CN111944831A

CN111944831A - Ceratopteris chinensis CYP18A1 gene and application thereof

Info

Publication number: CN111944831A
Application number: CN202010821539.9A
Authority: CN
Inventors: 陈航; 柳鹏飞; 凌晓霏; 陆沁; 张金稳; 王伟伟; 吴海霞; 陈晓鸣
Original assignee: Research Institute of Resource Insects of Chinese Academy of Forestry
Current assignee: Research Institute of Resource Insects of Chinese Academy of Forestry
Priority date: 2020-08-15
Filing date: 2020-08-15
Publication date: 2020-11-17

Abstract

The invention relates to a fraxinus chinensis CYP18A1 gene and application thereof, belonging to the technical field of genetic engineering. The nucleotide sequence of the fraxinus chinensis CYP18A1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2. The fraxinus chinensis CYP18A1 gene has obvious influence on the growth and development of larval stages of fraxinus chinensis, CYP18A1 expression is interfered, expression quantity is reduced, the development of fraxinus chinensis is slow, body length, body width and body surface area are all obviously smaller than those of a control group, and the results prove that the growth and development speed of fraxinus chinensis can be regulated and controlled through the gene or derivatives thereof, the wax secretion time of fraxinus chinensis can be prolonged, provenance is excellent, fine variety of fraxinus chinensis is cultivated, the yield of fraxinus chinensis is improved, dependence on climate and environment is eliminated in production of fraxinus chinensis, and the fraxinus chinensis gene has important significance for excavating new biological resources and promoting development of fraxinus chinensis industry.

Description

Ceratopteris chinensis CYP18A1 gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, particularly relates to a fraxinus chinensis CYP18A1 gene and application thereof, and particularly relates to regulation and control of the fraxinus chinensis ecdysone hydrolase CYP18A1 gene on development of fraxinus chinensis and application thereof.

Background

Ericerus pela channels is an important resource insect, belongs to the genus Ericerus of the superfamily Hemiptera (coccoidea) scale, and is distributed in the monsoon regions of northern subtropics, middle subtropics, and temperate zones (Zhang Changhai, 1991). The second-instar male nymphs of the wax insects secrete a large amount of biomass wax, the main components of the biomass wax are hexacosanoic acid (C25H51COOC26H53), the biomass wax has ecological values of protecting the growth and development of the wax insects and nutritional health-care and economic values, and is widely applied to the fields of printing, machinery, medicines, foods, cosmetics and the like (Xiaoming, 2011). The fraxinus chinensis has unique hermaphrodite development phenomena, namely, the female is in incomplete metamorphosis development, the male is in complete metamorphosis development, wherein the fraxinus chinensis nymphs of the first age migrate to leaves to feed as fixed leaves in the initial stage, and the fraxinus chinensis nymphs of the second age migrate to trunks as fixed stems in the initial stage. The life cycle of the female is as long as one year, and the main life process of the female is adult pregnant eggs; the life cycle of the male is five months, a large amount of white wax is generated to cover the larva bodies during the period of fixing the second-instar nymphs, the larva bodies under the wax layer complete the metamorphosis process of the second-instar male nymphs, prepupa, pupa and male adults, and the larva bodies are dead after mating with female worms in the male adult period of 10-15 days. In the late stage of the second-instar male nymphs of the fraxinus chinensis, the difference between the titer of Juvenile Hormone (JH) and the titer of ecdysone (20E) is large, the ecdysone effect is dominant, and the male nymphs are regulated and controlled to enter a complete metamorphosis development stage; the difference between the JH titer and the 20E titer of the second-instar female nymphs is small, juvenile hormone is dominant, and the female nymphs are regulated to enter an incomplete metamorphosis development stage.

Inactivation of ecdysone (20E) is essential for normal development, cytochrome P450 enzymes regulate insect ecdysone homeostasis, and CYP18a1 is a key enzyme in this process. CYP18a1 is a 26-hydroxylase of the CYP2 family, plays an important role in the catabolism of 20-hydroxyecdysone, is capable of converting 20E to 20, 26-dihydroxyecdysone while participating in xenobiotic metabolic processes, is conserved among most arthropods, and CYP18a1 has been shown to be induced by ecdysoid steroids, playing an important role in the preparation of metamorphosis and ecdysis. In drosophila, the knock-out of CYP18a1 results in the lethality of pupal, possibly as a result of impaired ecdysone degradation, and overexpression of CYP18a1 in PG results in nymph development arrest, indicating that CYP18a1 can inactivate all ecdysone and that a reduction in CYP18a1 transcriptional expression may impair the developmental stages of drosophila. In silkworms, CYP18A1 not only has an expression profile of time and tissue specificity, but also has an expression pattern closely related to the peak of ecdysone accumulation in haemolymph of silkworms, which shows that homologous CYP18A1 of insects is closely related to ecdysone homeostasis, the over-expression of CYP18A1 leads to the stunted development of terminal-age larvae, and the 20E titer in the insects over-expressing CYP18A1 is lower than that of a control group. CYP18a1 gene expression levels were significantly elevated prior to the transition from vitellogenesis to chorionic villus suggesting that it may lead to a reduction in the titer of this transition 20E. In Daphnia magna (Daphnia magna), the knockout of CYP18a1 gene leads to early embryo death before the first molting, possibly regulating the drop in molting steroid titer before molting and ovulation, and in bactrocera minax, the expression of CYP18a1 is relatively low throughout diapause.

