CN113025612A

CN113025612A - siRNA of high-efficiency lethal insect and application thereof

Info

Publication number: CN113025612A
Application number: CN202110265754.XA
Authority: CN
Inventors: 柴林; 张建琴; 李大琪; 史学凯; 高璐; 范云鹤
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2021-03-11
Filing date: 2021-03-11
Publication date: 2021-06-25

Abstract

The invention belongs to the technical field of biology, and particularly relates to siRNA of high-efficiency lethal insects and application thereof. The siRNA is an RNA interference core sequence, the length of the siRNA is 21bp, the nucleotide sequence of a sense strand is shown as SEQIDNO.1, the nucleotide sequence of an antisense strand is shown as SEQIDNO.2, and the insect is the tribolium castaneum. The siRNA is applied to preventing and treating the erythrozootic sitagliptin, the siRNA is copied to the recombinant dsRNA, the siRNA is positioned at any position in the sequence of the recombinant dsRNA, the recombinant dsRNA also comprises an auxiliary sequence, and the auxiliary sequence is a sequence with the similarity of less than 50% with the genome of the erythrozootic sitagliptin.

Description

siRNA of high-efficiency lethal insect and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to siRNA of high-efficiency lethal insects and application thereof.

Background

Tribocicloraceae (Triboliumcastaneum) belongs to Coleoptera, Bacteroideae, is an important storage pest in China, mainly harms flour, and can also be harmed to corn, rice, sorghum, beans, dried fruits, traditional Chinese medicinal materials and the like. In addition, the secretion of the pests contains carcinogen benzoquinone, so that flour is moldy and deteriorated, and the health of human beings is harmed. At present, domestic grain depots are generally prevented and treated by means of phosphine fumigation treatment. Researches report that main grain storage pests such as tribolium castaneum have resistance to phosphine, and research and development of novel control methods and strategies for controlling the pests are urgently needed.

RNA interference (RNAi) has been a hot spot for the last decade for new methods of pest control. Compared with chemical pesticides, the method has the advantages of strong specificity, greenness, safety and the like. The action mechanism is that exogenous double-stranded RNA (dsRNA) can be recognized by insect Dicer enzyme and is cut into small interfering RNA (siRNA), and post-transcriptional silencing of homologous sequence genes is caused under the action of RNA-induced silencing complex (RISC). Based on the action mechanism of RNAi, important physiological function genes of pests are subjected to targeted silencing and pest killing, so that the purpose of prevention and control is achieved.

The current research shows that the tribolium castaneum is sensitive to RNAi, and the pest control based on RNAi has great application value. Early studies show that the mortality rate of dsRNA of a segment of chitinase gene 10 of Tripsammophila to pests is 77 percent. How to improve the mortality of dsRNA to pests and achieve better pest control effect, and solving the scientific problem has important significance for efficiently controlling storage pests and reducing economic loss of grains. Through comprehensive analysis of research work on insect RNAi, the gene silencing efficiency is found to be related to the sequence specificity of dsRNA. Further based on the RNAi principle, it can be speculated that different sirnas may be a major factor in the difference in silencing efficiency of target genes. If siRNA with high-efficiency silencing function in dsRNA can be obtained by screening and the lethal efficiency of recombinant dsRNA containing the siRNA to pests is researched, the efficiency of dsRNA in pest control can be improved, and the application process of the dsRNA is accelerated.

Disclosure of Invention

The invention aims to provide siRNA for efficiently killing insects and application thereof, and the siRNA is used for preventing and treating pests through recombinant dsRNA containing the siRNA.

In order to achieve the purpose, the invention adopts the following technical scheme:

the siRNA of the high-efficiency lethal insect is an RNA interference core sequence, the length of the siRNA is 21bp, the nucleotide sequence of a sense strand is shown as SEQ ID NO.1, the nucleotide sequence of an antisense strand is shown as SEQ ID NO.2, the insect is tribolium castaneum, and the siRNA has the biological characteristics of blocking the ecdysis, pupation and eclosion of the tribolium castaneum, so that the insect is killed efficiently.

A recombinant dsRNA comprising siRNA to said lethal insect, further comprising an auxiliary sequence, said siRNA being located at any position in the recombinant dsRNA sequence, said auxiliary sequence being any non-homologous base sequence of the genome of tribolium castaneum. The auxiliary sequence can adjust the length and the base sequence characteristics according to different application requirements and conditions to protect and assist the siRNA interfering core sequence to enter insect cells more efficiently so as to play a role in gene silencing.

The siRNA is obtained by designing a plurality of different siRNAs and adding auxiliary sequences to construct recombinant dsRNA on the basis of a specific dsRNA sequence of the erythropoeis, wherein the dsRNA target silences the erythropoeis chitinase gene 10(TcCht10) with the length of 560bp, and the lethality rate to the erythropoeis reaches 77%, and the siRNA is a main factor of gene silencing caused by dsTcCht10 after injection, screening and verification. When the recombinant dsRNA sequence only contains 1 copy of the 21bp RNA interference core sequence, the gene silencing of the Totheezetimibe chitinase gene 10 can reach 53 percent, and the lethality rate of pests also reaches 64 percent; correspondingly, the silencing efficiency of other siRNAs is low, is only about 15-30%, and the mortality rate to pests is only about 10-20%.

