CN112941050B - Chimonanthus nitens GDSL lipase gene CpGLIP1 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of plant gene functions, and particularly relates to a chimonanthus nitens GDSL lipase gene CpGLIP1 and application thereof. The invention provides a chimonanthus nitens CpGLIP1 gene, and an amino acid sequence of an encoding protein of the chimonanthus nitens CpGLIP1 gene is shown as SEQ ID No. 1. The invention clones and obtains the chimonanthus nitens CpGLIP1 gene for the first time, performs expression characteristic analysis and prokaryotic expression, and verifies the function of the gene in a plant (arabidopsis thaliana) by a transgenic technology. The chimonanthus nitens CpGLIP1 gene is overexpressed in arabidopsis thaliana to improve the drought tolerance and cold resistance of arabidopsis thaliana, and the result provides a basis for researching the abiotic stress response of chimonanthus nitens and provides a reference for improving the abiotic stress tolerance of ornamental plants.

Description

Wintersweet GDSL lipase gene CpGLIP1 and application thereof

Technical Field

The invention belongs to the technical field of plant gene functions, and particularly relates to a chimonanthus nitens GDSL lipase gene CpGLIP1 and application thereof.

Background

Chimonanthus praecox (L.) Link is deciduous tree and shrub of Chimonanthus of Ceramiaceae, and is endowed with quality of aversion to severe cold, rigid and persistent, since ancient times, because it blooms in cold winter. The wintersweet is pruning resistant, barren resistant and stress resistant, and has strong tolerance to low temperature and drought. At present, the molecular biology research on wintersweet stress resistance has been advanced to a certain extent, but the overall situation is in the basic stage, the molecular mechanism of wintersweet for resisting various adversity stresses and the specific action path of related genes are not clear, and more related genes need to be excavated for further research.

GDSL lipases are a family of recently discovered lipolytic enzymes, are named because they have a conserved GDSL motif (Gly-Asp-Ser-Leu) at the N' -end of their proteins, have broad substrate specificity and region specificity, and are capable of hydrolyzing a variety of substrates such as thioesters, aryl esters, phospholipids, and amino acids. GDSL family members have wide functions and can play roles in various aspects such as plant morphological development, biotic/abiotic stress reaction, seed oil metabolism and the like. The research of the plant GDSL lipase is started later, the research on the GDSL participating in the abiotic stress is less, and the research report of the chimonanthus nitens GDSL lipase does not exist at present, and the research and application potential is certain.

Disclosure of Invention

The invention aims to provide a new choice for improving the stress resistance of plants.

The invention provides a chimonanthus nitens GDSL lipase gene CpGLIP1, and an amino acid sequence of a coding protein of the chimonanthus nitens GDSL lipase gene CpGLIP1 is shown as SEQ ID No. 1.

Specifically, the nucleotide sequence of the chimonanthus nitens GDSL lipase gene CpGLIP1 is shown as 44 th-1138 th bases of SEQ ID No. 2.

The invention also provides application of the GDSL lipase gene CpGLIP1 in plant adversity stress regulation.

Specifically, the plant is wintersweet, arabidopsis thaliana or poplar.

Specifically, the adversity stress is low temperature, high temperature, drought, high salinity or hormone treatment.

Further, the hormone is at least one of jasmonic acid JA, gibberellin GA3 or abscisic acid ABA.

Specifically, the low temperature is-4 to 4 ℃.

Specifically, the high temperature is 42 ℃.

Specifically, the drought is treatment by 30% of PEG6000 in mass fraction.

Specifically, the high salt is 150mM NaCl.

Specifically, the concentration of jasmonic acid JA in the treatment is 100 μ M.

Specifically, the concentration of the gibberellin GA3 in the treatment process is 10 mu M.

Specifically, the concentration of the abscisic acid ABA in the treatment is 50 mu M.

The invention firstly obtains the regulation and control of the expression of the gene by various stresses (high temperature, low temperature, hormone, high salt, drought and the like) through the expression pattern analysis. Further, functional verification is carried out through transgenic overexpression, and the expression of the gene is regulated and controlled by low temperature and drought. This also indicates that gene expression is only measured at the transcriptional level and that ultimately there may not be a direct correlation to transgenic plants or that changes in expression of individual genes may not be sufficient to affect all stress tolerance, but only that they are observed to affect cold and drought tolerance. Therefore, further functional verification of its function is required.

The invention has the beneficial effects that: the invention clones and obtains the chimonanthus nitens CpGLIP1 gene for the first time, performs expression characteristic analysis and prokaryotic expression, and verifies the function of the gene in a plant (arabidopsis thaliana) by a transgenic technology. The chimonanthus nitens CpGLIP1 gene is overexpressed in arabidopsis thaliana to improve the drought tolerance and cold resistance of arabidopsis thaliana, and the result provides a basis for researching the abiotic stress response of chimonanthus nitens and provides a reference for improving the abiotic stress tolerance of ornamental plants.

Drawings

FIG. 1 shows a multiple sequence alignment of a protein encoded by CpGLIP1 with GDSL proteins from other species; the sequence was derived from NCBI (https:// www.ncbi.nlm.nih.gov /): arabidopsis thaliana (NP-187079.1), tobacco (Nicotiana tabacum, XP-016440540.1), Populus tomentosa (Populus trichocarpa, XP-002325360.1), rice (Oryza sativa Japonica Group, XP-015636925.1), plum blossom (Prunus mume, XP-008218370.1), tomato (Solanum lycopersicum, NP-001305416.1), Pannelli tomato (Solanum pennellii, XP-015073680.1), orchid (Apotasia senzzhenica, PKA66465.1), potato (Solanum tuberosum, XP-006340481.1).

FIG. 2 is a dendrogram of CpGLIP1 protein with Arabidopsis GDSL protein.

FIG. 3 is an analysis of the expression of the CpGLIP1 gene in different tissues of Chimonanthus praecox (A) and at different stages of flower development (B).

FIG. 4 is an analysis of the expression of the CpGLIP1 gene under different abiotic stresses.

FIG. 5 is an analysis of the expression of the CpGLIP1 gene under different hormone treatments.

FIG. 6 shows the SDS-PAGE detection of CpGLIP1 fusion protein induced expression. M: protein Maker; 1: pET32a (+) empty vector was not induced; 2: pET32a (+) empty vector induction; 3: uninduced pET32a (+) -CpGLIP1 recombinant protein; 4: the induced pET32a (+) -CpGLIP1 recombinant protein.

FIG. 7 is an SDS-PAGE electrophoretic detection of CpGLIP1 fusion protein induced expression optimization. A: IPTG induction time optimization (0h, 1h, 2h, 4h and 6 h); b: IPTG induction temperature optimization (20 ℃, 28 ℃, 37 ℃); IPTG induction concentration optimization (0mM, 0.1mM, 0.5mM, 1mM, 2mM, 10 mM).

FIG. 8 is a soluble SDS-PAGE electrophoretic detection of CpGLIP1 gene prokaryotic expression products. M: protein Maker; 1: total protein after induction of pET32a (+) -CpGLIP 1; 2: pET32a (+) -CpGLIP1 induced supernatant; 3: pET32a (+) -CpGLIP1 induced precipitation; 4: total protein after induction with pET32a (+) empty vector; 5: pET32a (+) empty vector induced supernatant; 6: pET32a (+) empty vector induced precipitation; 7: uninduced pET32a (+) empty vector; 8: uninduced pET32a (+) -CpGLIP 1.

FIG. 9 is a relative expression analysis of the CpGLIP1 gene in transgenic Arabidopsis thaliana. 1-11: different single lines of CpGLIP1 transgenic Arabidopsis; WT: wild type Arabidopsis thaliana.

FIG. 10 shows germination rates (A) and normal growth rates (B) of wild type and transgenic Arabidopsis seeds treated with 0% and 20% PEG. OE3, OE8, 0E 7: three CpGLIP1 overexpression transgenic lines with high, medium and low expression levels; WT: wild type Arabidopsis thaliana.

FIG. 11 is the morphology of wild type and transgenic Arabidopsis seedlings grown under 20% PEG treatment. A: strain morphology comparison, OE3, OE8, 0E 7: three CpGLIP1 overexpression transgenic lines with high, medium and low expression, WT: wild type Arabidopsis thaliana. (ii) a B: normal growing seedling morphology (a), stunted growth seedling morphology (b).

