CN114316015A

CN114316015A - Insect-resistant protein hRI and coding gene and application thereof

Info

Publication number: CN114316015A
Application number: CN202111469961.3A
Authority: CN
Inventors: 李秀敏; 李继刚; 刘智
Original assignee: Hebei University
Current assignee: Hebei University
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2022-04-12

Abstract

The invention provides an insect-resistant protein hRI, and a coding gene and application thereof, wherein the amino acid sequence of the insect-resistant protein hRI is shown as a sequence 1 in a sequence table. The gene sequence is shown as a sequence 2 in a sequence table. The application of the insect-resistant protein hRI and the gene in improving the insect resistance of plants. hRI is applied to the insect-resistant research of plants for the first time, and the insect-resistant effect is good, and the inhibition rate (calculated according to the weight of the pests) of the transgenic insect-resistant plants on the growth of the pests is more than 60 percent; transgenic insect-resistant plants were subjected to very mild insect infestation compared to controls.

Description

Insect-resistant protein hRI and coding gene and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an insect-resistant protein hRI, and a coding gene and application thereof.

Background

Insect pests cause the loss of the yield and quality of crops, and seriously affect the stability and sustainable development of agricultural production. The chemical insecticide is adopted to control insect pests, so that the serious problems of pesticide residue, pest resistance evolution, natural enemy killing, environmental pollution and the like are brought while crops are protected. The transgenic insect-resistant crops developed by utilizing the transgenic technology have the characteristics of high efficiency, low cost, environmental protection and the like.

At present, the insect-resistant genes expressed on transgenic insect-resistant plants in agricultural production mainly comprise delta-endotoxin (Bt toxic protein) from bacillus thuringiensis, protease/amylase inhibitor (such as cowpea protease inhibitor CpTI and alpha-amylase inhibitor), plant-mediated RNAi and the like.

In the field of plant pest-resistant gene engineering, the types of pest-resistant genes available in the current production are very limited. The long-term use of the insect-resistant genes inevitably causes the resistance evolution of pests, so that the insect resistance of transgenic plants is reduced or lost, and the insect damage threat is confronted again by using agricultural production. With the popularization of the transgenic insect-resistant plants and the extension of the planting years, the problem of resistance evolution of pests is more and more prominent, and a new insect-resistant gene or means is urgently required to be searched.

Disclosure of Invention

The invention aims to provide an insect-resistant protein hRI, and a coding gene and application thereof, so as to solve the problems of single excellent insect-resistant gene and insect resistance evolution of the existing transgenic plant, and thus provide a new insect-resistant gene resource for plant insect-resistant genetic engineering.

The purpose of the invention is realized by the following technical scheme: an insect-resistant protein hRI, wherein the amino acid sequence of the insect-resistant protein hRI is shown as a sequence 1 in a sequence table.

The gene of the insect-resistant protein hRI is coded and has a sequence shown as a sequence 2 in a sequence table.

A recombinant vector containing the gene fragment. The recombinant vector is constructed by the following method: the gene fragment shown in the sequence 2 in the sequence table is connected to the pBIN438 plasmid, thereby constituting the plant expression vector pBIN 438-hRI.

The recombinant strain containing the recombinant vector is agrobacterium tumefaciens.

The application of the insect-resistant protein hRI and the gene in improving the insect resistance of plants.

The gene fragment is introduced into a target plant to obtain a transgenic plant, and the insect-resistant protein hRI is expressed in the transgenic plant, so that the insect resistance of the transgenic plant is higher than that of the target plant.

The above gene fragment is introduced into a target plant by the above recombinant vector.

The insect resistance refers to cotton bollworm resistance.

The target plant is upland cotton or tobacco.

The insect-resistant protein hRI used in the invention is a Human ribonucleic acid inhibitor (hRI) which is related to the degradation inhibition of RNA. hRI binds to RNase1, bovine RNase A and other ribonucleases very strongly (dissociation constant 3.5X 10)^-14M-4.5×10^-14M). hRI is applied to the insect-resistant research of plants for the first time, and the insect-resistant effect is good, and the inhibition rate (calculated according to the weight of the pests) of the transgenic insect-resistant plants on the growth of the pests is more than 60 percent; transgenic insect-resistant plants are very slightly damaged by pests compared to controls.

