CN117343935B

CN117343935B - Laodelphax striatellus lethal gene ubiquitin and application thereof in biological control

Info

Publication number: CN117343935B
Application number: CN202210791480.2A
Authority: CN
Inventors: 亓玉华; 卢刚; 李俊敏; 陈剑平
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2022-07-05
Filing date: 2022-07-05
Publication date: 2024-11-15
Anticipated expiration: 2042-07-05
Also published as: CN117343935A

Abstract

The invention discloses a lethal gene ubiquitin of gray leafhopper and an application thereof in biological control. Studies have found that the ubiquitin gene ubiquitin of gray leafhopper is essential for gray leafhopper to maintain life. After silencing ubiquitin in the body of gray leafhopper by RNA interference technology, the death of gray leafhopper can be caused. The method of the invention is safe and harmless, and provides a new approach for controlling gray leafhopper.

Description

Laodelphax striatellus lethal gene ubiquitin and application thereof in biological control

Technical Field

The invention relates to the field of biological control, in particular to a Laodelphax striatellus lethal gene ubiquitin and application of the Laodelphax striatellus lethal gene ubiquitin in biological control.

Background

Laodelphax striatellus (Laodelphax striatellus, fall en) belongs to the family of plant hoppers (DELPHACIDAE) of the order Hemiptera, and is a rice plant hopper which mainly endangers gramineae. The Laodelphax striatellus occurs in various provinces in China, wherein the lower reaches of the Yangtze river and the North China rice area have more occurrence and more serious harm, and the Laodelphax striatellus is widely distributed in east Asia, southeast Asia, europe, north Africa and the like, and is an important pest. The hazards of the laodelphax striatellus are mainly expressed in two aspects: direct hazards and indirect hazards. Direct harm means that the leaf, stem and phloem juice of the Gramineae plant is absorbed by the nymphs and adults of the Laodelphax striatellus, so that the moisture and the nutrient of the plant are lost, the yellow leaf and early blight of the plant are caused, and lodging is caused when serious, so that the yield and harvest of the rice field are greatly affected. Meanwhile, when the rice planthoppers are damaged, honeydew is secreted and covered on plants, fungi are caused to breed, rice ears are blackened and mildewed, and quality and yield are seriously affected. In addition to directly feeding juice to harm crops, laodelphax striatellus is also a transmission medium for various virus diseases in crops such as rice, corn, wheat and the like. In recent years, the indirect damage loss caused by the transmission of the toxin of the Laodelphax striatellus is far greater than the direct feeding damage of the Laodelphax striatellus, wherein the rice stripe disease and the rice black streaked dwarf disease transmitted by the Laodelphax striatellus are most serious. Under natural conditions, the two diseases are horizontally transmitted and spread by feeding healthy rice through the laodelphax striatellus, so that the diseases are caused to be pandemic, and the large-area yield reduction of the rice is caused.

For a long time, the control of the laodelphax striatellus takes chemical pesticides as the main component, the pesticide use amount is large, single, the times are large and unreasonable, the selection pressure of the pesticides is increased, the laodelphax striatellus generates drug resistance, the effective control effect can be achieved only by continuously increasing the pesticide use amount, serious environmental pollution is caused, and serious threat is generated to the safety of rice grains. Therefore, in actual production, prevention means other than chemical pesticides are demanded. With the development of biotechnology, pest control by biological means has become an important means for plant protection. Among them, RNA interference (RNAINTERFERENCE, RNAI) molecular mechanism is a posttranscriptional gene silencing mechanism mediated by double-stranded RNA (double-STRANDED RNA, DSRNA), and is a modern molecular tool for excavating gene functions. In organisms, some important genes are necessary for maintaining vital activities. In theory, if the RNA interference technology is used for interfering important genes in agricultural pests, the important genes can cause teratogenesis or death of the pests, so that the purpose of controlling the pests is achieved. Therefore, there is an urgent need for a safe and harmless method for establishing a new method for controlling pests by silencing and maintaining vital genes in the body of the Laodelphax striatellus through an interference technology.

Disclosure of Invention

Accordingly, one of the objects of the present invention is to provide a plant hopper lethal gene ubiquitin; the second purpose of the invention is to provide the application of the reagent for interfering and expressing the Laodelphax striatellus lethal gene ubiquitin in preparing the biological medicine for preventing and treating Laodelphax striatellus; the invention further aims to provide a method for preventing and controlling the Laodelphax striatellus. The invention utilizes the ubiquitin gene ubiquitin of the Laodelphax striatellus to be necessary for the Laodelphax striatellus to maintain life, and causes death of the Laodelphax striatellus after the ubiquitin is silenced in the Laodelphax striatellus by using an RNA interference technology.

