CN114621962B - Peanut AhBI-1 gene VIGS silencing system - Google Patents

Peanut AhBI-1 gene VIGS silencing system Download PDF

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CN114621962B
CN114621962B CN202210280701.XA CN202210280701A CN114621962B CN 114621962 B CN114621962 B CN 114621962B CN 202210280701 A CN202210280701 A CN 202210280701A CN 114621962 B CN114621962 B CN 114621962B
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vigs
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CN114621962A (en
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詹洁
罗锦
周敏
何龙飞
王爱勤
肖冬
张平刚
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Guangxi University
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    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield

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Abstract

The invention discloses a gene specific fragment for silencing peanut AhBI-1 gene, and constructs a corresponding VIGS virus vector and a VIGS silencing system. According to the invention, the pTRV2 virus silencing expression vector plasmid containing AhBI-1 gene specific fragments is constructed, and the peanut is transformed by an agrobacterium-mediated method, so that the endogenous AhBI-1 gene of the peanut is induced to silence, the expression level of the AhBI-1 gene of the peanut is effectively reduced, and the peanut is enabled to generate a dwarf phenotype. The inventor infects peanuts through a VIGS silencing technology, establishes a system for identifying gene functions by silencing peanut endogenous genes by pTRV-VIGS, has the advantages of rapidness, high flux and easiness in operation, and provides a new approach and technical support for developing peanut gene function research.