FIG. 12 is a phenotypic characterization of CpGLIP1 overexpressing transgenic lines versus wild-type Arabidopsis under drought stress. OE3, OE8, 0E 7: three CpGLIP1 overexpression transgenic lines with high, medium and low expression, WT: wild type Arabidopsis thaliana.

FIG. 13 is a statistics of plant survival rates of CpGLIP1 overexpression transgenic lines and wild type Arabidopsis thaliana under drought stress. OE3, OE8, 0E 7: three CpGLIP1 overexpression transgenic lines with high, medium and low expression, WT: wild type Arabidopsis thaliana.

FIG. 14 shows the results of determining the relative conductivities of transgenic plants and wild type Arabidopsis thaliana under normal conditions and drought stress conditions. OE3, OE8, 0E 7: three CpGLIP1 overexpression transgenic lines with high, medium and low expression, WT: wild type Arabidopsis thaliana.

FIG. 15 shows the water loss rate of excised leaves of CpGLIP1 overexpressing transgenic lines and wild type Arabidopsis thaliana. OE3, OE8, 0E 7: three CpGLIP1 overexpression transgenic lines with high, medium, and low expression, WT: wild type Arabidopsis thaliana.

FIG. 16 is a phenotypic characterization of CpGLIP1 overexpressing transgenic lines versus wild-type Arabidopsis under freezing stress at-4 ℃. OE3, OE8, 0E 7: three CpGLIP1 overexpression transgenic lines with high, medium and low expression, WT: wild type Arabidopsis thaliana.

FIG. 17 is a survival statistic of CpGLIP1 overexpressing transgenic lines versus wild type Arabidopsis under freezing stress at-4 ℃.

FIG. 18 is a measurement of physiological indices of CpGLIP1 overexpression transgenic lines and wild type Arabidopsis thaliana under normal conditions and low temperature conditions of 4 ℃. A: (ii) proline content; b: malondialdehyde content; c: chlorophyll SPAD value. OE3, OE8, OE 7: three CpGLIP1 overexpression transgenic lines with high, medium and low expression, WT: wild type Arabidopsis thaliana.

FIG. 19 is a PCR assay of CpGLIP1 gene in a partially transgenic poplar. M is DNA molecular weight standard DL 5000; 1-8: PCR detection of CpGLIP1 gene in part of transgenic poplar; 9: positive control (pGWB551-CpGLIP1 recombinant plasmid); 10: wild type poplar.

FIG. 20 is a relative expression analysis of the CpGLIP1 gene in transgenic poplar. 1-13: each CpGLIP1 transgenic poplar single plant; WT: wild type poplar.

FIG. 21 shows the water loss rate of isolated leaves of CpGLIP1 overexpressed transgenic poplar and wild-type poplar.

Detailed Description

Example 1 isolation of the Chimonanthus praecox CpGLIP1 Gene

Extracting the total RNA of the young leaves of the Chimonanthus praecox by a Trizol method, and synthesizing cDNA through reverse transcription. Based on the sequence fragments known in the Chimonanthus praecox transcriptome database (provided by the research center for flower engineering technology in Chongqing), primers were designed at both ends of the largest ORF frame using software Primer 5.0 for PCR amplification, and the sequences of the primers were as follows:

GLIP1-ORF-F：5'-gatcaagtgaccttagcccatc-3'(SEQ ID No.3)

GLIP1-ORF-R：3'-aggagatgagatgatactgaggc-5'(SEQ ID No.4)

the maximum ORF fragment of chimonanthus nitens CpGLIP1 is amplified by taking chimonanthus nitens cDNA as a template, and a PCR reaction system and reaction conditions are as follows: ddH ₂ O17.8. mu.L, 10 XHiFi Taq PCR Buffer II 2.5. mu.L, dNTP (10mM) 1.5. mu.L, GLIP1-ORF-F (10. mu.M) 1. mu.L, GLIP1-ORF-R (10. mu.M) 1. mu.L, Chimonanthus praecox cDNA 1. mu.L, TaKaRa Ex Taq ^TM 0.2. mu.L. Pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 1min for 10s, 30 cycles; extension at 72 ℃ for 10 min.

And recovering the PCR product, connecting the recovered PCR product to a pMD19-T vector, transforming escherichia coli competent Top10, and picking and sequencing recombinants.

The cDNA sequence of the chimonanthus nitens CpGLIP1 gene is shown as 44 th to 1138 th bases of SEQ ID No. 2.

The cDNA total length of the CpGLIP1 gene is 1457bp, comprises a 1095bp maximum open reading frame and encodes 364 amino acids; the balance is a non-coding region; the translation initiation site is at base 44. The chimonanthus nitens CpGLIP1 gene coding protein (SEQ ID No.1) has the highest similarity with GDSL protein from Jatropha curcas (Jatropha curcas, XP _012089388.1) and castor (Ricinus communis, XP _002522020.1), and the similarity reaches 80 percent and 78 percent. The CpGLIP1 protein has 4 amino acid residues Ser (S serine), Gly (G glycine), Asn (N aspartic acid) and His (H histidine) which play important roles in enzyme catalysis, the 4 conserved residues are respectively positioned in 4 conserved domains I, II, III and V, and GDSL-motif (GDSxxDxG) is positioned in the conserved domain I and accords with the structural characteristics of GDSL-like lipase (figure 1). The clade was constructed with the arabidopsis GDSL protein (fig. 2), showing that CpGLIP1 was clustered with CUTIN SYNTHASE CUS2(CUTIN SYNTHASE2), and AtLTL1 associated with salt stress, belonging to the second clade.

Note: RNA extraction kit, gel recovery kit and plasmid extraction kit used for experiments are purchased from Tiangen Biotechnology technology (Beijing) Co., Ltd; ExTaq high-fidelity polymerase,

RT-PCR Kit reverse transcription kits were purchased from TaKaRa, Japan (Dalian); the Escherichia coli competence Top10 was preserved by Chongqing flower engineering technology research center.

Example 2 analysis of the Gene expression characteristics of Chimonanthus praecox CpGLIP1

1 Collection of different stress treatment materials of different wintersweet development stages, tissues and seedlings thereof

The root, stem, cotyledon, young leaf, mature leaf, inner perianth sheet, outer perianth sheet, stamen and pistil of wintersweet are adopted to be quick-frozen by liquid nitrogen and then stored in a refrigerator at minus 80 ℃ for later use. The wintersweet flowers in the germination stage, the bud stage, the petal exposing stage, the initial blooming stage, the full blooming stage and the decay stage are respectively adopted, and samples of three parallel experiments in each stage are frozen by liquid nitrogen and then stored in a refrigerator at minus 80 ℃. Selecting healthy Chimonanthus nitens seedlings with consistent size cultured in greenhouse, and performing low temperature (4 deg.C), high temperature (42 deg.C), drought (30% PEG6000), high salt (150mM NaCl) treatment, JA (100 μ M), GA ₃ (10. mu.M), ABA (50. mu.M) hormone treatment. Placing the seedlings treated at low temperature into a low-temperature artificial climate incubator at 4 deg.C, and culturingPrepared NaCl and PEG solution is used to irrigate Chimonanthus praecox seedlings, JA and GA ₃ And ABA spraying wintersweet seedling leaves for abiotic stress, setting 5 time gradients of 0h, 2h, 6h, 12h, 24h and the like for each treatment, and designing three repeated numbers for each treatment.

2 extraction of Chimonanthus praecox total RNA and synthesis of cDNA first chain

The RNA extraction adopts a Trizol reagent method, and the specific operations are as follows:

(1) fresh plant tissues are taken and fully ground in liquid nitrogen, about 0.5mg is taken and put into a 1.5mL RNase-Free centrifuge tube, and 600 mu L Trizol is added and vortexed for 1min to uniformly mix the mixture by oscillation.

(2) And (3) standing the liquid after cracking at room temperature for 10min to fully separate nucleic acid from nucleoprotein.

(3) Add 120. mu.L of chloroform (20%), cover the tube, vortex for 30s, and let stand at room temperature for 10 min.

(4) Centrifuging at 12000rpm at 4 ℃ for 15min, dividing the sample into 3 layers, wherein the lower layer is organic, the middle layer and the upper layer are anhydrous, RNA mainly exists in a water phase, and transferring the water phase into a new RNase-Free centrifuge tube.

