CN112280757B - Application of anthrax fatty acid hydroxylase CsSCS7 - Google Patents

Abstract

The invention provides an application of Bacillus anthracis fatty acid hydroxylase CsSCS7 and a method for constructing a gene knockout vector and a gene knockout mutant, in particular to the application of Bacillus anthracis fatty acid hydroxylase CsSCS7 in degrading fungicides. The CsSCS7 gene knockout vector was obtained, and the CsSCS7 gene knockout mutant was obtained. After functional experiments, it was confirmed that the hydroxylase was very important for the growth of B. anthracis fungal hyphae, and was involved in regulating the degradation of fludioxonil by fungi. The function of degrading pyrrole fungicides such as fludioxonil has a good application prospect.

Description

Application of a fatty acid hydroxylase CsSCS7 from Bacillus anthracis

technical field

The invention relates to the field of biotechnology, in particular to an application of anthracis fatty acid hydroxylase CsSCS7 and a method for constructing a gene knockout vector and a gene knockout mutant.

Background technique

Sphingolipids ( sphingolipids ) are components of eukaryotic cytoplasmic membranes and are widely present in animals, plants and fungi. There are two complex sphingolipids in fungi: inositolphosphoceramides (IPC) and glucosylceramides (GlcCer). Experiments in phytopathogenic fungi have demonstrated that these two complex sphingolipids are involved in the growth and pathogenic process of a variety of fungi. For example, the inhibition of IPC synthase in Botrytis cinerea will seriously affect spore germination and hyphal growth; the ceramide synthase FgBar1 in Fusarium graminearum is specifically connected to the synthesis of GlcCer, and the deletion of GlcCer or the influence of methylation modification will cause Loss of pathogenicity of scab, etc. Previous studies have shown that the hydroxylation state of fungal sphingolipids is involved in the regulation of calcium ion homeostasis and the sensitivity to antifungal drugs, and is an important factor in maintaining cell growth under various stress conditions.

SCS7 is a fatty acid hydroxylase. The protein contains a cytochrome b5-like domain and a hydroxylase domain. It is known to function in yeast to regulate the hydroxylation of sphingolipids. It is a synthetic monohydroxylation The key enzyme of inositol phosphoceramide. Deletion of the SCS7 gene in yeast suppressed the calcium-sensitive phenotype of csg1 and csg2 mutants, but whether SCS7 affects yeast susceptibility to drugs is unknown.

Therefore, according to the existing research, it has not been proved that SCS7 is involved in the regulation of the sensitivity of bacteria to drugs, and there is no related function study of SCS7 in anthracis.

SUMMARY OF THE INVENTION

In view of this, the present invention proposes an application of anthracis fatty acid hydroxylase CsSCS7 and a method for constructing a gene knockout vector and a gene knockout mutant.

The technical scheme of the present invention is realized as follows:

The invention provides the application of anthrax fatty acid hydroxylase CsSCS7 in degrading fungicides.

To further illustrate, the application of the fatty acid hydroxylase CsSCS7 of B. anthracis in regulating the degradation of pyrroles by fungi.

It is further described that the protein encoded by the fatty acid hydroxylase CsSCS7 of B. anthracis, the gene knockout vector and the gene knockout mutant are used in regulating the degradation of pyrroles by fungi.

To further illustrate, the nucleotide sequence of the fatty acid hydroxylase CsSCS7 of B. anthracis is shown in SEQ ID NO: 1; the amino acid sequence of the protein encoded by the fatty acid hydroxylase CsSCS7 of B. anthracis is shown in SEQ ID NO: 2.

A method for constructing a Bacillus anthracis fatty acid hydroxylase CsSCS7 gene knockout vector, comprising the following steps:

(1) Design primer pairs CsSCS7-U-F/CsSCS7-U-R and CsSCS7-D-F/CsSCS7-D-R before and after the coding reading frame of the fatty acid hydroxylase CsSCS7 gene of B. anthracis;

(2) The upper arm sequence and the lower arm sequence after the C-terminus of the fatty acid hydroxylase CsSCS7 gene of B. anthracis were obtained by PCR amplification, and the upper arm and lower arm sequence were combined into the vector pCX62-S by homologous recombination to obtain a knockout Remove the carrier.

