CN103937822A

CN103937822A - Sequence of locust I type chitinase gene, and application of dsRNA of locust I chitinase gene

Info

Publication number: CN103937822A
Application number: CN201410069843.7A
Authority: CN
Inventors: 李大琪; 张建珍; 马恩波; 孙毅
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2014-02-28
Filing date: 2014-02-28
Publication date: 2014-07-23
Anticipated expiration: 2034-02-28
Also published as: CN103937822B

Abstract

The invention provides a sequence of locust I type chitinase gene (cht5), and an application of dsRNA of the locust I chitinase gene. The dsRNA can silence the I type chitinase gene to make locusts dead. The I type chitinase gene with the sequence represented by SEQ ID NO:1 is obtained by cloning the locust I type chitinase gene, and sequencing; and a gene fragment with the sequence represented by SEQ ID NO:2 is selected to synthesize double strand RAN (dsRNA). After the dsRNA is injected to the body cavity of the locust, the locust has delayed growth, and cannot successfully molt to enter a next age period, so the locust is dead. A new target is provided for the RNA interference based insect control in the invention.

Description

The sequence of locust I type chitinase gene and the application of dsRNA thereof

Technical field

The present invention relates to biological technical field.Be specifically related to the sequence of locust I type chitinase gene and the application of dsRNA thereof.

Background technology

Insect is the formidable enemy of China's agriculture production, and long-term application chemical insecticide causes that pesticide residue, environmental pollution are serious, resistance appears in locust, healthy etc. the series of problems of harm humans.Along with social development, in the urgent need to novel insect pest control method.

Disturb (RNAi) pest control technology just at high speed development based on RNA in recent years.The method has following advantage: 1) select the single-minded gene of insect is disturbed, to higher animal and and the mankind be safe; 2) pest-resistant have a specificity, to non-target organism without lethal effect; 3) nontoxic to environment.

For the phenomenon design pest control scheme of casting off a skin of locust uniqueness, both efficient kill pests, have avoided again environmental pollution, safe and reliable.Insect exoskeleton is mainly made up of chitin, and chitinase genes of insects is responsible for chitinous degraded, so that insect molting.Locust I type chitinase gene (cht5) plays a key effect in the time casting off a skin, and utilizes its important biomolecule to learn function and carries out pest control, time saving and energy saving, safe and efficient.

Summary of the invention

The object of this invention is to provide a kind of sequence and the application of dsRNA in lethal insect thereof of locust I type chitinase gene.

A kind of locust I type chitinase gene provided by the invention, its nucleotide sequence is SEQ ID NO:1.This sequence is the search by migratory locusts being transcribed to group, obtains 1 migratory locusts I type chitinase gene fragment, and after end sequence rapid amplifying (RACE), further clone obtains full length cDNA sequence.The long 1587bp of this sequence, comprises 1515bp open reading frame, coding 505 amino acid.

A kind of locust I type chitinase gene fragment provided by the invention, its nucleotide sequence is SEQ ID NO:2, be according to SEQ ID NO:1 design upstream primer SEQ ID NO:3 and downstream primer SEQ ID NO:4, obtain SEQ ID NO:2 by pcr amplification, it contains T7 promotor.Further according to T7RiboMAX ^tMexpress RNAi System(Promega) the synthetic dsRNA of test kit explanation in-vitro transcription.

The application in lethal locust of the synthetic dsRNA of SEQ ID NO:2: inject above-mentioned dsRNA to locust body cavity, result shows: this dsRNA mrna expression of reticent locust I type chitinase gene LmCht5-1 specifically, and cause migratory locusts cast off a skin difficulty and death.

Brief description of the drawings

Fig. 1: the difficulty that occurs casting off a skin after the synthetic dsRNA of migratory locusts injection in 5 ages SEQ ID NO:2 and dead phenotype.Left side is the contrast of injection dsGFP, and right side is the synthetic dsRNA of injection SEQ ID NO:2.

Fig. 2: migratory locusts are injected SEQ ID NO:2 after synthetic dsRNA24 hour, the reticent situation of the mRNA of migratory locusts I type chitinase gene LmCht5-1, β-actin is as reference gene.**P<0.01。

Embodiment

Embodiment 1: the sequence of migratory locusts I type chitinase gene and the acquisition of dsRNA thereof

1, the sequence of migratory locusts I type chitinase gene obtains

1) sequence of migratory locusts I type chitinase gene is transcribed the search of group database migratory locusts

Transcribe group database based on migratory locusts, adopt bioinformatics method to search for the sequence of migratory locusts I type chitinase gene, after sequential analysis and comparison, obtain altogether the sequence fragment of 1 migratory locusts I type chitinase gene, this cDNA sequence deletion 3' end.

