CircRNA (Ribonucleic Acid) primer group for detecting apis cerana infected by bee glomus as well as kit and application of circRNA primer group

Abstract

The invention relates to the technical field of biology, in particular to a circRNA primer group for detecting apis cerana infected by bee glomus as well as a kit and application of the circRNA primer group, and the primer group comprises the following primers: ac2651-F, ac2651-R, actin-F and actin-R; the nucleotide sequence of the ac2651-F is as shown in SEQ ID No. 1; the nucleotide sequence of the ac2651-R is as shown in SEQ ID No. 2; the nucleotide sequence of the actin-F is as shown in SEQ ID No. 3; and the nucleotide sequence of the actin-R is as shown in SEQ ID No. 4. The research finds and identifies a specific circRNA (Ribonucleic Acid) molecule ac2651 of which the expression is obviously changed after worker bee larvae of apis cerana are infected by bee glomus, the primer group provided by the research can be used for early diagnosis of the apis cerana chalkbrood disease, the diagnosis time is earlier than that of a traditional method, a diagnosis basis can be provided for whether bee colony larvae are infected by chalkbrood disease or not before the larvae are attacked, and the method has the advantages that the method is simple and convenient to operate and high in practicability. A new view angle and a new target spot are provided for clarification of a molecular mechanism of Chinese bee chalkbrood disease resistance, and a theoretical foundation is laid for development of a novel bee disease prevention and control technology based on RNA interference in the future.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a primer set for detecting circRNA in honeybees infected with Coccidioides apiae, a kit for the same, and its application. Background Technology

[0002] The Chinese honeybee (Apis cerana cerana) is the nominate subspecies of the Eastern honeybee (Apis cerana). It is endemic to my country and one of the main bee species used in beekeeping, possessing high ecological, economic, and research value. *Ascosphaera apis*, a deadly fungal pathogen, specifically infects larvae, causing chalkbrood and resulting in a significant decline in colony strength and productivity, leading to substantial losses in beekeeping. However, current diagnosis of *Ascosphaera apis* infection in bees primarily relies on observation of typical late-stage symptoms (such as...). Figure 4 As shown in the figure, there is a lack of early and sensitive molecular diagnostic methods, and current research on the role of circRNA in the disease resistance process of Chinese honeybees is scarce. Summary of the Invention

[0003] The purpose of this invention is to provide a primer set for detecting circRNA in honeybees infected with Coccidioidomyces apiaceus, along with its kit and application.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] This invention proposes a set of circRNA primers for detecting *Gastrocystis apiae* infection in honeybees.

[0006] The primer set includes the following primers: ac2651-F, ac2651-R, actin-F, and actin-R;

[0007] The nucleotide sequence of ac2651-F is shown in SEQ ID No. 1;

[0008] The nucleotide sequence of ac2651-R is shown in SEQ ID No. 2;

[0009] The nucleotide sequence of actin-F is shown in SEQ ID No. 3;

[0010] The nucleotide sequence of actin-R is shown in SEQ ID No. 4.

[0011] The present invention also includes a kit for detecting *Gastrocystis apiae* infection in honeybees, comprising the primer set described above.

[0012] The present invention also includes the application of the primer set as described above in the preparation of a reagent for detecting Coccidioidomyces beesiensis.

[0013] Compared with existing technologies, this study discovered and identified a specific circRNA molecule, ac2651, whose expression changed significantly after infection with Coccidioides apiae in worker bee larvae. The primer set provided in this study can be used for the early diagnosis of chalkbrood in honeybees, with a diagnostic time earlier than traditional methods. It can provide a diagnostic basis for whether bee colony larvae are infected with chalkbrood before the larvae develop symptoms, providing a new perspective and target for elucidating the molecular mechanism of chalkbrood resistance in honeybees, and laying a theoretical foundation for the future development of novel bee disease control technologies based on RNA interference. Attached Figure Description

[0014] Figure 1 A: Agarose gel electrophoresis of PCR amplification products, lane 1: DNA Marker, lane 2: PCR product of ac2651; B: Sanger sequencing peak diagram of amplified fragments.

