CN114316015B

CN114316015B - Insect-resistant protein hRI, and encoding gene and application thereof

Info

Publication number: CN114316015B
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: 2024-06-14
Anticipated expiration: 2041-12-03
Also published as: CN114316015A

Abstract

The invention provides an insect-resistant protein hRI, a coding gene and application thereof, and an 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. The invention applies hRI to plant insect resistance research for the first time, and obtains good insect resistance effect, and the inhibition rate (calculated according to the weight of the insect) of the transgenic insect-resistant plant to the growth of the insect is more than 60%; transgenic insect-resistant plants were very lightly infested as compared to the control.

Description

Insect-resistant protein hRI, and encoding 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

The insect damage causes the yield and quality loss of crops, seriously affects the stable and sustainable development of agricultural production. The chemical pesticide is used to control insect pest, so that the crop is protected, and serious problems such as pesticide residue, pest resistance evolution, natural enemy killing, environmental pollution and the like are brought. The transgenic insect-resistant crop developed by using the transgenic technology has the characteristics of high efficiency, low cost, environmental protection and the like.

The insect-resistant genes expressed on transgenic insect-resistant plants in agricultural production are mainly delta-endotoxin (Bt toxic protein) from Bacillus thuringiensis, protease/amylase inhibition (e.g. cowpea protease inhibitor CpTI, alpha-amylase inhibitor), plant-mediated RNAi, etc.

In the field of plant insect-resistant genetic engineering, the available insect-resistant gene types in production are very limited at present. 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 agricultural production is re-facing the insect damage threat. With the popularization of the transgenic insect-resistant plants and the extension of the planting period, the problem of pest resistance evolution is more and more prominent, and a new insect-resistant gene or means is required to be sought.

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 plants, thereby providing a new insect-resistant gene resource for plant insect-resistant genetic engineering.

The invention aims at realizing 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 for coding the insect-resistant protein hRI has a sequence shown as a sequence 2 in a sequence table.

Recombinant vectors containing the above gene fragments. The recombinant vector is constructed by the following method: the gene fragment shown in the sequence 2 in the sequence table is connected to a pBIN438 plasmid, so as to form a plant expression vector pBIN438-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-resistant property 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 means resistance to cotton bollworms.

The target plant is upland cotton or tobacco.

The insect-resistant protein hRI used in the invention is a human ribonucleic acid inhibitor (Human ribonuclease inhibitor, hRI) and is related to degradation inhibition of RNA. hRI binds very strongly to ribonucleases such as RNase1 and bovine RNase A (dissociation constant 3.5X10 ^-14M-4.5×10^-14 M). The invention applies hRI to plant insect resistance research for the first time, and obtains good insect resistance effect, and the inhibition rate (calculated according to the weight of the insect) of the transgenic insect-resistant plant to the growth of the insect is more than 60%; the damage of the transgenic insect-resistant plants was very slight compared to the control.

Degradation enzyme inhibitor genes for plant insect resistance research are reported to be aimed at starch and protein at present; the technology of the invention is based on long-term laboratory verification and analysis, has firm experimental evidence and better application value, can be used as reserve resources of the existing insect-resistant genes, and is used for plant insect-resistant genetic engineering.

Drawings

FIG. 1 inhibitory Activity of recombinant hRI on intestinal RNase Activity in Helicoverpa armigera. (a) hRI protective effect on dsRNA1 (b) protective effect of hRI on dsRNA 2; m: DNA MARKER;1: untreated dsRNA group; 2: dsRNA + hRI untreated group; 3-6: different amounts hRI experimental groups; 7: RRI (commercial recombinant RNA inhibitor, takara, 2313Q) control group; 8: EGFP control group.

FIG. 2pBIN438-hRI vector construction schematic.

FIG. 3 PCR identification of genomic DNA from a hRI transgenic tobacco plant. The number is transgenic plants and WT is wild type plants.

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

Figure 5 24h biting of tobacco leaves. hRI-2, hRI-11, hRI-12 are three tobacco plants expressing hRI and WT is a non-transgenic wild type control.

Fig. 6 feeding rotation hRI tobacco leaf bollworm weight change.

