Reference gene combination stably expressed by hermetia illucens under different conditions and application of reference gene combination

Abstract

The invention discloses a reference gene combination stably expressed by hermetia illucens under different conditions and application thereof, reference genes stably expressed by hermetia illucens in different development stages, different tissues, different genders and different genotypes are obtained through screening, and the reference gene combination is suitable for correction of hermetia illucens RT-qPCR detection results under different experimental conditions. Meanwhile, the invention further provides amplification primers for detecting the hermetia illucens reference genes, and the amplification primers are high in specificity and amplification efficiency. According to the invention, an early-stage foundation is laid for the quantification of related functional genes of hermetia illucens under different experimental conditions and the related research of molecular biology.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a combination of reference genes with stable expression in black soldier flies under different conditions and its application. Background Art

[0002] The use of quantitative real-time PCR (qRT-PCR, relative quantification) is a convenient, fast, low-cost, and efficient method for analyzing the expression and transcription of target genes. When performing qRT-PCR detection, different samples need to have the same RNA extraction, reverse transcription, cDNA concentration, and reverse transcription level, otherwise it will affect the true expression level of the target gene. To ensure reliable and accurate measurement of gene expression levels, it is crucial to use stable reference genes that are minimally affected by environmental factors and have consistent expression levels for calibration and standardization purposes.

[0003] However, research has shown that there is currently no universal reference gene that can be expressed relatively consistently under different experimental conditions, such as at different developmental stages, different treatment stages, and different tissues, etc. Misusing commonly used reference genes may also lead to errors or mistakes. Therefore, selecting appropriate reference genes is crucial for ensuring the accuracy of qRT-PCR result analysis in entomological research.

[0004] The black soldier fly belongs to the Diptera and is mainly distributed in tropical and subtropical regions. More and more researchers are conducting whole-genome sequencing on the black soldier fly. With the completion of the black soldier fly genome sequencing, researchers are focusing on elucidating its powerful digestion and absorption capabilities through genetic and molecular biology methods to improve its growth performance. However, most genetic and molecular biology studies require gene expression analysis, and the stability of reference genes may vary under different experimental conditions. Since black soldier fly species have been repeatedly introduced in many regions, researchers have roughly divided black soldier flies into three different genotypes. However, it is not clear whether the reference genes among different genotypes of black soldier flies are universal. Summary of the Invention

[0005] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments.

[0006] As one aspect of the present invention, the present invention provides a combination of reference genes with stable expression in black soldier flies under different conditions, wherein the combination of reference genes is any one of the following four groups of gene combinations:

[0007] (1) β-actin, 18S rRNA, and RPL13;

[0008] (2) RPL13, GST, and RP49;

[0009] (3) EF1-α, RPL13, and GST;

[0010] (4) RPL13, GAPDH, and 18S rRNA.

[0011] As a preferred embodiment of the combination of reference genes stably expressed by Hermetia illucens under different conditions described in the present invention: The nucleotide sequence of the amplification primer for β-actin is as shown in SEQ ID NO: 1; the nucleotide sequence of the amplification primer for 18S rRNA is as shown in SEQ ID NO: 10; the nucleotide sequence of the amplification primer for RPL13 is as shown in SEQ ID NO: 8.

[0012] As a preferred embodiment of the combination of reference genes stably expressed by Hermetia illucens under different conditions described in the present invention: The nucleotide sequence of the amplification primer for RPL13 is as shown in SEQ ID NO: 8; the nucleotide sequence of the amplification primer for GST is as shown in SEQ ID NO: 3; the nucleotide sequence of the amplification primer for RP49 is as shown in SEQ ID NO: 4.

[0013] As a preferred embodiment of the combination of reference genes stably expressed by Hermetia illucens under different conditions described in the present invention: The nucleotide sequence of the amplification primer for EF1-α is as shown in SEQ ID NO: 9; the nucleotide sequence of the amplification primer for RPL13 is as shown in SEQ ID NO: 8; the nucleotide sequence of the amplification primer for GST is as shown in SEQ ID NO: 3.