Further description, the sequence of the primer CsSCS7-U-F is:

5’-GTACCGGGCCCCCCCAGCTTCTCAGAATCCACATATCCAC-3’

The sequence of the primer CsSCS7-U-R is:

5’-CGATACCGTCGACCTCGAAGCTGCAGGTGGCGATCGTGAAT-3’

The sequence of the primer CsSCS7-D-F is:

5’-GCTCTCACCGCGGATCCTTCATCGACCAACGTTCATAC-3’

The sequence of the primer CsSCS7-D-R is:

5'-CTAGAACTAGTGGATCTCTCTAGGCCGGACATGTTGACTG-3'.

A method for constructing a CsSCS7 gene knockout mutant of Bacillus anthracis fatty acid hydroxylase, the knockout vector obtained by the above construction is introduced into the protoplast of Bacillus anthracis, screened by a DCM medium containing chlorsulfuron, and verified by PCR , to obtain the CsSCS7 gene knockout mutant of B. anthracis fatty acid hydroxylase.

Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention obtains a CsSCS7 gene knockout mutant by cloning the fatty acid hydroxylase CsSCS7 gene of the rubber tree anthracnose, and constructing a CsSCS7 gene knockout vector, and through functional experiments, It is confirmed that the hydroxylase is very important for the growth of mycelium of B. anthracis and participates in regulating the degradation of fludioxonil by fungi. The degradation of bactronil can be applied to the function of degrading pyrrole fungicides such as fludioxonil, and has a good application prospect.

Description of drawings

Fig. 1 is a schematic diagram of the gene knockout of fatty acid hydroxylase CsSCS7 of Bacillus anthracis of the present invention;

Figure 2 is the PCR verification of the gene deletion mutant ΔCsSCS7 of the present invention; the No. 1 lane is Marker, the verification primers of No. 2 and No. 3 lanes are CsSCS7-F/CsSCS7-R, and the verification primers of No. 4 and No. 5 lanes are CsSCS7 -Ou-F/ILV-R, the verification primers for lanes 6 and 7 are ILV-R/CsSCS7-Ou-R;

Figure 3 shows the colony growth morphology of wild-type HN08 strain and gene deletion mutant ΔCsSCS7 strain in CM medium with different fludioxonil concentrations (7 days);

Figure 4 shows the residues of fludioxonil after the wild-type HN08 strain and the gene deletion mutant ΔCsSCS7 strain were treated with fludioxonil.

Detailed ways

In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

The experimental methods used in the embodiments of the present invention are conventional methods unless otherwise specified.

Materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.

Example 1 - Cloning of the fatty acid hydroxylase CsSCS7 gene of Bacillus anthracis

The CsSCS7 gene sequence of C. anthracis was obtained by searching the NCBI database, and the homologous sequence was obtained by comparing the HN08 transcriptome sequence database of C. siamense (preserved in our laboratory) by the local BLAST method, and the primer pair CsSCS7-F ( 5'-CGCGGATCCATGCCGTCGAGAACTCTCCCTA-

3')/CsSCS7-R(5'-CCCAAGCTTTTTATTGCGTCTTGACAATGGGAG-3'). The DNA and cDNA of C. siamense HN08 were used as templates for amplification, respectively. According to sequence analysis, it was shown that the obtained sequence contained a complete coding open reading frame. The size of the DNA sequence is 1271bp, and the size of the cDNA sequence is 1215bp. The gene contains an intron, encoding 404 amino acids, a CytB5 domain, a low-complexity region, a transmembrane region, a hydroxylase, and Saccharomyces cerevisiae SCS7 homology, the gene named CsSCS7 gene, is a fatty acid hydroxylase.

Example 2-Construction of Bacillus anthracis fatty acid hydroxylase CsSCS7 gene knockout vector

Before and after the coding reading frame of the fatty acid hydroxylase CsSCS7 gene of B. anthracis, the primer pairs CsSCS7-U-F/CsSCS7-U-R and CsSCS7-D-F/CsSCS7-D-R were designed, and the upper arm sequence and the lower arm sequence after the C-terminus of the CsSCS7 gene were obtained by PCR amplification , using the method of homologous recombination, the upper arm and lower arm sequences were combined into the vector pCX62-S to obtain the knockout vector pCX62-S-CsSCS7, the schematic diagram is shown in Figure 1.

