CN114045294B

CN114045294B - Lipid transport protein gene and application thereof

Info

Publication number: CN114045294B
Application number: CN202111381894.XA
Authority: CN
Inventors: 年洪娟; 李拥; 陈麓; 李昆志
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2023-03-24
Anticipated expiration: 2041-11-22
Also published as: CN114045294A

Abstract

The invention discloses a lipid transport protein geneRTA1The nucleotide sequence is shown as SEQ ID NO. 1, and the protein of the amino acid sequence shown as SEQ ID NO. 2 is coded; the invention relates to a knockout gene fragment with a hygromycin gene marker and pYES3-RTA1The recombinant vector constructs the cryptococcus terrestris mutant and the transgenic INVSC1 yeast strain by an electrotransformation method, and further exploresRTA1The gene has the function of resisting acid and aluminum stress, and the experiment result shows that the acid and aluminum ion resisting capacity of the mutant strain is obviously weakened and transformedRTA1The gene yeast has increased aluminum resistance, and the gene has the potential of being applied to reducing aluminum toxicity in acid soil.

Description

Lipid transport protein gene and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a gene of cryptococcus terreus encoding lipid transporterRTA1And transgenesRTA1Yeasts, and their use in an aluminum environment.

Background

With the development of industrialization, a large amount of sulfide and other substances are discharged in the air, so that acid rain is formed, and the problem of soil acidification exists in many areas, such as red soil areas. Aluminum is one of the most abundant metal elements in the earth crust, accounting for about 8% of the total earth mass, and is widely and abundantly distributed. Aluminum is usually present in the form of sparingly soluble silicates or aluminas, which are only slightly toxic to organisms. With the increase of the acid rain phenomenon, the problem of soil acidification is more and more serious, and the content of aluminum ions in soil is increased. In which aluminum (Al) is used as an inorganic monomer in various chemical forms of aluminum ³⁺ ) The most obvious toxic action on various crops, al ³⁺ The presence of (a) is largely dependent on the ambient pH (Al at pH 3.0) ³⁺ Content of (B) is 100%, and Al is present at pH4.5 ³⁺ Content of (B) 80% and Al at pH 6.0 ³⁺ The content of the aluminum is less than 10 percent), so the aluminum toxicity is the main reason that the acid soil hinders the growth of crops. Aluminum can affect the cell membrane structure of organisms, affect the activity of transmembrane ion channels, and thus interfere with cellular ion exchange homeostasis. Researches show that the exchange amount of aluminum ions in soil accounts for about 20-80% of cations, so that excessive loss of the cations is caused, various nutrient elements in the soil such as potassium, calcium, magnesium and the like are deficient, the absorption of water and nutrient components by crops is indirectly influenced, and the yield of the crops is reduced.

How to scientifically and effectively treat the aluminum toxicity becomes a problem to be solved in the world, and researches find that the pH value of soil is improved by using lime so as to achieve the aim of reducing the aluminum toxicity, but the soil structure is easy to causeHardening, soil fertility reduction, water loss, soil microorganism reduction and the like. In acid high-aluminum soil, the survival rate of seedlings can be improved by inoculating certain mycorrhizal fungi, because the mycorrhizal fungi activate insoluble phosphate in rhizosphere soil and precipitate free aluminum ions, so that the harm of aluminum toxicity to crops is reduced. The reasons why mycorrhizal fungi improve the aluminium resistance of plants are: (1) The mycorrhizal fungi can increase the absorption range of nutrient elements through hypha growth, and reject or secrete organic acid and Al in vitro ³⁺ Chelating to precipitate and prevent the precipitate from being transported to plant roots to reduce the influence of aluminum toxin; (2) The specific structure of the hyphal cell wall of mycorrhizal fungi can separate toxic metal ions by reducing Al ³⁺ The content of Al is reduced ³⁺ Damage to plants. Therefore, it has been found that the use of a method of artificially inoculating a superior ectomycorrhizal fungus in the aspect of afforestation is the most effective countermeasure in a low pH and high alumina environment.

In plants, lipid transporters play a role in resistance to abiotic stress. Lipid transporters are involved in the response to various environmental stresses in wheat, such as low temperature, drought, oxidative stress, and the like. In other plants, such as maize, arabidopsis thaliana, sesame, and the like, the expression level of lipid transporters is also increased when subjected to the above-mentioned environmental stresses. The lipid transport protein of the fungus is generally a membrane protein consisting of six or seven transmembrane domains, has no homology with the lipid transport protein of animals and plants, is mainly researched on the aspect of fungal drug targets, and is not reported on the aspect of resisting abiotic stress of the fungus.