(5) 2 times of absolute ethyl alcohol (about 400 mu L) is slowly added into the transfer solution and mixed evenly.

(6) The mixture was centrifuged at 12000rpm for 10min, and the supernatant was aspirated to leave a white precipitate.

(7) Adding 700-800 mu L of 75% ethanol into the precipitate, rinsing, resuspending, standing for 2min, centrifuging at 12000rpm for 1min, sucking the supernatant and leaving a white precipitate.

(8) Repeat 7 times.

(9) And blowing the centrifuge tube with the precipitate on a super clean bench for 15min until the precipitate becomes transparent.

(10) Adding 30-50 mu L of RNase-Free ddH ₂ And O, standing for 5min, and carrying out electrophoresis detection.

Synthesis of first Strand cDNA of Chimonanthus praecox, first Strand cDNA was synthesized using PrimeScript RT-PCR Kit reverse transcription and extracted total RNA.

(1) Reaction for removing genomic DNA: 5 XgDNA Eraser Buffer 2. mu.L, gDNA Eraser 1. mu.L, Chimonanthus praecox total RNA 1. mu.L, RNase-free ddH ₂ O6. mu.L. Reacting at 42 deg.C for 5min to obtainObtaining reaction liquid I.

(2) Reverse transcription reaction: reaction solution I10. mu.L, 5 XPrimeScript Buffer 4. mu.L, Prime Script Enzyme Mix I1. mu.L, RT Primer Mix 1. mu.L, RNase-free ddH ₂ O4. mu.L. The reaction was carried out at 37 ℃ for 15min and at 85 ℃ for 5 s. The reaction product was stored in a refrigerator at-20 ℃.

3 fluorescent quantitation

(1) Design of real-time fluorescent quantitative PCR primer

The wintersweet Actin and Tublin genes are selected as double internal reference genes, specific primers required by amplification of internal reference genes CpAutin and CpTublin and a target gene CpGLIP1 are designed by using Primer Premier 6.0 software, and the sequences of the primers are shown in Table 1.

TABLE 1 primers

(2) CpGLIP1 gene real-time fluorescent quantitative PCR

And (3) carrying out real-time fluorescent quantitative PCR analysis on the expression conditions of the CpGLIP1 gene in different tissues and different flower development periods of the Chimonanthus praecox and seedlings under different abiotic stress treatment by taking cDNA obtained by reverse transcription as a template. Reaction system: 2 XSssoFastEva Green Supermix 5. mu.L, Primer-F (10. mu.M) 0.5. mu.L Primer-R (10. mu.M) 0.5. mu.L cDNA template 0.5. mu.L RNase Free dH ₂ O3.5 μ L; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s58 ℃ for 5s, and 39 cycles. Collecting fluorescence signals after each cycle, performing dissolution curve analysis at 65-95 ℃ after the reaction is finished, and analyzing the amplification specificity.

Data obtained from the assay were analyzed by Bio-Rad Manager ^TM Software (Version 1.1) analysis, using 2 ^-△△CT The method calculates the relative expression of the CpGLIP1 gene in different materials.

The expression mode of CpGLIP1 in the chimonanthus nitens is explored, and the CpGLIP1 has strong space-time expression specificity, has the highest expression level in the young leaves of the chimonanthus nitens, is not expressed basically in cotyledons and mature leaves, and has only trace expression in other tissues. At each stage of floral development, CpGLIP1 was expressed in the highest amount only during the emergence phase and only in trace amounts at other stages (fig. 3).

Further analyzing the expression patterns of the CpGLIP1 gene in abiotic stress and hormone, the results show that the gene can be induced to express by low temperature, high temperature, PEG, NaCl, JA and GA 3. After low-temperature PEG6000 simulated drought treatment, the expression level of CpGLIP1 is in an up-regulation trend. Under the PEG6000 treatment, the CpGLIP1 gene is quickly up-regulated in a short time, and although the expression is reduced in 6 hours, the expression is continuously increased subsequently, and the expression is maximized in 24 hours. During high temperature treatment, the expression of CpGLIP1 was on a continuous downward trend; in the high salt treatment CpGLIP1 also showed a downward expression trend over time, but subsequently rose back (fig. 4). After JA, GA3 treatment, CpGLIP1 exhibited up-regulated expression to varying degrees over time and fluctuated expression after ABA treatment (FIG. 5).

Note: the Trizol reagent used in this step is,

RT-PCR Kit reverse transcription Kit purchased from TaKaRa, Japan (Dalian); SsoFast ^TM The EvaGreen Supermix fluorescent quantitation kit was purchased from Bio-Rad, USA; the main equipment used is NANO DRAC2000 micro nucleic acid analyzer (U.S. Thermo), Bio-Rad CFX96 fluorescent quantitative PCR instrument, etc.

Example 3 prokaryotic expression analysis of the Chimonanthus praecox CpGLIP1 Gene

In order to verify whether the gene can be correctly translated into lipase protein to perform the function after transcription, a prokaryotic expression experiment is firstly carried out.

1 prokaryotic expression vector construction

A pair of specific primers is set according to the maximum ORF frame of CpGLIP1 gene, and the restriction sites BamH I and Hind III of the downstream primers and corresponding protective bases are added respectively.

The primer sequences are as follows:

PG-CpGLIP1-F:5'-cgggatccagacccatggccggttctt-3' (SEQ ID No.11), the restriction sites are underlined,

PG-CpGLIP1-R:5'-cccaagcttaggagatgagatgatactgaggc-3' (SEQ ID No.12) is underlined to indicate the cleavage site.

And carrying out PCR amplification by using plasmid DNA of a clone positive colony of the CpGLIP1 gene T vector as a template, wherein the plasmid DNA is correctly identified by sequencing. The PCR amplification system and conditions were as follows: ddH ₂ O17.8. mu.L, 10 XPCR Easy Taq Buffer 2.5. mu.L, dNTP (2mM) 2.0. mu.L, PG-CpGLIP1-F (10. mu.M) 1. mu.L, PG-CpGLIP1-R (10. mu.M) 1. mu.L, plasmid template 1. mu.L, ExTaqDNA polymerase 0.2. mu.L. Pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min for 10s, 28 cycles; extension at 72 ℃ for 10 min.

And (3) carrying out electrophoresis detection on the PCR product and 1% agarose gel, recovering the PCR product, connecting with a cloning vector pMD19-T, transforming the connecting product into escherichia coli Top10, and after PCR identification of the bacterial liquid, selecting positive bacterial liquid for sequencing. According to the sequencing results, the recombinant plasmid ligated with the gene of interest, CpGLIP1, was designated CpGLIP 1-T. Extracting recombinant plasmid and pET32a carrier plasmid, using BamHI and HindIII to respectively enzyme cut CpGLIP1-T plasmid and pET32a (+) carrier, the enzyme cut system is as follows: green Buffer 2.5. mu.L, BamHI 0.5. mu.L, Hind III 0.5. mu.L, CpGLIP1-T/pET32a (+) 15. mu.L, ddH ₂ O 6.5μL。

Enzyme digestion is carried out at 37 ℃ for more than 30min, inactivation is carried out at 80 ℃ for 10min, and agarose gel electrophoresis is carried out to recover CpGLIP1-T small fragment and pET32a (+) carrier large fragment in enzyme digestion products. Ligation of the target fragment to the expression vector was performed overnight at 4 ℃ according to the instructions of T4 ligase from Fermentas as follows: 10 XT ₄ 2.5. mu.L of DNA 9 Buffer, about 0.3pmol of Insert DNA, about 0.03pmol of vector DNA, 1. mu.L of T4DNA 9, plus ddH ₂ O to a total volume of 25. mu.L.

And (3) transforming the ligation product, detecting by PCR and extracting plasmids. The extracted plasmid was verified by double digestion with BamHI and HindIII. And (3) sending the positive plasmid with correct PCR detection and double enzyme digestion identification to a biological company for sequencing, and determining that no basic group mutation exists after comparison to obtain the prokaryotic expression recombinant plasmid of the CpGLIP1 gene, wherein the prokaryotic expression recombinant plasmid is named as pET32a (+) -CpGLIP 1.