The sequence of primer CsSCS7-U-F is:

5’-GTACCGGGCCCCCCCAGCTTCTCAGAATCCACATATCCAC-3’

The sequence of primer CsSCS7-U-R is:

5’-CGATACCGTCGACCTCGAAGCTGCAGGTGGCGATCGTGAAT-3’

The sequence of primer CsSCS7-D-F is:

5’-GCTCTCACCGCGGATCCTTCATCGACCAACGTTCATAC-3’

The sequence of primer CsSCS7-D-R is:

5'-CTAGAACTAGTGGATCTCTCTAGGCCGGACATGTTGACTG-3'.

Example 3-Construction of B. anthracis fatty acid hydroxylase CsSCS7 gene knockout mutant

The knockout vector pCX62-S-CsSCS7 obtained by the construction of Example 2 was introduced into the protoplast of rubber tree anthracnose ( C.siamense ) HN08 by using PEG-mediated protoplast transformation. ) DCM medium for screening, 6 batches were transformed, and 57 transformants were obtained.

The genomic DNA sequences of 57 transformants were extracted in batches and verified by PCR. The transformant △CsSCS7-51 was as expected. The primers of CsSCS7 gene CsSCS7-F(5'-CGCGGATCCATGCCGTCGAGAACTCTCCCTA-3')/CsSCS7-R(5'-CCCAAGCTTTTATTGCGTCTTGACAATGGGAG -3'), the wild type can amplify a target band with a size of about 1200bp, but the mutant does not amplify the target band. The upstream primer of CsSCS7 gene CsSCS7-Ou-F (5'-TGCTTTTGCATTGCTGTGACC-3') Using the internal primer ILV-R (5'-GTTCAACGCCGCCTTCCGACAAAAT-3') of the chlorsulfuron resistance gene ILV1, the mutant can amplify a target band with a size of about 2700bp, while the wild-type HN08 did not amplify to a band; The internal primer ILV-F (5'-GGCGGTGCTATCCTTCCCGTGTT-3') of the chlorsulfuron resistance gene ILV1 and the downstream primer CsSCS7-Ou-R (5'-ATGCCTTGTGTTCTTCCGGT-3') of the CsSCS7 gene, the mutant can be amplified to a size of one The target band of about 3000bp was not amplified in the wild-type HN08, as shown in FIG. 2 . Therefore, the PCR results preliminarily indicated that the CsSCS7 gene in the transformant ΔCsSCS7-51 had been replaced by the ILV1 gene.

Example 4-Sensitivity of the ΔCsSCS7 gene deletion mutant strain to the fungicide fludioxonil

In the susceptibility test of the ΔCsSCS7 gene deletion mutant strain to the fungicide fludioxonil, in the medium without fludioxonil, the growth rate of the ΔCsSCS7 gene deletion mutant was lower than that of the wild-type HN08, and with the concentration of fludioxonil When the concentration of fludioxonil increased to 0.1 μg/ml, the growth rate of the ΔCsSCS7 gene deletion mutant decreased significantly, as shown in Figure 3. The results indicate that CsSCS7 can realize the regulation of the sensitivity of fungi to pyrroles such as fludioxonil, and the expression of this gene can improve the resistance of fungi such as anthracis to pyrroles such as fludioxonil.

