CN109837291A - A kind of method and its special DNA fragment improving plant resistance to insect using RNA perturbation technique - Google Patents
A kind of method and its special DNA fragment improving plant resistance to insect using RNA perturbation technique Download PDFInfo
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Abstract
The present invention provides a kind of method and its special DNA fragment for improving plant resistance to insect using RNA perturbation technique, DNA fragmentation is as shown in the formula (I): SEQIt is positive‑X‑SEQReversely(I), wherein SEQIt is positiveIt is any one section including at least 21bp in the full-length cDNA segment of bollworm chitin deacetylase 5a gene；SEQReverselyWith the SEQIt is positiveReverse complemental；X is SEQIt is positiveWith SEQReverselyBetween intervening sequence, X and the SEQIt is positiveAnd SEQReverselyNot complementary, the full-length cDNA of the bollworm chitin deacetylase 5a gene is as shown in the sequence 1 in sequence table.The present invention is applied to cultivate in the genetically modified plants that insect resistace improves for the first time using bollworm chitin deacetylase 5a gene as RNA disturb target gene.Worm test result shows 1/10 of weight less than control group weight of 2 days to 5 days cotton bollworm larvaes after switching；Larval mortality on 5 days transgenic tobacco leafs reaches 68%, and wild-type tobacco only has 21%.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of side that plant resistance to insect is improved using RNA perturbation technique
Method and its special DNA fragment.
Background technique
Eukaryocyte takes in dsRNA(double-stranded RNA), the degradation of cellular endogenous homologous mRNA can be caused, to cause to correspond to
Gene expression is obstructed, and this phenomenon is referred to as RNA interference (RNA interference, RNAi).The RNAi skill of mediated plant
Art is by transgenic technology, the dsRNA(or hpRNA of expression insect genes, i.e. hairpin RNA) expression casette
It imported into plant, after this vegetable material of dsRNA/hpRNA containing pest gene of pests, the dsRNA/hpRNA meeting of intake
Cause the RNAi of pest, it is possible to can play the role of that insect growth development is inhibited even to kill off the insect pests, plant to reach protection
The purpose of object.
2007, Mao YB etc. expressed P450 gene (CYP6AE14) directly related with gossypol metabolic detoxification in cotton
DsRNA, after feeding bollworm, the expression of CYP6AE14 gene is significantly reduced, while bollworm subtracts the tolerance of gossypol
It is weak, improve gossypol to the toxicity of bollworm [non-patent literature: Mao YB, Cai WJ, Wang JW,et al.
Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi
impairs larval tolerance of gossypol. Nat Biotechnol, 2007, 25(11): 1307-
1313.].The same year, Baum etc. first pass through feeding dsRNA test and select from 290 candidate targets GeneScreens of diabroticavirgifera
Vacuole ATP enzyme gene (vacuolar ATPase, V-ATPase) is then transferred to the expression of corn render transgenic plant
DsRNA, the results showed that prevention and treatment diabroticavirgifera have good effect [non-patent literature: Baum JA, Bogaert T,
Clinton W et al. Control of coleopteran insect pests through RNA
interference. Nat Biotechnol, 2007, 25: 1322-1326.].Above-mentioned foundation sex work has started plant
One hot spot of Insect resistant gene engineer research field.
The RNAi technology of mediated plant has the advantages that anti insect gene source is wide, gene specific is strong, residual is low.However just
It is since gene source is extensive, seeking suitable target gene becomes the key point and difficult point of the technology.
Chitin deacetylase (chitin deacetylase, CDA) participates in the degradation and modification of insect chitin, urges
Change chitin and is converted into chitosan (hydrolysis of the acetyl group of N- acetyl glucosamine therein).CDA's develops insect growth
Important function have been confirmed.
Guo in 2005 etc. is separated to the cDNA sequence of CDA, this gene coding in cabbage looper for the first time in intestines cDNA library
Protein can be combined with each other with chitin, propose that the function of the albumen may be related with the formation of funnel and update.Hereafter,
Molecular cloning and its expression analysis research of CDA encoding gene have been carried out in various insects.
