Application of RNA (Ribonucleic Acid) interference cuticle protein gene in promoting conversion of cordyceps sinensis from bud spores to hyphae

Abstract

The invention discloses an application of RNA (Ribonucleic Acid) interference on a cuticle protein gene of a hepialus armoricanus larva to promotion of transformation of cordyceps sinensis from bud spores to hyphae. In order to solve the problems of long artificial tending period, high cost and the like of cordyceps sinensis caused by slow rigidification process of hepialus armoricanus larvae infected by cordyceps sinensis, the siRNA synthesized by related genes (flash, larva of larvae of hepialus armoricanus LCP-30, 26-hydroxylase, isoform B and probe citase 3) acts on the hepialus armoricanus larvae, so that the cordyceps sinensis in hemolymph of the hepialus armoricanus is promoted to be converted into hyphae from blastospores, and the cordyceps sinensis in the hemolymph of the hepialus armoricanus is promoted to grow into the cordyceps sinensis in the hemolymph of the hemolymph of the hemolymph of the hemolymph of the hemolymph of the hemolymph. The result shows that the related gene of the hepialus armoricanus larva can significantly promote the two-form transformation of the cordyceps sinensis, so that the bombyx batryticatus rate of the bacterium-carrying hepialus armoricanus larva is improved.

Description

[0001] This divisional application is for the patent application with the invention title: Application of RNA interference of genes related to Hepialus armoricanus larvae in promoting the transformation of Ophiocordyceps sinensis from blastospores to hyphae, patent application number: 202410767783.X, patent applicants Zhejiang Shouxian Valley Pharmaceutical Co., Ltd. and Zhejiang Yuewang Shengcao Biotechnology Co., Ltd., and the application date is June 14, 2024. Technical Field:

[0002] The present invention belongs to the technical field of artificial cultivation of Ophiocordyceps sinensis, and specifically relates to the application of RNA interference of the cuticleprotein 18.6, isoform B gene of Hepialus armoricanus larvae in promoting the transformation of Ophiocordyceps sinensis from blastospores to hyphae. Background Art:

[0003] Ophiocordyceps sinensis is a precious biological resource unique to the Qinghai-Tibet Plateau, which is a complex of insect and fungus formed by the infection of Hepialus armoricanus larvae by Ophiocordyceps sinensis. Due to the narrow production area, global warming and over-excavation, the output of wild Ophiocordyceps sinensis has been decreasing year by year. However, due to the sharp increase in consumption based on the functional effects of Ophiocordyceps sinensis, the production capacity of wild Ophiocordyceps sinensis can no longer meet the growing market demand. Great breakthroughs have been made in the artificial cultivation of Ophiocordyceps sinensis, and Ophiocordyceps sinensis comparable to wild ones can be produced by simulating the plateau environment in low-altitude areas, achieving a leap from the laboratory to the industry for rare biological resources. Factors such as the instability of the Ophiocordyceps sinensis strain, the high lethality rate of pathogenic bacteria to Hepialus armoricanus larvae, and the low rigidification rate of bacteria-carrying larvae seriously restrict the efficient artificial production of Ophiocordyceps sinensis. The key factor affecting the rigidification rate of bacteria-carrying larvae (i.e., the proportion of rigid insects formed after the transformation of blastospores into hyphae) lies in the efficiency of the transformation of Ophiocordyceps sinensis from blastospores to hyphae in the hemolymph of host insects.

[0004] Fungal dimorphism is a phenomenon in which the morphological forms of some fungi change between yeast-like and hyphal forms under the influence of environmental factors. Ophiocordyceps sinensis is a typical dimorphic fungus (blastospores - hyphae), and under the induction of specific factors, it can produce more blastospores through budding growth, or form slender prehyphae and hyphae through apical growth. N-acetylglucosamine, proline, farnesol, tyrosol, methyl farnesoate, and ecdysteroid can promote the transformation of Ophiocordyceps sinensis blastospores into hyphae to varying degrees in vitro. It is reported that insect ecdysteroid and juvenile hormone, and mannitol can promote the rigidification of Hepialus armoricanus larvae in vivo.