Through the literature search, the same public reports as the invention are not found.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provides a gene related to the growth and development of the fraxinus chinensis so as to regulate and control the development time of the fraxinus chinensis and increase the second-instar larva period of the fraxinus chinensis.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the first aspect of the invention provides a wax insect CYP18A1 gene, and the nucleotide sequence of the wax insect CYP18A1 gene is shown in SEQ ID NO. 1.

The second aspect of the invention provides a coded protein of the wax insect CYP18A1 gene, and the amino acid sequence of the coded protein is shown in SEQ ID NO. 2.

The third aspect of the invention provides an expression vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the fraxinus chinensis CYP18A1 gene.

The fourth aspect of the invention provides a recombinant vector containing a target gene in the fraxinus chinensis CYP18A1 gene, wherein the nucleotide sequence of the target gene is shown as SEQ ID NO. 3.

The fifth aspect of the invention provides application of the wax insect CYP18A1 gene in regulating the growth and development speed of the wax insect.

The sixth aspect of the invention provides application of the fraxinus chinensis CYP18A1 gene in prolonging the wax secretion time of fraxinus chinensis.

The seventh aspect of the invention provides an application of the fraxinus chinensis CYP18A1 gene in breeding of improved fraxinus chinensis seeds.

The eighth aspect of the invention provides application of the fraxinus chinensis CYP18A1 gene in increasing yield of fraxinus chinensis.

The fraxinus chinensis CYP18A1 gene is obtained through transcriptome, the nucleic acid sequence of the gene is shown as SEQ ID NO.1, the length of the sequence is 16997 bp, the Codon Adaptation Index (CAI) is 0.698, the number of effective codons (ENC) is 52.698, and the GC content: 45.27%, GC3s content: 57.65%, having 1 Open Reading Frame (ORF), length 1, 608bp, stop codon TAA.

The amino acid sequence of the protein coded by the wax insect CYP18A1 gene is shown in SEQ ID NO.2 according to the degeneracy and the preference of codons. The protein contains 535 amino acids, the highest content of the 535 amino acids is Leu (L) (58, accounting for 10.8 percent), and the protein does not contain two amino acids of Pyl (O) and Sec (U); the instability factor was 45.84, greater than 40. The target gene segment of the wax insect CYP18A1 for RNAi interference is shown in SEQ ID NO. 3. Exogenous vector plasmid L4440-CYP18A1 is constructed, DH5 alpha is transformed into recombinant vector plasmid recombinant plasmid target genes, a large amount of double-stranded RNA (dsRNA) of the target genes is expressed and accumulated in HT115, and the dsRNA of the target genes is sprayed on the wax insect to influence the growth and development of the wax insect.

Compared with the prior art, the invention has the beneficial effects that:

the white wax worm CYP18A1 gene has obvious influence on the growth and development of the larval stage of the white wax worm, CYP18A1 expression is interfered, the expression quantity is reduced, the white wax worm develops slowly, the body surface projection area is obviously smaller than a negative Control group CK (a natural group which is not treated) and a positive Control group Control (a group treated by HT 115/L4440), and the gene or a derivative thereof can be used for regulating the growth and development speed of the white wax worm, prolonging the wax secretion time of the white wax worm, screening excellent seed sources, cultivating fine white wax worm seeds, improving the yield of the white wax worm, enabling the white wax production to get rid of dependence on climate and environment, and having important significance on excavating new biological resources and promoting the development of the white wax industry.

Drawings

FIG. 1 is a hydropathic assay of the wax insect CYP18A1 protein; wherein, the maximum hydrophobic value of the amino acid at the 23 th position is 2.844, the minimum hydrophilic value of-2.744 appears between the amino acids at the 152 th position, the average hydrophobic property is-0.204, and the protein is amphiprotic hydrophilic protein;

FIG. 2 is an analysis of the transmembrane domain of the Ceratoptera chinensis CYP18A1 protein; note: CYP18a1 protein is a transmembrane bilayer membrane protein;

FIG. 3 is a functional locus of the Ceratoptera chinensis CYP18A1 protein, endoplasmic reticulum;

FIG. 4 is an analysis of Ceratoptera chinensis CYP18A1 protein belonging to the P450 superfamily family; the CYP18A1 protein contains a plurality of conserved structural domains, mainly belongs to the p450 superfamily family, the structural domain is positioned at the 40 th to 506 th amino acids, the region contains the most conserved haemoglobin binding domain, and the haemoglobin binding domain contains an absolutely conserved cysteine residue and is the fifth coordination ligand of the iron haemoglobin. (ii) a

