CN106614711A - Application of plant small molecular ribonucleoprotein RNA to pest control - Google Patents

Abstract

The invention discloses application of plant small molecular ribonucleoprotein RNA to pest control. During sequencing analysis on differential small molecular RNAs of pests spodoptera litura eating mustard, srRNA-348-25 of which the expression quantity is significantly reduced is found, and is identified as a conserved sequence ubiquitous in plants. srRNA-348-25 fragments are injected into bodies of the pests, the death rate of the spodoptera litura eating plant leaves significantly increases. The srRNA-348-25 is plant small molecular ribonucleoprotein RNA and mostly exists in plants rich in protein, and the fragments are safe to people, can be used as a novel pesticide for safely and effectively controlling herbivorous pests, and can be used directly or used for producing transgenic plants overexpressing the srRNA-348-25.

Description

A kind of applications of plant small molecular ribonucleoprotein RNA in control of insect

Technical field

The present invention relates to application of plant small molecular ribonucleoprotein RNA (srRNA-348-25) in control of insect, tool Body is related to plant origin srRNA-348-25 affects this function of insect growth, and so as to surround srRNA-348-25 developments base is turned Cause or inhibitor research and development, reach the purpose of control of insect.

Background technology

Since 1993 find small molecules interference RNA, a large amount of all kinds of microRNAs are found, include miRNAs, SiRNAs, piRNAs and snRNAs etc..MiRNA is the noncoding microRNA of the nucleotides of a class length about 21～24, is extensively deposited In being animal and plant body.Series of experiments shows that miRNA is probably that a class is guarded in evolution, has weight in organism Want the molecule of regulating and controlling effect.MiRNA is derived from the genetic transcription of nucleus interior coding miRNA into pri-microRNA, then by Drosha enzymes are cut into pre-miRNAs of the length about 70bp in hairpin, subsequently by caryoplasm/cytoplasm albumen from nucleus Inside it is transported in kytoplasm, is grown into the miRNA double helix complexs of 18-26bp by the shearing of Dicer enzymes afterwards.Wherein one solution spiral shell Rotation, and with AGO protein combinations, and form asymmetrical RISC compounds.This compound is by miRNA therein and said target mrna 3 ' UTR complementary pairings combine, target mRNA or block its translation so as to degrade, realize negative regulation to target gene. There is lot of documents to report, miRNA is played at aspects such as gene expression, signal transduction, development of plants, opposing stress, prevention diseases Vital effect.In recent years a class is found to have similar from the microRNA (srRNAs) of ribonucleoprotein DNA The function of miRNA.For example from the microRNA high-flux sequence interpretation of result of the mankind and mouse, it is found that substantial amounts of srRNAs is Can with ribonucleoprotein DNA pairing, while co-immunoprecipitation experiment shows that these srRNA can be combined with AGO；Experiment is also Show that some srRNA may take part in relevant metabolic pathway (Haibin Wei, the Ben Zhou, Fang of regulation and control diabetes Zhang,Yanyang Tu,Yanan Hu,Baoguo Zhang,Qiwei Zhai.Profiling and Identification of Small rDNA-Derived RNAs and Their Potential Biological Functions.PLOS ONE.2013.8(2):e56842).But the research of srRNA report is few, more do not have in insect and plant Research report in terms of thing relation.

The content of the invention

The present invention is when the difference microRNA of leaf mustard of taking food to insect prodenia litura carries out sequencing analysis, it was found that table Up to the srRNA-348-25 that amount is significantly lowered：5’-GUCGGGAGGGAAGCGGAUGGGGGCC-3’(SEQ ID NO:1) and identify For the conserved sequence of generally existing in plant.Injection srRNA-348-25 fragments enter polypide, the twill night of plant leaf blade of taking food The moth death rate is dramatically increased.Because srRNA-348-25 is plant small molecular ribonucleoprotein RNA, more is present in rich in egg In white plant, such as soybean, especially in the soybean of heating, therefore, the fragment is safe to people.Can be used as safely and effectively The novel pesticide for preventing and treating phytophagous insect, can be used directly and genetically modified plants.