[0005] After the fungus Ophiocordyceps sinensis infects the larvae of Hepialus armoricanus Oberthur, it grows in the hemolymph of the larvae in the form of blastospores for a long time and is regulated by the larvae of Hepialus armoricanus Oberthur. However, which genes of the larvae of Hepialus armoricanus Oberthur regulate the transformation of the blastospores of Ophiocordyceps sinensis in their hemolymph into hyphae has not been reported. Small interfering RNA (siRNA), sometimes called short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules with a length of 20-25 base pairs. It operates within the RNA interference (RNAi) pathway and interferes with the post-transcriptional degradation of mRNA of specific genes expressing complementary nucleotide sequences, thereby preventing translation. This patent reports the application of RNA interference of genes related to the larvae of Hepialus armoricanus Oberthur in promoting the transformation of Ophiocordyceps sinensis from blastospores into hyphae. Summary of the Invention:

[0006] Based on the regulation problem of the larvae of Hepialus armoricanus Oberthur in the dimorphic transformation of Ophiocordyceps sinensis, the purpose of the present invention is to provide the application of RNA interference of genes related to the larvae of Hepialus armoricanus Oberthur in promoting the transformation of Ophiocordyceps sinensis from blastospores into hyphae, so as to overcome the problems of low rigidity rate of the larvae of Hepialus armoricanus Oberthur carrying Ophiocordyceps sinensis and long cycle of artificial cultivation of Ophiocordyceps sinensis.

[0007] The present invention discovers through research that knocking down related genes of the larvae of Hepialus armoricanus Oberthur by RNAi technology can promote the transformation of Ophiocordyceps sinensis in the hemolymph of the larvae from blastospores into hyphae.

[0008] Therefore, the present invention provides genes in the larvae of Hepialus armoricanus Oberthur that can regulate the transformation of Ophiocordyceps sinensis from blastospores into hyphae, and the genes are as described in any of the following:

[0009] Flightin gene, whose nucleotide sequence is as shown in SEQ ID NO.8;

[0010] larval cuticle protein LCP-30 gene, whose nucleotide sequence is as shown in any of SEQ ID NO.11-15;

[0011] 26-hydroxylase (CYP18A1) gene, whose nucleotide sequence is as shown in SEQ ID NO.9 or 10;

[0012] cuticle protein 18.6, isoform B gene, whose nucleotide sequence is as shown in any of SEQ ID NO.21-27;

[0013] probable chitinase 3 gene, whose nucleotide sequence is as shown in any of SEQ ID NO.16-20.

[0014] The second purpose of the present invention is to provide siRNAs that knock down the expression of the above genes.

[0015] Preferably, the sequence of the siRNA is as shown in any of the following siRNAs or a combination of two or more:

[0016]

[0017]

[0018]

[0019]

[0020] The English letters represent the copy number of the gene, and the Arabic numerals represent the sense upper strand -1, sense lower strand -2, antisense upper strand -3, and antisense lower strand -4 of the siRNA in each copy of the gene.

[0021] The third aspect of the present invention is to provide the application of the above gene in regulating the transformation of Ophiocordyceps sinensis from blastospores to hyphae.

[0022] Preferably, it is the application of a preparation for reducing the expression level of the above gene in promoting the transformation of Ophiocordyceps sinensis from blastospores to hyphae.

[0023] Preferably, it is the application of promoting the transformation of Ophiocordyceps sinensis from blastospores to hyphae by knocking down the expression of the above gene through RNAi.

[0024] Preferably, it is the application of a preparation for reducing the expression level of the above gene in promoting the rigidification of the bat moth larvae carrying Ophiocordyceps sinensis, that is, in the preparation of a preparation for promoting the rigidification of bat moth larvae.

[0025] The fourth object of the present invention is to provide a method for promoting the rigidification of bat moth larvae, which is characterized in that the siRNA of the above gene is introduced into the bat moth larvae carrying Ophiocordyceps sinensis separately or in combination to promote the rigidification of the bat moth larvae.

[0026] Preferably, the Ophiocordyceps sinensis is Ophiocordyceps sinensis.

[0027] Preferably, the instar of the bat moth larvae is the 6th instar.