Insect CDA points are five classes (Group).1st class and the 2nd class contain three structural domains: chitin combination funnel A structure
Domain (Chitin binding Peritrophin-A domain, ChBD), low-density lipoprotein A receptoroid structural domain (Low-
Density lipoprotein receptor class A domain, LDLa) and polysaccharide deacetylase catalyst structure domain
(Polysaccharide deacetylase catalytic domain, CDA)；3rd class and the 4th class all do not have LDLa structure
There is CDA and ChBD structural domain in domain, but there are larger differences for the position of the two structural domain；5th class CDA only has CDA structural domain.
On this basis, the 1st class can be further separated into two subgroups (Ia, Ib), it was found that there are alternative splicings in Ib subgroup.Five
Class CDA reflects the evolutionary relationship of insect CDA, and plays an important role in the development of different plant species insect growth.
Bollworm (Helicoverpa armigera) it is a kind of important agricultural pests, it is a variety of to endanger cotton, corn etc.
Crops.The bollworm great outburst nineties in last century once brought extreme loss to Cotton in China production.Transgenic cotton against pests and its
The application of his measure is so that Cotton Production is able to steady development so far.It will be interior from the insecticidal proteins δ-of bacillus thuringiensis
Toxinencoding genes (Bt gene) imported into crops by transgenic technology, and crops is enable to express insecticidal proteins to reach
The purpose of pest control.Other than Bt gene, a kind of protease inhibitors (CpTI) gene from cowpea is also used for me
The Transgenic Cotton Varieties of state.But current transgenic cotton against pests are faced with that insect resistace is undesirable, insect-resistant evolves, is excellent
The problems such as good anti insect gene is more single, it would be highly desirable to develop new anti insect gene and method.
Inventor bollworm transcript profile Data Mining, gene cloning and mediated plant RNAi research in find, cotton
The chitin deacetylase 5a gene of earworm can become the ideal targets gene of the RNAi of mediated plant, currently, not yet will
Bollworm chitin deacetylase 5a gene improves the relevant report of plant resistance to insect as RNAi target gene.
Summary of the invention
The method of plant resistance to insect and its dedicated is improved using RNA perturbation technique it is an object of the invention to provide a kind of
DNA fragmentation, to solve, existing genetically modified plants insect resistace is undesirable, insect-resistant is evolved, excellent anti insect gene is more single is asked
Topic.
The purpose of the present invention is what is be achieved through the following technical solutions: a DNA fragmentation as shown in the formula (I):
SEQIt is positive-X-SEQReversely(I)
Wherein, SEQIt is positiveIt is to include at least 21bp in the full-length cDNA segment of bollworm chitin deacetylase 5a gene
Any one section；SEQReverselyWith the SEQIt is positiveReverse complemental；X is SEQIt is positiveWith SEQReverselyBetween intervening sequence, X and the SEQIt is positive
And SEQReverselyIt is not complementary, and length >=10bp of X；The full-length cDNA of the bollworm chitin deacetylase 5a gene is such as
Shown in sequence 1 in sequence table.
Further, the SEQIt is positiveNucleotide sequence as shown in sequence 2 in sequence table.
A kind of recombinant vector containing above-mentioned DNA fragmentation.
The recombinant vector constructs by the following method: by DNA fragmentation shown in sequence 2 in sequence table simultaneously with positive and
Reversed mode is inserted on pRNAi-GG, to constitute Genetic Transformation in Higher Plants carrier.
Bollworm chitin deacetylase 5a gene is improving answering in plant resistance to insect as RNA disturb target gene
With.
A method of plant resistance to insect is improved, above-mentioned DNA fragmentation is imported in purpose plant and obtains genetically modified plants,
The insect resistace of the genetically modified plants is higher than the purpose plant.
Further, above-mentioned DNA fragmentation is imported in purpose plant by the recombinant vector.
Further, the pest-resistant insect for referring to anti-Lepidoptera.Further, described pest-resistant to refer to anti-lepidopterous insects
Larva.Further, the pest-resistant larva for referring to bollworm resisting.
Further, the purpose plant is can be by the plant of above-mentioned insect institute feeding.Further, the purpose is planted
Object is upland cotton.
Method of the invention specifically includes the following steps:
1, gene order is obtained
By designing synthetic primer, internal control RT-PCR (RT-PCR) then is carried out to bollworm mRNA, Huo Zhetong
Chemical synthesis is crossed, bollworm chitin deacetylase 5a gene order (HaCDA5a gene, such as 1 institute of sequence in sequence table is obtained
Show).