It is an object of the invention to provide applications of the plant srRNA-348-25 in insecticide is prepared.

The technical solution used in the present invention is：

A kind of applications of plant small molecular ribonucleoprotein RNA in insecticide is prepared, the small molecular core ribonucleoprotein RNA is srRNA-348-25, its base sequence such as SEQ ID NO：Shown in 1.

Further, the insecticide can kill phytophagous polypide, or the growth for suppressing phytophagous polypide.

Further, the phytophagous polypide is Lepidoptera plant-feed insect, Homoptera plant-feed insect.

Further, the Lepidoptera and Homoptera insect are prodenia litura, silkworm, planthopper.

A kind of insecticide, in the insecticide srRNA-348-25 is contained, its base sequence such as SEQ ID NO：Shown in 1.

A kind of method of insect pest of the plant preventing and treating, it is characterised in that：Make plant overexpression small molecular core ribonucleoprotein RNA SrRNA-348-25, srRNA-348-25 base sequence such as SEQ ID NO：Shown in 1.

The invention has the beneficial effects as follows：

Present invention discover that a kind of plant small molecular ribonucleoprotein RNA (srRNA-348-25) has to the growth of phytophagous insect Have an inhibitory action, or even the death of phytophagous insect can be promoted, due to srRNA-348-25 be in human consumption plant into Point, to human security, can be used as safely and effectively preventing and treating the novel pesticide of phytophagous insect, can be used directly and transgenosis is planted Thing；Therefore srRNA-348-25 genetically modified plants (i.e. plant overexpression srRNA-348-25) carry out control of insect will have really The using value on border.

Description of the drawings

Fig. 1 is to inject the death condition after srRNA-348-25 after Spodoptera litura larvae feed leaf mustard；NC mimic in figure As NC (nonsense segment) control group, sr-348-25 is the abbreviation of srRNA-348-25；

Fig. 2 is the growing state for injecting Spodoptera litura larvae after srRNA-348-25 72h；Sr-348-25 is in figure The abbreviation of srRNA-348-25；

Fig. 3 is expressions of the srRNA-348-25 in different plants；Food is manually trained for prodenia litura polypide in figure Foster base, it is heated to form；Bean powder, wheat bran are the material not heated in figure；Leaf mustard is its blade；

The relative amount detection of srRNA-348-25 in Fig. 4 different materials；

Fig. 5 is that prodenia litura is taken food the inspection of leaf mustard (B.juncea) and culture medium (CK) middle intestines srRNA-348-25 contents afterwards Survey；

Fig. 6 enters detoxication enzyme SlGSTe1 expression after Spodoptera litura larvae and lowers for injection srRNA-348-25, and NC is in figure The control group of injection nonsense sequence；

Fig. 7 is the comparison of the prodenia litura growth of culture medium of the feeding containing srRNA-348-25 and leaf mustard respectively；

Fig. 8 is the comparative analysis of srRNA-348-25 sequences in various plants genome；

Fig. 9 is the silkworm phenotype for injecting 2 μ g srRNA-348-25 and nonsense sequence (NC) respectively；

Figure 10 is the death rate that srRNA-348-25 increases planthopper.

Specific embodiment

With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.

The research process of the present invention is as described below：

In order to study the mechanism that omnivorousness agricultural pests prodenia litura adapts to plant, we are respectively to feed containing soybean and wheat Intestines in the synthetic medium of bran and the prodenia litura of mustard leaf, have carried out the sequencing of high flux microRNA and analysis, find The maximum microRNA of intestines differential expression in prodenia litura, further study show that the microRNA not with prodenia litura base Because of a group sequence pairing, but exist in Plant Genome, be one section of sequence of 26S rRNA, belong to small molecular core ribonucleoprotein RNA, is named as srRNA-348-25.

SrRNA-348-25 contents in the high culture medium of protein content are high, and content is very low in leaf mustard leaf；And it Content in heating culture medium illustrates that it is probably the catabolite of protein complexes higher than culture medium is not heated.Therefore On the feed srRNA-348-25 contents are extremely low in intestines in the prodenia litura after leaf mustard, but if increasing it in prodenia litura body Content, prodenia litura feed leaf mustard leaf can cause death.