FIG. 5 is a tertiary structure analysis of the protein of Ceratoptera chinensis CYP18A 1; the tertiary structure of CYP18A1 is a monomer (monomer) formed by 253 alpha-helices, 26 Beta-turns, 55 extended chains and 201 random coils, and contains 1 BCD (Beta-Cyclodextrin) ligand, 1 HEM (PROTOPORPH YRIN IX CONTAINING FE) ligand, 1D 2V ((3S, 5Z, 7E, 22E) -9, 10-secoe rgosta-5, 7, 10, 22-tetraen-3-ol) ligand, and shorter extended peptide segments at the C and N terminals;

FIG. 6 is a PFKM value analysis of Ceratoptera chinensis CYP18A1 at 6 different developmental stages;

FIG. 7 shows the PCR amplification result (318bp) of the wax insect CYP18A1 target gene;

FIG. 8 shows the stable expression of wax insect CYP18A1 target gene reconstruction vector L4440-CYP18A1 in competent cell DH5 alpha, the left picture shows the single bacterial colony clone culture of DH5 alpha (L4440-CYP18A1), and the right picture shows the PCR result (318bp) of DH5 alpha (L4440-CYP18A1) bacterial liquid;

FIG. 9 shows the sequencing result of the recombinant plasmid CYP18A1/L4440-HT115, 1. Cluster-representing the transcriptome sequence, and 2.f-f representing the sequencing result sequence of the company

FIG. 10 shows the stable expression of wax insect CYP18A1 target gene recombinant vector L4440-CYP18A1 in HT115, the left picture shows the single colony clone culture of HT115(L4440-CYP18A1), and the right picture shows the PCR result (318bp) of HT115(L4440-CYP18A1) bacterial liquid;

FIG. 11 shows the in vivo expression abundance of the target gene CYP18A1 after the wax insect body is subjected to RNAi of the target gene CYP18A1, CYP18A1(30mg/ml) is an experimental group, and Control is a positive Control group (inconsistent with the marker in the figure);

FIG. 12 shows the change of growth state of Ceriporia chinensis in 30 days after the RNAi of the Ceriporia chinensis CYP18A1 target gene, CK is a negative control group (natural group), time is the sampling date, 6.02(6 months 2 days), 6.12(6 months 12 days), 6.22(6 months 22 days), and 7.02(7 months 2 days);

FIG. 13 is a graph showing the change in the growth rate of Ceriporia alba after 40 days from the RNAi of the Ceriporia alba CYP18A1 target gene; CYP18A1 is an experimental group, Control is a positive Control group, and CK is a natural group;

FIG. 14 is a schematic diagram of library construction.

Detailed Description

The present invention will be described in further detail with reference to examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.

First, the obtainment of Ceratopteris albidus CYP18A1 Gene

1. Material

The wax insect is sourced from Sichuan Showa (northern latitude: 27.33, east longitude: 103.72, altitude 1685m), the wax insect sample is collected in resource insect institute of Chinese forestry science research institute (northern latitude 25 degrees 3 '20', east longitude 102 degrees 45 '16', altitude 1950m), the parasitic tree is Ligustrum lucidum (Ligustrum lucidum), samples of six periods of FF, SF, EA, LA, FM and SM are collected in total, three groups of biological repetitions of each sample are collected, and the samples are frozen and stored at-80 ℃.

2. Extraction of fraxinus chinensis total RNA:

the total RNA of the white wax worm body is extracted by using an RNA extraction kit (EZ-10Tatal RNA Minin-presps kit) (Shanghai, Biotechnology engineering (Shanghai), Inc.), and the experimental steps are as follows;

(1) taking a clean small mortar, igniting with alcohol, heating and sterilizing to remove RNase;

(2) cooling the mortar by using liquid nitrogen until water vapor is condensed around the mortar;

(3) taking 20mg of fresh fraxinus chinensis insect bodies, adding into a mortar, and quickly grinding into powder;

(4) the powdery fraxinus chinensis tissues are quickly transferred into a 1.5ml RNase-free PE tube, 300 mul of RLT buffer is added, the mixture is slightly shaken and shaken evenly, and the mixture is kept stand for 5 minutes at room temperature;

(5) adding 10 μ l PK (protease K) and 590 μ l RNase-free water, mixing, and placing in 55 deg.C water bath for 10 min;

(6) centrifuging at 4 deg.C for 3 min at 12000g/min, collecting supernatant, adding into 1.5ml RNase-free PE tube, and removing precipitate;

(7) 1/2 supernatant (450-;

(8) 1/2 adding the above mixture into RNase-free adsorption column, centrifuging at 12000g/min for 2 min, and discarding the waste liquid; adding the rest 1/2 mixture into the same centrifugal column, and repeating the above steps;

(9) adding 350 mul DW (DW washing solvent), centrifuging at 12000g/min for 1min at room temperature, and discarding the waste liquid;

(10) adding 500 mul DW, centrifuging at 12000g/min for 1min, discarding the waste liquid, and repeating once;

(11) idling the centrifugal machine 12000g/min at room temperature, taking out the adsorption column beside the alcohol lamp, placing the adsorption column in a 1.5ml RNase-free PE tube, and standing for 2 minutes at room temperature;

(12) adding 50 mul of RNase-free water, and standing for 2 minutes at room temperature; centrifuge at 12000g/min for 1 min.