[0014] As a preferred embodiment of the combination of reference genes stably expressed by Hermetia illucens under different conditions described in the present invention: The nucleotide sequence of the amplification primer for RPL13 is as shown in SEQ ID NO: 8; the nucleotide sequence of the amplification primer for GAPDH is as shown in SEQ ID NO: 2; the nucleotide sequence of the amplification primer for 18S rRNA is as shown in SEQ ID NO: 10.

[0015] The present invention also provides the application of the combination of reference genes stably expressed by Hermetia illucens under different conditions in the preparation of a fluorescence PCR quantitative detection kit for Hermetia illucens: β-actin, 18S rRNA, and RPL13 are stably expressed in different developmental stages of Hermetia illucens from egg to adult;

[0016] RPL13, GST, and RP49 are stably expressed in different tissues of Hermetia illucens;

[0017] EF1-α, RPL13, and GST are stably expressed in different genders of Hermetia illucens;

[0018] RPL13, GAPDH, and 18S rRNA are stably expressed in different genotypes of Hermetia illucens.

[0019] Advantages of the present invention: The present invention discloses a combination of internal reference genes stably expressed in Hermetia illucens under different conditions and its applications. The present invention has screened out internal reference genes with stable expression in different developmental stages, different tissues, different genders, and different genotypes of Hermetia illucens, which are applicable to the correction of RT-qPCR detection results of Hermetia illucens under different experimental conditions. At the same time, the present invention also provides amplification primers for detecting these internal reference genes of Hermetia illucens, and the amplification primers have strong specificity and high amplification efficiency. The present invention lays a preliminary foundation for the quantification of related functional genes of Hermetia illucens under different experimental conditions and related research in molecular biology. Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments, where:

[0021] Figure 1 Ct values of candidate reference genes under different experimental conditions. A: Ct values of candidate reference genes in eggs, 3-day-old larvae, 8-day-old larvae, 13-day-old larvae, prepupae, pupae, and adults; B: Ct values of candidate reference genes in midgut, fat body, oral cavity, and head; C: Ct values of candidate reference genes in females and males; D: Ct values of candidate reference genes in different genotypes (Yunnan, Hubei, and Guizhou)

[0022] Figure 2 Expression stability of internal reference genes at different developmental stages. A - D: Analyze and evaluate the stability of internal reference genes and the required number of reference genes at different developmental stages of Hermetia illucens using geNorm, NormFinder, and BestKeeper respectively.

[0023] Figure 3 Expression stability of 11 internal reference genes in different tissues. A - D: Analyze and evaluate the stability of internal reference genes and the required number of reference genes in the midgut, fat body, oral cavity, and head tissues of Hermetia illucens using geNorm, NormFinder, and BestKeeper respectively.

[0024] Figure 4 Expression stability of 11 internal reference genes in different genders. A - D: Analyze and evaluate the stability of internal reference genes and the required number of reference genes for Hermetia illucens of different genders using geNorm, NormFinder, and BestKeeper respectively.

[0025] Figure 5Expression stability of reference genes in different genotypes. A - D: geNorm, NormFinder, and BestKeeper were used to analyze and evaluate the stability of reference genes and the required number of reference genes in three genotypes of Hermetia illucens.

[0026] Figure 6 To verify the stability of reference genes at different developmental stages of Hermetia illucens. Detailed implementation manners

[0027] To make the above - mentioned objects, features, and advantages of the present invention more obvious and understandable, the following specific embodiments are used to elaborate on the detailed implementation manners of the present invention.

[0028] Example 1: Design and detection of candidate reference gene primers

[0029] I. Experimental method:

[0030] 1. Extraction of total RNA and synthesis of cDNA from Hermetia illucens

[0031] Use TRIeasy total RNA extraction reagent TM (Yeasen, 19202ES60) to extract total RNA. Analyze the quality, concentration, and purity by measuring the absorbance using a spectrophotometer (Yeasen, 80480ES03). Total RNA (2 μg) is directly used as a template for cDNA synthesis, and cDNA is generated from mRNA by reverse transcription using a gDNA Eraser PrimeScript TM RT kit (Takara, RR047A). The first step is to remove genomic DNA by incubating at 42 °C for two minutes, the second step is to incubate the reaction at 37 °C for 15 minutes, and then incubate at 85 °C for 5 seconds. The finally synthesized cDNA is stored at - 80 °C for later use.