2 induced expression, condition optimization and solubility analysis of recombinant protein

2.1 inducible expression and electrophoretic detection

(1) The positive recombinant plasmid pET32a (+) -CpGLIP1 and the vector plasmid pET32a (+) are transformed into prokaryotic expression strain BL21(DE3) chemical ly component Cell respectively, inoculated on Amp resistant LB plate and cultured overnight at 37 ℃;

(2) selecting positive monoclonal, inoculating in LB liquid culture medium (containing Amp 50mg/L), shaking at 37 deg.C and 180rpm overnight;

(3) the culture is inoculated into LB liquid medium (containing Amp 50mg/L) at a ratio of 1: 100(v/v) for amplification culture, shake-cultured at 37 ℃ until OD600 is 0.6, and added with 1mM of isopropylthio-D-galactoside (IPTG) for induction. Meanwhile, a bacterial solution without an inducer is set as a control, and after the culture is continued for 4 hours, 1mL of each of pET32a (+) -CpGLIP11/BL21 bacterial solution and pET32a (+)/BL21 bacterial solution is collected (meanwhile, 1mL of each bacterial solution without the inducer is collected);

(4) centrifuging the sample, collecting thallus, adding 100 μ L PBS buffer solution, resuspending thallus, adding 4 × SDS-PAGE sample buffer solution at a ratio of 1:3(v/v), boiling in boiling water bath for 10min, and centrifuging at 12,000rpm for 2 min. 10 μ L of the supernatant was analyzed by 12% SDS-PAGE.

SDS-PAGE electrophoresis:

(1) preparing glue: preparing 12% separation glue according to the proportion, quickly and uniformly mixing, filling the mixture into a glue making groove, adding absolute ethyl alcohol to remove air bubbles, and flattening the glue surface; standing for 40min to completely solidify. After solidification, the absolute ethanol is poured out and the residual absolute ethanol is sucked dry by filter paper. Preparing 5% of concentrated glue according to a proportion, pouring the concentrated glue into a position which is 0.5cm away from the edge after the concentrated glue is quickly and evenly mixed, immediately inserting a comb with the thickness of 1.0mm into the position to avoid air bubbles, vertically placing the comb until the concentrated glue is solidified, pulling out the comb, and flushing a pore passage with electrolyte.

(2) Sample application: mu.L of protein sample is taken, 33 mu.L of 4 xSDS-PAGE loading buffer is added, boiling water bath is carried out for 10min, cooling to room temperature and then centrifugation is carried out for 10min at 12,000rpm, and 10 mu.L of supernatant is absorbed and loaded.

(3) Electrophoresis: pouring 1 Xelectrophoresis buffer solution into the electrophoresis tank, and keeping the voltage constant at 80V when the bromophenol blue indicator is used for concentrating gel; when the indicator is 0.5cm away from the bottom of the plate, the electrophoresis is stopped.

(4) Dyeing: and putting the gel into a culture dish containing a staining solution, and placing the gel on a low-speed shaking table for staining for 1 h.

(5) And (3) decoloring: rinsing the dyed gel with distilled water for several times, putting the gel into a culture dish filled with a destaining solution, placing the gel on a low-speed shaking table for destaining, and replacing the destaining solution for 1-2 times in the midway until the protein bands are clear. Observing and taking pictures on a film viewing lamp.

2.2 optimization of CpGLIP1 recombinant protein Induction expression conditions

(1) Induction time optimization

Activating Escherichia coli BL21(DE3) containing recombinant plasmid pET32a (+) -CpGLIP1, inoculating the culture into 50mL LB liquid medium (containing Amp 50mg/L) at a ratio of 1: 100(v/v) for amplification culture, performing shake culture at 37 ℃ until OD600 is 0.6 (about 3h), adding 1mM IPTG for induction, continuing induction culture, and collecting 1mL bacterial liquid at 0h, 1h, 2h, 4h and 6h respectively; centrifuging the samples collected at different times to obtain thallus, adding 100 μ L PBS buffer (phosphate buffer) to resuspend thallus, adding 4 xSDS-PAGE sample buffer at a ratio of 1:3(v/v), boiling in boiling water bath for 10min, and centrifuging at 12,000rpm for 2 min. 10 μ L of the supernatant was analyzed by 12% SDS-PAGE.

(2) Induction temperature optimization

Inoculating the culture into 50mL LB liquid culture medium (containing Amp 50mg/L) at a ratio of 1: 100(v/v), performing amplification culture, performing shake culture at 37 deg.C until OD600 is 0.6 (about 3h), adding 1mM IPTG for induction, and collecting 1mL bacterial liquid at 20 deg.C, 28 deg.C, 37 deg.C for 6h respectively; centrifuging samples collected under different temperature conditions to obtain thallus, adding 100 μ L PBS buffer solution to resuspend thallus, adding 4 xSDS-PAGE sample buffer solution at a ratio of 1:3(v/v), boiling in boiling water bath for 10min, and centrifuging at 12,000rpm for 2 min. 10 μ L of the supernatant was analyzed by 12% SDS-PAGE.

(3) IPTG inducer concentration optimization

Inoculating the culture into 50mL LB liquid medium (containing Amp 50mg/L) at a ratio of 1: 100(v/v), performing amplification culture, and performing shake culture at 37 deg.C until OD600 is 0.6 (about 3 h); the above-mentioned bacterial suspension was divided into 4 bottles of 10mL each, and IPTG was added thereto to give final concentrations of 0mM, 0.1mM, 0.5mM, 1.0mM, 2mM and 10mM, respectively, for induction expression for 6 hours. Collecting 1mL of bacterial liquid with different concentrations of an inducer; centrifuging the collected sample to obtain thallus, adding 100 μ L PBS buffer solution to resuspend thallus, adding 4 xSDS-PAGE loading buffer solution at a ratio of 1:3(v/v), boiling in boiling water bath for 10min, and centrifuging at 12,000rpm for 2 min. 10 μ L of the supernatant was analyzed by 12% SDS-PAGE.

2.3 solubility analysis of prokaryotic expression product of CpGLIP1 Gene

(1) BL21(DE3) strain containing the empty vector plasmid pET32a (+) and the recombinant plasmid pET32a (+) -CpGLIP1 was added to 10mL of LB liquid medium (containing 50mg/L Amp) and cultured overnight.

(2) Inoculating into 100mL LB liquid medium (containing 50mg/L Amp) at a ratio of 1: 100(v/v), performing amplification culture, performing shake culture at 37 deg.C and 200rpm until logarithmic phase (OD600 reaches about 0.6), adding IPTG to final concentration of 0.1mM, and performing shake induction at 37 deg.C and 180rpm for 6 h. Blank and negative control, namely BL21(DE3) bacterial liquid containing no-load plasmid pET32a (+) and recombinant plasmid pET32a (+) -CpGLIP1 without IPTG, are arranged at the same time, and the shaking culture is carried out at the same time.

(3) After induction, respectively collecting induction bacteria liquid, centrifuging at 4 ℃ and 12,000rpm for 10min, discarding the supernatant, inverting the centrifugal tube on absorbent paper, blotting, adding PBS buffer solution to resuspend the thalli, centrifuging at 4 ℃ and 12,000rpm for 10min, and discarding the supernatant (repeating for 1 time). Finally, 6-8 ml of PBS buffer solution is added to resuspend the thalli, and the thalli are preserved at the temperature of-80 ℃ for later use.

(4) The non-induced empty vector and recombinant plasmid were centrifuged at 12,000rpm for 10min at 4 ℃ for 1mL each, and the pellet was resuspended in 1mL PBS buffer, then 21. mu.L of the pellet was aspirated, added to 7. mu.L of 4 XSDS-PAGE loading buffer, and boiled for 5min to serve as blank and negative controls.

(5) And (4) repeatedly freezing and thawing the bacterial liquid obtained in the step (3) for 3 times by using liquid nitrogen, adding lysozyme to 1mg/mL, and incubating for 30min on ice.

(6) And (3) maintaining the lysate in an ice bath, carrying out ultrasonic crushing with the output power of 200-300W for 3s and the interval of 5s for 10min, and repeating for 2-3 times according to the crushing condition.

(7) After disruption, 200. mu.L of total protein solution was collected, the remaining disrupted sample was centrifuged at 4 ℃ and 12,000rpm for 30min, 90. mu.L of supernatant was collected to prepare an electrophoretic supernatant, and the remaining supernatant was transferred to a clean centrifuge tube and stored at-20 ℃. The pellet was resuspended in 5min PBS buffer, at 4 deg.C, 12,000rpm, and centrifuged for 10min (2 replicates). Finally, resuspending the precipitate with 2-4 mL of PBS buffer solution, and taking 90 mu L of the prepared sample on electrophoresis.

