stress of the facility is higher than that of the tomato starting plant.

In the above cultivation method, aquaporin gene SlPIP1;2, the method for transferring the tomato into the tomato starting plant comprises the following steps: polyethylene glycol method, agrobacterium mediated method or gene gun bombardment method.

The invention has the beneficial effects that:

the invention discovers that the aquaporin gene SlPIP in tomato is over-expressed for the first time; 2 can improve the resistance of tomatoes to continuous cropping soil of facilities, and lays a foundation for the facility adversity high-yield cultivation of tomatoes.

Drawings

Fig. 1: and (5) detecting the result of PCR amplified product fragment electrophoresis. Lane1: DL2000Plus marker; lane2,3: amplification products of the target gene.

Fig. 2: the structure of the expression vector pCAMBIA2301-35S-GFP is schematically shown.

Fig. 3: pCAMBIA2301-GFP-PIP1;2 transformation of Agrobacterium GV3101 colony PCR results. Lane1 DL2000Plus DNA MARKER; lane2-4:2301GFP-PIP1;2 transformation of Agrobacterium GV3101 colony PCR results.

Fig. 4: PCR results of positive seedlings were transformed.

Fig. 5: over-expression SlPIP1;2 effects on root knot index of tomato root system.

Fig. 6: over-expression SlPIP1;2 effects on tomato She Shuishi, root system hydraulic conductivity and transpiration rate.

Fig. 7: over-expression SlPIP1;2 effects on tomato stomatal conductance, mesophyll conductance and total CO ₂ transport conductance.

Fig. 8: over-expression SlPIP1;2 effects on the concentration profile of the stomatal lower lumen and chloroplast carboxylation site CO ₂.

Fig. 9: over-expression SlPIP1;2 effect on tomato photosynthetic-CO ₂ response curve.

Fig. 10: over-expression SlPIP1;2 effects on tomato individual fruit yield and total biomass.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. 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 application belongs.

In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, the technical scheme of the present application will be described in detail with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and are commercially available. Wherein:

culture medium for seed germination: MS powder, 4.43g L ^-1; sucrose 10g L ^-1; agar, 10g L ^-1; KOH regulates the pH to 5.8-6.0.

Pre-culture medium: MS culture medium +ZT2.0 mg/L +IAA0.2 mg/L.

Selection medium: MS culture medium +ZT2.0 mg/L +IAA 0.2mg/L +Kan kanamycin 100mg/L +Cb carbenicillin 500mg/L.

Stem elongation medium: MS culture medium +ZT1.0 mg/L +IAA 0.05mg/L +Kan kanamycin 100mg/L +Cb carbenicillin 500mg/L.

Rooting medium: MS culture medium+IAA 0.1mg/L+Kan kanamycin 50mg/L+Cb carbenicillin 300mg/L.

Example 1: slPIP1;2 gene over-expression strain obtaining method

Slpip1;2 Gene amplification

(1) Design of synthetic amplification primers

Primer name	Primer sequences	Numbering device
			PIPF1	GAGAACACGGGGGACTCTAGAATGGAGGGGAAAGAAGAGGA	SEQ ID NO.2
PIPR1	GCCCTTGCTCACCATGGTACCGTTTCCACTCTTGAATGGAA	SEQ ID NO.3

(2) RNA extraction and reverse transcription

RNA of tomato seedlings is extracted, detected by electrophoresis and subjected to reverse transcription to obtain cDNA.

(3) Amplification of target Gene

Using cDNA as template, high-fidelity enzyme is used to amplify target gene, the system is as follows:

the PCR reaction conditions were: 94 ℃ for 2 minutes; 98 ℃ for 10 seconds; 55 ℃,5 seconds; 72 ℃,55 seconds (35 cycles of amplification); 72℃for 5min.

The PCR products were electrophoretically detected and cut into gel, and the target products were recovered using a gel recovery kit (FIG. 1).

2. Expression vector construction

(1) Linearizing a carrier: the vector pCAMBIA2301-GFP plasmid (commercially available, with a schematic structure shown in FIG. 2) was double digested with Xba I and Kpn I, and the double digested system was as follows:

After water bath at 37 ℃ for 4 hours, electrophoresis, gel cutting recovery and electrophoresis detection are carried out to recover products.