[0032] 2. Quantitative real - time PCR (qRT - PCR)

[0033] Dilute the cDNA 5 - fold for PCR reaction. Use Hieff qRT-PCR reactions were performed using Universal Blue qPCRSYBR Green Master Mix (Yeasen, Shanghai, 11184ES25) and Applied Biosystems 7500 Real-Time PCR System (Applied Biosystem, CA, ABI7500). Eleven candidate reference genes (GAPDH, β-actin, GST, tubulinα-1A, tubulinβ-4A, tubulinβ, RP49, RPL13, EF1-α, 18S RNA, 28S RNA) were selected. qRT-PCR primers were designed using Primer Premier 5 software. The qRT-PCR primer sequences are shown in Table 1.

[0034] Table 1 Information on RT-qPCR Primer Sequences of Candidate Reference Genes

[0035]

[0036]

[0037] II. Experimental Results:

[0038] The results are shown in Table 2. The correlation coefficients (R 2 ) and PCR amplification efficiencies were 0.981 - 0.999 and 90% - 110% respectively. The results indicate that the primers of the 11 pairs of candidate reference genes all have stability and specificity.

[0039] Table 2 PCR Amplification Efficiencies and Correlation Coefficients of Candidate Reference Genes

[0040]

[0041]

[0042] Example 2: Screening of the Most Stable Reference Genes in Hermetia illucens under Different Conditions

[0043] I. Experimental Methods:

[0044] 1. Collection of Hermetia illucens Samples under Different Treatment Conditions:

[0045] All stages of black soldier flies were sampled and disinfected with alcohol in DEPC water. The number of individuals per replicate for each developmental stage was as follows: eggs, 3-day-old larvae, 8-day-old larvae, and 13-day-old larvae, prepupae (PP), pupae (P), and adults (A, 10 males and 10 females). Ten to 15 individuals of each stage were collected, ground, and mixed, and 50 mg was obtained. Different tissue samples were collected: 50 mg each of the midgut, fat body, oral cavity of 13-day-old larvae, and head of adults. Three different genotypes of black soldier flies were collected and preserved in Yunnan, Hubei, and Guizhou earlier in this experiment. Therefore, 10 13-day-old larvae from Yunnan, Hubei, and Guizhou were selected to represent these different genotypes. Ten individuals were collected, ground, and mixed, and 50 mg was obtained from each. These samples were immediately placed in liquid nitrogen and stored at −80°C.

[0046] 2. RT-qPCR amplification:

[0047] Following the methods of Example 1, RNA extraction, cDNA template preparation, and RT-qPCR amplification were performed on samples collected under different conditions. After completion of the reaction, the cycle threshold (Ct value) of each candidate internal reference gene in each treated sample was derived. The stability of the candidate internal reference genes under different treatment conditions was evaluated using the ΔCt method, GeNorm, BestKeeper, and NormFinder. RefFinder was used to rank the expression stability of the candidate internal reference genes.

[0048] 2. Experimental results:

[0049] 1. Expression changes of reference genes under different experimental conditions:

[0050] In this study, 11 reference genes were selected and named GAPDH, β-actin, GST, tubulinα-1A, tubulinα-4A, tubulinβ, RP49, RPL13, EF1, 18S RNA, and 28S RNA. The stability of the reference genes in different developmental stages, different tissues, and different sexes of black soldier flies was analyzed. When analyzing the stability of reference genes in different genotypes, 13-day-old larvae from Yunnan, Hubei, and Guizhou were selected. The raw Ct values of the reference genes ranged from 8.99 (18S RNA) to 36.23 (tubulinα-4A) ( Figure 1 AC). However, when selecting reference genes for different genotypes, the qRT-PCR results of tubulinα-1A and tubulinα-4A reference genes in Guizhou samples showed "undefined". Therefore, after removing tubulinα-1A and tubulinα-4A, the reference genes of different genotypes ( Figure 1 D).