2, the selection of target sequence and the bollworm 5a type special dsRNA expressed sequence structure of chitin deacetylase are set
Meter
(1) after obtaining above-mentioned DNA sequence dna, partial sequence therein is chosen as target sequence, it is desirable that length is not shorter than 21 bases
To (bp), expand to obtain target sequence by round pcr；
(2) DNA recombinant technique is utilized, target sequence is built into plant hpRNA(hairpin RNA) expression structure, expression structure is such as
Shown in formula (I):
SEQIt is positive-X-SEQReversely(I)
Wherein, the SEQIt is positiveNucleotide sequence as shown in sequence 2 in sequence table, SEQReverselyAnd SEQIt is positiveFor sequence is identical, direction
Opposite sequence, X sequence are that length is not shorter than 10 nucleotide pairs (bp) and and SEQIt is positiveAnd SEQReverselyNot complementary any DNA
Sequence.
3, vector construction and Plant Transformation
Recombinant expression carrier containing above-mentioned expression structure is passed through into agrobacterium-mediated transformation, particle bombardment, pollen tube passage method etc.
Mature Genetic Transformation in Higher Plants technological sourcing is into target plant, to improve purpose plant to the resistance in relation to pest.Expression
Carrier framework can be pRNAi-GG, and DNA fragmentation shown in sequence 2 in sequence table is inserted into a manner of forward and reverse simultaneously
On pRNAi-GG, so that building contains the recombinant expression carrier of above-mentioned expression structure.
4, insect resistace verification test and applicable object
The plant that the present invention is suitable for is any plant that can be carried out transgeneic procedure.Common genetically modified plants object includes cotton
Flower, corn, rice, wheat, tomato, potato, soybean, poplar etc..Controlling object can be any feeding genetically modified plants and
It can cause the pest of RNAi effect.Common pest include from Lepidoptera, coleoptera, Isoptera, Diptera, Hymenoptera,
The plant-feed insect or agricultural pests of the monoids such as Orthoptera, Semiptera, Thysanoptera.Detailed process of the invention is shown in Figure 1.
The present invention is applied to for the first time using bollworm chitin deacetylase 5a gene as RNA disturb target gene
It cultivates in the genetically modified plants that insect resistace improves.The present invention has preferable insect resistace, Neng Gou great to the larva of lepidopterous insects
The big infringement for reducing insect to plant, more specific aim.It is demonstrated experimentally that the cotton bollworm larvae growth being inoculated on wild-type tobacco
Breeding is normal, and the cotton bollworm larvae growth and development on transgene tobacco is heavily suppressed, 2 days to 5 days bollworms after switching
The weight of larva is less than the 1/10 of control group weight；By wild type and transgenic tobacco leaf mixing feeding cotton bollworm larvae, cotton
Earworm shows extremely strong food refusal phenomenon to transgenic tobacco leaf；Larval mortality on 5 days transgenic tobacco leafs of feeding
Reach 68%, wild-type tobacco only has 21%.
Detailed description of the invention
Fig. 1 is the flow diagram of the method for the present invention, in figure: being 1. and 2. target sequence, the sequence same direction is on the contrary, 3.
For the intron sequences for playing interval action.
Fig. 2 is pRNAi-GG-HaCDA5a plasmid map.
Fig. 3 is the PCR testing result of pRNAi-GG-HaCDA5a transgene tobacco, in figure: "+": positive control；1-9: to
It detects plant and is kalamycin resistance positive plant；M:Marker DL2000；N1: wild-type tobacco negative control；N2: nothing
Template-negative controls.
Fig. 4 is the RT-PCR testing result of transgene tobacco, in figure: "+": positive control；1-11: plant to be detected and it is
Positive plant；M:Marker DL2000；N1: wild-type tobacco；N2: without template.
Fig. 5 is to sting the photo for biting tobacco leaf in bollworm administering transgenic.
Fig. 6 is feeding Nicotiana gossei and transgene tobacco cotton bollworm larvae changes of weight figure.
Fig. 7 is the bollworm death rate pair after continuous 5 days Nicotiana gosseis of feeding and pRNAi-GG-HaCDA5a transgene tobacco
Than figure.
Fig. 8 is the photo that Nicotiana gossei and transgene tobacco compare feeding experiment.Wherein: left figure is transgenosis (5a, under)
12 hours photos of cotton bollworm larvae are fed with non-transgenic (WT, on) tobacco leaf, and right figure is transgenosis (5a, under) and non-turn
Gene (WT, on) tobacco leaf feeding 60 hours photos of cotton bollworm larvae.