(2) Connecting the target gene recovery product with a linearized vector, wherein the connection adopts a recombination connection kit, and the connection system is as follows:

2*Mix 5μL

Gene recovery product 1. Mu.L

Carrier recovery product 4. Mu.L

After 30 minutes of connection at 50 ℃, DH5 alpha is converted into a competent product, LB plates containing kanamycin (the kanamycin content is 50 mu g/mL) are coated, bacterial liquid PCR screening is carried out, positive bacterial liquid is verified by sequencing, and plasmids are extracted from the positive bacterial liquid with correct sequencing for standby.

3. Agrobacterium transformation

Agrobacterium GV3101 was transformed with the constructed plasmid, plated with LB plates containing kanamycin (kanamycin content 50. Mu.g/mL), and positive bacterial suspensions were screened by bacterial suspension PCR (FIG. 3).

The primers were screened as follows:

PCR system:

The reaction procedure:

95℃、2min；

95℃、10s，55℃、10s，72℃、35s，35cycle。

4. genetic transformation of tomato

(1) Obtaining aseptic seedlings: selecting full, uniform and fresh tomato seeds, repeatedly washing with sterile water for several times, sterilizing the tomato seeds with 70% alcohol for 30s and 10% sodium hypochlorite for 10min, washing with sterile water for 4-5 times, sucking with sterile filter paper, and inoculating into seed germination culture medium. After the seeds are dark cultured until most of the seeds germinate and are exposed to the white, the seeds are placed under the conditions of illumination for 16 hours every day, illumination intensity of 1600-1800lx and temperature (24+/-2) DEG C for culture.

(2) Culturing agrobacterium tumefaciens: and taking agrobacterium tumefaciens competence stored at-80 ℃ and inserting the agrobacterium tumefaciens competence into ice when the agrobacterium tumefaciens competence is partially melted at room temperature or at palm of a hand for a while and is in an ice water mixed state. Every 100 mu L of competent plasmid DNA is added with 0.1 mu g (volume is not more than 10 mu L), the mixture is stirred by hands at the bottom of a tube, and the mixture is mixed uniformly, and then placed on ice for 5 minutes, liquid nitrogen for 5 minutes, water bath at 37 ℃ for 5 minutes and ice bath for 5 minutes in sequence. 700. Mu.L of LB liquid medium without antibiotics is added, and the culture is carried out for 2 to 3 hours at 28 ℃ under shaking at 200 rpm. Centrifuging at 6000rpm for one minute to collect bacteria, collecting about 100 μl supernatant, gently blowing to resuspension bacteria block, coating on LB plate containing corresponding antibiotics, and culturing in 28 deg.C incubator for 2-3 days. 2-3 single colonies are randomly selected and used for colony PCR, and correct agrobacterium single clones are identified and marked for later use. The labeled Agrobacterium was picked with a sterile gun head and inoculated into 5ml LB liquid medium containing the corresponding antibiotic (50 ml blue-cap centrifuge tube) and cultured with shaking at 28℃and 200rpm for 24 hours. Centrifuging at 20 ℃ for 15min at 4,000rpm, and collecting thalli; the cells were uniformly blown with a transformation Buffer and resuspended to an OD ₆₀₀ =about 0.5.

(3) Explant preparation: the transformation is carried out by selecting the true leaves, cotyledons and hypocotyls as explants. Cutting the leaf tip and the leaf stalk of the real leaf and the cotyledon, and cutting the rest part into leaf blocks with the size of 0.5cm multiplied by 0.5 cm; the hypocotyl was cut into pieces of about 0.5-0.6mm in length, placed horizontally on a preculture medium, and 15-20 pieces per dish. Culture conditions were the same as above, and preculture was performed for 1 day.

(4) Agrobacterium inoculation and co-cultivation of explants: taking the explant out of the pre-culture medium, placing the explant into a culture dish which is poured with agrobacterium which is diluted by a conversion Buffer to OD ₆₀₀ =0.5, converting for 30min, taking the explant out, sucking the explant on sterile paper, placing the explant into the pre-culture medium again, and co-culturing for 1-2 days.