Disclosure of Invention

The invention provides a lipid transport protein geneRTA1The nucleotide sequence is shown as SEQ ID NO. 1, and the protein of the amino acid sequence shown as SEQ ID NO. 2 is coded; it is derived from cryptococcus terreus (C. humicolus) BSLL1-1 strain, gene sequence full length 1146bp, coding 381 amino acids composed of lipid transport protein, molecular weight approximately 42kDa.

Another object of the present invention is to provide the above-mentioned lipid transporter geneRTA1Application in enhancing acid stress resistance of microorganismsOr/and aluminum stress ability.

In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions:

1. cryptococcus terrestris isolated from acidic soil in the rhizosphere of tea trees in tea gardens around Longling county, baoshan, yunnan province (C. humicolus) Extracting genome DNA from BSLL1-1 strain, and sequencing genome in Shanghai human genome research center to obtainRTA1A gene sequence. Designing a primer according to the sequence, and carrying out PCR amplification by taking the cryptococcus oxytetralis cDNA as a templateRTA1A gene fragment; connecting the target fragment to pMD-19T vector to obtain recombinant vector pMD19-T-RTA1And heat shock transformation is carried out on the obtained product to escherichia coli competent cell DH5 alpha, a plate containing ampicillin is coated on the obtained product, a positive colony is selected for sequencing, and a gene for coding RTA1 protein is identified, wherein the sequence of the gene is shown as SEQ ID NO. 1, the protein is coded by 381 amino acids, and the amino acid sequence is shown as SEQ ID NO. 2;

construction of pYES3-RTA1A recombinant vector; then pYES 3-containing cells were added to the INVsc1 competent yeast cellsRTA1Recombining carrier, then electric shock transforming, smearing the mixed liquid on Trp lacking plate to screen and obtain transgeneRTA1A yeast.

2、RTA1Construction of Gene recombination fragment

Will be provided withRTA1The three independent segments of the front 1120bp nucleotide (the front 1120 nucleotide outside the RTA1 gene), the back 1080bp nucleotide (the back 1080 nucleotide outside the RTA1 gene) and the hygromycin gene of the gene are respectively provided with an amplification primer, and a reverse primer of the front gene and a forward primer of the back gene are both provided with a section of homologous arm; are respectively provided withRTA1The forearm and hygromycin gene are taken as templates and are obtained by amplificationRTA1Front arm: the first 2/3 hygromycin fragment; the hygromycin gene andRTA1the rear arm is used as a template, and the subsequent 2/3 hygromycin is obtained by amplification:RTA1a hind arm recombinant fragment; then, overlapping extension PCR is carried out by taking the two recombinant fragments as templates to obtain recombinant fragmentsRTA1Front arm: hygromycin gene:RTA1rear arm, named RTA1F: hyg: RTA1R;

3. then adding a recombinant fragment RTA1F with correct sequencing into the cryptococcus terreus competent cells: hyg: RTA1R, then carrying out electric shock transformation, coating the mixed solution on a YPD plate containing 300 mu g/mL hygromycin, and screening the mutant strain of the cryptococcus terreus which generates homologous recombination; picking a single colony from a hygromycin plate, and detecting whether the knockout is successful by using a PCR (polymerase chain reaction) and real-time PCR method;

4. the invention compares the wild type strain of cryptococcus terreus and the mutant strain of cryptococcus terreus in the transgene in step 1RTA1Growth of Yeast and non-transgenic Yeast under aluminum stress

Preparing GM solid culture media with pH values of 3.5, 4.5, 5.5, 6.5 and 7.5 respectively;

preparing a GM solid culture medium with a pH of 4.5 and containing aluminum ions, wherein the final concentration of the aluminum ions in the GM solid culture medium is 50mmol/L, 100mmol/L and 150mmol/L;

inoculating cryptococcus terrestris wild type strains and cryptococcus terrestris mutant strains into a YPD liquid culture medium for shake cultivation overnight; inoculating to fresh YPD liquid medium at an inoculation amount of 1% (v/v), and adjusting the concentration of the bacterial liquid to OD ₆₀₀ Is 1; respectively diluting the bacterial liquid to 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 Respectively taking 1.5 mu L of bacterial liquid with each concentration and inoculating the bacterial liquid to GM solid plates with different pH values; respectively inoculating 2.5 mu L of bacterial liquid with each concentration on a GM solid culture medium containing aluminum ions, setting three plates for each type for repetition, culturing at 28 ℃, and observing the growth conditions of different strains under aluminum acid stress;

initial OD ₆₀₀ Wild type strain of Cryptococcus oxytoca and mutant strain of Cryptococcus oxytoca with pH of 4.5 and 50mmol/L Al content ³⁺ Culturing at 28 ℃ and 200rpm in GM liquid medium (K), and measuring OD every 2 hours ₆₀₀ Drawing a growth curve, and observing the cell morphology of the cryptococcus terrestris strain and the mutant strain in a GM liquid culture medium which does not contain aluminum ions and has a pH value of 4.5 and a final concentration of the aluminum ions of 50mmol/L under a microscope;