3. Sequencing transcriptome data: sequencing by Seno Nuo grass genesis Limited liability company, comprising the following steps (1) white wax insect Total RNA sample detection

The detection of RNA samples by a Nordheim source mainly comprises 4 methods:

A. analyzing the degradation degree of RNA and whether pollution exists by agarose gel electrophoresis;

purity of the Nanodrop detection RNA (OD260/280 ratio);

c, accurately quantifying the RNA concentration by the qubit;

agilent 2100 accurately detects RNA integrity;

(2) wax insect cDNA library construction and library inspection

After the sample is qualified, the magnetic beads with oligo (dT) are used for enriching the eukaryotic mRNA. Then fragmentation buffer is added to break mRNA into short fragments, mRNA is used as a template, hexabasic random primers (random hexamers) are used for synthesizing single-strand cDNA, buffer solution, dNTPs, DNA polymerase I and RNase H are added to synthesize double-strand cDNA, and AMPure XP beads are used for purifying double-strand cDNA. The purified double-stranded cDNA is subjected to end repair, A tail is added and a sequencing joint is connected, and then the AMPureXP beads are used for fragment size selection. And finally, carrying out PCR amplification, and purifying the PCR product by using AMPure XP beads to obtain a final library. After the library is constructed, firstly using Qubit2.0 to carry out preliminary quantification, diluting the library to 1.5ng/ul, then using Agilent 2100 to detect the insert size of the library, and after the insert size meets the expectation, using a Q-PCR method to accurately quantify the effective concentration of the library (the effective concentration of the library is more than 2nM) so as to ensure the quality of the library. The schematic diagram of library construction is shown in FIG. 14.

(3) Sequencing on machine

And after the library is qualified, carrying out Illumina HiSeq sequencing on different libraries according to the effective concentration and the requirement of the target offline data volume.

(4) Analysis and Assembly of original sequences

And after the sequencing data is qualified, performing transcript splicing, assembling and analyzing. We obtained the Ceratoptera chinensis CYP18A1 gene.

Wax insect CYP18A1 analysis

1. Wax insect CYP18A1 gene and expression protein analysis

Through transcriptome analysis and screening, the obtained fraxinus chinensis CYP18A1 gene is 16997 bp long, the Codon Adaptation Index (CAI) is 0.698, the number of effective codons (ENC) is 52.698, and the GC content: 45.27%, GC3s content: 57.65%, has 1 Open Reading Frame (ORF), length 1608bp, stop codon TAA, codes 535 amino acids, and has the highest content of Leu (L) (58, 10.8%), and does not contain two amino acids of Pyl (O) and Sec (U); the instability factor was 45.84, greater than 40. The protein showed an amphipathic hydrophilic protein with a maximum hydrophobicity of 2.844 at amino acid position 23 and a minimum hydrophilicity of-2.744 between amino acids 152, with a peak distribution below 0 and a peak distribution above 0, and an average hydrophobicity of-0.204 (FIG. 1). The CYP18a1 protein is a double-transmembrane protein (fig. 2), located in the endoplasmic reticulum, functioning in concert with the endoplasmic reticulum (fig. 3), and contains a plurality of conserved domains, mainly belonging to the P450 superfamily family, located at amino acids 40-506 (fig. 4), which contains the most conserved hemoglobin binding domain, which contains an absolutely conserved cysteine residue, is the fifth coordinating ligand for hemoglobin iron, and contains the highly conserved P450 motif F-X-G-X-C-X-G, having a total of 73 phosphorylation sites (34 for Ser, 22 for Thr, and 17 for Tyr), and the activity and function of regulatory proteins may be regulated by phosphorylation reactions at these sites. The protein has a tertiary structure of monomers (monomer) formed by 253 alpha-helices, 26 Beta-turns, 55 extended chains and 201 random coils, and contains 1 BCD (Beta-Cyclodextrin) ligand, 1 HEM (PROTOPORPHYRIN IX CONTAINING FE) ligand, 1D 2V ((3S, 5Z, 7E, 22E) -9, 10-secoergosta-5, 7, 10, 22-tetraen-3-ol) ligand, and shorter extended peptide segments (EP) at the C and N ends (FIG. 5).