2) full length cDNA sequence of migratory locusts I type chitinase gene obtains

Based on obtaining migratory locusts I type chitinase gene fragment, adopt primer premier5.0 software design 3 ' RACE primer.All primers are synthetic by the English Weihe River, the Shanghai biological company limited of prompt base.Extract the total RNA of migratory locusts nymph in 5 age, by the synthetic RACE template of RACE test kit (clontech), transfer for full length cDNA sequence.After PCR, will obtain product purification, clone is transformed in intestinal bacteria, sends to order-checking.After interpretation of result, splicing obtains migratory locusts I type chitinase gene LmCht5-1cDNA full length sequence, and its sequence is SEQ ID NO:1.

2, migratory locusts I type chitinase gene dsRNA is synthetic

1) design of migratory locusts I type chitinase gene dsRNA primer

Migratory locusts I type chitinase gene sequence SEQ ID NO:1 based on obtained, adopts primer premier5.0 software design.Design dsRNA synthetic primer, its sequence is respectively SEQ ID NO:3 and SEQ ID NO:4.All primers are synthetic by the English Weihe River, the Shanghai biological company limited of prompt base.

2) migratory locusts I type chitinase gene dsRNA's is synthetic

Above-mentioned primer SEQ ID NO:3 and SEQ ID NO:4 synthesize PCR product, warp sV Gel and PCR Clean-Up System(Promega) after test kit purifying, according to T7RiboMAX ^tMexpress RNAi System(Promega) the synthetic dsRNA of test kit explanation in-vitro transcription.DsRNA concentration adopts microplate reader SpectraMax190 to measure, and to be concentrated into final concentration be 3 μ g/ μ l.

Embodiment 2: migratory locusts I type chitinase gene dsRNA lethal 5 age migratory locusts

1, migratory locusts I type chitinase gene dsRNA injection

By 1 μ l(3 μ g) dsRNA of SEQ ID NO:2 be expelled between 5 age migratory locusts the 2nd age in days nymph two, three uromeres with 25 μ l specification microsyringes, inject altogether 40, male and female half and half.The dsGFP of injection same volume concentration is to control group body.Migratory locusts after injection are placed in to 30 DEG C of constant temperature biochemical cultivation cases to be raised.

2, inject after dsRNA 5 age migratory locusts phenotype observation

5 age nymph after injection dsRNA, injection dsGFP control group starts to cast off a skin after 6 days and all successfully sloughs off to adult, on average the time of casting off a skin is 15-20 minute, sloughs off to polypide form vigor after adult normally, and after half a day, starts normal feed.Totally 40 of the treatment group nymphs of injection dsLmCht5-1, compared with control group nymph, wherein have 26 nymphs finally can not complete to cast off a skin and cause death, and mortality ratio reaches 65.0%.There are three kinds of different phenotypes (Fig. 1) in 26 dead nymphs: the first be the nymph of a locust while casting off a skin only pronotum and head ftracture a little along crestal line, the visible new epidermis of cracking part, back protuberance, wing bud opens, and continues to death always; The second phenotype is that the new and old epidermis of appendage cannot separate, and new wing also cannot be sloughed off from old epidermis wing bud; The third is that pronotum crestal line cracking part increases, and the new and old epidermis of abdomen end can separate, and can obviously see ghost, and whole polypide distortion is until death.The probability that above three kinds of phenotypes occur is basic identical.

3, migratory locusts I type chitinase gene is reticent detects

After dsRNA injection in 5 ages, select the rear 24h silence efficiency of injection to detect, whole worm polypide is extracted object as RNA, and every group arranges 3 biology and repeats.Total RNA the reverse transcription of extracting each sample become the first chain cDNA, and the relative expression quantity of Real-time PCR testing goal gene (LmCht5-1) and house-keeping gene (β-actin), to calculate the silence efficiency (Fig. 2) of goal gene.

Claims

1. a locust I type chitinase gene, its nucleotide sequence is SEQ ID NO:1.

2. a locust I type chitinase gene fragment, its nucleotide sequence is SEQ ID NO:2.

3. the dsRNA of locust I type chitinase gene segment condense as claimed in claim 2.

4. the application of dsRNA as claimed in claim 3 in lethal locust.