Description

Peanut AhBI-1 gene VIGS silencing system
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a peanut AhBI-1 gene VIGS silencing system.
Background
Virus induced silencing GENE SILENCING (VIGS) refers to that after a plant is infected by a virus carrying a target gene fragment, mRNA of a specific induction sequence homologous gene is degraded or is modified by methylation and the like along with replication and transcription of the virus, so that endogenous gene silencing of the plant is caused, phenotype or physiological index change is caused, and then the function of the target gene is researched according to the phenotype variation. VIGS is a gene transcription technology developed based on plant defense mechanisms against RNA viruses to characterize plant gene function, whose inherent molecular basis may be post-transcriptional gene silencing (post-TRANSCRIPT GENE SILENCE). Compared with the traditional gene function analysis method, the VIGS can silence and analyze the function of the target gene in the current generation of infected plants; there is no need to develop stable transformants and there is the potential to silence single or multiple gene family members.
Peanut is one of five oil crops in the world, is an economic crop for eating and pressing oil, is a source of vegetable fat and protein required by people living, and plays an important role in world agricultural production and trade. Asia is the most important peanut planting area, the planting area accounts for 63.4% of the total world area, and China is the peanut producing country only in india. However, in the current production work, the technical means for peanut genomics function research are very limited, and the method for obtaining the transgenic material is rare and difficult to succeed. The agrobacterium transformation method is the most commonly used method in the current plant genetic engineering research, and has the advantages of low cost, low copy number of the introduced genes and the like. Peanut belongs to dicotyledonous plants, is one of natural hosts of agrobacterium and is self-pollinated, and although genetic transformation of peanut has been advanced in recent years, the currently constructed genetic transformation system of peanut has the problems of long transformation period, low transformation efficiency and the like, so that an efficient and rapid genetic transformation method is an important technological breakthrough for researching the gene function of peanut.
It has been reported and previously studied that the peanut AhBI-1 gene (the full-length nucleotide sequence is shown in a sequence table SEQ.ID.NO. 1) is related to programmed cell death of peanuts and is a regulatory gene for inhibiting programmed cell death of peanuts.
Disclosure of Invention
The invention aims to provide a peanut AhBI-1 gene VIGS silencing system which provides technical support for peanut functional genomics research.
In order to solve the technical problems, the invention adopts the following technical scheme:
The gene specific fragment for silencing the peanut AhBI-1 gene is AhBI-1vigs, and has a base sequence of a sequence table SEQ.ID.NO. 2.
The VIGS virus vector for silencing peanut AhBI-1 gene is pTRV2 vector containing the specific fragment of the gene.
The VIGS viral vector for silencing the peanut AhBI-1 gene is a recombinant viral vector pTRV2-AhBI-1VIGS, and is constructed by connecting the gene specific fragment to a VIGS viral backbone vector pTRV 2.
Peanut AhBI-1 gene VIGS silencing system comprising the above gene-specific fragment and VIGS viral vector.
The construction method of the peanut AhBI-1 gene VIGS silencing system mainly comprises the following steps:
Construction of the vector (I)
(II) Agrobacterium transformation
And (III) infecting peanut materials.
The step (I) is carried out according to the following steps:
(1) Amplifying AhBI-1 gene full-length sequence from peanut cDNA, and amplifying specific fragment AhBI-1vigs from AhBI-1 gene full-length sequence;
(2) Extracting pTRV2 plasmid, selecting BamHI and EcoRI enzyme cutting sites, double enzyme cutting carrier plasmid, and connecting target fragment and linearized pTRV2 carrier by using homologous recombination method;
(3) E.coli DH5 alpha is transformed, and monoclonal colony is selected for bacterial liquid PCR identification to be positive.
Step (II) is carried out according to the following steps: transforming the constructed pTRV2-AhBI-1vigs recombinant plasmid into Agrobacterium GV3101 competent cells by a freeze thawing method; then inoculating to LB solid culture medium with corresponding resistance, picking monoclonal colony, and PCR identification positive.
Step (III) is carried out according to the following steps: mixing pTRV2, pTRV2-AhBI-1vigs and pTRV2-AHPDSVIGS resuspended bacteria liquid with PTRV resuspended bacteria liquid gently in equal volume, and incubating at room temperature for 3-5h; when the third true leaf of the peanut seed just appears, carrying out infection work, injecting pre-cultured peanut lower epidermis leaf, wherein the infection area is at least more than 75% of the leaf area; dark culturing for 36-48h, normal light culturing, observing plant leaf phenotype change, and detecting expression of AhBI-1 by taking new leaves at the moment when yellow She Xianxiang with different degrees appears in positive comparison of about two weeks, namely constructing a peanut AhBI-1 gene silencing system.
The application of the gene specific fragment, the VIGS virus vector and the VIGS silencing system in silencing peanut AhBI-1 genes.
Based on previous studies, the inventors designed a gene-specific fragment for silencing the peanut AhBI-1 gene and constructed a corresponding VIGS viral vector and VIGS silencing system. According to the invention, the pTRV2 virus silencing expression vector plasmid containing AhBI-1 gene specific fragments is constructed, and the peanut is transformed by an agrobacterium-mediated method, so that the endogenous AhBI-1 gene of the peanut is induced to silence, the expression level of the AhBI-1 gene of the peanut is effectively reduced, and the peanut is enabled to generate a dwarf phenotype. The inventor infects peanuts through a VIGS silencing technology, establishes a system for identifying gene functions by silencing peanut endogenous genes by pTRV-VIGS, has the advantages of rapidness, high flux and easiness in operation, and provides a new approach and technical support for developing peanut gene function research.