Degrading enzyme inhibitor genes reported to be used for plant insect-resistant research are all directed to starch and protein; the technology of the invention is based on long-term laboratory verification and analysis, has solid experimental evidence and better application value, can be used as a reserve resource of the existing insect-resistant gene, and is used for plant insect-resistant gene engineering.

Drawings

FIG. 1 shows the inhibitory activity of recombinant hRI on the activity of midgut RNase in Heliothis armigera. (a) hRI protective effect on dsRNA1 (b) protective effect of hRI on dsRNA 2; m: DNA Marker; 1: dsRNA untreated group; 2: dsRNA + hRI untreated group; 3-6: different amounts of hRI experimental groups; 7: RRI (commercial recombinant RNA inhibitor, Takara, 2313Q) control group; 8: EGFP control group.

FIG. 2 is a schematic diagram of the vector construction of pBIN 438-hRI.

FIG. 3 PCR identification of genomic DNA of transgenic hRI tobacco plants. The number designation is transgenic plant, WT is wild type plant.

FIG. 4 RT-PCR identification of hRI transgenic tobacco plants. The number designation is transgenic plant, WT is wild type plant.

FIG. 524 h shows biting of the tobacco lamina. hRI-2, hRI-11, hRI-12 are three tobacco plants expressing hRI, WT is a non-transgenic wild type control.

FIG. 6 shows weight changes of Helicoverpa armigera with tobacco leaf taken as feed hRI.

Detailed Description

The technical solution of the present invention will be described in detail with reference to specific examples. The test conditions and operations not mentioned in the examples of the present invention are performed according to the conventional methods in the art, and those skilled in the art can refer to the related art books, such as: guillain, SammBruk et al, molecular cloning, Experimental Manual, 4 th edition (2017), et al.

Example 1

Activity analysis of RNase inhibitor hRI protein

Adopting RT-PCR technology to amplify human lung tissue cDNA to obtain hRI coding gene sequence, wherein the primer sequence is:

Hrif：5'-CACTCTTCACCTCCACCA-3'

hrir：5'-CTGAGCGTTTCTCTTCAAACC-3'

PCR reactivity program: pre-denaturation (95 ℃, 3min), 35 amplifications (95 ℃ 10 s; 52 ℃ annealing 30s, 72 ℃ extension 90s), and re-extension (72 ℃, 10 min). Gel electrophoresis, after the gel recovery of the target band, cloning to pUCm-T vector by enzyme linkage, and verifying through transformation, screening and sequencing test.

Preparing a prokaryotic expression vector pET17b-TC capable of carrying out TC directional cloning by using a TC cloning method (refer to Chinese invention patent: TC vector for gene directional cloning and preparation and use methods thereof, patent number CN 201210436709.7); the plasmid vector fragment is recovered by XcmI digestion. Using primers:

HRPepf:5'-ATGAGCCTGGACATCCAGAGCCTGGACATCCAGTGTG-3'

HRPepr:5'-TCAGGAGATGACCCTCAGGGATGGCTTGTCCTTCTCCA-3'

the hRI gene sequence on pUCm-T was amplified, recovered by gel electrophoresis, and ligated to pET-17b-TC large fragment. The hRI gene prokaryotic expression plasmid is obtained after transformation, screening and identification, and then BL21(DE3) pLysS escherichia coli competence is introduced.

hRI protein isolation and purification were performed using His-Tag technology: inducing hRI protein expression by IPTG, and detecting hRI protein expression product by SDS-PAGE; resuspending the induced bacteria with 10mL of Tris-HCl lysate (containing lysozyme), standing at 4 ℃ for 30min to lyse the bacteria, and then carrying out ultrasonication; the cleaved samples were subjected to imidazole washing and Ni column elution according to the High Affinity Ni-NTA Resin instructions, and the resulting purified recombinant hRI protein was finally examined by SDS-PAGE.