Fig. 9 is the relative expression quantity comparison diagram of different time bollworm HaCDA5a gene in embodiment 5.
Specific embodiment
Technical solution of the present invention is described in detail combined with specific embodiments below.It is not mentioned in the embodiment of the present invention
Experimental condition and operation by conventional method in that art carry out, those skilled in the art can refer to the relevant technologies books implementation,
Such as: Green, Pehanorm Brooker write " Molecular Cloning:A Laboratory guide " the 4th edition (2017), etc..
Embodiment 1
Using RT-PCR(internal control RT-PCR) technology obtains gene order.
1, PCR primer designs
Bollworm chitin deacetylase 5a gene (HaCDA5a) PCR primer sequence is CDA5aF:5'-
ATGAAGTTGTTCGGGCTTCTTG-3', CDA5aR:5'-CAGTTGTAGATTTATTGTCCAAG-3'.
2, bollworm Total RNAs extraction and cDNA synthesis and amplification
5 age bollworm total serum IgEs are extracted with TRIzol reagent, synthesize the first chain of cDNA with reverse transcriptase.PCR reaction system: 2 μ L
10 × Buffer, it l μm of ol/L up/down trip primer, 0.1 mmol/L dNTPs, 1 units of Taq polymerase, 0.5 μ L cDNA, uses
ddH2O complements to 20 μ L.PCR response procedures: 94 DEG C of 3 min of initial denaturation, 94 DEG C of 30 s of denaturation, 52 DEG C of 30 s of annealing, 72 DEG C are prolonged
Stretch 2 min, 35 circulations；It is final to extend 72 DEG C, 10 min.
3, PCR product recycling, TA clone and identification
PCR gel electrophoresis product is separately recovered using silicagel column method, is then connect with carrier T.Linked system is (10 μ L): 1 μ
L carrier T, 1 μ L T4 DNA ligase, 1 μ L 10 × T4 ligase Buffer, 7 μ L recovery products.By prepared connection
System is placed 16 DEG C and is carried out after staying overnight connection, is transformed into Escherichia coli (DH5 α) with heat shock method.With primer CDA5aF/
CDA5aR antagonism bacterium colony carries out PCR identification, send biotech firm to be sequenced PCR positive colony, the gene sequence can be obtained
It arranges (as shown in sequence 1).
Embodiment 2
1, the preparation of target sequence
Round pcr is used to expand above-mentioned positive colony to prepare target sequence, primer sequence is respectively as follows:
CDAI5aF:5'-accaggtctcaggagATGAAGTTGTTCGGGCTTCTTG-3',
CDAI5aR:5'-accaggtctcatcgtTACTCCAAGCCATATTCCTG-3 ',
The lower-case portion of 15 bases in the end primer 5' is partial sequence required for Golden gate clone technology.The target of amplification
Gene order size is 578bp(HaCDA5a, in sequence table shown in sequence 2).
2, PCR amplification obtains target gene segment
PCR amplification system is equal are as follows: plasmid template 0.6 μ L, corresponding each 2.4 μ L, 2 × Taq MsterMix 15 of upstream and downstream primer
μ L, ddH2O supplies 30 μ L；PCR amplification program are as follows: 95 DEG C, 3min；95 DEG C, 30s；52 DEG C, 30s；72 DEG C, 40s, 35 circulations；
72 DEG C, 10min.The PCR product of amplification is subjected to agarose gel electrophoresis, is met according to the recycling of gel reclaims kit specification
It is expected that the DNA electrophoretic band of size.
Embodiment 3
The building of plant hpRNA expression vector
Plant expression vector skeleton used in the present embodiment is pRNAi-GG, has a hpRNA expression cassette on the vector plasmid:
The driving of CaMV35S promoter, Nos terminator terminate transcription；There are two the bacterium ccdB being spaced apart by intron sequences for transcriptional domain
Gene；Respectively there are two Bsa I restriction endonuclease (IIS type) recognition sites at each ccdB gene order both ends.