5. preparing a GM induction solid culture medium with pH of 4.5 and 7.5;

preparing a GM induction solid culture medium with the pH of 4.5 and containing aluminum ions, wherein the final concentration of the aluminum ions in the GM solid culture medium is 0.1mmol/L, 0.2 mmol/L and 0.5 mmol/L;

transferring INVSC1 yeast strain, non-loaded INVSC1 strain (I-P-T), and non-loaded INVSC1 strainRTA1Gene INVSC1 strain (I-P-RTA1) Inoculating to YPD liquid culture medium, and shake culturing overnight; inoculating to fresh GM induction liquid culture medium with pH4.5 at 1%, and adjusting the concentration of the culture solution to OD ₆₀₀ Is 1; respectively diluting the bacterial liquid to 10 ⁰ 、10 ^-1 、10 ^-2 、10 ^-3 Respectively taking 1.5 muL of bacterial liquid with each concentration on GM induction solid plates with different pH values for inoculation; inoculating 2.5 mu L of bacterial liquid with each concentration on GM induction solid plates with different aluminum ion concentrations, arranging three plates for each type to be used as a repeat, carrying out inverted culture in an incubator at 28 ℃, observing the size of bacterial colonies, and observing the growth conditions of different strains under aluminum stress.

The invention has the following advantages and technical effects:

the invention provides a gene for resisting aluminum sulfate stress in soilRTA1，RTA1The resistance of the gene mutant strain to aluminum stress is obviously reduced, and the gene mutant strain is transformed by treating the gene mutant strain in environments with aluminum ions with certain concentration and different pH valuesRTA1When the strain is genetically transformed with INVSC1, the strain is transformedRTA1The growth of the gene INVSC1 strain has little influence. The gene has the function of resisting the stress of aluminum, and can be used for modifying microorganisms to resist the stress of acidity and aluminum toxicity in the environment by utilizing the gene engineering technology.

Drawings

FIG. 1 shows a lipid transporter geneRTA1The amplification results of (1) are shown schematically, wherein lane 1 isRTA1Gene, lane M is 2000 Marker;

FIG. 2 isRTA1The PCR results of the transgenic yeast are shown schematically, wherein lane M is 2000 marker, lane 2 isRTA1A transgenic yeast;

FIG. 3 shows the wild type strain of Cryptococcus oxytoca and the wild type strain of Cryptococcus oxytoca at 50mmol/L of aluminum without aluminum treatmentAt the ion concentrationRTA1The result of gene expression;

FIG. 4 shows the present inventionRTA1F：hyg：RTA1The amplification of the recombinant gene fragment of R is shown in the drawing, wherein A in the left drawing isRTA1Forearm: the first 2/3 hygromycin recombinant fragment, B is the second 2/3 hygromycin:RTA1a hind arm recombinant fragment; in the right figure C isRTA1F：hyg：RTA1R recombinant gene segment; in the figure, M is a DNA Marker;

FIG. 5 shows the present inventionRTA1PCR and real-time PCR detection maps of gene knockout, wherein the map A is a PCR detection map of knockout strains, wherein lane 1 is a wild strain, lane 2 is a knockout strain, and M is a DNA molecular weight standard; b is the gene transcription of the knockout strain detected by real-time PCR, wherein the wild type-50 is the wild type strain treated with 50mmol/L Al ³⁺ Treating, mutant-50 is a knockout strain transferred into recombinant fragment by 50mmol/L Al ³⁺ Processing;

FIG. 6 shows the wild type strains of Cryptococcus terrestris andRTA1detecting the acid stress resistance of the mutant strain;

FIG. 7 shows the wild type strains of Cryptococcus terrestris andRTA1detecting the aluminum stress resistance of the mutant strain;

FIG. 8 shows the wild type strains of Cryptococcus terrestris andRTA1growth curve chart of mutant strain treated by 50mmol/L aluminum ion;

FIG. 9 is a graph showing the microscopic examination results of the untreated wild type and RTA1 mutants (upper row) and the wild type and RTA1 mutants (lower row) treated with 50mmol/L aluminum ion in the present invention;

FIG. 10 is a view of a rotary tableRTA1The detection results of the acid stress resistance of the gene INVSC1 strain, the INVSC1 yeast strain and the transferred no-load INVSC1 strain are obtained;

FIG. 11 is a view of a rotary tableRTA1The results of the detection of the aluminum stress resistance of the gene INVsc1 strain, the INVsc1 yeast strain and the non-load transfer INVsc1 strain.