2. Ceratoptera chinensis CYP18A1 expression analysis result

By analyzing the PFKM values of CYP18a1, it was found that CYP18a1 was low expressed in first-instar male larvae (FM), first-instar female larvae (FF), early female adults (EA), late female adults (LA), and high expressed in second-instar male larvae (SM) and second-instar female larvae (SF) (fig. 6).

2.1 wax insect CYP18A1 exogenous vector construction and expression

(1) Primer design

Screening a CYP18A1 transcript sequence according to a fraxinus chinensis transcriptome database, analyzing the base sequence of the CYP18A1 gene through interference site prediction software (siDirect version 2.0), finding out a fragment with more interference sites, wherein the length of the fragment is 318bp, and the off-target effect is avoided. Primers were designed using Premier 6.0. Sal I and Sac I enzyme sites (SalI and SacI, underlined) were added to the upstream primer and the downstream primer of the synthesized CYP18A1 gene primer (Table 1).

TABLE 1

(2) Amplification of candidate Gene (FIG. 7)

The synthesized primer CYP18A1 (Table 1) was diluted to 10. mu.M for PCR amplification, and the specific reaction system (Table 2) and procedure for the amplification of the target gene were as follows:

TABLE 2

The PCR amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.

(3) Gel recovery of PCR amplification products

After PCR, 1% agarose Gel electrophoresis is used for detecting CYP18A1 target gene fragments (adding an electrophoretogram), then a Gel block of a CYP18A1 gene band is cut off in an ultraviolet instrument, and a SanPrep Column type DNA Gel recovery Kit (SanPrep Column DNA Gel Extraction Kit) is used for recovery, wherein the experimental steps are as follows:

A. cutting off a gel block containing the CYP18A1 gene strip by using a sharp blade in an ultraviolet gel cutting instrument;

B. adding 5 times volume of Buffer B2, performing water bath for 10 minutes at 50 ℃, and performing manual mixing every few minutes in the process;

C. adding 1/3 volumes of isopropanol and mixing;

D. transferring into a centrifugal column, centrifuging at 8000 Xg/min for 30s, and discarding waste liquid;

E. adding 500 μ l of Wash Solution, centrifuging at 9000 Xg/min for 30s, and discarding the waste liquid;

F. repeating the step E1 times;

G. centrifuging the empty adsorption column at room temperature at 9000 Xg/min for 1 min;

H. transferring the adsorption column into a 1.5ml centrifuge tube, adding 30 mul of Elution Buffer, standing for 1 minute at room temperature, and centrifuging for 1 minute at 9000 Xg/min;

I. sucking the DNA solution in the centrifugal tube into the centrifugal column again for 9000 Xg for centrifugation for 1 minute; collecting a DNA solution;

J. the ultramicro spectrophotometer is used for measuring the concentration and purity of the target gene solution, and the target gene solution is stored in a refrigerator at the temperature of 20 ℃ below zero for later use.

(4) Enzyme digestion of target fragment and L4440 interference vector

The CYP18A1 rubber cutting obtained above is subjected to double digestion by Sac I and Sal I in an ice bath environment to recover a product and an L4440 plasmid vector.

A. The cleavage system is shown in Table 3:

TABLE 3

The mixture was gently flicked and the enzyme was allowed to act at 37 ℃ for 10 minutes.

B. Gel recovery of the cleavage products

After the enzyme digestion is finished, detecting double enzyme digestion products of CYP18A1 gene or L4440 plasmid by gel electrophoresis, then recovering the gel, measuring the concentration and purity of the recovered products, and storing at-20 ℃ for later use.

(5) T4 DNase ligation reaction

The T4 DNA ligase cuts the recovered CYP18A1 and L4440 plasmids, the experiment is carried out according to the reaction system shown in the table 4, and the overnight ligation is carried out, thus obtaining the recombinant plasmid exogenous vector CYP18A1+ L4440.

TABLE 4

Reagent	Amount of the composition used
		CYP18A1 target fragment	9.5μl
L4440 vector	1.0μl
		T4 ligase Buffer	2.0μl
T4 ligase	0.5μl
		dH₂O	7.0μl
Total	20.0μl

(6) Transformation of recombinant plasmids

A. Transferring the exogenous vector CYP18A1+ L44405 ul into competent cell DH5 alpha (20 ul DH5 alpha), and keeping ice bath for 30 minutes;

B. after the ice bath is finished, quickly transferring the mixture into a water bath heat shock kettle with the constant temperature of 42 ℃ for 70 seconds;

C. it was quickly placed on ice and left to stand for 3 minutes;

D. add 400 μ l SOC liquid medium (with Amp +); wherein the SOC liquid culture medium (containing Amp +) comprises the following components: 34g/L SOC powder, 100mg/L Amp +, Trypton e 2% (w/v), Yeast extract 0.5% (w/v), NaCl 10mM, KCl 2.5mM, MgCl₂ 10mM，Glucose 20mM；

E.160r/min, shake fungus for 1h at 37 ℃;

F. coating part of the bacterial liquid on an SOC solid culture medium containing Amp +; wherein, the components of the SOC solid culture medium containing Amp + are 12g/L agar powder, 34g/L SOC powder, 100mg/L Amp +, Tryptone 2% (w/v), Yeast extract 0.5% (w/v), NaCl 10mM, KCl 2.5mM, MgCl₂ 10mM，Glucose 20mM。

G.37 ℃ for 14 hours.