Inhibition assay of recombinant hRI protein on intestinal juice ribonuclease activity in Helicoverpa armigera: by taking a commercial RNA inhibitor as a control, green fluorescent protein (EGFP) or His-hRI with different amounts is respectively mixed with 0.5 mu L of midgut digestive juice of cotton bollworm and buffer solution, protein eluent is supplemented to 7 mu L, after 30min of metal bath at 37 ℃, 60ng of dsRNA is respectively added and rapidly mixed, 1h of metal bath at 37 ℃ and 5min of water bath at 85 ℃. RNA electrophoresis was performed on a 1% agarose gel prepared with DEPC water to examine the degradation of dsRNA. The results are shown in FIG. 1.

As shown in FIG. 1, under the same experimental conditions, the results of the negative control group (lane 1) showed that the buffer used in the experiment had no degradation effect on dsRNA; the dsRNA of the experimental group (lane 8) without hRI is degraded more completely, which reflects that the midgut enzyme of cotton bollworm has stronger destructive effect on feeding dsRNA under natural condition; partial degradation of the dsRNA occurred with the addition of the commercial inhibitor RRI (lane 7); in the experimental groups (

lanes

3, 4, 5 and 6) with different hRI dosages, the degradation degree of dsRNA is decreased with the increase of the dosage (0, 0.25, 0.5 and 1 muL) of hRI, and the activity of intestinal enzyme in cotton bollworm is obviously inhibited by the recombinant hRI protein, which shows that hRI has obvious degradation inhibition effect on dsRNA. The results show that the prokaryotic expression of hRI has obvious effect in inhibiting degradation of dsRNA by mesenteric enzymes.

Example 2

Obtaining of transgenic tobacco with ribonuclease gene

Construction of plant expression vectors: designing specific primers of hripBf and hripSr, wherein the hripBf introduces a Kozak sequence and a BamH I enzyme cutting site, and the hripSr introduces a Sal I enzyme cutting site:

hripBf:5'-CGCGGATCCAACAATGGCTAGCCTGGACATCCAGA-3'

hripSr:5'-GCAGGTCGACCCTCAGGAGATGAC-3'

hRI gene was PCR amplified using the primers and recovered by gel electrophoresis. The pBIN438-X plasmid and the recovered fragment were each double-digested with restriction enzymes BamH I and Sal I. The digestion system (50. mu.L) was: restriction enzymes are respectively 0.8 mu L and 4 mu L of corresponding endonuclease Buffer, 34.3 mu L of plasmid is cut by enzyme, the plasmid is cut by enzyme overnight at 37 ℃, a carrier framework and a target segment are recovered by electrophoresis, and the concentration of the recovered product is estimated. Enzyme-linked, transformed, screened and identified according to the molar ratio of the vector fragment to the target fragment of 1:3-1: 10. The PCR identification primer is hripBf/Sr, and the restriction enzymes used for enzyme digestion identification are EcoR I and Hind III. The structure of the constructed plant expression vector pBIN438X-hRI is shown in figure 2.