[0015] Figure 2 The relative expression level of ac2651 in the intestine of 4-, 5- and 6-day-old worker bees infected with Coccidioides apiae was determined.

[0016] Figure 3 The incidence of chalkbrood in worker bee larvae of the Chinese honeybee after inoculation with Coccidioides apiae.

[0017] Figure 4 For visual observation, A represents uninfected larvae; B represents infected larvae. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0019] Example 1

[0020] (1) Select strong and healthy Chinese honeybee colonies (artificially raised), quickly transport the combs to the laboratory, and use a clean transfer needle to transfer 2-day-old larvae into a 48-well plate containing 50 μL of feed (63% royal jelly, 30% sterilized water, 6% honey, and 1% yeast extract) (1 larva / well); (2) Place the 48-well plate in a constant temperature and humidity chamber at 35±0.5℃ and 90% relative humidity (RH) for rearing; (3) When the larvae reach 3 days old, the control group larvae are fed feed without Corydalis melitica (50 μL / larvae), while the treatment group is fed feed containing Corydalis melitica spores (final concentration 1×10⁻⁶). 6The control and treatment groups were fed a diet of 50 μL / head (number of larvae per mL), and then the diet without spores of Coccidioides apiae was changed every 24 hours until they were 6 days old; (4) the intestinal tissue of larvae in the control, 5 and 6-day-old control and treatment groups was dissected in a clean bench. Each 3 larvae's intestines constituted a biological replicate, and 3 biological replicates were set up for each age.

[0021] (2) Based on the circRNA molecule novel_circRNA_002651 (named ac2651; the DNA copy sequence obtained by sequencing is shown in SEQ ID No. 5) previously detected and identified from the intestinal tissue of honeybee larvae, back-to-back primers (Divergent primers) across the reverse splicing site were designed and synthesized using Primer Premier 6 software. Total RNA was extracted from the intestinal samples, digested with RNase R enzyme (3U / mg) to remove linear RNA, treated at 37℃ for 15 min, and then reverse transcribed using random primers. The resulting cDNA was used as a template for PCR amplification. The PCR amplification reaction system and procedure are shown in Tables 1 and 2, where the upstream primer in Table 1 is ac2651-F in Table 5, and the downstream primer in Table 1 is ac2651-R in Table 5. The amplified products were detected by 2% agarose gel electrophoresis and observed and photographed under a gel imaging analysis system. The target fragment was recovered from the gel and TA cloned. The bacterial culture with positive PCR results was sent to Sangon Biotech (Shanghai) Co., Ltd. for Sanger sequencing.

[0022] Agarose gel electrophoresis results as follows Figure 1 As shown in Figure A, the ac2651-specific fragment amplified by back-to-back primers is the expected size (approximately 109 bp). Sanger sequencing results are as follows... Figure 1 As shown in B, the target fragment sequence is completely consistent with the next-generation sequencing results, and the target fragment contains the BS site. These results confirm the existence of the BS site in ac2651.

[0023] Table 1 PCR reaction system

[0024]

[0025] The PCR Mix used in Table 1 is from Yisheng. PCR Master Mix (With Dye), Catalog No.: 10102ES03; Contains TaqDNAPolymerase, dNTP mixture, MgCl2, and an optimized buffer system.