[0028] Preferably, for the siRNA, the inoculation dose per larva is 4 μg.

[0029] In view of the problems that blastospores in the larvae of Hepialus armoricanus infected by Ophiocordyceps sinensis can be maintained for a long time, the sclerotization process is time-consuming and the sclerotization rate is low, resulting in a long artificial cultivation cycle and high cost of Ophiocordyceps sinensis, the siRNAs of the flightin, larval cuticle protein LCP-30, 26-hydroxylase, cuticle protein 18.6, isoform B, and probable chitinase 3 genes of Hepialus armoricanus larvae are injected into the bacteria-carrying Hepialus armoricanus larvae separately or in combination. The proportions of blastospores transformed into prehyphae in the hemolymph of Hepialus armoricanus larvae are flightin (83.33±11.02), larval cuticle protein LCP-30 (78.25±3.55%), 26-hydroxylase (CYP18A1) (40.48±6.30%), cuticle protein 18.6, isoform B (39.81±2.31%), and probable chitinase 3 (34.52±7.81%), respectively, while only blastospores are present in the hemolymph of Hepialus armoricanus larvae in the control. This shows that knocking down the relevant genes of Hepialus armoricanus larvae can significantly increase the proportion of Ophiocordyceps sinensis bacteria transformed from blastospores into hyphae in the hemolymph of larvae. Description of the Drawings:

[0030] Figure 1 It is a picture of blastospores carried in the hemolymph of Hepialus xiaojinensis larvae and transformed into prehyphae or hyphae. It is the blastospores (marked by yellow arrows, white in grayscale) and prehyphae (marked by red arrows, gray in grayscale) in the hemolymph of larvae 120 h after injecting the dsRNA of the relevant genes of the larvae (400-fold fluorescence microscope).

[0031] Figure 2 It is the detection of RNAi efficiency by qRT-PCR 120 h after injecting siRNA;

[0032] Note: 1: Pupal cuticle protein; 2: Ecdysone-induced protein 78C; 3: Coactosin-like protein; 4: Flightin; 5: Larval / pupal rigid cuticle protein 66; 6: 26-hydroxylase (CYP18A1); 7: Larval cuticle protein LCP-30; 8: Multidrugresistance protein 1; 9: Probable chitinase 3; 10: Cuticle protein 18.6, isoform B. Detailed implementation method:

[0033] The following examples further illustrate the present invention, rather than limiting the present invention.

[0034] Example 1:

[0035] The Ophiocordyceps sinensis (number: KD) was isolated from wild Ophiocordyceps sinensis in Kangding, Sichuan by tissue isolation method. After molecular identification, it was identified as Ophiocordyceps sinensis and stored at -80 °C. The liquid PMG medium for Ophiocordyceps sinensis: 200 g of potato juice, 20 g of maltose, 10 g of peptone, 3 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate, 0.02 g of vitamin B1, and 5 g of grinding liquid of artificially reared fresh Galleria mellonella larvae. After mixing, it was made up to 1 L with distilled water, dispensed into 250 mL Erlenmeyer flasks, 150 mL per flask, and autoclaved at 121 °C for 30 min. After cooling, it was used. On the ultra-clean workbench, the solid-cultured Ophiocordyceps sinensis blocks (about 0.5 cm 3 ) were inoculated into the above liquid medium, cultured on a shaker at 120 rpm and 13 °C for 45 days. The liquid culture was filtered through three layers of sterile lens paper, and the filtrate was collected into a 50 mL sterilized centrifuge tube. It was centrifuged at 8000 rpm and 10 °C for 15 min, the supernatant was discarded, resuspended with sterile phosphate buffer (PBS; pH = 7.0), and centrifuged again to discard the supernatant. The collected blastospores were diluted with sterile PBS to 6×10 6 cells / mL for injection infection of the 6th instar larvae of Thitarodes xiaojinensis.

[0036] The larvae of Thitarodes xiaojinensis were reared in a low-altitude laboratory with Daucus carota and Potentilla anserina as feed. Thitarodes xiaojinensis is a common Hepialus insect.