Detailed Description

The technical scheme of the invention is described in detail below with reference to specific embodiments. Test conditions and operations not mentioned in the examples of the present invention are carried out according to conventional methods in the art, and those skilled in the art can refer to related art books, for example: greens, sambrook et al, 4 th edition (2017) of the guidance on molecular cloning experiments, et al.

Example 1

Activity assay of ribonuclease inhibitor hRI protein

The coding gene sequence hRI is obtained by amplifying cDNA of human lung tissue by adopting RT-PCR technology, and the used primer sequence is as follows:

Hrif：5'-CACTCTTCACCTCCACCA-3'

hrir：5'-CTGAGCGTTTCTCTTCAAACC-3'

PCR reactivity procedure: pre-denaturation (95 ℃,3 min), 35 amplifications (95 ℃,10 s;52 ℃, 30s annealing, 72 ℃, 90s extension), and re-extension (72 ℃,10 min). After gel electrophoresis and recovery of target band gel, the target band gel is cloned to pUCm-T vector through enzyme linkage, and the target band gel is verified through transformation, screening and sequencing.

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

HRPepf:5'-ATGAGCCTGGACATCCAGAGCCTGGACATCCAGTGTG-3'

HRPepr:5'-TCAGGAGATGACCCTCAGGGATGGCTTGTCCTTCTCCA-3'

The hRI gene sequence on pUCm-T was amplified and recovered by gel electrophoresis and ligated with the pET-17b-TC large fragment. The hRI gene prokaryotic expression plasmid is obtained after transformation, screening and identification, and then BL21 (DE 3) pLysS escherichia coli competence is introduced.

HRI protein isolation and purification were performed using His-Tag technology: inducing hRI protein expression by IPTG, and checking hRI protein expression products by SDS-PAGE; the induced cells were resuspended in 10mL Tris-HCl lysate (containing lysozyme), allowed to stand at 4℃for 30min to lyse the cells, followed by sonication; the cleaved samples were subjected to imidazole washing and Ni column elution according to HIGH AFFINITY NI-NTA Resin instructions, and finally the resulting purified recombinant hRI protein was checked by SDS-PAGE.

Inhibition assay of the intestinal ribonuclease activity of the recombinant hRI protein in bollworm: taking commercial RNA inhibitor as a control, mixing green fluorescent protein (EGFP) or His-hRI with different amounts of 0.5 mu L of cotton bollworm midgut digestive juice and buffer solution respectively, supplementing 7 mu L with protein eluent, after 30min of 37 ℃ metal bath, respectively adding 60ng dsRNA, quickly mixing, 1h of 37 ℃ metal bath and 5min of 85 ℃ water bath. RNA electrophoresis was performed on 1% agarose gel with DEPC water to examine 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) show that the buffer used in the experiment has no degradation effect on dsRNA; the dsRNA of the experimental group (lane 8) without hRI is degraded more thoroughly, which reflects that the intestinal enzyme of the cotton bollworm has stronger damage to feeding dsRNA under natural conditions; the dsRNA was partially degraded by 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 hRI dosages (0, 0.25, 0.5 and 1 mu L), and the intestinal enzyme activity of the cotton bollworms is obviously inhibited by the recombinant hRI protein, which shows that hRI has obvious degradation inhibition effect on dsRNA. The results show that hRI of prokaryotic expression has obvious effect in inhibiting degradation of dsRNA by intestinal fluid enzymes.

Example 2

Acquisition of ribonuclease Gene transgenic tobacco

Construction of a plant expression vector: specific primers hripBf and hripSr were designed, wherein hripBf introduced a Kozak sequence and BamH I cleavage site, hripSr introduced a Sal I cleavage site:

hripBf:5'-CGCGGATCCAACAATGGCTAGCCTGGACATCCAGA-3'

hripSr:5'-GCAGGTCGACCCTCAGGAGATGAC-3'

The hRI gene was amplified by PCR using the primer and recovered by gel electrophoresis. The pBIN438-X plasmid and the recovered fragment were double digested respectively with the restriction enzymes BamHI and SalI. The cleavage system (50. Mu.L) was: restriction enzymes 0.8. Mu.L each, corresponding endonuclease Buffer 4. Mu.L, plasmid 34.3. Mu.L to be digested, overnight digestion at 37℃were used, and the vector backbone and target fragment were recovered by electrophoresis, and the concentration of the recovered product was estimated. And (3) carrying out enzyme ligation, transformation and screening identification according to the proportion of the carrier fragment to the target fragment of 1:3-1:10. The PCR identification primer is hripBf/Sr, and restriction enzymes used for enzyme digestion identification are EcoR I and Hind III. The structure of the plant expression vector pBIN438X-hRI is shown in FIG. 2.