(8) Adding 4 xSDS-PAGE loading buffer solution into the collected total protein solution, supernatant and precipitate at a ratio of 1:3(v/v), boiling in a boiling water bath for 10min, and centrifuging at 12,000rpm for 2 min. 10 μ L of the supernatant was analyzed by 12% SDS-PAGE.

The experimental result shows that the fusion protein pET32a (+) -CpGLIP1 has obvious specific expression at the position of about 58kD (figure 6); the expression level of the fusion protein reaches the maximum within 2h (FIG. 7A), the difference between 4h and 2h is not large, and the expression level is reduced within 6 h; under a certain time and inducer concentration, the expression level of the fusion protein increases along with the increase of the induction temperature, and the expression level is the highest at 37 ℃ (FIG. 7B); under the induction condition of a certain time and temperature, the fusion protein has a small amount of background expression without an inducer, the fusion protein can be expressed in a large amount by adding the inducer, and the influence of the concentration of the inducer on the expression amount is not large (figure 7C).

pET32a (+) -CpGLIP1 fusion protein was specifically expressed in the pellet after disruption and centrifugation after IPTG induction, and not expressed in the supernatant. Indicating that the target protein is mainly present in the precipitate, and the pET32a (+) -CpGLIP1 fusion protein is preliminarily judged to be present in an insoluble inclusion body form in Escherichia coli (FIG. 8).

Note: prokaryotic expression strain BL21(DE3) chemical ly Cell used in the experiment was purchased from Beijing Quanji gold Biotechnology, Inc., and prokaryotic expression vector pET32a (+) was provided by Chongqing flower engineering technology research center. The apparatus mainly comprises SCIENTA-II D type ultrasonic cell crusher sound insulation box (Ningbo Xinzhi Biotechnology GmbH), POWER PAC 3000 electrophoresis apparatus.

Example 4 Agrobacterium-mediated transformation of Arabidopsis thaliana with CpGLIP1 Gene

Construction of 1 chimonanthus nitens CpGLIP1 gene plant overexpression vector

Gateway was used to construct a CpGLIP1 gene plant overexpression vector. The specific primer of CpGLIP1 spanning the maximum ORF frame was designed, and attB1 and attB2 site sequences were added to the 5' ends of the upstream and downstream primers, respectively. The primer sequences are as follows:

attB1-CpGLIP1-F：5’-ggggacaagtttgtacaaaaaagcaggctgatcaagtgaccttagcccatc-3' (SEQ ID No.13) is underlined attB1,

attB2-CpGLIP1-R：5’-ggggaccactttgtacaagaaagctgggtaggagatgagatgatactgaggc-3' (SEQ ID No.14) is underlined as attB2 site.

Carrying out PCR amplification by taking the plasmid of the CpGLIP1 gene obtained by extraction as a template, wherein the amplification system and conditions are as follows: ddH ₂ O17.8. mu.L, 10 XPCR Easy Taq Buffer 2.5. mu.L, dNTP (2mM) 2.0. mu.L, attB1-CpGLIP1-F (10. mu.M) 1. mu.L, attB2-CpGLIP1-R (10. mu.M) 1. mu.L, plasmid template 1. mu.L, ExTaq DNA polymerase 0.2. mu.L. Pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 1min for 10s, 30 cycles; extension at 72 ℃ for 10 min.

After the PCR product was electrophoresed on a 1% agarose gel, the target fragment was recovered. Extracting plasmid of entry vector pDONR221

The BPClonase TM II Enzyme Mix instructions perform the BP reaction of the fragment of interest with the entry vector. The reaction system is as follows: 1-7. mu.L of attB-PCR product (. gtoreq.10 ng/. mu.L), 1. mu.L of pDONR221 plasmid (150 ng/. mu.L), 2. mu.L of BP Clonase TM II Enzyme Mix, and TE buffer (pH 8.0) to a total volume of 10. mu.L.

And (3) incubating for 4-6h at 25 ℃, adding 1 mu L of protease K solution into the reaction system, mixing uniformly by vortex, and incubating for 10min at 37 ℃ to stop the reaction. Taking 1 microliter BP reaction product to transform into colon bacillus Top10, carrying out positive clone screening on an LB plate containing 50mg/L Kan antibiotic, carrying out PCR identification on bacterial liquid, selecting a single clone to carry out sequencing, and obtaining the entry clone pDONR221-CpGLIP1 successfully when the sequencing is correct.

Extracting the constructed entry cloning vector plasmid and over-expression vector pGWB551 plasmid

LR Clonase TM II Enzyme Mix protocol the LR reaction was performed as follows: pDONR221-CpGLIP1 (50-150 ng/. mu.L) 1-7. mu.L,pGWB551 plasmid (150 ng/. mu.L) 1. mu.L, LR Clonase TM II Enzyme Mix 2. mu.L, TE buffer (pH 8.0) supplemented to a total volume of 10. mu.L.

After incubation for 4-6h at 25 ℃, 1 μ L of protease K solution was added to the reaction system, vortexed and mixed, and incubated at 37 ℃ for 10min to terminate the reaction. And (3) transforming 1 mu L of LR reaction product into escherichia coli Top10, carrying out positive clone screening on an LB plate containing 50mg/L Spec antibiotic, carrying out PCR identification on bacterial liquid, selecting a single clone for sequencing, and successfully constructing the plant over-expression vector pGWB551-CpGLIP1 after the sequencing is correct.

The plasmid pGWB551-CpGLIP1 was extracted and the recombinant plasmid was electrotransferred into Agrobacterium GV 3101.

2pGWB551-CpGLIP1 transformation of Arabidopsis thaliana

(1) Cultivation of Arabidopsis thaliana

Taking a proper amount of arabidopsis thaliana seeds in a 1.5mL centrifuge tube, adding a 16.7% sodium hypochlorite solution for disinfection for 7min, fully oscillating the mixture in the period, and then washing the mixture for 5-6 times by using sterile water. Absorbing seeds by using a pipette, uniformly sowing the seeds on an MS culture medium, vernalizing the MS culture medium in a refrigerator at 4 ℃ for 2-3 days, and then transferring the MS culture medium to an environment with 16 h/8 h of illumination, 2000Lux illumination, 22 ℃ and 70% of air humidity for culture. Transplanting the seedlings into a soil culture substrate (peat: vermiculite: 1) after about 12 days. The initial terminal inflorescence can be cut off during the cultivation process to promote the lateral shoot growth. When the arabidopsis grows to the length of 5cm from the scape, the infection can be carried out.

(2) Method for transforming arabidopsis thaliana by inflorescence infection method

The inoculating needle is dipped with Agrobacterium solution containing pGWB551-CpGLIP1 plasmid, streaked on YEB plate containing corresponding antibiotic (50mg/L Gen, 50mg/L Spec), and cultured in dark at 28 ℃ for 36-48 h.

A single colony was picked up in YEB liquid medium containing the corresponding antibiotic (50mg/L Gen, 50mg/L Spec) and cultured at 28 ℃ for 24 hours with shaking at 200 r.

After the bacterial liquid is detected to be correct by PCR, 500 mul of the bacterial liquid is inoculated in 50ml of YEB liquid culture medium, and the bacterial liquid is subjected to shaking culture at the temperature of 28 ℃ and at the speed of 200r until the OD600 value is 1-1.2.

Transferring the bacterial liquid into a 50ml centrifuge tube, centrifuging for 15min at 5000r, discarding the supernatant, resuspending the precipitate with an infection solution (sterile water + 5% sucrose + 0.5% Silwet L-77), and diluting to OD600 value of about 0.8.

Watering is needed to keep soil moist one day before infection, flowers which are already opened are cut off when the infection is carried out for the first time, and only buds which just appear white are left. Soaking flower buds into the dye solution for about 10s, taking out, placing the flower pot in a paper box (the inner wall of the paper box is sprayed with a spray can in advance), covering the paper box with black cloth to shield light, culturing at a constant temperature of 22 ℃ for 24h, taking out the Arabidopsis, and transferring to a normal growth environment for culturing.

After one week of primary infection, secondary infection is carried out according to the growth state of arabidopsis thaliana so as to improve the transformation efficiency.