The gene expression profile in middle intestines after analysis prodenia litura feed culture medium and leaf mustard, finds prodenia litura with albumen The high protein of hydrolase reply soybean, the plant secondary substance that toxicity is tackled with detoxication enzyme.The target base of analysis srRNA-348-25 Cause, it is found that it may suppress detoxifying gene, and not action protein hydrolase.Therefore, prodenia litura feed high-load srRNA- The culture medium of 348-25 is not dead, but the death rate is big after the leaf mustard of the feed of the prodenia litura containing high dose srRNA-348-25 in vivo Increase.So preventing and treating prodenia litura using srRNA-348-25.

Due to srRNA-348-25, content is extremely low in leaves of plants, then whether srRNA-348-25 can be used for preventing and treating Take food other plant-feed insectsWe inject respectively srRNA-348-25 into lepidopterous silkworm and the planthopper of Homoptera, The death rate increases after their feed plants.Because srRNA-348-25 is the composition in human consumption plant, to human security, because This srRNA-348-25 genetically modified plants carries out control of insect will actual using value.

Embodiment 1srRNA-348-25 suppresses growth and survival after prodenia litura feed leaf mustard

Experimental technique：

The consistent first day five age Spodoptera litura larvae of in the same size, health status is chosen, the weight of the son per cephalont is weighed, SrRNA-348-25 treatment groups and NC control groups (nonsense fragment) are randomly divided into, 40 per group, repeat experiment 3 times.Follow along blood Ring flow direction, with micro syringe between the abdominal foot of flank portion first and second of Spodoptera litura larvae, injects respectively SrRNA-348-25 and NC (nonsense fragment), injection volume is the μ g of every cephalont 4, feeding leaf mustard after injection.See within the 1st, 2,3 days after injection The metamorphosis of insect and evidence obtaining of taking pictures are examined, daily the death rate of statistics insect.

Above-mentioned srRNA-348-25 sequences are：5’-GUCGGGAGGGAAGCGGAUGGGGGCC-3’(SEQ ID NO：1).

Experimental result：

As depicted in figs. 1 and 2, Fig. 1 is Spodoptera litura larvae feed mustard after injection srRNA-348-25 to laboratory test results Death condition after dish；It can be seen that injection srRNA-348-25 Spodoptera litura larvae on the feed leaf mustard 24h, 48h and The death rate after 72h respectively up to 77.50%, 92.45% and 92.45%, significantly larger than 6.91%, the 8.69% of control group (NC) With 8.69%.Fig. 2 for injection srRNA-348-25 or nonsense fragment (NC) Spodoptera litura larvae life on the feed after leaf mustard 72h Long situation, it can be seen that the larva slight of stature survived in srRNA-348-25 treatment groups, and the larva of NC control groups is obvious It is loose many.

The above results explanation srRNA-348-25 suppresses growth and survival after prodenia litura feed leaf mustard.

The source of embodiment 2srRNA-348-25 and its impact to prodenia litura polypide

In order to study the mechanism that microRNA regulation and control omnivorousness agricultural pests prodenia litura adapts to plant, respectively to feed Intestines in the prodenia litura of synthetic medium and mustard leaf containing soybean and wheat bran, have carried out the sequencing of high flux microRNA With analysis, it was found that a maximum microRNA of intestines differential expression in prodenia litura, the small molecule is further study show that RNA is not matched with prodenia litura genome sequence, but is existed in Plant Genome, is one section of sequence of 26S rRNA, is belonged to Small molecular core ribonucleoprotein RNA, is named as srRNA-348-25.