Obtaining sterile seedlings and genetically transforming: selecting plump wild tobacco seeds to remove impurities, sequentially sterilizing with alcohol and NaClO solution, and uniformly planting in MS solid culture medium (Murashige and Skoog solid culture medium); placing the plant in a plant growth box for culturing under the following conditions: the temperature is 28 +/-1 ℃, the relative humidity is 65 +/-5%, and the photoperiod is 16L: 8D. Under aseptic conditions, tobacco seedlings of appropriate size were transferred to tissue culture flasks containing MS solid medium. And when the height of the aseptic seedling reaches about 10cm, taking the leaf to perform agrobacterium-mediated leaf disc transformation. The method comprises the following steps: (1) pre-culturing leaves: shearing well-grown sterile wild tobacco leaves into squares (about 1cm multiplied by 1cm in size), uniformly paving the leaves on an MS solid culture medium containing no antibiotics with forceps in a manner that the back faces the culture medium, and culturing for 2d in an artificial climate box; (2) and (3) agrobacterium tumefaciens expanding culture: agrobacterium containing recombinant plasmid pBIN438X-hRI is taken from an ultralow temperature refrigerator at minus 80 ℃, activated at 220rpm/min at 28 ℃ for 24 hours and then transferred into 150mL LB liquid medium for amplification culture until logarithmic phase; (3) concentrating bacterial liquid: centrifuging the thalli in the growth period of the mobile phone at 4 ℃, washing the thalli for 2 times by using an MS liquid culture medium, and then suspending the thalli in 100mL of MS liquid culture medium; (4) infection: gently clamping the pre-cultured leaf into MS liquid heavy suspension, shaking for infection for 15min, placing the leaf on sterilized filter paper, sucking off excessive liquid, clamping the leaf with the back facing downwards, placing into a plate containing co-culture medium, and wrapping the plate with newspaper for dark culture for 2 d; (5) cleaning leaves and selectively culturing; sterile ddH is used first₂O washing the leaves for 3 times, finally washing the leaves for 1 time by using an MS liquid culture medium,placing the leaf with the back face facing downwards on a selective differentiation culture medium for selective culture; (6) and (4) continuing culturing: cutting off the differentiated bud, inserting the bud into a tissue culture bottle for rooting culture, finally, when the transgenic tobacco grows to about 10cm, lightly taking out the regenerated plant from the tissue culture bottle by using long tweezers, washing the root by using flowing cold water, removing the residual tissue culture medium, and then transferring the regenerated plant into soil.

Molecular identification of transgenic tobacco: extracting DNA of tobacco leaves by a CTAB method, amplifying the DNA by using a specific identification primer, and detecting hRI whether the DNA is integrated into a tobacco genome, wherein the primer sequence is as follows:

RNH1igF:5'-CAGCAGTGCCAAGTGGTCAG-3'

RNH1igR:5-'CAATGCCGCACAGGTCCC-3'

identification of transcription levels: extracting total RNA of fresh tobacco leaves by using TRIzol reagent, carrying out reverse transcription, and using wild type tobacco total RNA as a control. The primers are used for RT-PCR amplification to identify whether the target gene is transcribed. The identification process needs to be independently extracted at least twice, and PCR or RT-PCR identification is carried out twice each time to ensure the accuracy of the result. Transgenic plants were characterized at the genomic level (FIG. 3) and at the transcriptional level (FIG. 4).

Example 3

Insect resistance analysis of transgenic tobacco

Cutting the artificial feed into small pieces of 0.5cm × 0.5cm, placing in a sterile culture dish, inoculating the bollworm egg larva to the feed, and culturing to 2-year-old (body length of 0.42cm-0.62cm) for insect test analysis. Selecting 3 plants from T1 generation single copy transgenic plant line, selecting 3 tobacco leaves with consistent growth vigor for feeding bollworm larvae, and selecting leaves with consistent growth vigor from wild tobacco planted at the same period for feeding the bollworm as a control. 3 single copies of tobacco of the same gene were set as an inter-group repeat to reduce errors due to different gene insertion sites; 3 leaves of the same tobacco plant are set to be subjected to repeated tests in a group, so that human errors caused by leaf selection are reduced; each group of bollworm was weighed 3 times to reduce the occasional error in weight measurement, and the average was taken during subsequent data processing. 16 cotton bollworm larvae with the same growth vigor and weight are selected from the cotton bollworms of 2 instars and put into a locker box with blades. Weighing the weight of the cotton bollworms every 24 hours, counting the residual quantity of the cotton bollworms, and counting the data to 120 hours. And observing the biting condition of the leaves at 24h and taking pictures for recording. During the period of worm test, the lockbox is cleaned and the fresh tobacco leaf is replaced every 24 h. And finally, calculating the mean weight of the cotton bollworms according to the statistical data, and comparing the difference between the mean weights of the cotton bollworms in different groups through single-factor variance analysis by using IBM SPSS Statistics 20 software. And drawing a mean body weight line graph of the cotton bollworms according to the statistical analysis result by using Excel 2016 software.