Further, the auxiliary sequence is a sequence with the similarity of the genome of the tribolium castaneum lower than 50% so as to avoid off-target effect.

Furthermore, the number of the siRNA is more than or equal to 2, and the silencing efficiency and the fatality rate of the Totheezetimibe chitinase gene 10 can be improved by increasing the copy number of the siRNA to recombine the siRNA. When the siRNA contains more than 2 copies, the gene silencing efficiency can be improved to more than 94 percent, and the lethality rate to pests is improved to 100 percent.

Application of recombinant dsRNA containing siRNA of the lethal insect in preventing and treating the tribolium castaneum.

Compared with the prior art, the invention has the following advantages:

1) compared with chemical pesticides, the method has the advantages of strong specificity, greenness, safety and the like. On one hand, the problem of comprehensive treatment after the pesticide pollutes the environment is reduced, and on the other hand, the siRNA kills insects efficiently, thereby greatly saving the cost for preventing and controlling pests. Accords with the concept of national green innovation and development.

2) Compared with the traditional method of applying dsRNA to pest control, the method screens and obtains the core sequence siRNA interfered by RNA, and reduces the off-target effect of dsRNA.

3) According to the invention, through constructing the recombinant dsRNA, the constraints of low efficiency and high cost of siRNA in the tribolium castaneum are eliminated; on the other hand, by increasing the copy number of the core sequence siRNA, pests can be killed efficiently, and the purpose of prevention and control is achieved.

Drawings

FIG. 1 shows mRNA silencing and lethality of chitinase gene 10(TcCht10) following red-pseudogluteline injection of different types of recombinant dsRNA (siRNA1-5), dsGFP and dsTcCht 10;

FIG. 2 shows mRNA silencing and mortality of chitinase gene 10(TcCht10) after recombinant single-copy siRNA3, double-copy siRNA3-2, triple-copy siRNA3-3, four-copy siRNA3-4 and dsGFP are injected into 6 th larvae of Tripsammosile castanea;

FIG. 3 is a phenotype of stunted larvae after injection of dsGFP, siRNA3-2 and dsTcCht10 into 6 th instar larvae of Tripsammosile castanea.

Detailed Description

Example 1

An siRNA killing insects is an RNA interference core sequence, the length of the siRNA is 21bp, the nucleotide sequence of a sense strand is shown as SEQ ID NO.1, the nucleotide sequence of an antisense strand is shown as SEQ ID NO.2, and the insects are tribolium castaneum.

The recombinant dsRNA comprises the siRNA of the lethal insect and also comprises an auxiliary sequence, wherein the siRNA is positioned at any position of the sequence of the recombinant dsRNA, the auxiliary sequence is a sequence with the similarity of the genome of the Trimerella erythropolis being lower than 50%, and the number of the siRNA is more than or equal to 2.

The application of the recombinant dsRNA is applied to preventing and treating the tribolium castaneum.

Example 2 obtaining of highly lethal siRNA to Tripsammosile castanea

1. Obtaining dsRNA with lethal function of tribolium castaneum

1) Analysis of amino acid sequence of chitinase gene 10 of Tripsammophila castanea

Based on the functional research of the chitinase gene of the tribolium castaneum, the chitinase gene 10(TcCht10) influencing the molting of the tribolium castaneum is selected as a target gene. Through searching the genome database of the tribolium castaneum, the mRNA full-length sequence of TcCht10 is determined, and the Genbank accession number is NM-001042602.1. Further domain analysis was performed on the SMART website (http:// SMART. embl-heidelberg. de /), to predict the protein sequence and catalytic domains of chitinase activity, chitin binding domains.

2) Obtaining double-stranded RNA of chitinase gene 10 of Tripsammosilene castanea

The dsTcCht10 sequence is designed in the area of the coding chitinase activity catalytic domain, the start site is 7448 th, the end site is 8007 th, and the length is 560 bp. Primeremier 5.0 software was used to add the T7 promoter sequence and design double-stranded RNA synthesis primers for delivery to Shanghai Biotechnology, Inc. Using mRNA as template, using double-stranded RNA primer to synthesize PCR product

SV Gel and PCR Clean-Up System (Promega) kit after purification according to T7 RiboMAX^TMExpress RNAi System (Promega) kit demonstrates in vitro transcription synthesis of dsTcCht 10. The concentration of dsTcCht10 was measured using a microplate reader SpectraMax 190 and concentrated to a final concentration of 1. mu.g/. mu.L.

3) Dstcht 10 lethal 6-instar Chinemys castanea larvae

400ng dsTcCht10 was injected into 6 th instar Tripsammosile castanea larvae for biological functional verification, with dsGFP as the control group. Experiments show that: compared with a control group, the silencing efficiency of the dsTcCht10 on the target gene chitinase gene 10 is 71%, the larvae of the tribolium castaneum have a phenotype that the old epidermis can not be detached, and the lethality rate is 77%. The dsTcCht10 has certain control effect on the red-simulated paddy beetle, but the lethality of pests needs to be further improved.