First, expressions of the srRNA-348-25 in different plants

Experimental technique：

Respectively by prodenia litura polypide synthetic medium (100g containing analysis for soybean powder, wheat bran 80g, dusty yeast in every liter of culture medium 26g, casein 8g, vitamin C 8g, Choline Chloride 1g, sorbic acid 2g, cholesterol 0.2g, inositol 0.2g and agar powder 26g), Bean powder, wheat bran, mustard leaf press Trizol method extracted total RNAs, and 2 μ g total serum IgEs are taken after measure concentration carries out reverse transcription (reference The One Step PrimeScript miRNA cDNA Synthesis Kit specifications of TAKARA are carried out), the cDNA for obtaining is produced For the template of qRT-PCR after thing dilution, expression water of the further qRT-PCR detections srRNA-348-25 in each sample It is flat.Interpretation of result determines the relative of the relative internal standard gene (GAPDH, HQ012003) of target gene using the method for relative quantification Expression multiple.Using SPSS16.0 statistical analysis softwares, using ANOVA (comparing two-by-two between multiple process) or independent sample T Inspection (two sample rooms compare) is analyzed come the comparison in difference between being processed.

Experimental result：

Testing result as shown in figure 3, there it can be seen that the srRNA-348-25 contents in synthetic medium (food) most Height, the srRNA-348-25 contents in bean powder show that the high-load srRNA-348-25 in culture medium can also apparently higher than wheat bran Can mainly caused by bean powder；But the srRNA-348-25 contents in mustard leaf are very low.

Experimental technique：

In order to srRNA-348-25 contents may increase after further clear and definite bean powder heating, take respectively 0.1g bean powderes, 0.1g wheat bran, 0.1g press 1:The bean powder of 1 mixing and the mixture of wheat bran (are wherein respectively provided with not heating and add by culture medium Hot mode process after bean powder and wheat bran mixture, 0.1g rice leafs, 0.1g peanut leafs, 0.1g yeast, carry out with it is above-mentioned The operations such as identical RNA extractions, reverse transcription and PCR, 1 μ g RNA are respectively taken respectively carries out the PCR of reverse transcription and same volume.

Experimental result：

Testing result is as shown in figure 4, there it can be seen that the expression of the srRNA-348-25 in bean powder is far above in wheat In bran, peanut and rice leaf；SrRNA-348-25 expressions are not heated relatively in bean powder and wheat bran mixture after heating Obvious increase, show in bean powder albumen heating after, compared with polyribosome RNA generate microRNA.

The above results prompting srRNA-348-25 contents in plant leaf blade are very low；In the high culture medium of protein content Content is high, and the content in culture medium after the heating, higher than culture medium is not heated, further illustrating srRNA-348-25 can It can be the catabolite of protein complexes.

2nd, the relative amount of srRNA-348-25 is detected after prodenia litura feed leaf mustard

The prodenia litura in second day five age is divided into into 2 groups, per group of difference feeding mustard leaf (B.juncea) and manually training Foster base (CK), the 12h and 48h after feeding detects the content situation of srRNA-348-25 in intestines in prodenia litura.

Testing result as shown in figure 5, there it can be seen that either take food after 12h or 48h, prodenia litura feed leaf mustard Afterwards the content of its internal srRNA-348-25 being lacked compared with feed culture medium.Because srRNA-348-25 contents are far high in culture medium Content (Fig. 3) in leaf mustard leaf, thus this result show after prodenia litura feed in middle intestines the content of srRNA-348-25 with Its content of srRNA-348-25 in food is directly proportional.Because insect defines not in long-term evolution to different food With adaptation mechanism, prodenia litura take food respectively the culture medium containing different content srRNA-348-25 and/or leaf mustard piece when not Can be dead.But our research finds, as srRNA-348-25 contents (hormone srRNA- in increase prodenia litura body Take food again after 348-25) leaf mustard when, the prodenia litura suppressed and death rate of growth increases (see Fig. 1 and Fig. 2).

Embodiment 3srRNA-348-25 suppresses prodenia litura after feed leaf mustard to grow and promote its dead mechanism

First, the action target of srRNA-348-25

Suppress prodenia litura growth and promote its dead mechanism to further study srRNA-348-25, we Will prodenia litura take food culture medium after rotate into again food leaf mustard, then respectively rotate into food leaf mustard after 0,6,48h collection sample enter Row high-flux sequence, analyzes difference expression gene, as shown in Table 1 and Table 2.