(3) Screening and characterization of transgenic Arabidopsis thaliana

Harvesting mature T approximately 1 month after transformation ₀ Seeds were used and dried in an oven at 37 ℃ for about two weeks before being stored in a refrigerator for subsequent experiments.

Will T ₀ And (3) disinfecting the generation transgenic seeds, sowing the seeds on an MS culture medium containing 25mg/L Hyg, performing vernalization in a refrigerator at 4 ℃ for 3d under a dark condition, and then transferring the seeds to a normal environment for culture, wherein transgenic arabidopsis thaliana can grow normally on the culture medium containing Hyg.

Transplanting the normally growing seedlings to obtain several different strains, continuously culturing, and treating the T of each strain ₁ 、T ₂ The generation seeds are continuously screened until the seeds harvested from all the strains are green resistant seedlings after sowing, generally T ₃ The generation seeds are the transgenic homozygous lines.

The DNA of plant leaves of a transgenic line and a wild type line is extracted by using a CTAB method, a pair of specific primers of CpGLIP1 genes is used for PCR identification, and pGWB551-CpGLIP1 plasmid is used as a positive control. The results showed that the T3 transgenic line amplified a band of interest that was consistent in size with the positive control, whereas the wild-type and negative controls failed to amplify the band of interest, indicating that the CpGLIP1 gene had been successfully inserted into the Arabidopsis genome.

(4) Fluorescent quantitative PCR detection of transgenic arabidopsis

Extracting total RNA of transgenic arabidopsis leaves with positive DNA detection, carrying out reverse transcription on the extracted RNA to obtain a cDNA first chain, using the AtActin gene of arabidopsis as an internal reference gene, and detecting the expression quantity of the CpGLIP1 gene in different transgenic line species by adopting real-time fluorescent quantitative PCR. The primer sequences are shown in Table 2:

TABLE 2 primers

The detection results show that the CpGLIP1 gene has different expression degrees in transgenic Arabidopsis thaliana, wherein the expression level of No. 3 is the highest, the expression levels of No.2, No. 4, No. 8 and No. 9 are slightly lower, the expression levels of No.1 and No.11 are the lowest, and the expression of the target gene is not detected in wild Arabidopsis thaliana (FIG. 9). According to the expression conditions of the target gene in different strains, selecting No. 3 with the highest expression quantity, No. 8 with the second expression quantity and No. 7 with the lower expression quantity for subsequent experiments.

Note: for experiments

BP Clonase ^TM II Enzyme Mix、

LR Clonase ^TM II enzymeMix, Proteinase K solution from Invitrogen; silwet L-77 is available from Sigma; SsoFast ^TM The EvaGreen Supermix fluorescent quantitation kit is purchased from Bio-Rad company; the main equipment used is NANO DRAC2000 micro nucleic acid analyzer from Thermo company, CFX96 fluorescent quantitative PCR instrument from Bio-Rad company.

Example 5 functional analysis of transgenic Arabidopsis

1 study of drought stress

(1) MS plate PEG simulation drought stress

Three transgenic T3 generation lines of OE3, medium OE7 and low OE3 with high expression levels, as well as seeds of wild type lines were sown on MS plates containing 0%, 20% PEG6000, and germination and growth were observed. The results show that 20% PEG6000 stress does not affect the germination rate of Arabidopsis seeds (FIG. 10A), but the observation of the subsequent growth state shows that part of the seedlings have extended leaves and can continue to grow normally; some seedlings were severely stressed, the plants were dwarf, yellow and growth was inhibited (FIG. 11). Transgenic lines OE3, OE8 showed approximately 50% of normal seedlings and OE7 showed approximately 30% of normal seedlings, whereas wild type Arabidopsis seedlings were severely stressed (FIG. 10B) with only about 10% of normal seedlings and 20% PEG6000 stress resulted in better growth status of transgenic lines than wild type.

(2) Soil drought stress

And (3) carrying out soil drought treatment on the wild type and the transgenic arabidopsis thaliana which are two weeks old after transplantation for two weeks, observing and photographing to record the phenotype of the wild type and the transgenic arabidopsis thaliana, observing the survival condition of the plants after rehydration for 4 days, photographing, and counting the survival rate. After the treatment, the conductivity of Arabidopsis thaliana was measured.

The phenotype is observed, and the wild type and the transgenic arabidopsis plants have damage phenotypes with different degrees after 12 days of water cut (figure 12), which are expressed as leaf dehydration and yellowing and plant wilting. Wild type and OE7 plants are most severely dehydrated, and leaves almost completely wilt; high and medium expression OE3 and OE8 plants have light wilting degree, and partial leaves are still full of water. After 14 days of water cut-off, a part of wild type and OE7 plants wither, and the rest plants wither to death nearly seriously; plants OE3 and OE8 have a lower wilting degree than wild type plants. The arabidopsis thaliana is subjected to rehydration treatment, part of arabidopsis thaliana cannot be reactivated due to severe water loss, most of plants of OE3 and OE8 are fresh green in leaves and full in water, normal growth can be recovered, the survival rate reaches about 80%, the survival rate of an OE7 strain is second, the wild arabidopsis thaliana is more dead, and the survival rate is lowest (fig. 13). Therefore, under drought stress conditions, the survival rate of the CpGLIP1 transgenic line was higher than that of the wild type.

The relative conductivity was determined, and the conductivity of each line of arabidopsis was increased after drought treatment compared to before treatment, with the relative conductivity of wild type arabidopsis being significantly higher than that of transgenic arabidopsis (fig. 14). From the change amplitude of the relative conductivity, the wild type and 3CpGLIP1 transgenic lines, namely Arabidopsis thaliana, respectively have 12.56%, 8.33%, 9.39% and 11.73% of the increase compared with the respective control, and the increase amplitude of the wild type is larger than that of the transgenic line.

(3) Water loss rate of leaves in vitro

Selecting wild type arabidopsis thaliana and transgenic arabidopsis thaliana with good leaf growth conditions about 3 weeks after transplantation as research objects, taking 5 lotus throne leaves with consistent growth states of the wild type arabidopsis thaliana and the transgenic arabidopsis thaliana, respectively measuring the weight (X0) of the lotus throne leaves by using an electronic balance, then placing the lotus throne leaves in 4 culture dishes for natural dehydration for 160min at room temperature, measuring the weight (Xt) of the leaves every 20min, calculating the water loss rate of the leaves at each time point, and drawing a line drawing according to data. The water loss rate in vitro is (X0-Xt)/X0X 100%

The water loss line graph in vitro of the leaves shows that the water loss speed of the wild type arabidopsis is higher than that of each CpGLIP1 gene-transferred strain arabidopsis within 160min (figure 15), which indicates that the water retention capacity of the CpGLIP1 gene-transferred strain is higher than that of the wild type arabidopsis.

In conclusion, the CpGLIP gene is related to plant drought resistance, and the drought resistance of plants can be improved by over-expressing the CpGLIP1 gene in arabidopsis thaliana.

2 Low temperature stress study

(1) Arabidopsis thaliana-4 ℃ freezing stress phenotype

And (4) carrying out freezing stress treatment on the transgenic arabidopsis thaliana and the wild type arabidopsis thaliana which are two weeks old after transplantation. And (3) placing each strain of arabidopsis thaliana in a low-temperature artificial climate box at the temperature of-4 ℃ for freezing treatment for 6 hours, then taking out the arabidopsis thaliana and placing the arabidopsis thaliana in a normal culture environment at the temperature of 22 ℃ for recovery, and observing and counting the growth condition and the survival rate of the plant in the process. The criteria for survival were: the leaves do not completely wilt and the plant can continue to grow.

The results show that after 6 hours of treatment at-4 ℃, both wild type arabidopsis thaliana and transgenic arabidopsis thaliana are subjected to freezing damage, the leaves are curled, and the leaf area is reduced, wherein the leaf color of the wild type arabidopsis thaliana is especially dull and yellowish. After freezing treatment, the arabidopsis thaliana is taken out immediately, unfreezing and growth recovery are carried out at 22 ℃, most wild arabidopsis thaliana is wilted and yellowed, even withered and dead, and part of leaves of the arabidopsis thaliana with the CpGLIP1 gene are green, and even new leaves can grow from the growing point (figure 16). After continuous culture, the survival rate of the plants is counted, the survival rates of the transgenic lines OE3, OE8 and OE7 are 57.1%, 50% and 33.3% respectively, and the survival rate of the wild type Arabidopsis is the lowest and is only 19.1% (FIG. 17).