Example 5 - Determination of the degradation function of pyrrole fungicides such as fludioxonil

To carry out the residual determination of fludioxonil after the wild-type HN08 strain and the gene deletion mutant ΔCsSCS7 strain are treated with fludioxonil, the present invention uses the spore fluid of the wild-type HN08 strain and the gene deletion mutant ΔCsSCS7 strain to inoculate the spore fluid containing 1.204µg/ml of fludioxonil After culturing for 3 days in the CM liquid medium of bacteriocin, use a liquid chromatograph to measure the residual amount of fludioxonil, and use the CM liquid medium without adding spore liquid to add the same amount of agent as a control, and the spore liquid is configured to 10 in advance. ⁵ /ml concentration. The results showed that the concentration of fludioxonil in the medium containing the wild-type HN08 strain and the gene deletion mutant ΔCsSCS7 strain was significantly reduced, and both could degrade fludioxonil. The degradation amount of the ΔCsSCS7 strain was less than that of the HN08 strain, as shown in Figure 4 The results show that the expression of the fatty acid hydroxylase CsSCS7 gene of B. anthracis can increase the amount of degraded fludioxonil by B. anthracis, and can be applied to the degradation of fludioxonil and other pyrrole fungicides.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of the present invention. within the scope of protection.

sequence listing

<110> Hainan University

<120> Application of anthracis fatty acid hydroxylase CsSCS7 and method for constructing gene knockout vector and gene knockout mutant