As a result find to rotate into after food leaf mustard, many proteolysis expression of enzymes of intestines lower (table 1) in prodenia litura, and many The expression of the detoxication enzyme of reply leaf mustard secondary substance raises (table 2).And then will have in srRNA-348-25 and Tables 1 and 2 The target-gene sequence of differential expression carries out pair analysis, detects the target that srRNA-348-25 may be acted on, and as a result finds SrRNA-348-25 and the proteolysis enzyme gene with differential expression for detecting do not have and are combined with possibility and (are unworthy of completely It is right)；But with the possibility that the gene such as some detoxication enzymes detected in table 2 specifically has combination；Further investigation revealed that, SrRNA-348-25 may (this be one and ensures that prodenia litura tackles the weight of leaf mustard secondary substance with detoxifying gene Slgste1 Gene is wanted, it is hardly expressed when prodenia litura takes food culture medium, can be found in Xiaopeng Zou, Zhibin Xu, Haiwang Zou,Jisheng Liu,Qili Feng,Sichun Zheng.Glutathione S-transferase SlGSTE1in Spodoptera litura may be involved in feeding preference of host Plants.Insect Biochemistry and Molecular Biology, 2016,70:3 ' noncoding regions tool 32-43) There is combination.

The prodenia litura of table 1. rotates into the significant proteolysis enzyme gene number system of differential expression after food leaf mustard from feed culture medium Meter

The prodenia litura of table 2. rotates into the significant detoxifying gene number statistics of differential expression after food leaf mustard from feed culture medium

In order to further study whether srRNA-348-25 acts on detoxifying gene Slgste1, we are by srRNA- 348-25 injections detect the expression in transcriptional level of Slgste1 genes into the Spodoptera litura larvae in five ages；Detection knot Fruit is as shown in fig. 6, there it can be seen that inject after srRNA-348-25, the transcription of Slgste1 genes in Spodoptera litura larvae body Expression is substantially reduced, and illustrates that srRNA-348-25 suppresses Slgste1 gene expressions.

2nd, phenotype analytical

Experimental technique：

5 groups of experiments are set altogether, and each group disposition is as follows：

Control group (simple water)：Give prodenia litura simple fed with water, such as Fig. 7 A；

Experimental group (simple water srRNA)：Give prodenia litura simple feeding srRNA-348-25, compare with water, it was demonstrated that Whether srRNA-348-25 is non-toxic, such as Fig. 7 A；

Control group (culture medium)：Make prodenia litura feed synthetic medium, such as Fig. 7 B；

Experimental group (2 μ g srRNA+ culture mediums)：Make prodenia litura artificial culture of the feed containing 2 μ g srRNA-348-25 Base, such as Fig. 7 B and Fig. 7 C；

Experimental group (2 μ g srRNA+ leaf mustard)：Make prodenia litura leaf mustard of the feed containing 2 μ g srRNA-348-25, such as Fig. 7 C；

According to above-mentioned process, growth and the death condition of each group Spodoptera litura larvae are observed respectively.

Observation result is as shown in fig. 7, as can be seen that 2 simple μ g srRNA-348-25 do not affect twill night from Fig. 7 A Growing for moth, illustrates that srRNA-348-25 may not act on any gene related to growth；Can be seen that from Fig. 7 B Compared with the prodenia litura of feed media alone, growth is simultaneously for the prodenia litura of culture medium of the feed containing 2 μ g srRNA-348-25 It is not suppressed, shows that srRNA-348-25 does not regulate and control the table of the gene related to medium component such as proteolytic enzyme yet Reach；As can be seen that the prodenia litura of culture medium of the feed containing 2 μ g srRNA-348-25 and leaf mustard, the growth of the latter from Fig. 7 C The former is significantly less than, illustrates that eating leaf mustard again when polypide is enriched with 2 μ g srRNA-348-25 can suppress the growth of prodenia litura, The death of polypide, mechanism of action therein is promoted to be likely due to srRNA-348-25 and inhibit the detoxication enzyme base such as Slgste1 Because of the expression of (polypide decomposes enzyme necessary to poisonous plants secondary substance), making the toxicity of leaf mustard increases, and ultimately results in polypide Feed and growth retardation, so as to promote the death of polypide.