(7) Screening and identification of expression vectors (FIG. 8)

A. Selecting SOC solid culture medium for cultureThe single bacterial beads of (2) were transferred to SOC liquid medium (containing Amp at 100. mu.g/ml)⁺) Culturing at 37 ℃ and 160r/min for 14h to obtain CYP18A1/L4440-HT115 bacterial liquid.

B. After PCR of the cultured bacterial liquid, electrophoresis is carried out for 30 minutes by using 1% agarose gel under the condition of 120V, a strip is detected,

C. the CYP18A1/L4440-HT115 bacterial liquid is subjected to amplification culture, plasmids are extracted, and the plasmids are sent to a company for sequencing, and the sequencing result is shown in figure 9.

Wax insect CYP18A1 gene target fragment expression and RNAi interference experiment

Inducible expression of dsRNA

The recombinant plasmid (CYP18A1-L4440) was transformed into HT115 competent strain, spread on SOC solid medium (containing 100. mu.g/ml ampicillin and 50. mu.g/ml tetracycline), cultured for 14h, picked single beads were transferred into SOC liquid medium (containing 100. mu.g/ml ampicillin and 50. mu.g/ml tetracycline), cultured at 37 ℃ on a shaker for 12h, and HT115 bacterial fluid containing the desired gene was selected (FIG. 10), and then the culture was added to 2XYT liquid medium containing 75. mu.g/ml ampicillin and 12.5. mu.g/ml tetracycline, cultured at 160r/min on a shaker at 37 ℃ until OD₆₀₀And (3) when the concentration is 0.4-0.5, adding IPTG (concentration is 0.64mmol/L) for induction.

The SOC solid culture medium comprises 12g/L agar powder, 34g/L SOC powder, Tryptone 2% (w/v), Yeast extract 0.5% (w/v), NaCl 10mM, KCl 2.5mM, MgCl₂ 10mM，Glucose 20mM。

The SOC liquid culture medium comprises the following components: 34g/L SOC powder, Tryptone 2% (w/v), Yeast extract 0.5% (w/v), NaCl 10mM, KCl 2.5mM, MgCl₂ 10mM，Glucose 20mM；

The 2xYT liquid medium comprises the following components: 1.6% (W/V) Tryptone, 1% (W/V) Yeast extract, 0.5% (W/V) NaCl.

dsRNA transfection

The bacterial strain is diluted by DEPC water, CYP18A1/L4440-HT115 bacterial liquid with the concentration of 30ng/ml is prepared as an experimental group (CYP18A1), an empty plasmid L4440 is transferred into HT115 competent cells, and the L4440-HT115 is constructed as an experimental positive Control group (Control), and a negative Control group which is not treated is a natural group (CK). In the transfection period, the initial stage of the second-instar male nymphs of the fraxinus chinensis is selected, and because the initial stage of the second-instar male nymphs secretes a small amount of wax, the surface of the worm body is not coated with the fraxinus chinensis, and the bacterial liquid can directly act on the surface of the worm body and enter the body through the wax glands and pores on the surface of the fraxinus chinensis. The CYP18A1/L4440-HT115 bacterial liquid and the L4440-HT115 bacterial liquid are directly sprayed on the polypide by a spray bottle, the polypide is treated for 1 time at 9 o 'clock every morning and is continuously treated for 3 times, experimental samples are collected at intervals of 12h, 24h, 48h and 72h from the 9 o' clock on the third day, and the abundance in the CYP18A1 polypide is detected by qRT-PCR.

qRT-PCR (FIG. 11)

(1) Primer design

Gene-specific primers for qPCR were designed using Primer 6.0 (table 5).

TABLE 5

Diluting cDNA of all samples to 5 ng/mu l, diluting primers to 10 mu M, quantitatively detecting gene expression quantity by qRT-PCR, selecting beta-actin gene as internal reference, and performing 3 times of biological repetition on each sample.

(2) The reaction system is shown in Table 6:

TABLE 6

qPCR reaction: 95-30 s (pre-denaturation), 95-5 s (denaturation), 60-40 s (annealing), and 39 cycles of the second step.

The results show that the expression level of the target gene CYP18A1 is significantly lower than that of the control group 48 hours after the interference.

4. Detection of external morphology

By means of a body microscope VXH2000, changes of the body morphology and the growth condition of the wax worms were monitored by sampling every 10 days, changes of the external morphology (fig. 12), the metamorphosis development process and the projection area thereof were detected (13), and 5 heads were measured per experimental group.