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1271

<212> DNA

<213> Artificial Sequence

<400> 1

atgccgtcga gaactctccc taccttcacc cgcgccgagg tcgaggcaca taattcgaag 60

aagtcctgtt atgttacgat tggcaaaaat gtctacgacg taaccgactt cgcccaggac 120

catcccggtg gcgccgacct cgtcttcgac tacggcggca aggacatcga atccatcctg 180

cgcgatccga catcccaccc ccactccgag gctgcgtacg aagttctcga cgactccttg 240

gttggattcg tcatttctca aaagtcaatc aatgggacgg ccaacaagcc caacggaaaa 300

gcgaatggac atgtgaacgg gaatgcgaac ggcaatggca acagcgctgc caagacccag 360

gagcatgagg acgagccaaa gatgaatgag aatggcgagc tctgggatgg tgagcgatgg 420

gttcatcctc gcactggcat ggctagcgag gaggatttga gcaaggac ggattacacc 480

aatgactaca agaaacacaa gttcctcgac ttgagccgcc cactgttccc tcagatctgg 540

tacgggggct tcagcaagga gttctacctt gatcaggttc atcgccctcg ccattacaag 600

ggcggcgagt ctgcaccact atttggaaac tttctcgagc ccctttccaa gacgccttgg 660

tgggttgtgc ctgtggcgtg gctcccccca gttgcatacg gtacctattt ggctagagag 720

ggcatggaca gcaccttcca ggaggtctgc tattgggggc tcggcttctt tctctggagc 780

ttgatcgagt acatcttgca ccgcttcctc ttccaccttg acaagtaagc tgcccctatg 840

atgtgctatg caattccaga taaccaacag cgaaaaccag gtggcttccg gacaaccgag 900

ttggcattac catgcatttc ctcctccacg gcatccatca ttacctgcct atggacaagt 960

atcgcctcgt catgcctcct acactctttg ttgttctcgc cacgccgttc tacaagctgg 1020

ctcattgggt tttttcgtac agctggcacg ccgctaccgc tgtttattgc ggaggcatct 1080

ttggttacat ctgctacgac ttgacgcact acttccttca ccaccagaac ttgccgctct 1140

ggtacaagga attgaagaag taccaccttc agcaccactt ccttgactat gagcttggct 1200

ttggcgtcac cagccggttc tgggatagca ttttcggcac cgaactgcct cccattgtca 1260

agacgcaata a 1271

<210> 2

<211> 404

<212> PRT

<213> Amino acid sequence

<400> 2

Met Pro Ser Arg Thr Leu Pro Thr Phe Thr Arg Ala Glu Val Glu Ala

1 5 10 15

His Asn Ser Lys Lys Lys Ser Cys Tyr Val Thr Ile Gly Lys Asn Val Tyr

20 25 30

Asp Val Thr Asp Phe Ala Gln Asp His Pro Gly Gly Ala Asp Leu Val

35 40 45

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

50 55 60

Ser His Pro His Ser Glu Ala Ala Tyr Glu Val Leu Asp Asp Ser Leu

65 70 75 80

Val Gly Phe Val Ile Ser Gln Lys Ser Ile Asn Gly Thr Ala Asn Lys

85 90 95

Pro Asn Gly Lys Ala Asn Gly His Val Asn Gly Asn Ala Asn Gly Asn

100 105 110

Gly Asn Ser Ala Ala Lys Thr Gln Glu His Glu Asp Glu Pro Lys Met

115 120 125

Asn Glu Asn Gly Glu Leu Trp Asp Gly Glu Arg Trp Val His Pro Arg

130 135 140

Thr Gly Met Ala Ser Glu Glu Asp Leu Ser Lys Glu Thr Asp Tyr Thr

145 150 155 160

Asn Asp Tyr Lys Lys His Lys Phe Leu Asp Leu Ser Arg Pro Leu Phe

165 170 175

Pro Gln Ile Trp Tyr Gly Gly Phe Ser Lys Glu Phe Tyr Leu Asp Gln

180 185 190

Val His Arg Pro Arg His Tyr Lys Gly Gly Glu Ser Ala Pro Leu Phe

195 200 205

Gly Asn Phe Leu Glu Pro Leu Ser Lys Thr Pro Trp Trp Val Val Pro

210 215 220

Val Ala Trp Leu Pro Pro Val Ala Tyr Gly Thr Tyr Leu Ala Arg Glu

225 230 235 240

Gly Met Asp Ser Thr Phe Gln Glu Val Cys Tyr Trp Gly Leu Gly Phe

245 250 255

Phe Leu Trp Ser Leu Ile Glu Tyr Ile Leu His Arg Phe Leu Phe His

260 265 270

Leu Asp Lys Trp Leu Pro Asp Asn Arg Val Gly Ile Thr Met His Phe

275 280 285

Leu Leu His Gly Ile His His Tyr Leu Pro Met Asp Lys Tyr Arg Leu

290 295 300

Val Met Pro Pro Thr Leu Phe Val Val Leu Ala Thr Pro Phe Tyr Lys

305 310 315 320

Leu Ala His Trp Val Phe Ser Tyr Ser Trp His Ala Ala Thr Ala Val

325 330 335

Tyr Cys Gly Gly Ile Phe Gly Tyr Ile Cys Tyr Asp Leu Thr His Tyr

340 345 350

Phe Leu His His Gln Asn Leu Pro Leu Trp Tyr Lys Glu Leu Lys Lys

355 360 365

Tyr His Leu Gln His His Phe Leu Asp Tyr Glu Leu Gly Phe Gly Val

370 375 380

Thr Ser Arg Phe Trp Asp Ser Ile Phe Gly Thr Glu Leu Pro Pro Ile

385 390 395 400

Val Lys Thr Gln

<210> 3

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 3

gtaccgggcc cccccagctt ctcagaatcc acatatccac 40

<210> 4

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 4

cgataccgtc gacctcgaag ctgcaggtgg cgatcgtgaa t 41

<210> 5

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 5

gctctcaccg cggatccttc atcgaccaac gttcatac 38

<210> 6

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 6

ctagaactag tggatctcta ggccggacat gttgactg 38

<210> 7

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 7

cgcggatcca tgccgtcgag aactctccct a 31

<210> 8

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 8

cccaagcttt tattgcgtct tgacaatggg ag 32

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 9

tgcttttgca ttgctgtgac c 21

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 10

gttcaacgcc gccttccgac aaaat 25

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 11

ggcggtgcta tccttcccgt gtt 23

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 12

atgccttgtg ttcttccggt 20

Claims (3)

1. The application of the anthrax fatty acid hydroxylase CsSCS7 is characterized in that: the application of the anthrax fatty acid hydroxylase CsSCS7 in degrading pyrrole bactericide, wherein the nucleotide sequence of the anthrax fatty acid hydroxylase CsSCS7 is shown as SEQ ID NO: 1 is shown.

2. The use of the anthrax fatty acid hydroxylase CsSCS7 of claim 1, wherein: the anthrax fatty acid hydroxylase CsSCS7 is applied to positively regulating the degradation of the fungi to the pyrrole medicaments.

3. The use of the anthrax fatty acid hydroxylase CsSCS7 of claim 1, wherein: the amino acid sequence of the protein coded by the anthrax fatty acid hydroxylase CsSCS7 is shown as SEQ ID NO: 2, respectively.

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