Plant expression vector is constructed using Goldgate technology.By target sequence pcr amplification product and pRNAi-GG plasmid
It is placed in a pipe, while Bsa I is added and carries out Golden with T4 DNA ligase (connecting enzyme reaction buffer solution using T4)
Gate reaction, reaction system are as follows: 350 U of 200 ng of pRNAi-GG plasmid, target sequence PCR product 50 ng, T4 ligase
(TAKARA company), 1 μ L(TAKARA company of T4 ligase buffer), 5 U(NEB company of Bsa I), ddH2O supplies 10
μL.Identified later by conversion, sequencing and etc., the hpRNA expression vector of target sequence can be built into.Wherein pRNAi-
GG-HaCDA5a map is as shown in Figure 2.
Embodiment 4
Plant expression vector transformation of tobacco
1, using the tobacco genetic transfoumation of mediated by agriculture bacillus
The plasmid vector being built into is imported into Agrobacterium LBA4404 competent cell using Electroporation conversion, to carry out agriculture
The tobacco genetic transformation experiment that bacillus mediates.The plant that the present embodiment is converted is tobacco, and transgenosis object of the invention is unlimited
In tobacco.
It obtains tobacco aseptic seedling: wild-type tobacco seed being taken to be placed in 1.5mL microcentrifugal tube the processing that carries out disinfection: is first
30s first is cleaned with the ethanol solution that volume fraction is 70%, then three times with aseptic water washing, the secondary chlorine for being later 10% with concentration
Acid sodium solution concussion cleaning 10min, finally with sterile water wash five times for cultivating sterile tobacco plant.To sterile tobacco plant
Blade is grown to after a certain size, and cutting area in superclean bench with knife and tweezers is about 1cm2Aseptic blade is trained for tissue
It supports.
It takes Agrobacterium (LBA4404) bacterium solution containing pRNAi-GG-HaCDA5a plasmid to be activated, then transfers into body
It is cultivated on the LB culture medium that product is 300mL, to Agrobacterium OD600Bacterium solution is dispensed when=0.8 to be centrifuged, and thallus is enriched with, it will
It is configured to certain density agrobacterium suspension for infecting tobacco leaf.By preculture, dark culture and selection culture rank
Section, obtains the adventitious bud being differentiated to form from callus, moves it into and carry out culture of rootage in root media, to its point
It is transplanted into soil when metaplasia root and blade quantity length to 4-6 piece and continues culture for Insect resistance assay.
2, the PCR detection of transgene tobacco
The genomic DNA for extracting transgenic tobacco plant carries out PCR detection.It is identified by PCR, acquisition turns HaCDA5a base
Because of 9 plants of positive plant.Positive transgenic tobacco gene group DNA amplified band size and positive plasmid amplified band size one
It causes, wild-type tobacco genomic DNA and the positive band without Template-negative controls without the corresponding size of appearance.Turn pRNAi-
GG-HaCDA5a carrier tobacco plant testing result is as shown in figure 3, template is followed successively by positive plasmid (+), transgenosis cigarette to be detected
Blade of grass piece genome DNA(1-11), wild-type tobacco leaves genomic DNA (N1) and without Template-negative controls (N2).
3, the RT-PCR detection of transgene tobacco
The blade of transgenic tobacco plant and wild-type tobacco plants is taken to be fully ground in liquid nitrogen, according to TRIzon(Bei Jingkang
For ShiJi Co., Ltd) total RNA extraction reagent specification extraction tobacco leaf total serum IgE.It carries out Ago-Gel (1%) electrophoresis and observes RNA
Quality；With the value of light absorption value and A260/280 and A260/230 of the nucleic acid analyzer measurement RNA at 260nm and 280nm.
Take satisfactory tobacco RNA synthesis the first chain of cDNA of two kinds of detection method detections.It is carried out using the first chain of cDNA as template
RT-PCR detection (wild-type tobacco cDNA and without template system be negative control), the primer be amplifying target genes spy
Specific primer, to identify transgenic tobacco plant on transcriptional level.Turn the detection of pRNAi-GG-HaCDA5a carrier tobacco plant
As a result as shown in Figure 4.
Embodiment 5
1, HaCDA5a hpRNA expresses plant twig and shoot pest
Pest involved by the present embodiment is bollworm, and controlling object of the present invention is not limited to bollworm.
By transgenic tobacco plant seed kind in the MS resistant panel containing kanamycins, statistics credit is carried out to germination percentage
Analysis sifts out the T2 of transgenosis list copy for transgenic tobacco plant.It is consistent and for single copy insertion to randomly choose 3 plants of growing ways
Transgenic tobacco plant carries out bollworm administering transgenic, and using wild-type tobacco plants as control strain, one is divided into 2 groups (in fact
Test group and control group).In tests, bollworm eggs are put in and carry out being protected from light hatching in the growth cabinet that condition of culture is set,
Worm examination analysis is carried out according to above-mentioned two groups of different disposals after it is just incubated.