Drawings
FIG. 1 is a diagram showing electrophoresis results of AhBI-1 gene amplification, in which: marker is 2000bp, and 1 and 2 are all target fragments amplified from peanut root tip cDNA.
FIG. 2 is a schematic representation of Agrobacterium-mediated pollen tube channel method transformation of peanuts, wherein: a, injecting and selecting the lower epidermis of peanut leaves; b, performing contact injection by using a 1mL injector without a needle tube; c peanut hypodermis infestation of the leaf after injection.
Figure 3 is a graph of peanut growth phenotype observations two weeks after VIGS infestation, in which: CK is wild type of Zhonghua No. 2, ahPDS positive is peanut positive control pTRV2-AHPDSVIGS, yellow leaf phenotype appears, ahBI-1 is peanut pTRV2-AhBI-1vigs plant.
Fig. 4 is a phenotypic view of peanut at sampling test, in which: WT is wild type in Zhonghua No. 2, pTRV 2-empty vector is a plant which does not contain target gene and only transfers pTRV2 empty vector into peanut, pTRV2-AHPDSVIGS is positive control plant of peanut silencing AhPDS gene, pTRV2-AhBI-1vigs is experimental plant of peanut silencing AhBI-1.
FIG. 5 is a graph showing the results of gene expression level detection of AhBI-1: CK is normal culture of Zhonghua No. 2, pTRV 2-empty vector is a plant which does not contain target gene and only transfers pTRV2 empty vector into peanut, and 1-24 is AhBI-1 infected peanut. Expression significance at test level 0.05, abc represents a significance analysis
Detailed Description
In the following examples: if not specified, the implementation methods are all conventional methods; the materials used are all purchased from conventional biochemical reagent stores; in the embodiment, the mass percentages are all unless otherwise specified; wherein the quantitative test is carried out by setting three times of repeated experiments, and the results are averaged. The primers involved in the experiments are shown in Table 1:
TABLE 1 primer list
EXAMPLE 1 obtaining of Positive bacterial liquid
1. Extraction of peanut root tip/leaf RNA
The RNA extraction method is described with reference to the general RNA extraction kit instructions of Promega (Shanghai) biological products Co., ltd. The operation steps are as follows:
(1) Preparing special gun heads and centrifuge tubes, mortar rod, medicine spoon and other devices, and sterilizing at 200 deg.c for 4 hr.
(2) Before using the mortar, precooling the sample by using liquid nitrogen, placing about 0.2g of the sample into the precooled mortar, adding enough liquid nitrogen, lightly squeezing the sample by using a mortar rod, quickly grinding the sample to be fine powder when a small amount of liquid nitrogen remains, and repeating the steps for 1-2 times (the liquid nitrogen is not volatilized to prevent RNA degradation when the sample is ground).
(3) Transferring the ground sample powder into a precooled centrifuge tube, adding 500 mu L of lysate, blowing and mixing uniformly, adding 500 mu L of diluent, blowing and mixing uniformly, and standing for 5min at room temperature.
(4) Centrifuging at 12,000rpm for 5min, sucking the supernatant, transferring to a new 1.5mL centrifuge tube, adding 500 μl of absolute ethanol, blowing, and mixing; the mixture was subjected to centrifugation at 12,000rpm for 45sec (the mixture was subjected to column chromatography several times when the sample amount was large).
(5) 600. Mu.L of the eluate was added thereto, and the mixture was centrifuged at 12,000rpm and 4℃for 45sec.
(6) Mu.L of incubation (nuclease free water: 40. Mu.L, DNase I: 5. Mu.L, 10 XDNase I buffer: 5. Mu.L) was added and incubated at room temperature for 15min.
(7) 600. Mu.L of the eluate was added thereto, and the mixture was centrifuged at 12,000rpm and 4℃for 45sec.
(8) The elution was repeated once and idling was carried out for 2min.
(9) The column was relocated to a new elution tube, 40. Mu.L of nuclease-free water was added, and the mixture was allowed to stand at room temperature for 2min at 12,000rpm and centrifuged for 1min.
(10) The eluate was again added to the column and eluted once, and the concentration was measured.
2. CDNA Synthesis
According to Northenan Biotech Co LtdIII RT SuperMix for qPCR (+ GDNA WIPER) kit instructions, reverse transcription was performed to obtain cDNA, steps were as follows:
(1) RNA template, 5× HiScript III qRT Supermix, 4× GDNA WIPER and RNase-Free ddH 2 O were dissolved and placed in ice box for use.
The reaction system was formulated as follows in table 2:
TABLE 2 reaction system
Gently beating and mixing by a pipetting gun. 42 ℃ for 2min.
(2) Directly adding 5X HiScript III qRT Supermix into the reaction tube of the first step
TABLE 3 first step reaction System
The mixture was gently beaten and mixed by a pipette, and reverse transcription was performed under the following conditions.
TABLE 4 reverse transcription procedure
3. Construction of pTRV2-AhBI-1vigs expression vector
Specific primers (pTRV 2-AhBI-1R/F and pTRV2-AhBI-1vigs R/F) are designed according to the gene sequence information, peanut cDNA is used as a template to amplify target fragments (see SEQ ID No.2 of the sequence table), the reaction system is shown in the following table 5, and the PCR reaction program is referred to 2×)Max Master Mix (Dye Plus) instruction.
TABLE 5 PCR reaction system (total volume 20. Mu.L)
The reaction procedure was as follows:
After the PCR product is detected by 1% agarose gel electrophoresis, the target gene fragment is purified and recovered by a DNA purification and recovery kit, and the concentration of the target gene fragment is measured.
Extracting pTRV2 plasmid, selecting BamHI and EcoRI enzyme cutting sites, designing specific primers, adding homologous arm sequences with proper length, and carrying out double enzyme cutting on the vector plasmid; the target fragment and the linearized pTRV2 vector are connected by homologous recombination.
E.coli DH5 alpha is transformed, and monoclonal colony is selected for bacterial liquid PCR identification to be positive.
4. GV3101 competence transformed with recombinant plasmid
Preparation of GV3101 competent cells
(1) Melting GV3101 strain stored at-80deg.C in ice box, streaking on YFP plate containing resistance (Rif: 25mg/L; str:25 mg/L), and culturing in a constant temperature incubator at 28deg.C for 2-3d to obtain single colony.