The results show that, after the interference, the second-instar male nymphs of the wax beetles develop malformations and develop slowly.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> resource insect research institute of China forestry science research institute

Paralichthys olivaceus CYP18A1 gene and application thereof

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<170> SIPOSequenceListing 1.0

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<213> Artificial sequence ()

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gaccttgatt aacaaaatgc cagaacatta gtgggaaccg actggcaacc ttttcgtgtt 120

caaaaaatcg ggtaacgatt cgtcagcgat tatttcacca tcgaatggtc tttcaatgag 180

ttgtatctta tagctggcgg gagttaaggt gatgccgaaa atgccttcta aatctggaag 240

tgattgacct tcgggcaatt taaggttgaa cgtgtgcaaa atcgaagaaa aaaataaaaa 300

tatttccatg cgagcgagaa attcacccaa acaatttcgt cgaccgacgc caaacggtat 360

aaaaaaatcg ggtttaacaa ctctgccttc ggaattgata aagcgacttg gacgaaattc 420

atccggtgac tcccacaatt ctggatcatt attgacagcg taaattaacg gaatgatttc 480

cgtatttttt ggtatggtaa atcctttgaa atttacgtct ttcatggtgg cgtgcgagtt 540

gcccaaaggt acgacactcg atttgcgcag cacctccaaa atggtcgcct ccgtgatggg 600

caaatagggt aaatcgtcaa aggtgggcaa ccgtttgcgg ccaacgacca tgtccagctc 660

agcctgaatt gaacgagcca gctccggata gtggaccatg tagatcaacg accagtgcaa 720

cgtggttttg gttgtctcca tgccggcggt gaacaggtcg acgacgattt ggcggatctg 780

cgagttgtgg tccttgccct ggaacagatt ttcgctgttt ccggacagct cggctcgccg 840

gatctccatc agatagcagt cgagcacgtc gcgcgtcttc gactcttcga acgtgcgtcg 900

atggtcgtcg atgatcttct ggaagaagtt gtgcgttttc gccttgttct cgtcgatttt 960

cttcatctcc ttcaggaagt agggaaagtg gcgcagaaac gggaaatagt tgaggaagac 1020

gatgccgcca aacagctgga agccttcttc gattttctcc atcagtcgcc ggaaaatggg 1080

atcgttgaag ctgaagcgga cgctcatcag cagcgagcag atgacgttgc tcaccgagat 1140

gaccagactg tggttcaaat cgatggcgcg tcctttttga cttttcaaat tgtccagcaa 1200

gtaggaaacc tcgcccataa ttctgctctc cagatgattg ttgtttttct tattaaagat 1260

ggtcattcca aagcaacgta aacacgaatg cacaaatcga cgttgctctt tccatatatt 1320

tccttgcgtg tttataattc catagccacc gagtaatttg taaaattcat tcgacggttt 1380

accactgaat tcttctttat tgaacgcctc cttaatccac ttcggatcgc tcaatatgat 1440

cacatttttc aagcccaaat tcaccgaaaa tattttacca tatttctgcg acaaagccaa 1500

atattgacga tgaacgtttt gacctttgga aatgaaatat aggaaaccga tgactggcag 1560

accccaaggt ccgggaggca tatttctgta ttttctgtac ttcattagca tgtacagaca 1620

taagatggtg cctatcagca gacgtgtgtc gcagctattc caaaatcgtc caaatttcat 1680

ttttgtatag tcgaaca 1697

<210> 2

<211> 535

<212> PRT

<213> Artificial sequence ()