It is connected to just an instar bollworm grub is incubated on man-made feeds.It is to be grown to two ages, choose the close larva of size
It is connected to fresh transgenosis, fed on wild-type tobacco blade.Blade is placed in glass culture dish, and each culture dish connects 12
Item, experimental group and control group set three groups of repetitions respectively, and 36 altogether.Fresh tobacco leaves are replaced during feeding daily and are recorded
Cotton bollworm larvae changes of weight, while the death rate is calculated, feeding result is as shown in Figure 5.Three biology of experimental setup repeat.It is real
Verify bright, the cotton bollworm larvae growth and breeding being inoculated on wild-type tobacco is normal, and turns on HaCDA5a hpRNA genetic tobacco
Cotton bollworm larvae growth and development it is heavily suppressed, after switching the weight of 2 days to 5 days cotton bollworm larvaes be less than control group weight
1/10(it is as shown in Figure 6)；Larval mortality after 5 days on transgenic tobacco leaf reaches 68%, and wild-type tobacco only has 21%
(as shown in Figure 7)；By wild type and transgenic tobacco leaf mixing feeding cotton bollworm larvae, bollworm is to transgenic tobacco leaf
Show extremely strong food refusal phenomenon (as shown in Figure 8).
2, the silence efficiency detection of cotton bollworm larvae target gene
The instar larvae just hatched is connected on bollworm man-made feeds, when growing to for two ages to larva, is chosen big
Small identical cotton bollworm larvae is inoculated in fresh transgenosis respectively, is fed on wild-type tobacco blade, every 12h replacement one
Secondary fresh blade, collect for 24 hours, the cotton bollworm larvae of 48h, 60h, every time collect 3 be placed in centrifuge tube, rapidly freeze in -80 DEG C
In refrigerator.Three biology are arranged to repeat.
The RNA for extracting the cotton bollworm larvae collected carries out the first chain of cDNA that reverse transcription obtains bollworm, concrete operations step
Rapid reference specification.Fluorescent quantitative PCR experiment detection feeding transgenosis cigarette is carried out with Beacon Designer software Design primers
Grass is to the silence efficiency of target gene, and reference gene is β-actin, using SYBR Green method.Fluorescence PCR primer sequence:
CDA5aqF:5'-AGAACTCCTCAAACCTACTG-3', CDA5aqR:5'-TACTCCAAGCCATATTCCTG-3'.Bollworm
β-actin sequence: primer β-ActinF:5'-CTGACCGTATGCAGAAGGAG-3', β-ActinR:5'-
CACAAGCGTAATTTGAGCC-3'。
Fluorescent PCR system are as follows: 10 μ L SYBR Green Master Mix, 0.5 μm of ol/L up/down swim primer, 0.5 μ
L cDNA, uses ddH2O supplies 20 μ L.Fluorescent PCR program are as follows: 5 DEG C of initial denaturation 5 min, 95 DEG C of 10 s of denaturation, 53 DEG C of annealing 10
S, 72 DEG C；Extend 15 s, 45 circulations.Each sample carries out 3 biology and repeats, and makees with no responsive transcription template and without template
Negative control.2-△△CtMethod analyze the change of relative expression quantity of the different bollworm HaCDA5a genes in different feeding times
Change, with minimum expression quantity be 1, experimental data is handled with Microsoft Excel(2007), SPSS software to data into
Row significance analysis, P < 0.05 are significant difference.Target gene silencing maximum is close to 55%.As a result as shown in Figure 9.
Comparative example 1
Using HaCDA1 gene as target gene, HaCDA1 hpRNA expression plant is obtained simultaneously referring to the operating procedure of embodiment 1 ~ 5
Its insect resistace is studied, wherein the cDNA synthetic primer of HaCDA1 gene is HaCDA1 gene ICD1f:5'-
GGTTTTGTTCATAATGTC-3'；ICD1r:5'-ATCAACAACAACAACATC-3'.HaCDA1 gene target sequence synthesized primer
Object is CDAI1F:5'--accaggtctcaggag GGTTTTGTTCATAATGTC-3',
CDAI1R:5'-accaggtctcatcgtATCAACAACAACAACATC-3', resulting target sequence such as sequence
Shown in 3, the target-gene sequence size of amplification is 643bp.Cotton boll is carried out to the finally obtained HaCDA1 hpRNA genetic tobacco that turns
Worm administering transgenic, as a result as shown in Figure 5.The insect resistace of genetically modified plants has not predictability it can be seen from contrast effect,
The method for improving plant resistance to insect using HaCDA5a gene of the invention has insect resistant effect outstanding.