In order to achieve the above purpose, the present invention provides the following technical solutions:

1. The amino acid coded by the Laodelphax striatellus lethal gene ubiquitin is shown as SEQ ID NO. 2.

Preferably, the nucleic acid sequence of the Laodelphax striatellus lethal gene ubiquitin is shown as SEQ ID NO.1 or a sequence which has 90% homology with SEQ ID NO.1 and codes amino acid shown as SEQ ID NO.2 after substitution.

2. The primer pair is used for amplifying the Laodelphax striatellus lethal gene ubiquitin, and the nucleotide sequence of the primer pair is shown as SEQ ID NO.3 and SEQ ID NO. 4.

3. Application of a reagent for interfering expression of the Laodelphax striatellus lethal gene ubiquitin in preparation of biological Laodelphax striatellus prevention and treatment medicines.

Preferably, the laodelphax striatellus are adults and nymphs.

Preferably, the reagent for interfering with expression of the Laodelphax striatellus lethal gene ubiquitin is double-stranded RNA for interfering with the Laodelphax striatellus lethal gene ubiquitin.

Preferably, the double-stranded RNA is synthesized by using SEQ ID NO.7 and SEQ ID NO.8 as primers and using the Laodelphax striatellus cDNA as a template after amplification by using an in vitro transcription kit.

4. A method for preventing and controlling Laodelphax striatellus, which can interfere and express the expression of a Laodelphax striatellus lethal gene ubiquitin the Laodelphax striatellus.

The invention has the beneficial effects that: the invention discloses a Laodelphax striatellus lethal gene ubiquitin, researches show that the Laodelphax striatellus ubiquitin gene ubiquitin is necessary for the Laodelphax striatellus to sustain life, and after the Laodelphax striatellus is silenced in the Laodelphax striatellus body by an RNA interference technology, the Laodelphax striatellus death can be caused.

Drawings

In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:

FIG. 1 is a nucleic acid, amino acid sequence and conserved domain analysis of the Laodelphax striatellus ubiquitin gene;

FIG. 2 is a phylogenetic tree of the plant hopper ubiquitin genes;

FIG. 3 shows the results of interference of the gene of Laodelphax striatellus (A: detection of interference efficiency of Laodelphax striatellus in the three-stage nymph stage, B: statistics of survival rate of Laodelphax striatellus after interference of Laodelphax striatellus in the three-stage nymph stage with the ubaquistatin).

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.

Example 1 acquisition of full Gene ubiquitin, construction of a evolutionary Tree and Synthesis of double-stranded RNA

The Laodelphax striatellus population used in this study was maintained on conventional japonica rice (Xiushui 128), raised in a climatic chamber and established. The temperature of the artificial climate chamber is set to 26+/-1 ℃ and the photoperiod is 12h:12h (light: dark), relative humidity 75%. The seedlings were replaced every 7 days.

Collecting 5 female insects and 5 male Laodelphax striatellus, immediately placing the two female insects and the 5 male Laodelphax striatellus into liquid nitrogen for quick freezing, extracting total RNA of the Laodelphax striatellus according to a Trizol reagent specification, and carrying out reverse transcription by a two-step method by taking the extracted RNA as a template and referring to a reverse transcription kit HISCRIPT II Q SELECT RT SuperMix for qPCR specification of Novozan company after the quality and the concentration of the detected RNA meet the requirements. The complete open reading frame sequence was obtained by specific upstream primers: 5'-gacgtaccagattacgctcatatgcagatattcgtgaaaacg-3' (SEQ ID NO. 3), specific downstream primer: 5'-gcagctcgagctcgatggatccttagttggttccaccacgaag-3' (SEQ ID NO. 4), amplifying by polymerase chain reaction (Polymerase Chain Reaction, PCR) with the cDNA as template, connecting to pGAD-T7 vector, performing copy screening, sequencing, and encoding amino acid sequence as shown in SEQ ID NO. 2.

atgcagatattcgtgaaaacgttgacaggaaagaccattacattggaggtggagccctcagacacgattgagaacgtgaaggccaagattcaagataaggaaggaatccccccagaccagcagcgtctcattttcgctggtaaacagctggaggatggcaggacattgtcagactacaatattcagaaagaatccactctccatcttgtgttgcgtctccgtggtggctaa(SEQ NO.1)

MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG(SEQ NO.2)

The correctly sequenced Laodelphax striatellus ubiquitin sequence is compared with genes with higher homology to other species in NCBI. Sequence alignment was first performed using Muscle on MEGA7.0 software, and then the relationship between homologous genes was analyzed using the Nearest-Neighbor Method (Nearest-Neighbor Method), and the results are shown in fig. 2. The results show that the plant hoppers ubigstin has a close relationship with the bipedal dust mites (Halotydeus _destructor) ubigstin-60S ribosomal protein L40, the diptera bifidus (Cloeon dipterum) Hypothetical predicted protein and the western corn rootworm (Diabrotica VIRGIFERA VIRGIFERA) ubigstin-60Sribosomal protein L40, and are aggregated into a branch with the ubigstin of other insect species, indicating that the gene has higher conservation in other species.