<400> 2

Phe Asp Tyr Thr Lys Met Lys Phe Gly Arg Phe Trp Asn Ser Cys Asp

1 5 10 15

Thr Arg Leu Leu Ile Gly Thr Ile Leu Cys Leu Tyr Met Leu Met Lys

20 25 30

Tyr Arg Lys Tyr Arg Asn Met Pro Pro Gly Pro Trp Gly Leu Pro Val

35 40 45

Ile Gly Phe Leu Tyr Phe Ile Ser Lys Gly Gln Asn Val His Arg Gln

50 55 60

Tyr Leu Ala Leu Ser Gln Lys Tyr Gly Lys Ile Phe Ser Val Asn Leu

65 70 75 80

Gly Leu Lys Asn Val Ile Ile Leu Ser Asp Pro Lys Trp Ile Lys Glu

85 90 95

Ala Phe Asn Lys Glu Glu Phe Ser Gly Lys Pro Ser Asn Glu Phe Tyr

100 105 110

Lys Leu Leu Gly Gly Tyr Gly Ile Ile Asn Thr Gln Gly Asn Ile Trp

115 120 125

Lys Glu Gln Arg Arg Phe Val His Ser Cys Leu Arg Cys Phe Gly Met

130 135 140

Thr Ile Phe Asn Lys Lys Asn Asn Asn His Leu Glu Ser Arg Ile Met

145 150 155 160

Gly Glu Val Ser Tyr Leu Leu Asp Asn Leu Lys Ser Gln Lys Gly Arg

165 170 175

Ala Ile Asp Leu Asn His Ser Leu Val Ile Ser Val Ser Asn Val Ile

180 185 190

Cys Ser Leu Leu Met Ser Val Arg Phe Ser Phe Asn Asp Pro Ile Phe

195 200 205

Arg Arg Leu Met Glu Lys Ile Glu Glu Gly Phe Gln Leu Phe Gly Gly

210 215 220

Ile Val Phe Leu Asn Tyr Phe Pro Phe Leu Arg His Phe Pro Tyr Phe

225 230 235 240

Leu Lys Glu Met Lys Lys Ile Asp Glu Asn Lys Ala Lys Thr His Asn

245 250 255

Phe Phe Gln Lys Ile Ile Asp Asp His Arg Arg Thr Phe Glu Glu Ser

260 265 270

Lys Thr Arg Asp Val Leu Asp Cys Tyr Leu Met Glu Ile Arg Arg Ala

275 280 285

Glu Leu Ser Gly Asn Ser Glu Asn Leu Phe Gln Gly Lys Asp His Asn

290 295 300

Ser Gln Ile Arg Gln Ile Val Val Asp Leu Phe Thr Ala Gly Met Glu

305 310 315 320

Thr Thr Lys Thr Thr Leu His Trp Ser Leu Ile Tyr Met Val His Tyr

325 330 335

Pro Glu Leu Ala Arg Ser Ile Gln Ala Glu Leu Asp Met Val Val Gly

340 345 350

Arg Lys Arg Leu Pro Thr Phe Asp Asp Leu Pro Tyr Leu Pro Ile Thr

355 360 365

Glu Ala Thr Ile Leu Glu Val Leu Arg Lys Ser Ser Val Val Pro Leu

370 375 380

Gly Asn Ser His Ala Thr Met Lys Asp Val Asn Phe Lys Gly Phe Thr

385 390 395 400

Ile Pro Lys Asn Thr Glu Ile Ile Pro Leu Ile Tyr Ala Val Asn Asn

405 410 415

Asp Pro Glu Leu Trp Glu Ser Pro Asp Glu Phe Arg Pro Ser Arg Phe

420 425 430

Ile Asn Ser Glu Gly Arg Val Val Lys Pro Asp Phe Phe Ile Pro Phe

435 440 445

Gly Val Gly Arg Arg Asn Cys Leu Gly Glu Phe Leu Ala Arg Met Glu

450 455 460

Ile Phe Leu Phe Phe Ser Ser Ile Leu His Thr Phe Asn Leu Lys Leu

465 470 475 480

Pro Glu Gly Gln Ser Leu Pro Asp Leu Glu Gly Ile Phe Gly Ile Thr

485 490 495

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

500 505 510

Gly Glu Ile Ile Ala Asp Glu Ser Leu Pro Asp Phe Leu Asn Thr Lys

515 520 525

Arg Leu Pro Val Gly Ser His

530 535

<210> 3

<211> 318

<212> DNA

<213> Artificial sequence ()

<400> 3

tcatcagcag cgagcagatg acgttgctca ccgagatgac cagactgtgg ttcaaatcga 60

tggcgcgtcc tttttgactt ttcaaattgt ccagcaagta ggaaacctcg cccataattc 120

tgctctccag atgattgttg tttttcttat taaagatggt cattccaaag caacgtaaac 180

acgaatgcac aaatcgacgt tgctctttcc atatatttcc ttgcgtgttt ataattccat 240

agccaccgag taatttgtaa aattcattcg acggtttacc actgaattct tctttattga 300

acgcctcctt aatccact 318

Claims

1. The fraxinus chinensis CYP18A1 gene is characterized in that the nucleotide sequence of the fraxinus chinensis CYP18A1 gene is shown in SEQ ID NO. 1.

2. The fraxinus chinensis CYP18a1 gene-encoding protein of claim 1, wherein the amino acid sequence of the encoding protein is represented by SEQ ID No. 2.

3. An expression vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the fraxinus chinensis CYP18a1 gene of claim 1.

4. The recombinant vector containing the target gene in the fraxinus chinensis CYP18a1 gene of claim 1, wherein the nucleotide sequence of the target gene is shown in SEQ ID No. 3.

5. The wax insect CYP18A1 gene of claim 1, for regulating the growth and development speed of wax insect.

6. The wax insect CYP18A1 gene of claim 1, for prolonging wax secretion time of wax insect.

7. The use of the wax insect CYP18A1 gene of claim 1 for breeding improved varieties of wax insect.

8. The use of the fraxinus chinensis CYP18a1 gene of claim 1 to increase production of fraxinus chinensis.