SEQUENCE LISTING
<110>University Of Hebei
<120>a kind of method and its special DNA fragment that plant resistance to insect is improved using RNA perturbation technique
<130> 2-2
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1173
<212> DNA
<213> HaCDA5a
<400> 1
atgaagttgt tcgggcttct tgctttgttg ctggtagctt cggccttcgc tgatgactct 60
tcgtcttcag aagaagaaaa tgagggcctg cctttggcag aggcctgtga ccaggaagct 120
tgcagcttgc ctgactgcgg atgttccagc accaacattc ccggaggatt gaacccacga 180
gatataccac agttcgtaac tgtcaccttc gacgatggtg ttaacgtgaa caacattatc 240
acttaccgca acatcctgta caaccgactg aactccaacg gctgtcccgc tggagttacc 300
ttcttcgtca gccacgagta caccaactat gctctcatca acgagctcta taaccagggc 360
ttcgaaattg ccctacactc catcagtcac agaactcctc aaacctactg gttcgaagcc 420
accaaagaag ttatcaagga agaaattgct gaccagaaag cccagatggc tcacttcgct 480
aacattcctc ccagcgccat taaaggtgtt cgcatgccct tcctccaatt ggctggcaac 540
gctagcttcg aagtcatgca ggaatatggc ttggagtacg attgcacttg gcctacgatc 600
gcccacacaa acccaggact atggccttac accctggact acgcttcgac ccaggactgc 660
atcattcctc cctgcccatc tgcctccatc cctggagttt gggttaagcc tatggttgcc 720
tggtctgacc tcaacggagt tccttgctca atggttgacg cttgcttctt catccctgac 780
cgcgaaaatg aagaggaatg gtacaagttc atcctcagca acttcgagag gcactacttg 840
ggcaaccgtg ctcccttcgg cttctacgtc cacgaggcct tcttggctgc caaccctgcc 900
gtcaaccgtg cgctagttcg cttcatggat ctggtcaaca atctgaatga tgctttcatg 960
gtgaacgccc acgaagtcgt cgactgggtt aagaacccga agccactcaa tgagtacagg 1020
agccaaggct gccgcagctt cagcccttca acctgcaacc ccaacaactg tggtcctctc 1080
ttctccacac acaatcaatt ggcttactac atgcaagtat gcagcgcttg tcctaacaac 1140
tacccatggg tcggcaatcc tcttggacaa taa 1173
<210> 2
<211> 578
<212> DNA
<213> HaCDA5a
<400> 2
atgaagttgt tcgggcttct tgctttgttg ctggtggctt cggtcttcgc tgatgactct 60
tcgtcgtcag aagaagaaaa tgagggcctg cctttggcag aggcctgtga ccaggaagct 120
tgcagcttgc ctgactgcag atgttccagc accaatattc ccggaggatt gaatccacga 180
gatgtaccac aattcgtaac tgtcaccttc gacgatggtg ttaacgtgaa caacattatc 240
acttaccgca acatcctgta caaccgcttg aactccaacg gctgtcctgc cggagtcacc 300
ttcttcgtca gtcatgagta caccaactat gctctcatca acgagcttta taaccagggc 360
ttcgaaatcg ccctgcactc catcagccac agaactcctc aaacctactg gtttgaagcc 420
accaaagagg ttatcaagga agaaatcgct gaccagaaag cccagatggc tcacttcgct 480
aacattcctc ccagtgccat taagggtgtt cgcatgccct tcctccaatt ggcaggcaac 540
gctagcttcg aaatcatgca ggaatatggc ttggagta 578
<210> 3
<211> 643
<212> DNA
<213> HaCDA1
<400> 3
atcaacaaca acaacatcga actttacaaa gagatcttca acggaaaacg taaaaacccc 60
aacggttgcg acattaaggc cacatacttt atttcgcaca agtacaccaa ctactcggct 120
gttcaggaaa ctcacagaaa gggacatgaa atcgctgtac actcaatcac gcacaacgat 180
gacgaacgct tctggagcaa cgctaccgtt gacgactggg gcaaggagat ggccggtatg 240
agagtcatca tcgagaagtt ctcgaacatc actgacaaca gtgtcgtggg tgtgcgagcg 300
ccgtacctcc gtgtcggtgg taacaaccag ttcaccatga tggaagagca agccttcttg 360
tatgacagca ccatcactgc tcccctgtcc aacccgccgc tatggccata cactatgtac 420
ttcagaatgc ctcaccgttg tcacggaaac ctgcagagct gccccaccag gagccacgcc 480
gtgtgggaga tggtgatgaa cgaacttgac cgtcgtgagg accccaccaa cgatgagtac 540
ttgcctggat gcgccatggt tgactcttgt tctaacattt tgacaggaga tcagttctac 600
aacttcctca accacaactt cgacagacat tatgaacaaa acc 643
Claims (10)
1. a DNA fragmentation as shown in the formula (I):
SEQIt is positive-X-SEQReversely(I)
Wherein, SEQIt is positiveIt is to include at least 21bp in the full-length cDNA segment of bollworm chitin deacetylase 5a gene