[0037] The siRNA of the genes of Hepialus armoricanus larvae was synthesized by the following method (the siRNA sequences of each gene are shown in Table 1). The primers were designed with the help of the website http: / / rnaidesigner.thermofisher.com / rnaiexpress / design.do and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The siRNA was synthesized with reference to the instructions of the M5 HiPer T7 In Vitro Transcription T7 kit. The DNA annealing buffer, Oligonucleotide #1, and #2 were added at 95°C to prepare the annealed oligonucleotide DNA template. The annealed oligonucleotide DNA template and 2xT7 In Vitro Transcription Mix were incubated in a PCR instrument. After incubation, DNase I was used to treat to eliminate potential genomic DNA in all samples. The separate sense and antisense reactions were combined. The purification was carried out by centrifugation with 3M sodium acetate (pH 5.2) and isopropanol, and the precipitate was washed with 70% cold ethanol. The concentration and quality of the synthesized siRNA of each gene were detected by ultraviolet spectrophotometer and agarose electrophoresis.

[0038] The siRNA of each gene (with a concentration of 4 μg / μL and an inoculation dose of 4 μg siRNA per larva. If there are multiple copies of the gene, the siRNAs of each copy are mixed in equal mass before injection) was injected into 30 larvae of Thitarodes xiaojinensis carrying Ophiocordyceps sinensis. After injection, the mortality rate, the rate of becoming stiff worms, the pupation rate, and the molting rate of the larvae were examined. Five days after injection, in an operating room at 13 - 16°C, hemolymph was collected from the treated Thitarodes xiaojinensis larvae. The original hemolymph was mixed with a fluorescent dye in equal proportion, and then observed and photographed under an inverted fluorescence microscope at 400 times magnification. Under the fluorescence microscope, the spore morphology (blastospores and prehyphae), the viability of the mycelium in the larvae were examined and photographed, and further diluted with sterile double-distilled water and counted using a hemocytometer. The whole experiment was repeated twice.

[0039] The defaunated bat moth larvae were ground into fine powder in liquid nitrogen and then total RNA was extracted. The purity and quantity of RNA were detected using a OneDrop OD-1000+ spectrophotometer. The integrity of RNA was detected by 1% agarose gel electrophoresis. Only RNA samples with high integrity, an A260 / A280 ratio between 2.000 and 2.124, and an A260 / A230 ratio greater than 2.0 were used to generate the first-strand cDNA. Reverse transcription was performed according to the instructions of the ToloScript All-in-one RT EasyMix for qPCR. 1 μg of total RNA was extracted from a total volume of 20 μl to synthesize the first cDNA, which was stored in a -20°C refrigerator. Primer design for real-time fluorescence quantitative PCR: Primers were designed using primer-blast of NCBI and synthesized by Sangon Biotech (Shanghai) Co., Ltd. qRT-PCR: Experiments were carried out according to the instructions of 2×SYBR Green qPCR Premix (Universal). Template 1 μL, F-Primer (10 μM) 0.4 μL, R-Primer (10 μM) 0.4 μL, 2×TransStart TipGreen qPCR SuperMix 10 μL. Two technical replicates were set for each treatment; the cDNA obtained by reverse transcription was diluted 5-fold as the template for qPCR reaction, with 3 replicates for each sample. Reaction conditions: 95°C for 30 s, 95°C for 10 s, 55°C for 15 s, 72°C for 10 s, 40 cycles. After the reaction, melting curve analysis was performed starting from 65°C to 95°C to ensure the consistency and specificity of the amplification products. All reactions were carried out on a CFX96 system according to the manufacturer's instructions. Data analysis identified and excluded outliers through a PCR instrument, and the relative expression levels were calculated using the 2-ΔΔCt method.