2. Obtaining of highly effective lethal siRNA of tribolium castaneum

1) Obtaining of chitinase gene siRNA of Tripsammosilene ruber

Based on the RNA interference principle, the nucleotide sequence of the dsTcCht10 with the length of 560bp is analyzed, a segment with the GC content of 45-55% is selected to design siRNA with the length of 21bp, 5 siRNAs are obtained and handed over to Shanghai Biotechnology Limited company for synthesis.

2) Functional verification of chitinase gene siRNA of tribolium castaneum

5 siRNA are injected into 6 th instar larva of the tribolium castaneum, the dosage is 400ng, and the result shows that the siRNA only injected does not play a silencing role on the target gene. In order to make siRNA function, it was found through extensive literature research that: most insects have a better gene silencing response to dsrnas of longer sequence length. Accordingly, helper sequences are added to protect and assist the siRNA from silencing. A dsGFP sequence with the length of 560bp is selected as an auxiliary sequence, siRNA replaces nucleotide sequences with the same length on the auxiliary sequence, 5 different recombinant dsRNA (siRNA1-5) is constructed, and injection is performed to verify the silencing efficiency and the biological function. The experimental results show that the siRNA1-5 can silence target genes to different degrees, but the silencing efficiency is greatly different, and the lethality rate is also different. Wherein, siRNA3 can silence the Todarussis rubefaciens chitinase gene 10 to 53 percent, and the lethality rate to pests also reaches 64 percent; correspondingly, other siRNAs were less effective in silencing, only 15-30%, and only 10-20% of the pests were lethal (FIG. 1). Thus, siRNA3 is a major factor in chitinase gene 10 gene silencing and is highly lethal to tribolium castaneum larvae.

Example 3 acquisition of highly lethal recombinant dsRNA of Tripsammosile castanea

siRNA3 with different copy numbers is constructed into a 560bp dsGFP auxiliary sequence, and how to improve the silencing efficiency of the recombinant dsRNA on a target gene and the lethality of the recombinant dsRNA on the tribolium castaneum is explored. 1-4 copies of siRNA3 randomly replace auxiliary sequences with different sites with equal length to form recombinant dsRNA, which is named as siRNA3-1, siRNA3-2, siRNA3-3 and siRNA 3-4. The recombinant dsRNA template is synthesized by Shanghai Biotechnology Limited, synthesized into dsRNA through in vitro transcription, and injected into 6-instar Trimerella erythropolis to observe the influence on the growth and development of the larvae. The results show that: when the recombinant dsRNA contains 1 interfering core sequence siRNA3 (siRNA3-1), the target gene is silenced by 53 percent, and the mortality rate to pests is 64 percent; when 2 siRNAs 3 are contained (siRNA3-2), the target gene is silenced to 94 percent, and the mortality rate to pests is increased to 100 percent; when 3 or 4 siRNAs 3 are contained (siRNA3-3 and siRNA3-4), the silencing effect on target genes reaches about 95 percent, and the lethality rate on pests is stable to 100 percent (figure 2). Thus, the silencing efficiency of siRNA3 on target gene and the lethality to pests are improved with the increase of copy number, and the peak value can be reached when 2 copies of interfering core sequence are contained. The discovery solves the problem of low mortality rate of the Triplostegian dsRNA and improves the application value of the Triplostegian dsRNA.

FIG. 3 shows that after 6 th larva of Tripsammosile castanea is injected with dsGFP, siRNA3-2 and dsTcCht10, the larva molting is hindered, and it can be seen that the phenotype of siRNA3-2 is approximately the same as that of dscht10, namely the larva has the biological characteristics of blocking the molting, pupating and eclosion of the Tripsammosile castanea, thereby efficiently killing pests.

Sequence listing

<110> university of Shanxi

<120> siRNA for highly effective lethal insect and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA

<213> Tribolium castaneum)

<400> 1

ttaacgccaa gtcgtacggc c 21

<210> 2

<211> 21

<212> DNA

<213> Tribolium castaneum)

<400> 2

ggccgtacga cttggcgtta a 21

Claims

1. The siRNA for efficiently killing insects is characterized in that the siRNA is an RNA interference core sequence, the length of the siRNA is 21bp, the nucleotide sequence of a sense strand is shown as SEQ ID No.1, the nucleotide sequence of an antisense strand is shown as SEQ ID No.2, and the insects are tribolium castaneum.

2. A recombinant dsRNA comprising a siRNA to the lethal insect of claim 1, further comprising a helper sequence, said siRNA being located at any position in the sequence of the recombinant dsRNA, said helper sequence being any non-homologous base sequence of the genome of tribolium castaneum.

3. The recombinant dsRNA of claim 2, wherein the helper sequence is a sequence with a similarity to the genome of Tripsammomyza rubescens of less than 50%.

4. The recombinant dsRNA of claim 2, wherein the number of siRNAs is 2 or more.

5. The use of the recombinant dsRNA of claim 2 in the control of tribolium castaneum.