Any one section；SEQReverselyWith the SEQIt is positiveReverse complemental；X is SEQIt is positiveWith SEQReverselyBetween intervening sequence, X and the SEQIt is positive
And SEQReverselyIt is not complementary, and length >=10bp of X；The full-length cDNA of the bollworm chitin deacetylase 5a gene is such as
Shown in sequence 1 in sequence table.
2. DNA fragmentation according to claim 1, which is characterized in that the SEQIt is positiveNucleotide sequence such as sequence table in sequence
Shown in column 2.
3. containing the recombinant vector of DNA fragmentation as claimed in claim 1 or 2.
4. recombinant vector according to claim 3, which is characterized in that the recombinant vector constructs by the following method: will
DNA fragmentation shown in sequence 2 is inserted on pRNAi-GG in a manner of forward and reverse simultaneously in sequence table, to constitute plant
Genetic transformation carrier.
5. bollworm chitin deacetylase 5a gene is improving answering in plant resistance to insect as RNA disturb target gene
With.
6. a kind of method for improving plant resistance to insect, which is characterized in that DNA fragmentation of any of claims 1 or 2 is imported purpose
Genetically modified plants are obtained in plant, the insect resistace of the genetically modified plants is higher than the purpose plant.
7. the method according to claim 6 for improving plant resistance to insect, which is characterized in that of any of claims 1 or 2
DNA fragmentation is imported in purpose plant by recombinant vector described in claim 3 or 4.
8. the method according to claim 6 or 7 for improving plant resistance to insect, which is characterized in that described pest-resistant to refer to anti-squama
Wing purpose insect.
9. the method according to claim 6 or 7 for improving plant resistance to insect, which is characterized in that described pest-resistant to refer to anti-cotton
The larva of earworm.
10. the method according to claim 6 or 7 for improving plant resistance to insect, which is characterized in that the purpose plant is land
Ground cotton.
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| CN110628789A (en) * | 2019-10-25 | 2019-12-31 | 邯郸市农业科学院 | Breeding method of insect-resistant low-phenol cotton variety |
| CN113061623A (en) * | 2021-05-06 | 2021-07-02 | 湖北大学 | Preparation method of transgenic plant for expressing cell nucleus transformation of virus-like particles and application of transgenic plant in resisting cotton bollworm |
| CN115011627A (en) * | 2022-05-10 | 2022-09-06 | 新疆大学 | Preparation method of cotton bollworm-resistant transgenic cotton |
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| CN107586785A (en) * | 2017-11-13 | 2018-01-16 | 贵阳学院 | The application of lasioderma serricorne chitin deacetylase gene 1 and its dsRNA in control of insect |
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| CN110628789A (en) * | 2019-10-25 | 2019-12-31 | 邯郸市农业科学院 | Breeding method of insect-resistant low-phenol cotton variety |
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| CN113061623A (en) * | 2021-05-06 | 2021-07-02 | 湖北大学 | Preparation method of transgenic plant for expressing cell nucleus transformation of virus-like particles and application of transgenic plant in resisting cotton bollworm |
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