(2) Determination of arabidopsis thaliana low-temperature stress physiological index at 4 DEG C

Dividing each plant of arabidopsis thaliana after transplanting into a control group and a treatment group, normally culturing the control group at 22 ℃, placing the treatment group in a 4 ℃ artificial climate box for low-temperature treatment for 3 days, and keeping other culture conditions consistent with those of the control group.

And then, measuring related physiological indexes (proline, malondialdehyde and chlorophyll SPAD) of the arabidopsis thaliana strains of the control group and the treatment group.

The result of the physiological indexes is shown in figure 18, and the proline content has no obvious difference for untreated wild type and transgenic arabidopsis thaliana. After treatment at 4 ℃, the proline content of the arabidopsis thaliana is increased, and the proline content of the arabidopsis thaliana with the CpGLIP1 transgenic gene is obviously higher than that of the wild type arabidopsis thaliana. The wild type and transgenic arabidopsis thaliana had low MDA levels without treatment and had no significant difference. After 3 days of treatment at 4 ℃, MDA in OE3 and OE8 slightly rises, MDA in wild type arabidopsis thaliana is accumulated in a large amount, and the content of MDA is remarkably higher than OE3 and OE8 and slightly higher than OE 7. The SPAD value of each strain of Arabidopsis thaliana in the treatment group is slightly increased compared with that of the control group, and the SPAD value of a wild type is slightly lower than that of a transgenic strain, but no significant difference is caused.

The results of the physiological indexes under the conditions of phenotype under the freezing stress and low-temperature stress show that the injury degree of the CpGLIP1 transgenic arabidopsis thaliana is smaller than that of the wild arabidopsis thaliana under the cold condition, and the fact that the CpGLIP1 gene is overexpressed in the arabidopsis thaliana can improve the cold resistance of the plant.

Note: the DDS-309+ intelligent conductivity meter used for the experiments was purchased from Kyoto Scientific Co., Ltd, the Varioskan Flash full-wavelength scanning multifunctional microplate reader was purchased from Thermo Scientific Co., Ltd, and the SPAD-502 chlorophyll meter was purchased from KONICA MMINOLTA, Japan.

Example 6 Agrobacterium mediated transformation of the 717 Populus tremula with the CpGLIP1 Gene

(1) Preparation of invaded dye liquor

The Agrobacterium plate containing the recombinant plasmid pGWB551-CpGLIP1 was streaked and activated, picked into YEB medium (containing 50mg/LSpec +50mg/L Gen), cultured on a shaker at 28 ℃ and 200rpm, and shaken again to OD 6000.5-0.8.

Centrifuging the bacterial liquid at 5000rpm for 20min, discarding the supernatant, re-suspending with 100ml MS-IM (containing 100 μ M AS) to obtain the infection liquid, and oscillating and activating the infection liquid on a shaker at 100rpm for 1h for later use.

(2) Infection by infection

Selecting a poplar tissue culture seedling with good growth vigor about 2 months after growing in a multiplication culture medium, cutting 0.8-1.5 cm of stem without axillary buds, and scratching for 2-3 times along a longitudinal axis by using a blade to form a wound (or cutting a poplar tissue culture seedling with 0.1-0.5 cm of axillary buds) ² A petiole of the blade; or cutting leaves, cutting a wound at the intersection of the main vein and the lateral vein), and carrying out oscillating infection for 1h in the agrobacterium infection solution.

(3) Co-cultivation

The infected explants were blotted dry with sterile paper scraps and cultured in CIM medium at 25 ℃ for 2 days in the dark.

(4) Callus culture

The inoculated explants were washed three times with sterile deionized water, then three times with sterile deionized water containing 350mg/L Cef, and placed on sterile filter paper to blot the residue.

Explants were transferred to CIM + H + C medium for dark culture to screen transformed calli. The culture medium was changed every 10 days for about 4 times.

(5) Culturing for bud

Transferring the explants with the callus to a 2SIM + H + C culture medium for bud culture, and subculturing every 3-4 weeks. When young buds grow on the surface of the green callus, the callus and the buds are transferred to a 2SIM + H + C culture medium with the TDZ concentration reduced by 25 times for continuous culture, and vitrification is prevented.

(6) Shoot elongation culture

When the shoots grow to about 1cm and axillary buds appear, explants containing multiple shoots are transferred to SB0.1+ H + C medium to promote shoot elongation.

(7) Rooting culture

When the bud grows to about 2cm, the part above the callus is cut off by scissors, rooting culture is carried out on Red Medium, and resistance screening is further carried out on the regenerated bud.

(8) Genome PCR detection of transgenic poplar

The regeneration poplar which can normally grow in Red Medium is respectively taken to extract genome DNA from leaves and wild leaves, pGWB551-CpGLIP1 plasmid is taken as a positive control, wild DNA and ddH2O are taken as a negative control, and PCR amplification is carried out by using a specific primer of CpGLIP 1. Electrophoresis results show that the transgenic line can amplify a target band with the size consistent with that of the positive control, while the wild-type and the negative control cannot amplify the target band (FIG. 19), and the CpGLIP1 gene is preliminarily judged to be successfully inserted into the 717 poplar genome.

(9) Real-time fluorescent quantitative PCR detection of transgenic poplar

Transplanting the transgenic poplar with positive PCR detection into soil to be cultured for 3 weeks, taking leaves of the transgenic poplar to extract total RNA, carrying out real-time fluorescence quantitative PCR after reverse transcription into cDNA, and detecting the expression condition of the CpGLIP1 gene in the transgenic poplar. The fluorescent quantitation primer sequences are shown in Table 3.

TABLE 3 primers

The results show that the tested transgenic samples all detected different degrees of expression of the target gene, while the expression of the target gene was not detected in WT (FIG. 20), indicating that several CpGLIP1 transgenic poplar individuals were successfully obtained.

The formula of the culture medium is as follows:

FV vitamin (500 mL): 0.05g of nicotinic acid, 0.05g of pyridoxine hydrochloride, 0.05g of calcium pantothenate, 0.05g of thiamine nicotinate, 0.05g of L-cysteine and 5.0mL of biotin.

Proliferation medium (1L): 0.25g of MES, 0.1g of inositol, 2.315g of MS salt (without organic), 0.20g of L-glutamine, 10.0ml of FV vitamin, 20.0g of sucrose, 7.0g of agar and pH 5.8.

MS-IM (1L): 0.25g of MES, 0.1g of inositol, 2.315g of MS salt (organic free), 0.20g of L-glutamine, 10.0mL of FV vitamin, 1.80g of D (+) -galactose, 1mL of AS (100mM), pH 5.0.

CIM (1L) 0.25g MES, 0.1g inositol, 2.315g MS salt (without organic), 10mL FV vitamin + L-glutamine, 1mL NAA (10mM), 1mL 2ip (5mM), 1mL AS (100mM), 30.0g sucrose, 7.0g agar, pH 5.8.

CIM + H + C (1L): 0.25g of MES, 0.1g of inositol, 2.315g of MS salt (without organic), 10mL of FV vitamin + L-glutamine, 1mL of NAA (10mM), 1mL of 2ip (5mM), 200. mu.L of Hyg (50mg/mL), 2.0mL of Cef (200mg/mL), 30.0g of sucrose, 7.0g of agar, and pH 5.8.

2SIM + H + C (1L): 0.25g of MES, 0.1g of inositol, 2.315g of MS salt (organic free), 10mL of FV vitamin + L-glutamine, 400. mu.L/16. mu.L of TDZ (0.5mM), 200. mu.L of Hyg (50mg/mL), 2.0mL of Cef (200mg/mL), 30.0g of sucrose, 7.0g of agar, and pH 5.8.

SB0.1+ H + C (1L): 0.25g of MES, 0.1g of inositol, 2.315g of MS salt (organic free), 10mL of FV vitamin + L-glutamine, 45. mu.L of BAP (0.5mg/mL), 200. mu.L of Hyg (50mg/mL), 2.0mL of Cef (200mg/mL), 30.0g of sucrose, 7.0g of agar, and pH 5.8.

Red Medium (1L): 0.25g of MES, 0.1g of inositol, 2.315g of MS salt (organic free), 10mL of FV vitamin, 0.2g of L-glutamine, 100. mu.L of IBA (1mg/mL), 200. mu.L of Hyg (50mg/mL), 2.0mL of Cef (200mg/mL), 30.0g of sucrose, 7.0g of agar, and pH 5.8.