[0040] The results showed (Table 2) that 120 hours after the bacteria-carrying live larvae were injected with siRNAs of related genes (flightin, larval cuticle protein LCP-30, 26-hydroxylase (CYP18A1), cuticle protein 18.6, isoform B, and probable chitinase 3), the proportions of blastospores in the hemolymph transformed into prehyphae (Table 2) were 83.33 ± 11.02, 78.25 ± 3.55%, 40.48 ± 6.30%, 39.81 ± 2.31%, and 34.52 ± 7.81%, respectively. However, no prehyphae were found in the hemolymph after the bacteria-carrying live larvae were injected with siRNAs of pupal cuticle protein, ecdysone-induced protein 78C, and multidrug resistance protein 1 genes, and no prehyphae appeared in the hemolymph of the controls injected with siGFP and sterile ultrapure water. The qRT-PCR verification results showed that after the injection of siRNAs, the expression levels of the genes interfered with in the bacteria-carrying Hepialus larvae decreased to varying degrees (the inhibition rate ranged from 38.64% to 91.54%) ( Figure 2 ). These results indicate that RNAi-mediated interference with the flightin, larval cuticle protein LCP-30, 26-hydroxylase (CYP18A1), cuticle protein 18.6, isoform B, and probable chitinase 3 genes in Hepialus larvae promotes the transformation of blastospores in the hemolymph of bacteria-carrying larvae into prehyphae, which further leads to the stiffening of bacteria-carrying larvae.

[0041] Table 1. siRNA sequences of Hepialus larvae genes

[0042]

[0043]

[0044]

[0045]

[0046] Note: In the table, English letters (such as A, B, C, D, etc., where A1, A2, A3, A4 represent the same siRNA, and the same applies to B, C, D, E) represent the copy number numbers of the genes (2 copies of Pupal cuticle protein, 1 copy of Ecdysone-induced protein 78C, 3 copies of Multidrug resistance protein 1, only 1 copy of Flightin, 2 copies of 26-hydroxylase (CYP18A1), 5 copies of Larval cuticle protein LCP-30, 5 copies of Probable chitinase 3, 3 copies of Cuticle protein 18.6, isoform B); Arabic numerals represent the sense upper strand (1), sense lower strand (2), antisense upper strand (3), and antisense lower strand (4) of siRNA in each copy of the gene.

[0047] Table 2. Percentage of larvae with blastospores and prehyphae in hemolymph 120 h after injecting dsRNA of related genes of larvae

[0048]

[0049] Note: The above data are the average + SD 120 hours after injecting dsRNA. The larval genes were knocked down by injecting dsRNA of different genes (4 μg / μL of siRNAs in 4 μL of RNase-free water) into the hemolymph of larvae containing only blastospores. Different letters in each column indicate significant differences in data (Duncan test, p < 0.05).

Claims

1. A gene that can regulate the transformation of Cordyceps sinensis from blastospores to hyphae, characterized in that: The gene is cuticle protein 18.6, isoform B gene, and its nucleotide sequence is shown in any one of SEQ ID NO.21-27.

2. An siRNA for knocking down the expression of the gene according to claim 1.

3. The siRNA according to claim 2, characterized in that The sequence of the siRNA is as shown in any of the following siRNAs or a combination of two or more: The English letters represent the copy number of the gene, and the Arabic numerals represent the sense upper strand -1, sense lower strand -2, antisense upper strand -3, and antisense lower strand -4 of the siRNA in each copy of the gene.

4. Use of the gene according to claim 1 in regulating the transformation of Cordyceps sinensis from blastospores to hyphae.

5. The use according to claim 4, characterized in that: The invention relates to the use of a preparation for reducing the expression level of the gene described in claim 1 in promoting the transformation of Cordyceps sinensis from blastospores to hyphae.

6. The use according to claim 5, characterized in that: The invention relates to the use of RNAi to knock down the gene expression of claim 1 to promote the transformation of Cordyceps sinensis from blastospores to mycelium.

7. Use of a preparation for reducing the expression level of the gene according to claim 1 in a preparation for promoting rigidification of bat moth larvae carrying Cordyceps sinensis.

8. A method for promoting the rigidification of bat moth larvae, characterized in that: The siRNA of the gene described in claim 1 is introduced separately or mixed into the bat moth larvae carrying Cordyceps sinensis to promote the rigidification of the bat moth larvae.

9. The method according to claim 8, characterized in that The siRNA is the siRNA according to claim 3 10. The method according to claim 9, characterized in that The cordyceps sinensis is Ophiocordyceps sinensis; the bat moth larvae are at the sixth instar.

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