(5) Selection and culture: the co-cultured explants were transferred to selection medium for selection culture, and after several days of selection culture, cotyledons began to thicken and hypocotyls began to thicken. Transformed explants will form callus and adventitious buds on selection medium. And subculturing every two weeks.

(6) And (3) adventitious bud elongation culture: calli with shoot primordia were cut into small pieces and transferred to stem elongation medium for subculture, once every two weeks. If necessary in a shorter time.

(7) Rooting culture of adventitious buds: when the adventitious bud length is about 1cm, healthy regenerated buds are selected to grow, the basal callus and the culture medium are completely excised, and the basal callus and the culture medium are transferred to a rooting culture medium for culture, so that complete plants are formed.

(8) Training seedlings, identifying and transplanting and culturing: after lateral roots of seedlings grow out, the bottle cap is opened, a small amount of sterile water (3-5 mm of culture medium is overflowed) is poured into the culture bottle, the culture bottle is placed in a cool and ventilated place for seedling training, and meanwhile, sampling is carried out for positive seedling identification.

PCR system: 2 x mix 10 μl

Primer PHB50F (10. Mu.M) 0.8. Mu.L

Primer PIP12R (10. Mu.M) 0.8. Mu.L

DNA 0.5μL

ddH₂O 7.9μL

Reaction conditions: 95 ℃ for 2min;

95℃10s

55℃10s

72℃35s 35cycle

the electrophoresis detection PCR products are shown in FIG. 4.

Example 2: slPIP1; functional research for improving resistance of tomato root system to facility secondary salinization by 2 genes

1. The test method comprises the following steps:

1.1 cultivation conditions:

The cultivation facility is a sunlight greenhouse, the soil is continuous cropping soil for more than 10 years for continuously planting tomatoes, the soluble salt is accumulated at the height of the soil surface layer, the content of the soluble salt exceeds 1-2 g Kg ^-1, the secondary salinization is serious, and osmotic stress is caused; soil hardening, poor aggregate structure, a volume weight of 1.42g cm ^-3, compaction, poor ventilation and water permeability; the pH is 6.4, and the pH is weak acid; in addition, diseases such as root knot nematodes are also serious. Because of secondary salinization and soil hardening, the root system is difficult to absorb moisture and mineral elements, the tomato plants grow weaker, and the yield is seriously reduced.

The tomato plants planted are respectively: wild tomato plants and SlPIP a 1;2 tomato plants over-expressed by the genes (SlPIP 1; construction of tomato plants over-expressed by the genes of 2) was carried out in the same manner as in example 1).

During the test, wild type tomato plants and SlPIP a 1;2, the cultivation and management method of the tomato plants with the over-expressed genes is kept consistent.

1.2 Phenotypes and assay methods:

Transpiration rate and pore conductance: 3 mature leaves which are strong and grow are selected in the middle of each test plant, a Li-6400 type photosynthesis system manufactured by Li-cor company in the United states is used, the photosynthesis effective radiation intensity is controlled by an LED light source, the concentration of CO ₂ in a leaf chamber is controlled to be 400 mu molmol ^-1 by installing a high-pressure concentration CO ₂ small steel cylinder, and the gas flow rate is 400 mu mols ^-1, so that the gas exchange parameters of the leaves are measured.

She Shuishi: she Shuishi of the same blade is synchronously measured by adopting a PSYPRO dew point water potential meter, the blade is taken by a puncher, the water potential of the blade is measured by placing the blade into a C-52 sample chamber, data is recorded every 5 minutes, and the continuous recording and the average value is taken as the blade water potential at the moment. The stem water potential is measured by adopting a transpiration inhibition method of adjacent section leaves, and the stem water potential is estimated approximately.