The comparative analysis of srRNA-348-25 sequences in the different Plant Genomes of embodiment 4

Because srRNA-348-25 sequences cannot find the sequence of pairing in prodenia litura genome, therefore we distinguish Have chosen several Plant Genomes and compare analysis, include soybean (GenBank sequences number：Dbj | LB172039.1), on Sheep's hay (GenBank sequences number：gb|AOCO010307372.1)；Arabidopsis (gene group #：TAIRIO).Comparison result such as Fig. 8 Shown, srRNA-348-25 is guarded very much in plant, is one section of sequence of 26s rRNA, this explains why it is in high egg Content is higher in white soybean, and content is all low in the blade of all kinds of plants.Therefore, transgenosis srRNA-348- in theory 25, increase the blade of all kinds of plants its content, being likely to arrive causes to take food bad anti-of insect generation of such plant Should.

Embodiment 5srRNA-348-25 suppresses the growth of silkworm

Due to srRNA-348-25, content is extremely low in leaves of plants, then whether srRNA-348-25 can be used for preventing and treating Take food other plant-feed insectsThe present embodiment has carried out corresponding research to lepidopterous silkworm.

After having injected whether also the silkworm in 3 days 5 ages, 2 μ g srRNA-348-25 of injection and nonsense control sequence are taken respectively, Will be taken food mulberry leaf to silkworm, observe the growing state of silkworm.

Observation result is as shown in figure 9, there it can be seen that inject 72 days after srRNA-348-25, the growing state of silkworm Substantially it is suppressed, growth inhibition ratio is 100%.

Embodiment 6srRNA-348-25 promotes the death of planthopper

Due to srRNA-348-25, content is extremely low in leaves of plants, then whether srRNA-348-25 can be used for preventing and treating Take food other plant-feed insectsThe present embodiment injects respectively 0.2 μ g srRNA- to the planthopper of the primary pest Homoptera of paddy rice 348-25 and nonsense sequence (control group), volume injected is 0.2 μ l, and whether will also take food Rice Leaf after having injected to planthopper (planthopper is put in paddy rice after having injected), observes the growing state of planthopper.

Observation result is as shown in Figure 10, there it can be seen that 2 days after injection srRNA-348-25, srRNA-348-25's The death rate is 86%, and the death rate of control group is only 33%.

The above results explanation increases the content of srRNA-348-25 in planthopper body, can also promote planthopper feed Rice Leaf The death rate afterwards, further proves that srRNA-348-25 can promote the death rate of the insect of phytophagous.

Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment Limit, other any Spirit Essences without departing from the present invention and the change, modification, replacement made under principle, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

SEQUENCE LISTING

<110>South China Normal University

<120>A kind of applications of plant small molecular ribonucleoprotein RNA in control of insect

<130>

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 25

<212> RNA

<213>Artificial sequence

<400> 1

gucgggaggg aagcggaugg gggcc 25

Claims (6)

1. applications of a kind of plant small molecular ribonucleoprotein RNA in insecticide is prepared, the small molecular core ribonucleoprotein RNA For srRNA-348-25, its base sequence such as SEQ ID NO：Shown in 1.

2. application according to claim 1, it is characterised in that：The insecticide can kill phytophagous polypide, or suppress The growth of phytophagous polypide.

3. application according to claim 2, it is characterised in that：The phytophagous polypide is Lepidoptera plant-feed insect, same Wing mesh plant-feed insect.

4. application according to claim 3, it is characterised in that：The Lepidoptera and Homoptera insect be prodenia litura, silkworm, Planthopper.

5. a kind of insecticide, it is characterised in that：Contain srRNA-348-25 in the insecticide, its base sequence such as SEQ ID NO： Shown in 1.

6. a kind of method that insect pest of the plant is prevented and treated, it is characterised in that：Make plant overexpression small molecular core ribonucleoprotein RNA SrRNA-348-25, srRNA-348-25 base sequence such as SEQ ID NO：Shown in 1.