[0051] 2. Expression stability of 11 reference genes at different developmental stages of Hermetia illucens:

[0052] The expression stability values calculated by the geNorm algorithm indicated that β-actin, EF1, and RPL13 were suitable reference genes during development ( Figure 2 A). According to NormFinder analysis, the stable reference genes at different developmental stages were β-actin and 18S RNA, with stability values less than 0.5, and the most unstable gene was GST ( Figure 2 C). According to BestKeeper analysis, the top three stable sequences of the selected reference genes were 18S RNA, EF1, and RPL13 ( Figure 2 D). The ΔCt rankings were RPL13, 18S RNA, β-actin, EF1, 18S RNA, GST, RP49, GADPH, tubulinβ, tubulinα-4A, and tubulinα-1A. The online tool RefFinder was used to compare and rank the reference genes by combining the above methods (Table 3). The results showed that the most unstable reference gene at different developmental stages was tubulibα-4A, and the three most stable reference genes were β-actin, 18S RNA, and RPL13. Therefore, β-actin, 18S RNA, and RPL13 could be used as the best reference gene combination for detecting target genes at different developmental stages of Hermetia illucens.

[0053] 3. Expression stability of 11 reference genes in different tissues and genders of Hermetia illucens:

[0054] Similarly, we used the methods for evaluating candidate reference genes at different developmental stages. The geNorm, NormFinder, BestKeeper, and ΔCt methods were used to analyze and evaluate the stability of reference genes in different tissues and genders. The results of geNorm analysis showed that EF1, RP49, β-actin, and RPL13 were relatively stable reference genes in different tissues ( Figure 3 A); EF1, RPL13, GST, and β-actin were relatively stable reference genes in different genders ( Figure 4 A). The results of NormFinder analysis showed that the stability values of RP49, β-actin, and EF1 in different tissues were less than 0.5 ( Figure 3 C); the stability values of tubulinα-4A, 18S RNA, 28S RNA, β-actin, and GST in different genders were all less than 0.5 ( Figure 4 C). The results of BestKeeper analysis showed that the top three rankings of the stability of reference genes in different tissues were RPL13 > GST > β-actin > ( Figure 3D); The top three genes with the highest stability among reference genes of different genders were tubulinα-1A > RPL13 = EF1( Figure 4 D). Combining the above results with the ΔCt ranking (Table 3), comprehensive ranking was performed using RefFinder. The stability of 28S RNA and GAPDH was the lowest in different tissues and genders respectively. Finally, we recommended the most suitable reference genes as RPL13, GST, and RP49 for different tissues, and EF1, RPL13, and GST for different genders.

[0055] 4. Analysis of the stability of reference genes of Hermetia illucens with different genotypes:

[0056] The results of geNorm analysis showed that GADPH, RPL13, 18S RNA, and RP49 were relatively stable reference genes among different genotypes( Figure 5 A). The paired variation analysis showed that V7 / 8 was less than 0.15( Figure 5 B), so GADPH, RPL13, 18S RNA, RP49, GST, tubulinβ, and EF1 were the most suitable reference genes. The results of NormFinder analysis showed that the stability values of 18S RNA, GADPH, RPL13, and EF1 were all lower than 0.5( Figure 5 C), indicating that they were relatively stable reference genes among different genotypes. The results of BestKeeper analysis showed that the top three genes with the highest stability among reference genes of different genotypes were GAPDH, RPL13, and EF1( Figure 5 D). Combining the above results with the ΔCt ranking (Table 3), comprehensive ranking was performed using RefFinder. The results showed that the expression of β-actin was the least stable, and it was recommended to use RPL13, GAPDH, and 18S RNA as the most suitable reference genes for research on different genotypes.

[0057] Table 3 Ranking of candidate internal reference genes under different experimental conditions

[0058]

[0059]

[0060]

[0061] Note: De: Different developmental stages; Ti: Different tissues; Se: Different genders; Ge: Different genotypes.

[0062] Example 3: Verification of the stability of internal reference genes of Hermetia illucens at different developmental stages

[0063] I. Experimental method:

[0064] 1. Reference gene verification:

[0065] Research has shown that the expression of lipase 3-like is dynamic during different developmental stages of the black soldier fly, and the transcriptional level of lipase in the 5th instar (5L) larval stage is significantly higher than that in the prepupal stage (PP). Therefore, we selected the black soldier fly lipase 3-like (Hi-Lip-3L) (LOC119647546) as the target gene to evaluate the stability of candidate reference genes in different developmental stages (Hi-Lib-3L-F: 5′-CTTGAGGACTGGG-3′, Hi-Lip-3-R: 5′-TTGTTGGACTGGGGGGG-3′). Using 2 -ΔΔCt methods to measure the relative expression levels of Hi-Lip-3L in the larval and prepupal stages. The data were statistically analyzed using GraphPad Prism 8.0 software. The differences between the experimental results were analyzed by one-way analysis of Student's t-test, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p > 0.05 (ns).