Detailed Description

The present invention will be described in further detail with reference to the following examples, but the present invention is not limited thereto, and the methods in the examples are carried out in accordance with conventional methods unless otherwise specified, and reagents and bacterial species used therein are commercially available reagents or bacterial species, or reagents prepared in accordance with conventional methods, unless otherwise specified.

Example 1: cryptococcus oxytetracycline (C. humicolus) Extraction of total RNA and cDNA synthesis of BSLL1-1 strain

Extraction of yeast total RNA (on ice) was performed using TRIZOL kit (TaKaRa Co.) with the following steps: taking 0.1g of cryptococcus oxytoca thalli, adding the cryptococcus oxytoca thalli into a mortar, grinding the cryptococcus oxytoca thalli into powder by using liquid nitrogen, then adding 1mL of TRIZOL extracting solution, and continuously grinding the powder until the powder is clear. Transferring the grinding liquid to an EP tube, standing at room temperature for about 5min, adding 0.2mL of chloroform, reversing and uniformly mixing, wherein the whole body without differentiation phenomenon is in a milk shake shape, and then placing the sample on ice for 5min, and centrifuging at 12000rpm and 4 ℃ for 10min; the supernatant was transferred to a new EP tube and extracted once with 0.2mL of chloroform. Taking the supernatant, adding isopropanol with the same volume, standing at-20 ℃ for 1h, and centrifuging at 12000rpm at 4 ℃ for 15min; discarding the supernatant, washing twice with 1mL of 75% ethanol, centrifuging at 12000rpm at 4 ℃ for 5min, pouring off the ethanol, naturally drying, dissolving with 20-40 mu L of DEPC-treated water, and storing at-80 ℃.

Using PrimeScript ^TM The RT reagent Kit with gDNA Eraser reverse transcription Kit carries out reverse transcription on the extracted total RNA;

1. preparing the following template RNA/primer mixed solution in a tube;

2. keeping the temperature at 42 ℃ for 2min, and then quickly putting on ice;

3. the following transcription reaction solution was prepared in the above tube

5. The cDNA solution obtained after incubation at 37 ℃ for 15min, heating at 85 ℃ for 5s and then placing on ice can be directly used for fluorescent quantitative PCR analysis.

Example 2:RTA1cloning and sequencing of the coding Gene

Taking Cryptococcus oxytoca cDNA as a templateRTA1PCR amplification of gene, the primer for amplification is positive:AAGCTTATGCCCGCACCCGCGCCACT (underlined)HindIII cleavage site), in reverse:GGATCCTTACCATGGGGGAGGGCCTTG (underlined)BamHI cleavage site). Reaction conditions are as follows: pre-denaturation at 94 deg.C for 3min, then performing 35 cycles at 94 deg.C, 30s,56 deg.C, 30s,72 deg.C, 1min, and extending at 72 deg.C for 5min after the cycle is over; subjecting the obtained PCR amplification product to agarose gel electrophoresis (FIG. 1), and purifying the target band with a DNA gel recovery kit; connecting the target fragment to pMD19-T vector to obtain recombinant vector pMD19-T-RTA1(ii) a Transforming into Escherichia coli competent cell DH5 alpha by thermal stimulation, spreading on LB solid plate containing ampicillin, and performing inverted culture at 37 deg.C for about 12 hr; selecting single colony, culturing in liquid LB culture medium for 12 hr, PCR verifying, extracting plasmid with the amplified bacterial liquid, sequencing with recombinant vector,RTA1the nucleotide sequence of the gene is shown in a sequence table SEQ ID NO 1.