Acquisition of sterile seedlings and genetic transformation: selecting full wild tobacco seeds, removing impurities, sequentially sterilizing with alcohol and NaClO solution, and uniformly planting in MS solid culture medium (Murashige and Skoog solid culture medium); culturing in a plant growth box under the following conditions: the temperature is 28+/-1 ℃, the relative humidity is 65+/-5%, and the photoperiod is 16L:8D. Under aseptic conditions, suitably sized tobacco seedlings were transferred to tissue culture flasks containing MS solid medium. When the height of the aseptic seedling reaches about 10cm, taking the leaves for agrobacterium-mediated leaf disk transformation. The method comprises the following steps: (1) leaf preculture: cutting sterile wild tobacco leaves with good growth vigor into cubes (the size is about 1cm multiplied by 1 cm), uniformly spreading the leaves on MS solid culture medium containing antibiotic-free according to the back surface direction by forceps, and culturing in a climatic chamber for 2d; (2) agrobacterium expansion culture: the agrobacterium containing the recombinant plasmid pBIN438X-hRI is taken from an ultralow temperature refrigerator at the temperature of minus 80 ℃ and is transferred into 150mL LB liquid culture medium for expansion culture after being activated for 24 hours at the temperature of 28 ℃ at 220rpm/min, and the culture is carried out until the logarithmic phase; (3) bacterial liquid concentration: centrifuging the cell in the growth period of the mobile phone at 4 ℃ and 4000rpm, washing the cell for 2 times by using an MS liquid culture medium, and then re-suspending the cell in 100mL of the MS liquid culture medium; (4) infestation: lightly clamping the precultured leaf into MS liquid heavy suspension, vibrating and infecting for 15min, placing the leaf on sterilized filter paper, sucking redundant liquid, clamping the back of the leaf downwards, placing the leaf into a flat plate containing co-culture medium, wrapping the flat plate with newspaper, and culturing for 2d in a dark state; (5) cleaning and selecting culture of the leaves; firstly, cleaning the leaves for 3 times by using sterile ddH ₂ O, finally cleaning the leaves for 1 time by using an MS liquid culture medium, and placing the back surfaces of the leaves on a selective differentiation culture medium downwards for selective culture; (6) continuing to culture: cutting off the differentiated buds, inserting the buds into a tissue culture bottle for rooting culture, and finally, when transgenic tobacco grows to about 10cm, slightly taking out regenerated plants from the tissue culture bottle by using long tweezers, flushing roots by flowing cold water, removing residual tissue culture medium and transferring the roots into soil.

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

RNH1igF:5'-CAGCAGTGCCAAGTGGTCAG-3'

RNH1igR:5-'CAATGCCGCACAGGTCCC-3'

Identification of transcript levels: total RNA from fresh leaves of tobacco was extracted with TRIzol reagent and reverse transcribed, and wild-type tobacco total RNA was used as a control. RT-PCR amplification is performed by using the primers, and whether the target gene is transcribed or not is identified. In the identification process, the method is at least extracted twice independently, and PCR or RT-PCR identification is carried out twice each time to ensure the accuracy of the result. Genomic and transcriptional levels were identified for transgenic plants (FIG. 3) and (FIG. 4).