The dsRNA was synthesized by PCR using the above-described correctly sequenced plasmid as a template and adding a T7 promoter to both ends of the target gene sequence according to the kit instructions of T7RiboMAX Express RNAI SYSTEM (Promega, USA), dsGFP was used as a control gene, and the sequence of the dsGFP upstream primer used was: 5'-taatacgactcactatagggagaacaagttctccgttccggc-3' (SEQ ID NO. 5); the downstream primer sequences were: 5'-taatacgactcactatagggagatggttccttgatgccgttct-3' (SEQ ID NO. 6), the template is the DNA sequence of green fluorescent protein GFP gene; dsubiquitin the upstream primer sequence is: 5'-taatacgactcactatagggagaatgcagatattcgtgaaaacg-3' (SEQ ID NO. 7), the downstream primer sequence is: 5'-taatacgactcactatagggagattccttccttatcttgaatc-3' (SEQ ID NO. 8), wherein the template is Laodelphax striatellus cDNA, and the synthesized double strand is detected by using 1% agarose gel and stored at-80 ℃ in a refrigerator.

Example 2 double-stranded RNA injection interfering with ubiquitin in Laodelphax striatellus

The capillary was drawn into a glass needle suitable for microinjection at 62.5 ℃ by a one-step method using a needle drawing machine, and the above-described synthesized double-stranded RNA was injected into the glass needle. Selecting three-instar Laodelphax striatellus nymphs with consistent instar, anesthetizing the Laodelphax striatellus by using carbon dioxide, placing the Laodelphax striatellus under a stereoscopic vision mirror, and injecting the nymphs between the second abdominal node and the third abdominal node by using a microinjection instrument, wherein the recommended parameters of the microinjection instrument are injection pressure (injection pressure, pi) 1000hPa, pressure maintenance time (ti) 0.5s, and compensation pressure (compensation pressure, pc) 20hPa.

Example 3 interference condition detection

Feeding the injected Laodelphax striatellus nymphs on rice seedlings, detecting interference conditions by quantitative PCR and WB after 48 hours, wherein the quantitative primers of qRT-ubiquitin upstream are as follows: 5'-cccacctgaccagcagcgcctc-3' (SEQ ID NO. 9), qRT-ubiquitin downstream quantitative primers are: 5'-ttagccaccacggagacgca-3' (SEQ ID NO. 10), the results of which are shown in FIG. 3A.

The results show that ubiquitin expression was significantly down-regulated on average at mRNA levels and protein levels after injection dsubiquitin.

Example 4 mortality statistics and phenomenon recording

The injected nymphs of the Laodelphax striatellus were raised on rice seedlings, the survival of the nymphs was counted every 24 hours, and the statistics were carried out, the results are shown in tables 1 and 2 (2 experimental replicates), and the survival rate is shown in FIG. 3B.

TABLE 1 mortality statistics

Treatment/time	24h	48h	96h	120h	144h
						dsGFP	42	34	32	30	26
dsubiquitin	38	21	2	0	0

TABLE 2 mortality statistics

Treatment/time	24h	48h	96h	120h	144h
						dsGFP	82	78	72	67	60
dsubiquitin	95	56	19	4	0

The results show that: after the three-instar interference gene ubiquitin of the Laodelphax striatellus, the Laodelphax striatellus is dead, and the Laodelphax striatellus is all dead within 6 days.

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims

1. The application of a reagent for interfering expression of a Laodelphax striatellus lethal gene ubiquitin in preparation of a biological medicine for preventing and treating Laodelphax striatellus is characterized in that: the laodelphax striatellus is an adult or a nymph; the reagent for interfering and expressing the Laodelphax striatellus lethal gene ubiquitin is double-stranded RNA interfering the Laodelphax striatellus lethal gene ubiquitin; the double-stranded RNA is synthesized by using SEQ ID NO.7 and SEQ ID NO.8 as primers and using Laodelphax striatellus cDNA as a template after amplification by using an in vitro transcription kit.

2. A method for preventing and controlling Laodelphax striatellus is characterized in that: the reagent for interfering the expression of the laodelphax striatellus lethal gene ubiquitin in the laodelphax striatellus is double-stranded RNA interfering the laodelphax striatellus lethal gene ubiquitin; the double-stranded RNA is synthesized by using SEQ ID NO.7 and SEQ ID NO.8 as primers and using Laodelphax striatellus cDNA as a template after amplification by using an in vitro transcription kit.