Note: the reagents MS (without organic and sucrose) used in the experiment were purchased from Qingdao Haibo biotechnology, Inc., acetosyringone AS from Sigma, MES, inositol, L-glutamine, D (+) -galactose, biotin from Solambio, Beijing, Thidiazuron TDZ, isopentenyl adenine 2ip, NAA, BAP, IBA, and cefuromycin Cef from biotech, Inc., Beijing Dingguo.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and scope of the present invention as defined in the appended claims.

Sequence listing

<110> university of southwest

<120> chimonanthus nitens GDSL lipase gene CpGLIP1 and application thereof

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 364

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Ala Gly Ser Ser Ala Pro Phe Ala Ser Leu Leu Thr Leu Val Ala

1 5 10 15

Leu Cys Thr Phe Thr Leu Gln Val Asp Ala Arg Ala Phe Phe Val Phe

20 25 30

Gly Asp Ser Leu Val Asp Asn Gly Asn Asn Asn Tyr Leu Ala Thr Ser

35 40 45

Ala Arg Ala Asp Ser Pro Pro Tyr Gly Ile Asp Tyr Pro Thr Arg Arg

50 55 60

Pro Thr Gly Arg Phe Ser Asn Gly Leu Asn Ile Pro Asp Ile Ile Ser

65 70 75 80

Glu Ser Leu Gly Thr Glu Ser Thr Leu Pro Tyr Leu Ser Pro Gln Leu

85 90 95

Thr Gly Glu Arg Leu Leu Val Gly Ala Asn Phe Ala Ser Ala Gly Ile

100 105 110

Gly Ile Leu Asn Asp Thr Gly Val Gln Phe Leu Asn Ile Ile Arg Ile

115 120 125

Ser Gln Gln Leu Thr Tyr Phe Glu Gln Tyr Gln Lys Arg Leu Ser Gly

130 135 140

Leu Val Gly Pro Asp Lys Ala Gln Gln Leu Val Asn Gln Ala Leu Val

145 150 155 160

Leu Ile Thr Leu Gly Gly Asn Asp Phe Val Asn Asn Tyr Tyr Leu Val

165 170 175

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

180 185 190

Tyr Ile Ile Ser Glu Tyr Arg Lys Ile Leu Arg Arg Leu Tyr Asp Leu

195 200 205

Gly Ala Arg Arg Val Leu Val Thr Gly Thr Gly Pro Met Gly Cys Val

210 215 220

Pro Ala Glu Leu Ala Gln Arg Ser Arg Asn Gly Asn Cys Asp Pro Glu

225 230 235 240

Leu Met Arg Ala Ala Gly Leu Phe Asn Pro Gln Leu Glu Gln Met Leu

245 250 255

Asn Gly Leu Asn Ser Glu Ile Gly Ser Asp Ile Phe Ile Ala Ala Asn

260 265 270

Thr Arg Leu Met Asn Ser Asp Phe Ile Gln Asn Pro Gln Ala Phe Gly

275 280 285

Phe Val Thr Ala Lys Val Ala Cys Cys Gly Gln Gly Pro Tyr Asn Gly

290 295 300

Leu Gly Leu Cys Thr Val Leu Ser Asn Leu Cys Pro Asn Arg Asn Val

305 310 315 320

Tyr Ala Phe Trp Asp Ala Phe His Pro Ser Glu Arg Ala Asn Arg Phe

325 330 335

Ile Val Asp Lys Ile Leu Arg Gly Ser Thr Gln Tyr Met Lys Pro Met

340 345 350

Asn Leu Ser Thr Ile Leu Glu Met Asp Ser Arg Thr

355 360

<210> 2

<211> 1457

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

caaagggtga taagagatca agtgacctta gcccatcaga cccatggccg gttcttcagc 60

tccctttgct tctctactaa ccctggtggc actctgcacc ttcaccttgc aggttgatgc 120

tcgcgcattc tttgtcttcg gagattcact tgtcgacaat ggaaacaaca attacttggc 180

cacctctgct cgtgcagatt cgcctcctta cggtatcgac tacccaactc gccgaccaac 240

aggccgcttc tccaatggac ttaacatccc agacatcatc agtgagagtc ttggtacaga 300

gtctacattg ccttacctga gcccacagct caccggggag agactattag tcggcgctaa 360

ctttgcctct gctggaattg gaatacttaa tgacacgggg gttcaatttc ttaacataat 420

caggatctcc caacagctga cttactttga acaataccag aagcggttaa gcggccttgt 480

cggtccagat aaggcccaac aacttgtaaa tcaagcactc gttctgatca ccctgggcgg 540

caatgacttc gttaacaact attacttagt tccattttca gcgaggtcgc ggcaattttc 600

attgccggat tacgttgtct acattatctc cgagtatcgg aaaattctaa ggagattata 660

cgatttaggt gctcggaggg tccttgttac tggaactggg cccatgggct gtgttccggc 720

tgagcttgcc cagagaagtc ggaatggaaa ctgcgacccg gaattgatgc gggctgcagg 780

gctattcaac cctcagctcg agcagatgtt gaacgggctc aacagcgaaa ttggatcaga 840

catcttcatc gccgctaata cccgactaat gaattcagat ttcattcaga atccacaggc 900

attcggtttt gtgactgcaa aagttgcgtg ttgcgggcaa gggccttata atgggcttgg 960

actatgcaca gttttgtcaa atctatgtcc aaataggaat gtctacgcat tctgggatgc 1020

ctttcatcct tcagagaggg caaacaggtt tatcgtcgac aagatattac gtggttcgac 1080

tcagtacatg aagcccatga acttgagcac catcctagaa atggattcaa ggacttagcc 1140

tcctaagggc ctcagtatca tctcatctcc tttacttgtt tgtttggtgt ttttcttgtc 1200

tctcccaatc tctcatcaat gtcatgtata atgtatctca taaatgcatt tgaagaccca 1260

aaataagata atttttctac cttcgcaatt ataaacatat gcaagtattt tctaagttgt 1320

gtttgtcttt gcatatctat ttctacattg atgtcatgta actttgttta tgaatccaaa 1380

atgataagaa atggtatcca agtgtaactt tttttttctt tacatctaat agagttactt 1440

tagtgagttg aaaaaaa 1457

<210> 3

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gatcaagtga ccttagccca tc 22

<210> 4

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aggagatgag atgatactga ggc 23

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aactgcgacc cggaattgat 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gcaacacgca acttttgcag 20

<210> 7

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gttatggttg ggatgggaca gaaag 25

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gggcttcagt aaggaaacag ga 22

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tagtgacaag acagtaggtg gaggt 25

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gtaggttcca gtcctcactt catc 24

<210> 11

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cgggatccag acccatggcc ggttctt 27

<210> 12

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cccaagctta ggagatgaga tgatactgag gc 32

<210> 13

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ggggacaagt ttgtacaaaa aagcaggctg atcaagtgac cttagcccat c 51

<210> 14

<211> 52

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggggaccact ttgtacaaga aagctgggta ggagatgaga tgatactgag gc 52

<210> 15

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cttaggtctc ggtcgcag 18

<210> 16

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atcggtggtt agggacac 18

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tccaagacaa ggaaggcatc c 21

<210> 18

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

agcaccaagt gaagggttga ctc 23

Claims (6)

1. The chimonanthus nitens GDSL lipase gene CpGLIP1 is characterized in that the amino acid sequence of the encoded protein is shown as SEQ ID No. 1.

2. The chimonanthus nitens GDSL lipase gene CpGLIP1 as claimed in claim 1, wherein the nucleotide sequence of the chimonanthus nitens CpGLIP1 gene is shown as the 44 th to 1138 th bases of SEQ ID No. 2.

3. The use of the chimonanthus nitens GDSL lipase gene CpGLIP1 in plant stress control as claimed in claim 1, wherein the stress is low temperature or drought treatment.

4. Use according to claim 3, wherein the plant is Chimonanthus praecox or Arabidopsis thaliana.

5. The use according to claim 3 or 4, wherein the low temperature is-4 to 4 ℃.

6. The use as claimed in claim 3 or claim 4 wherein the drought is 30% mass fraction PEG6000 treatment.

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