The biting condition of the tobacco leaves for 24h was recorded by photographing, and the result is shown in the figure (fig. 5). The results show that the transgenic hRI experimental group and the control group (wild type WT) all have different degrees of bite damage after being fed with the second-instar cotton bollworm larvae for 24h, wherein the bite condition of wild type tobacco leaves is more serious, and the bite condition of the transgenic hRI tobacco leaves is less. The insect resistance of the hRI transgenic tobacco is obviously improved.

The mean weight gain of the bollworms in five time periods of 24h, 48h, 72h, 96h and 120h in the wild-type tobacco and the transformed hRI tobacco was counted (Table 1) and a line graph of the mean weight gain of the bollworms was plotted (FIG. 6).

Table 1: mean body weight (g) of bollworm in different time periods with feeding rotor hRI

Note: different lowercase letters in the same column indicate that there is a significant difference in the level P < 0.05; different capital letters in the same column indicate that the difference is extremely significant in the P < 0.01 level difference.

The weight growth trend shows that: in the same time period, the feeding time of the tobacco group of the transgenic hRI tobacco is obviously lower than that of the wild type tobacco, the feeding time of the tobacco group of the transgenic hRI tobacco is obviously different from that of the wild type tobacco at 48h, the difference reaches an extremely obvious level at the beginning of 72h, and the result shows that the growth of the cotton bollworm larvae of the transgenic hRI tobacco has obvious inhibiting effect. There was no significant difference between the mean body weights of the 3 groups of Helicoverpa armigera fed transgenic hRI tobacco, indicating that the insect-inhibitory ability of transgenic hRI tobacco was not affected by the gene insertion site in the insect test analysis.

Sequence listing

<110> university of Hebei

<120> insect-resistant protein hRI, and coding gene and application thereof

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cagaccccct cctgcaagat ccagaagctg agcctccaga actgctgcct gacgggggcc 300

ggctgcgggg tcctgtccag cacactacgc accctgccca ccctgcagga gctgcacctc 360

agcgacaacc tcttggggga tgcgggcctg cagctgctct gcgaaggact cctggacccc 420

cagtgccgcc tggaaaagct gcagctggag tattgcagcc tctcggctgc cagctgcgag 480

cccctggcct ccgtgctcag ggccaagccg gacttcaagg agctcacggt tagcaacaac 540

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aggtttctcc tggagctaca gataagcaac aacaggctgg aggatgcggg cgtgcgggag 1080

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gatgtgagtg acagcagctg cagcagcctc gccgcaaccc tgttggccaa ccacagcctg 1200

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tcctga 1386

Claims

1. An insect-resistant protein hRI, which is characterized in that the amino acid sequence of the insect-resistant protein hRI is shown as the sequence 1 in the sequence table.

2. The gene for coding the insect-resistant protein hRI of claim 1, wherein the gene sequence is shown as a sequence 2 in a sequence table.

3. A recombinant vector comprising the gene fragment of claim 2.

4. The recombinant vector according to claim 3, wherein the recombinant vector is constructed by: the gene fragment shown in the sequence 2 in the sequence table is connected to the pBIN438 plasmid, thereby constituting the plant expression vector pBIN 438-hRI.

5. A recombinant strain comprising the recombinant vector of claim 2 or 3, wherein the recombinant strain is agrobacterium tumefaciens.

6. Use of the insect-resistant protein hRI of claim 1 and the gene of claim 2 to improve insect resistance in a plant.

7. The use of claim 6, wherein the gene fragment of claim 2 is introduced into a plant of interest to obtain a transgenic plant, and wherein the insect-resistant protein hRI is expressed in the transgenic plant, thereby rendering the transgenic plant more insect-resistant than the plant of interest.

8. The use according to claim 7, wherein the gene fragment of claim 2 is introduced into a plant of interest by the recombinant vector of claim 3 or 4.

9. Use according to claim 7 or 8, wherein said insect resistance is against cotton bollworm.

10. The use according to claim 7 or 8, wherein the plant of interest is upland cotton or tobacco.