Root system hydraulic conductivity: the root system hydraulic conductivity was measured by a pressure chamber method. Cutting tomato seedlings from root base parts, sealing the whole root system by silica gel, and slowly pressurizing a steel bottle until 0.1MPa to 0.5MPa are added each time. Each pressure gradient was maintained at steady state outflow for 1 minute, juice was extracted with absorbent cotton for 1 minute, and then the effluent mass was measured on a ten-thousandth scale. Root system hydraulic conductivity L _pr(m s^-1MPa^-1) is expressed in terms of the flow rate per unit surface area per unit time: l _pr＝V×S^-1×p^-1×t^-1. V is the volume of water flow (m ³) through the root system during the t(s) period; s is the root section cross-sectional surface area (m ²); p is the steel cylinder applied pressure (MPa). And slope fitting is carried out according to a relation curve of the water flow rate and the pressure difference in the unit area in the pressurized process.

Root knot index: grading the disease according to the percentage of root knots in the total root system:

photosynthetic parameters: selecting functional blades with the same section position, controlling the leaf chamber environment to perform gas exchange and chlorophyll fluorescence measurement by using a photosynthetic apparatus-fluorescent leaf chamber (LI-6400), controlling the photosynthetic effective radiation intensity to 1100 mu mol m ^-2s^-1 by using an LED light source, wherein the temperature is 28 ℃ and the air relative humidity is 70%; the leaf chamber CO ₂ concentration was controlled to 400. Mu. Molmol ^-1 by installing a high pressure concentrate CO ₂ small cylinder with a gas flow rate of 400. Mu. Mols ^-1. Photosynthetic CO ₂ transport is resolved from a "source- > stream- > sink" perspective, and according to the gas diffusion law, the partial pressure difference drives CO ₂ from the high partial pressure "source" -atmosphere to the stomatal subchamber, via mesophyll to the low partial pressure "sink" -chloroplast carboxylation site. According to the general laws of physics that characterize fluid and substance diffusion, the overall mesophyll conductance g _m from the stomatal subchamber to the chloroplast carboxylation site can be calculated from the photosynthetic CO ₂ assimilation rate (a) and the CO ₂ concentration gradient (C _i-C_c) from the intercellular (Ci) to the Rubisco carboxylation site (Cc) in the chloroplast:

g_m＝A/(C_i-Cc)

The intercellular CO ₂ concentration C _i and the net photosynthetic rate A are measured by gas exchange, and the energy assimilated by CO ₂ can be quantified through the electron transfer rate, so that the concentration Cc of CO ₂ in the Rubisco enzyme carboxylation reaction substrate chloroplast can be reversely obtained:

J, electron transfer rate, j=Φ _PSII·P·αβ;R_d, mitochondrial respiration rate under light (dark respiration rate under light); Γ, chloroplast CO ₂ compensation point without mitochondrial respiration; p, photosynthetic light quantum flux density; alpha, the absorption efficiency of the blade to the photons; beta, the distribution proportion of light quanta among the optical systems; phi _PSII, the optical quantum efficiency of the optical system II. The measurement and calculation of mesophyll resistance are mature in the earlier study of applicant, and specific steps of parameter estimation such as leaf chamber environment setting, dark respiration rate under light, CO ₂ compensation point and the like can be referred to the earlier study of applicant (Zhang D,Li Y,Li Y.The potential implications of a plasma membrane aquaporin in improving CO2 transport capacity,photosynthetic potential and water use efficiency under contrasting CO2 source in Solanum lycopersicum(tomato)[J].Scientia Horticulturae,2021,283:110122.).

2. Test results:

The test results are shown in FIGS. 5-10. Over-expressing SlPIP <1> in continuous cropping soil with serious hardening, secondary salinization and root-knot nematodes; 2 can induce tomato resistance, improve root resistance to root knot nematodes, and significantly reduce root knot index of root knot nematode infestation (figure 5); can improve the water conductivity of the root system, enhance the water absorbing capacity of the root system, increase the transpiration rate and the water potential of the leaves, and improve the water status of plants (figure 6). Over-expression SlPIP1;2 can improve the transmission conductivity of stomata and mesophyll tissues to CO ₂ (figure 7) and the effective concentration of CO ₂ at chloroplast carboxylation sites (figure 8) after improving the moisture condition of plants, and improve the transmission efficiency of CO ₂ from the atmosphere to the chloroplast carboxylation sites in the photosynthesis process. The photosynthetic-CO ₂ response curve shows that SlPIP is overexpressed; 2 increases the photosynthetic capacity of the tomato leaf (figure 9). Over-expression SlPIP1;2 significantly improved fruit yield and total biomass per plant of tomato plants in continuous cropping soil, promoting plant growth (fig. 10).