Example 3:RTA1construction of transgenic INVSC1 Yeast

Recombinant vector pMD19-T-RTA1And pYES3/CT vectorHinddiii andBamh, carrying out double enzyme digestion on the H I, and carrying out enzyme digestion for 6 hours at 37 ℃; then, the materials are subjected to glue recovery, and a connection system is prepared: 2 mu L of glue recoveryRTA1Gene fragments, pYES3/CT vector recovered from 3 muL glue, and 5 muL solution I, and connecting the system at 16 ℃ overnight. It was transformed into E.coli competent cells DH 5. Alpha. By heat stimulation, and then spread on LB solid plates containing ampicillin, and cultured upside down at 37 ℃ for about 12 hours. Selecting a single colony to culture in a liquid LB culture medium for about 12 hours, carrying out PCR verification, then extracting plasmids from the bacterial liquid of the amplified target segment, and adding 20 mu L of pYES3-RTA1Recombinant vector, placed on ice for 15min; then adding the mixed solution into an electric rotating cup with ice pre-cooling of 0.2cm, carrying out electric shock (electric shock parameters: 25 muF, 2500V and 200 omega), adding 400 muL of pre-cooled sorbitol after the electric shock, and mixing the mixed solution on an ultra-clean workbenchUniformly spreading the mixed solution on an SD culture medium plate lacking Trp, and placing the plate in an incubator at 28 ℃ for inverted culture for 2-3 days; picking single colony from the Trp-lacking plate, adding YPD liquid culture medium to culture at 28 ℃ overnight, collecting thallus and carrying out PCR detection; the result is shown in fig. 2, where a band between 1000 and 2000 was detected.

Example 4: fluorescent quantitative PCR analysis of cryptococcus terrestrisRTA1Expression of genes under 50mmol/L aluminum stress

Design of 18S rRNA of Cryptococcus terrestris andRTA1the real-time PCR primer of the gene and the 18S rRNA gene are used as internal references, and the primer sequences are as follows:

using SYBRPremix ExReal-Time PCR was performed with TaqII (Tli RNaseH Plus) kit, reaction System 20 μ L (see table below), using a two-step PCR reaction program with Applied Biosystems 7500 Fast Real-Time PCR System: stage 1: pre-denaturation, reps: 30s at 1,95 ℃; stage 2: PCR reaction, reps:2, 95 ℃ for 5s, 60 ℃ for 30s; confirming the amplification curve and the melting curve of real-time PCR after the reaction is finished, and preparing a standard curve when PCR quantification is carried out.

Using 18S rRNA gene as internal reference, using gene expression quantity of wild cryptococcus oxytetracycline cultured by adding 50mmol/L aluminum ions as control, and applying 2 ^-ΔΔCt The method analyzes the experimental result, and the formula is as follows:

（1）Ct _{treatment of} – Ct _{Internal reference (treatment)} = ΔCt _{Treatment of} ；Ct _{Control of} – Ct _{Internal reference (contrast)} = ΔCt _Control ；

（2）ΔCt _{Treatment of} – ΔCt _Control = ΔΔCt；

(3) Fold difference = 2 ^-ΔΔCt ；

According to the formula, the Comparison (CK) is '1', and the wild type of cryptococcus oxytoca without aluminum treatment and the wild type of cryptococcus oxytoca can be calculated under the aluminum ion concentration of 50mmol/LRTA1Differences in expression of the genes; lipid transporter gene under aluminum stressRTA1The real-time PCR analysis result of (A) showed that of Cryptococcus oxytoca without aluminum treatmentRTA1Gene is not expressed, and the cryptococcus oxytetracycline highly expressed lipid transporter gene treated by 50mmol/L aluminum ionsRTA1(FIG. 3).

Example 5: overlapping PCR amplificationRTA1Homologous recombination gene fragment

To pairRTA1The 1120bp and the last 1080bp nucleotide of the gene and the hygromycin gene are respectively designed with amplification primers F1 and R1, F2 and R2, F3 and R3, and the sequences are as follows:

F1：:CACTGCAGACCATCTCCGAC，

R1：TGCTCCTTCAATATCATCTTCTGTCGGCGGGGTGTGTGTCGCT；

F2：GGATCCACTTAACGTTACTGAAATCCACACATTAACTGTAGTCC，

R2：ATCTGCGCAACAAGGTGG；

F3：AAAGTTCGACAGCGTCTCCG，

R3：TCCATACAAGCCAACCACGG。

the homology arms are underlined.