Example 3

Insect resistance analysis of transgenic tobacco

Cutting artificial feed into small pieces of about 0.5cm×0.5cm, placing in sterile culture dish, inoculating larva of cotton bollworm egg to feed, and standing to 2 years (body length 0.42cm-0.62 cm) for insect test analysis. Selecting 3T 1 generation single copy transgenic lines, wherein each plant selects 3 tobacco leaves with consistent growth vigor to feed cotton bollworms, and simultaneously selects leaves with consistent growth vigor in wild tobacco planted in the same period to feed cotton bollworms as a control. 3 single copy tobacco plants of the same gene are set as repeat groups to reduce errors caused by different gene insertion sites; 3 leaves of the same tobacco are set as repeated tests in a group, so that human errors caused by selecting the leaves are reduced; the bollworm weights in each group were weighed 3 times to reduce occasional errors in weight measurement, and the average value was taken during subsequent data processing. Selecting 16 cotton bollworm larvae with the same growth vigor and the same weight from cotton bollworms in the 2 nd age, and placing the cotton bollworms larvae into a locker box with leaves. The body weight of the cotton bollworms is weighed every 24 hours, the residual quantity of the cotton bollworms is counted, and the data is counted to 120 hours. The status of blade bite at 24h was observed and recorded for photographing. During the insect test, the lockers were cleaned every 24 hours and fresh tobacco leaves were replaced. And finally, calculating the average weight of the cotton bollworms according to the statistical data, and comparing the differences among the average weights of the cotton bollworms among different groups by using IBM SPSS STATISTICS software through single-factor analysis of variance. An average cotton bollworm weight line graph was drawn with Excel 2016 software based on the results of the statistical analysis.

The biting condition of 24h tobacco leaves was recorded by photographing, and the result is shown in the graph (fig. 5). The results show that the experimental group (hRI) and the control group (wild WT) are subjected to different degrees of bite damage after being fed with the second-instar larvae of cotton bollworms for 24 hours, wherein the bite situation of wild tobacco leaves is serious, and the bite situation of hRI tobacco leaves is light. It can be seen that the insect resistance of the transgenic hRI gene tobacco is obviously improved.

Average body weight gain of cotton bollworms (table 1) and a line graph of average body weight gain of cotton bollworms (fig. 6) were plotted for five time periods of feeding wild type tobacco and transgenic hRI tobacco for 24h, 48h, 72h, 96h, 120 h.

Table 1: average body weight (g) of cotton bollworms at different time periods of feeding rotation hRI leaves

Note that: different lowercase letters in the same column indicate significant differences at the level P < 0.05; the different capital letters in the same column indicate that the difference is very significant at the level P < 0.01.

The trend of weight gain showed that: in the same time period, the feeding rotation hRI tobacco group is obviously lower than the feeding wild type tobacco group, the feeding rotation hRI tobacco group is obviously different from the feeding wild type tobacco group in 48 hours, the starting difference of 72 hours reaches an extremely obvious level, and the result shows that the rotation hRI tobacco bollworm larva has obvious inhibition effect. There was no significant difference between the average weights of the 3 groups of cotton bollworms feeding the hRI tobacco, indicating that the insect inhibitory ability of the hRI tobacco in the insect test analysis was not affected by the gene insertion site.

Sequence listing

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ctcaacctgc gcagcaacga gctgggcgat gtcggcgtgc attgcgtgct ccagggcctg 240

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

gacatcaatg aggctggcgt ccgtgtgctg tgccagggcc tgaaggactc cccctgccag 600

ctggaggcgc tcaagctgga gagctgcggt gtgacatcag acaactgccg ggacctgtgc 660

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aggtttctcc tggagctaca gataagcaac aacaggctgg aggatgcggg cgtgcgggag 1080

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tcctga 1386

Claims

1. The application of the insect-resistant protein hRI or the gene thereof in improving the insect resistance of plants; 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 the sequence table, and the insect resistance refers to bollworm resistance.

2. The use according to claim 1, wherein the transgenic plant is obtained by introducing a gene fragment of the insect-resistant protein hRI into the plant of interest, and wherein the insect-resistant protein hRI is expressed in the transgenic plant, such that the insect-resistance of the transgenic plant is higher than that of the plant of interest.

3. The use according to claim 1, wherein the gene fragment of the insect-resistant protein hRI is introduced into the plant of interest by means of a recombinant vector of the gene fragment of the insect-resistant protein hRI; the recombinant vector is constructed by the following method: the gene fragment shown in the sequence 2 in the sequence table is connected to a pBIN438 plasmid, so as to form a plant expression vector pBIN438-hRI.

4. Use according to claim 2 or 3, wherein the plant of interest is upland cotton or tobacco.