[0066] 2. Sample collection:

[0067] Ten 5L larvae and ten prepupal larvae (PP) of the black soldier fly were collected respectively. After disinfection, 50 mg of fat body tissue samples were immediately placed in liquid nitrogen and stored at -80°C.

[0068] 3. RT-qPCR amplification:

[0069] According to the methods of Example 1 and 2.

[0070] II. Experimental results:

[0071] Hi-Lip-3L was selected as the target gene, and the expression levels of Hi-Lip-3-L were normalized using the two most stable reference genes β-actin and 18S RNA and the least stable reference gene Tubulinα-4A in different developmental stages evaluated in Example 2. The results showed that when β-actin and 18S RNA were used as reference genes, the expression profiles showed similar trends, that is, the expression level of the Hi-Lip-3L gene in the 5L stage was significantly higher than that in the PP stage. In contrast, when Tubulinα-4A was used as the reference gene, there was no significant difference in the expression levels of Hi-Lip-3L between the 5L and PP stages ( Figure 6 ). It indicates that the reference genes screened and evaluated by this method are reliable.

[0072] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A combination of reference genes stably expressed by Hermetia illucens under different conditions, characterized in that: The internal reference gene combination is any one of the following four groups of gene combinations: (1) β-actin, 18S rRNA, and RPL13; (2) RPL13, GST, and RP49; (3) EF1-α, RPL13, and GST; (4) RPL13, GAPDH, and 18S rRNA.

2. The combination of reference genes stably expressed by Hermetia illucens under different conditions according to claim 1, characterized in that: The nucleotide sequence of the amplification primer for β-actin is shown in SEQ ID NO: 1; the nucleotide sequence of the amplification primer for 18S rRNA is shown in SEQ ID NO: 10; the nucleotide sequence of the amplification primer for RPL13 is shown in SEQ ID NO:

8.

3. The combination of reference genes stably expressed by Hermetia illucens under different conditions according to claim 1, characterized in that: The nucleotide sequence of the amplification primer for RPL13 is shown in SEQ ID NO: 8; the nucleotide sequence of the amplification primer for GST is shown in SEQ ID NO: 3; the nucleotide sequence of the amplification primer for RP49 is shown in SEQ ID NO:

4.

4. The reference gene combination stably expressed by Hermetia illucens under different conditions according to claim 1, characterized in that: The nucleotide sequence of the amplification primer for EF1-α is shown in SEQ ID NO: 9; the nucleotide sequence of the amplification primer for RPL13 is shown in SEQ ID NO: 8; the nucleotide sequence of the amplification primer for GST is shown in SEQ ID NO:

3.

5. The combination of reference genes stably expressed by Hermetia illucens under different conditions according to claim 1, characterized in that: The nucleotide sequence of the amplification primer for RPL13 is shown in SEQ ID NO: 8; the nucleotide sequence of the amplification primer for GAPDH is shown in SEQ ID NO: 2; the nucleotide sequence of the amplification primer for 18S rRNA is shown in SEQ ID NO:

10.

6. Use of the combination of reference genes stably expressed by Hermetia illucens under different conditions according to claim 1 in the preparation of a fluorescence PCR quantitative detection kit for Hermetia illucens, characterized in that: β-actin, 18S rRNA, and RPL13 are stably expressed in different developmental stages of Hermetia illucens from egg to adult; RPL13, GST, and RP49 are stably expressed in different tissues of Hermetia illucens; EF1-α, RPL13, and GST are stably expressed in different genders of Hermetia illucens; RPL13, GAPDH, and 18S rRNA are stably expressed in different genotypes of Hermetia illucens.

7. Use of the internal reference gene combination stably expressed by Hermetia illucens under different conditions according to claim 1 in the fluorescence PCR quantitative detection of Hermetia illucens.

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