(2) Single colonies were picked and inoculated into 5mL YFP liquid medium (Rif: 25mg/L; str:25 mg/L), 28℃at 180rpm, and shake-cultured overnight.
(3) According to the following steps of 1:50 were inoculated into a new 100mL of resistant (Rif: 25mg/L; str:25 mg/L) YEP liquid medium and cultured until OD600 was 0.5.
(4) The bacterial liquid after the culture is ice-bathed for 30min, and simultaneously, reagents and materials (centrifuge tubes, EP tubes, gun heads and the like) required by competence are prepared by precooling.
(5) And (3) subpackaging the bacterial liquid into a 50mL centrifuge tube in an ultra-clean bench, centrifuging at 4 ℃/5000rpm for 10min, and discarding the supernatant.
(6) After the residual liquid was removed as much as possible, the supernatant was discarded by gentle resuspension with pre-chilled 20mM CaCl 2 (10% glycerol), centrifugation at 4℃at 5000rpm for 10min.
(6) Adding appropriate amount of 20mM CaCl 2 (containing 10% glycerol) to resuspend thallus, sub-packaging 200 μl per tube, vertically placing tweezers into liquid nitrogen for quick freezing, and preserving at-80deg.C for use.
(II) transformation of GV3101 competent cells
(1) The clone competent cells (DH 5. Alpha. Competent cell) were thawed on ice.
(2) 10. Mu.L of the recombinant product was added to 100. Mu.L of competent cells in liquid form, and the mixture was gently stirred (mixed without shaking) and allowed to stand on ice for 30min.
(3) After heat shock in a water bath at 42 ℃ for 60sec, the mixture is immediately placed on ice for cooling for 3-5min.
(4) 900. Mu.L of liquid LB medium (without antibiotics) was added and resuscitated at 37℃for 1h (rotation speed 200 rpm).
(5) At the same time, LB solid medium plates of the corresponding resistance (Kana) 100 ug/ml+rifampin (Rif) 25 ug/ml+streptomycin (Str) 25 ug/ml) were pre-heated in an incubator at 37 ℃.
(6) The bacteria were resuspended by pipetting, 100. Mu.L of the resuspended bacteria were gently spread on plates containing the correct resistance with sterile spreading bars, the plates were sealed and labeled.
(7) Culturing in an incubator at 37deg.C for 12-16 hr.
(8) Single colonies with consistent growth vigor on the transformation plate are selected for bacterial liquid PCR identification, and a reaction system for preserving positive clone bacterial liquid (50% glycerol 1:1 preservation) is shown in the following table 6.
TABLE 6 PCR reaction system (total volume 10. Mu.L)
And detecting the PCR amplification product by 1% agarose gel electrophoresis, selecting bacterial liquid with correct bands for carrying out detection, carrying out sequence comparison, and carrying out subsequent experiments with correct sequences.
Results: the positive bacterial liquid containing the target gene is successfully obtained.
Example 2 Agrobacterium-mediated pollen tube channel method for transformation of peanuts
1. Injection of
Selecting full peanut seeds (variety: zhonghua No. 2), husking, accelerating germination for 3-4d in wet perlite at the temperature of 26+/-2 ℃, peeling the seed coats after the seeds germinate, transferring the seeds into an improved Hoagland culture solution for water culture for 3-4d, wherein the culture condition is 26+/-2 ℃, illumination is 30-50 mu mol m -2s-1, and illumination period is 16h/8h, and carrying out infection work when a third true leaf appears.
Preparing fresh dyeing liquor (pTRV 1, pTRV2-AhBI-1vigs, pTRV 2-AHPDSVIGS)
(1) Culture broths (pTRV 1, pTRV2-AhBI-1vigs, pTRV 2-AHPDSVIGS): taking fresh bacterial liquid (positive after PCR) and respectively culturing (28 ℃ C., 200 rpm) in 200mL of LB liquid culture medium (containing Kana:100 mug/mL, rif:25 mug/mL, str:25 mug/mL) overnight to make OD 600 reach about 0.8-1.0;
(2) Centrifuging at 5000rpm for 10min, and collecting thalli;
(3) Washing the thalli with a small amount of LB liquid, and centrifuging again;
(4) The cells were resuspended in LB liquid medium (10 mM MES (2-morpholinoethanesulfonic acid), 10mM MgCl 2 (magnesium chloride) and 100 mM AS (acetosyringone) to an OD 600 of about 1.0.
And (3) re-suspending the bacterial liquid, and gently mixing the re-suspended bacterial liquid with PTRV1 re-suspended bacterial liquid in equal volume, and incubating for 3-5h at room temperature. The pre-cultured peanut hypodermis leaf is injected in the early morning, and the infection area is at least 75% of the leaf area. Dark culturing for 36-48h, normal light culturing, observing plant leaf phenotype change, and detecting expression of AhBI-1 by taking new leaves at the moment when yellow She Xianxiang with different degrees appears in positive comparison of about two weeks, namely constructing a peanut AhBI-1 gene silencing system.
2. Verification of Positive plants
Two weeks after injection when yellow She Xianxiang was observed in the positive control, new leaves were taken to detect AhBI-1 expression. RNA from the new leaves was extracted, reverse transcribed into cDNA, and the amount of expression of AhBI-1 was quantitatively determined by fluorescence, and qPCR (primer: qAhBI-1R/F) was mainly performed using ChamQ Universal SYBR QPCR MASTER Mix (Vazyme). The specific reaction system is shown in Table 7, and the reaction procedure is shown in Table 8.
TABLE 7 qPCR reaction system (total volume 20. Mu.L)
Table 8 qPCR reaction system (total volume 20. Mu.L)
The specific annealing temperature and the required extension time are adjusted and set by referring to the specific information of each gene. Three biological replicates were set and data processing was calculated using the 2 -ΔΔCt method.
Results: the peanut plants containing the target genes are successfully screened, the positive rate conversion rate is 62.5%, the difference of growth phenotypes is obvious, and the peanut plants can be used for subsequent research of related functions of the genes.
Sequence listing
<110> University of Guangxi
<120> Peanut AhBI-1 Gene VIGS silencing System
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ctatgttgcg ctgtggttgc ttcagctgtt ggtgcttacc ttcatgtgct gtggaatatt 180
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ccttttgaag agcaaaagag ggttactttg ttgatggttt cggccctgtt tcaaggtgcc 300
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atggattctt ttacttcgtt cttcgattct tcccgaaccc gctggagcta cgatgctctc 60
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tggaggtcta ctcactgct 19
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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aagataaggc ttggttca 18