The overlap extension PCR procedure for amplifying each fragment was as follows:

1. transport protein gene by lipidRTA1Adding primers F1 and R3 at 94 deg.C for 3min; 30s at 94 ℃; 35s at 55 ℃; amplifying at 72 deg.C for 2min for 35 cyclesRTA1Front arm: the first 2/3 hygromycin recombinant fragment;

2. the hygromycin gene andRTA1using the rear arm of the gene as a template, adding primers F3 and R2 at 94 ℃ for 3min; 30s at 94 ℃; 35s at 55 ℃;72 ℃ for 35 cycles at 2min, 2/3 hygromycin after amplification:RTA1a hind arm recombinant fragment;

3. performing overlap extension PCR by using the recombinant fragments obtained in the previous two steps as templates, and firstly, adding no primer at 94 ℃ for 3min; 30s at 94 ℃; 8 cycles of 3min at 72 ℃; then adding primers F1 and R2 at 94 ℃,3min, 94 ℃ and 30s; 35s at 55 ℃; performing 35 cycles at 72 deg.C for 3min, and amplifying to obtainRTA1F：hyg：RTA1R recombinant gene fragment (fig. 4).

Example 6:RTA1construction of Gene-knocked-out Strain (mutant Strain)

Adding 20 mu.L of cryptococcus terrestris competent cells with correct sequencing into 80 mu.L of cryptococcus terrestris competent cellsRTA1F：hyg：RTA1Placing the R recombinant fragment on ice for 15min; then adding the mixed solution into an electric rotating cup with ice pre-cooling of 0.2cm, carrying out electric shock (electric shock parameters: 25 muF, 2500V and 200 omega), adding 400 muL of pre-cooled sorbitol after the electric shock, and uniformly coating the mixed solution on 300 mug-mL on an ultra-clean workbench ^-1 Placing on YPD plate with hygromycin content, and inversely culturing in 28 deg.C incubator for 2-3d; from 300. Mu.g.mL ^-1 Picking single colony on YPD plate with hygromycin content, adding YPD liquid culture medium to culture overnight at 28 ℃, collecting thallus to carry out PCR and real-time PCR detection; the PCR results showed (FIG. 5A) that the recombinant fragment-transferred strain was not amplifiedRTA1Gene, wild type amplifiedRTA1Genes, which indicate that the recombinant fragment carrying the hygromycin gene has successfully undergone homologous recombination, in the strain into which the recombinant fragment was transferredRTA1The gene was successfully knocked out. The results of Real-time PCR detection (FIG. 5B) showed that the concentration of Al was from 50mmol/L ³⁺ Treating wild-type and mutant strainsRTA1From the expression level of the gene, 50mmol/L Al ³⁺ The mutant strain was treated to show little expression, indicating that there was little expression in the mutantRTA1The gene has been knocked out.

Example 7: detection of acid and aluminum stress resistance of cryptococcus terrestris mutant strain

Adding aluminum ions into GM solid culture medium with pH of 4.5 to make final concentration of 50mmol/L, 100mmol/L, 150mmol/L, and using solid culture medium without aluminum ions as control; simultaneously preparing GM solid culture media with the pH values of 3.5, 4.5, 5.5, 6.5 and 7.5 respectively; inoculating wild type yeast and mutant yeast into YPD liquid culture medium, and shakingThe culture was carried out overnight in a bed, and then transferred to fresh GM broth to adjust the concentration of the bacterial liquid to OD ₆₀₀ Is 1; then diluting to 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 (ii) a Respectively taking 1.5 mu L of each concentration bacterial liquid and inoculating the bacterial liquid to GM solid plates with different pH values; respectively inoculating 2.5 mu L of bacterial liquid with each concentration on a GM solid culture medium containing aluminum ions, setting three plates for each type for repetition, culturing at 28 ℃, and observing the growth conditions of different strains under aluminum acid stress;

the results are shown in FIG. 6, and the results in FIG. 6 show that the growth of mutants was hindered in GM solid medium pH 3.5, whereas the mutants grew normally as the wild type in GM solid medium pH 4.5-7.5, but the growth vigor of the mutant colonies was weaker than the wild type; under the treatment of 50, 100 and 150mmol/L aluminum ions, the mutant can not grow as well as the wild type, and under the treatment of 150mmol/L aluminum ions, the mutant can not grow (FIG. 7);

initial OD ₆₀₀ Wild type strain of Cryptococcus oxytoca and mutant strain of Cryptococcus oxytoca with pH of 4.5 and 50mmol/L Al content ³⁺ Culturing at 28 ℃ and 200rpm in GM liquid medium (K), and measuring OD every 2 hours ₆₀₀ Values, growth curves (FIG. 8) are plotted in Al ³⁺ In GM culture medium with final concentration of 50mmol/L,RTA1the mutant has a lag phase in the presence of,RTA1the mutant is delayed for 24 hours; and observing the cell morphology of the strains Cryptococcus oxytoca and mutant at pH4.5 and a final aluminum ion concentration of 50mmol/L in GM liquid medium containing no aluminum ion at pH4.5 under a microscope (FIG. 9), and observing the cell morphology of the strains at pH4.5 and no aluminum ion in GM liquid mediumRTA1Mutants tend to aggregate into clumps compared to wild type; in GM liquid medium with pH4.5 and final aluminum ion concentration of 50mmol/LRTA1The growth conditions of the mutant and the wild type are basically consistent, and no thallus aggregation phenomenon exists.