[0026] Table 2 PCR reaction procedures

[0027]

[0028] (3) Total RNA was reverse transcribed using random primers to obtain cDNA of ac2651. At the same time, Oligo dT primer and Random 6mers Primer were mixed at a ratio of 1:1 and reverse transcribed to obtain cDNA of internal control actin (NCBI reference sequence number: XM_017059068.2). According to the reaction system in Table 3, real-time quantitative PCR was performed on an automated PCR analysis system according to the procedure in Table 4. This included two parts: ① Detecting the relative expression level of the circRNA molecule ac2651 using the reaction system in Table 3 on the automated PCR analysis system according to the procedure in Table 4. The upstream primer in Table 3 is ac2651-F from Table 5, and the downstream primer is ac2651-R from Table 5. ② Simultaneously, according to the reaction system in Table 3, detecting the expression level of the internal reference gene actin using the procedure in Table 4 on the automated PCR analysis system. This was used to correct for variations between samples (such as RNA quality, reverse transcription efficiency, etc.). The upstream primer in Table 3 is actin-F from Table 5, and the downstream primer is actin-R from Table 5. Through 2- ΔΔCt Relative expression levels were calculated using the Ct method (valid range: 15-35). GraphPad Prism 8 software was used for data analysis and plotting. Experimental data are expressed as Mean ± SD (Sudent's t-test, ns: P>0.05, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001).

[0029] The results are as follows Figure 2 As shown, compared with the control group, the expression level of ac2651 in the intestine of 4-, 5-, and 6-day-old larvae in the treatment group was significantly downregulated, by 82.0%, 20.2%, and 51.5%, respectively (P<0.01). The difference was statistically significant, and the experiment was performed in three technical replicates and three biological replicates.

[0030] When using this invention to detect whether bee larvae are infected with Coccidioides apis, the expression level of ac2651 in the experimental group (the bee larvae being tested) is more than 20% lower than that in the control group (artificially raised, uninfected bee larvae), which is considered a positive infection.

[0031] Table 3 Reaction System

[0032]

[0033] The qPCRMix used in Table 3 is from Yisheng. qPCR SYBR Green Master Mix (LowRox Plus), catalog number: 11202ES03; contains dNTPs and Mg.2+ , Champagne Taq DNA Polymerase, SYBR GreenI, Specific ROX Reference Dye, etc.

[0034] Table 4 Reaction Procedure

[0035]

[0036] Table 5 Primer Information

[0037]

[0038]

[0039] Example 2

[0040] Statistics on the incidence of chalkbrood

[0041] Three-day-old worker bee larvae of the Chinese honeybee were inoculated with *Gnaphalium affineum*. Twenty-four larvae constituted one biological replicate, with a total of three replicates. Feed was changed every 24 hours for seven consecutive days. Before feed changes, the disease incidence rate of the worker bee larvae was measured, measured by the appearance of white mycelium on their body surface. The formula for calculating the chalkbrood disease incidence rate was: (Number of larval corpses with white *Gnaphalium affineum* mycelium on their body surface in each biological replicate / 24) × 100%.

[0042] Statistical results showed that the incidence of chalkbrood in larvae was 0% at 1-2 days post-contraction (dpi); the average incidence rates at 3-7 days post-contraction (dpi) were 2.78%, 26.39%, 47.22%, 51.39%, and 54.17%, respectively. Figure 3 ).

[0043] As can be seen from the morbidity curve, the larvae show signs of disease at 3 dpi (i.e., 6-day-old larvae), while with the present invention, infection can be detected in the intestines at 1 dpi (4-day-old larvae).

[0044] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the invention. Modifications and variations made by those skilled in the art in accordance with the spirit of the invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A primer set for detecting *Gnaphalium apiae* infection in honeybees, characterized in that: The primer set includes the following primers: ac2651-F, ac2651-R, actin-F, and actin-R; The nucleotide sequence of ac2651-F is shown in SEQ ID No. 1; The nucleotide sequence of ac2651-R is shown in SEQ ID No. 2; The nucleotide sequence of actin-F is shown in SEQ ID No. 3; The nucleotide sequence of actin-R is shown in SEQ ID No.

4.

2. A kit for detecting *Gastrocystis apiae* infection in honeybees, comprising the primer set described in claim 1.

3. The application of the primer set as described in claim 1 in the preparation of a reagent for detecting Coccidioides beesiensis.

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