Thus, slPIP1 is overexpressed; 2 can effectively improve the resistance to root-knot nematodes, enhance the water absorption capacity of root systems, effectively respond to poor soil aeration and water permeability caused by hardening and osmotic stress caused by secondary salinization, improve the water condition of plants, further improve the photosynthetic rate of leaves, promote plant growth and improve yield. Over-expression SlPIP1;2 is an effective means for improving the soil resistance of tomato plants to continuous cropping soil of a facility.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong agricultural university

<120> Application of aquaporin gene SlPIP, 2 in improving resistance of tomato to facility continuous cropping soil

<130> 2022

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 858

<212> DNA

<213> Aquaporin Gene SlPIP1, 2

<400> 1

atggagggga aagaagagga tgtgaaggtt ggagcaaaca agtattcaga aaggcagcca 60

ttagggactt cagcacagag caaggactac aaggaggcac caccagcacc attatttgag 120

gctggtgagc tacattcttg gtctttttgg agagctggga ttgcagagtt catggctact 180

tttttgttcc tttacataac tgtattgact gttatgggtt attcaagggc taacagcaaa 240

tgtagtactg ttggtgttca aggcattgct tgggcttttg ggggtatgat ttttgccctt 300

gtttactgca ctgctggcat atcaggtgga cacattaacc ctgctgtgac atttggttta 360

tttctggcaa ggaaattgtc cttaaccagg gcagtgttct acattgtgat gcagtgcctt 420

ggtgcaatct gtggtgctgg tgttgtcaaa gggttccagc catctttgtt tgagactaag 480

ggtggaggtg ccaatgttgt tgcccatggt tacaccaagg gagatggcct tggtgctgag 540

attattggca cttttgttct tgtctacact gtcttctctg ctactgatgc caagagaaat 600

gctagagact cccatgtccc tattctggct cctctcccaa ttggatttgc agtgttcttg 660

gttcatttgg ctacaatccc tattacaggc accggcatta accctgctag aagcctaggc 720

gctgccattg tctacaacaa agaacatgca tgggatgatc attggatttt ctgggtggga 780

ccattcattg gagctgcact tgctgcctta taccaccaag ttatcataag agccattcca 840

ttcaagagtg gaaactga 858

<210> 2

<211> 41

<212> DNA

<213> Artificial sequence

<400> 2

gagaacacgg gggactctag aatggagggg aaagaagagg a 41

<210> 3

<211> 41

<212> DNA

<213> Artificial sequence

<400> 3

gcccttgctc accatggtac cgtttccact cttgaatgga a 41

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<400> 4

aagatggatt gcacgcaggt 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

agcttcaaag cagatccaag 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<400> 6

agttcatttc atttggagag 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

ccagcaccac agattgcacc 20

Claims

1. Aquaporin gene SlPIP1;2 in improving the resistance of tomatoes to continuous cropping soil;

The aquaporin gene SlPIP1;2 is shown as SEQ ID NO. 1;

The improvement of the resistance of tomatoes to facility continuous cropping soil is as follows: reducing root knot index of tomato root knot nematode infection.

2. Contains aquaporin gene SlPIP1;2, the application of the recombinant expression vector, the transgenic cell line or the engineering bacteria in improving the resistance of tomatoes to continuous cropping soil of facilities;

The aquaporin gene SlPIP1;2 is shown as SEQ ID NO. 1;

The improvement of the resistance of tomatoes to facility continuous cropping soil is as follows: reducing root knot index of tomato root knot nematode infection;

The recombinant expression vector is constructed by taking pCAMBIA2301 as a starting plasmid, and is added with a 35S promoter and GFP.