Example 8: rotating shaftRTA1Detection of resistance to acid aluminum stress of gene INVSC1 strain

Preparing a GM induction solid culture medium with the pH of 4.5 and 7.5; preparing GM inducing solid culture with pH4.5 and aluminium ionThe final concentration of aluminum ions in the GM solid culture medium is 0.1mmol/L, 0.2 mmol/L and 0.5 mmol/L; the INVSC1 yeast, the empty vector yeast I-P-T and the empty vector yeast I-P-T are transferredRTA1Gene INVSC1 Yeast I-P-RTA1Inoculating to YPD liquid culture medium, and shake culturing overnight; inoculating to fresh GM induction liquid culture medium at 1% inoculation amount, and adjusting the bacterial liquid concentration to OD ₆₀₀ Is 1; then diluting to 10 ⁰ 、10 ^-1 、10 ^-2 、10 ^-3 (ii) a Inoculating a certain amount of culture of each concentration gradient bacterial liquid to a solid plate, setting three plates for each type for repetition, carrying out inverted culture in an incubator at 28 ℃, and observing the size of a bacterial colony;

the results are shown in 10 and 11; INSVc1, I-P-T growth on GM-induced solid Medium, and I-P-T growth failure in GM-induced solid Medium at pH7.5, I-P-RTA1Normal growth was observed in GM-induced solid media at ph4.5 and 7.5 (fig. 10); under the treatment of 0.1, 0.2 and 0.5mmol/L of aluminum ions, the concentration of the aluminum ions is increased compared with that without the addition of the aluminum ions, and I-P-RTA1The growth conditions of (a) were all better than those of INVSc1 and I-P-T. (FIG. 11).

Sequence listing

<110> university of Kunming science

<120> lipid transport protein gene and application thereof

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1146

<212> DNA

<213> Cryptococcus terrestris BSLL1-1 (C. Humicola BSLL 1-1)

<400> 1

atgcccgcac cccgcgcact ccgcaacgca gccctcttcc tcgtggtagc agggaaagcc 60

ctcgcccgca cgtgccccga cgcgaccgac gaccccgcca ccgatccgtg caaccagctg 120

cgctacctcc cgagccagta cggcgcgggc gtgtgtctcg cgctgtggct ggtcctcgcg 180

ctggccttca cgtgcgacca ggtcgcgtgg ccgtacttcc gcgtactgcc cgccctcctc 240

ggcgcgtgga ccctcgcggg cggcctgctc atccgcgcgc ggtaccatgc ccgcccgcac 300

tgggtcggcg ggtttgtcgg catggcgatc ctgcactggg tcgggggctg gatgatgact 360

ccggcgatca cggtcgtgct cgcgtacctc gtgtcgcatc tcggcgcgga ggcgtacctc 420

cccggccagt ggagcacgcg caagctactg catgtgtatc tctggaccga cgccctctgg 480

ctgctgctgc agtccctcgg cgggttcatc gggtcgctcg tgccctctgg cgcgcggtcg 540

accggcgagg cgttctacct catcggcctg ctgggccagc tcgtctccct cgtgctgttt 600

accgcacagt acgccgcctt catgctgcgt gttcgcaagg acacccgggt gtgggcggcg 660

cacgcggcgt ggcgcccgct ggcgtacgcc ctggcgtgga gcctcttctg gctgctccag 720

cgcgcggcgt accgcgccgt cctcttccgc ggcgggtaca agggctacct ccgccgtccg 780

gacgcgcagt ttctggcgct tgacgtcgtg tcgctctggc tcgcgtttgt accgctcgcg 840

tggcactggc ccggccgggt gctgcggagc cgcgtgcccc aggcggtgta cgaggagggc 900

gcgtcgcaga ccgtgtccct tgcttctgtc gtccagccca ccatgcgcga cacccacgcc 960

gtgcccatgc ccccgggctt catctacaaa accagcccag agtacctcca gcgcgtcgat 1020

gagttgcgtg ccgagatcaa gaggctcgaa gacgacgcga agcacgccga ggcgcaagcc 1080

gaggccagag ccgctgccac ccgctcacct cgctcgtcca cgcgcaaggc ccctccccag 1140

tggtaa 1146

<210> 2

<211> 341

<212> PRT

<213> Cryptococcus terrestris BSLL1-1 (C. Humicola BSLL 1-1)

<400> 2

Met Pro Ala Pro Arg Ala Leu Arg Asn Ala Ala Leu Phe Leu Val Val

1 5 10 15

Ala Gly Lys Ala Leu Ala Arg Thr Cys Pro Asp Ala Thr Asp Asp Pro

20 25 30

Ala Thr Asp Pro Cys Asn Gln Leu Arg Tyr Leu Pro Ser Gln Tyr Gly

35 40 45

Ala Gly Val Cys Leu Ala Leu Trp Leu Val Leu Ala Leu Ala Phe Thr

50 55 60

Cys Asp Gln Val Ala Trp Pro Tyr Phe Arg Val Leu Pro Ala Leu Leu

65 70 75 80

Gly Ala Trp Thr Leu Ala Gly Gly Leu Leu Ile Arg Ala Arg Tyr His

85 90 95

Ala Arg Pro His Trp Val Gly Gly Phe Val Gly Met Ala Ile Leu His

100 105 110

Trp Val Gly Gly Trp Met Met Thr Pro Ala Ile Thr Val Val Leu Ala

115 120 125

Tyr Leu Val Ser His Leu Gly Ala Glu Ala Tyr Leu Pro Gly Gln Trp

130 135 140

Ser Thr Arg Lys Leu Leu His Val Tyr Leu Trp Thr Asp Ala Leu Trp

145 150 155 160

Leu Leu Leu Gln Ser Leu Gly Gly Phe Ile Gly Ser Leu Val Pro Ser

165 170 175

Gly Ala Arg Ser Thr Gly Glu Ala Phe Tyr Leu Ile Gly Leu Leu Gly

180 185 190

Gln Leu Val Ser Leu Val Leu Phe Thr Ala Gln Tyr Ala Ala Phe Met

195 200 205

Leu Arg Val Arg Lys Asp Thr Arg Val Trp Ala Ala His Ala Ala Trp

210 215 220

Arg Pro Leu Ala Tyr Ala Leu Ala Trp Ser Leu Phe Trp Leu Leu Gln

225 230 235 240

Arg Ala Ala Tyr Arg Ala Val Leu Phe Arg Gly Gly Tyr Lys Gly Tyr

245 250 255

Leu Arg Arg Pro Trp His Trp Pro Gly Arg Val Leu Arg Ser Arg Val

260 265 270

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

275 280 285

Ser Val Val Gln Pro Thr Met Arg Asp Thr His Ala Val Pro Met Pro

290 295 300

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

305 310 315 320

Glu Ala Arg Ala Ala Ala Thr Arg Ser Pro Arg Ser Ser Thr Arg Lys

325 330 335

Ala Pro Pro Gln Trp

340

<210> 3

<211> 26

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

aagcttatgc ccgcaccccg cgcact 26

<210> 4

<211> 28

<212> DNA

<213> Artificial sequence (Artificial)

<400> 4

ggatccttac cactggggag gggccttg 28

<210> 5

<211> 19

<212> DNA

<213> Artificial sequence (Artificial)

<400> 5

atgctgaaaa gccccgact 19

<210> 6

<211> 18

<212> DNA

<213> Artificial sequence (Artificial)

<400> 6

attccccgtt acccgttg 18

<210> 7

<211> 17

<212> DNA

<213> Artificial sequence (Artificial)

<400> 7

ccgtgtccct tgcttct 17

<210> 8

<211> 16

<212> DNA

<213> Artificial sequence (Artificial)

<400> 8

tcggcacgca actcat 16

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (Artificial)

<400> 9

cactgcagac catctccgac 20

<210> 10

<211> 43

<212> DNA

<213> Artificial sequence (Artificial)

<400> 10

tgctccttca atatcatctt ctgtcggcgg ggtgtgtgtc gct 43

<210> 11

<211> 44

<212> DNA

<213> Artificial sequence (Artificial)

<400> 11

ggatccactt aacgttactg aaatccacac attaactgta gtcc 44

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence (Artificial)

<400> 12

atctgcgcaa caaggtgg 18

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence (Artificial)

<400> 13

aaagttcgac agcgtctccg 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence (Artificial)

<400> 14

tccatacaag ccaaccacgg 20

Claims

1. Lipid transport protein geneRTA1The nucleotide sequence is shown in SEQ ID NO. 1.

2. The lipid transporter gene of claim 1RTA1In enhancing cryptococcus terrestris: (C. humicolus) The ability to resist acid stress or/and aluminum stress.