Claims (3)

1. The application of a gene specific fragment for silencing a peanut AhBI-1 gene in constructing a peanut dwarfing phenotype is characterized in that the gene specific fragment is AhBI-1vigs and is a base sequence of a sequence table SEQ.ID.NO. 2.
2. Use of VIGS viral vector for silencing peanut AhBI-1 gene in constructing peanut dwarf phenotype, characterized in that the VIGS viral vector is pTRV2 vector containing the gene specific fragment of claim 1, which is recombinant viral vector pTRV2-AhBI-1VIGS, constructed by ligating the gene specific fragment of claim 1 into VIGS viral backbone vector pTRV 2.
3. Use of a peanut AhBI-1 gene VIGS silencing system product in the construction of a peanut dwarf phenotype, characterized in that the silencing system product comprises a gene-specific fragment as defined in claim 1 or a VIGS viral vector as defined in claim 2.
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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1419541A (en) * 2000-03-27 2003-05-21 应用研究系统Ars股份公司 Pharmaceutically active pyrrolidine derivatives as BAX inhibitors
DE102004024184A1 (en) * 2004-05-13 2006-01-26 Basf Plant Science Gmbh Novel nucleic acid sequences and their use in methods for achieving pathogen resistance in plants
BRPI0408286A (en) * 2003-03-12 2006-03-07 Basf Plant Science Gmbh process for generating or increasing plant resistance to at least one biotic or abiotic stress factor, polypeptide sequence encoding a bi1 protein, nucleic acid sequence, recombinant expression cassette, recombinant vector and organism
WO2011084718A1 (en) * 2009-12-17 2011-07-14 The Curators Of The University Of Missouri Plant genes associated with seed oil content and methods of their use
CN102660555A (en) * 2012-05-03 2012-09-12 广西大学 Peanut senescence gene for regulating programmed cell death, coding sequence and application
CN102892890A (en) * 2010-03-18 2013-01-23 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and method for making the same
CN102939383A (en) * 2009-12-30 2013-02-20 先锋国际良种公司 Methods and compositions for targeted polynucleotide modification
CN103740753A (en) * 2013-12-28 2014-04-23 杭州师范大学 Application of BI-1 gene in promoting growth of catharanthus roseus cells and method
CN111328287A (en) * 2017-07-04 2020-06-23 库瑞瓦格股份公司 Novel nucleic acid molecules
CN113736819A (en) * 2021-09-02 2021-12-03 浙江大学 Method for constructing butterfly flower gene silencing system

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8014957B2 (en) * 2005-12-15 2011-09-06 Fred Hutchinson Cancer Research Center Genes associated with progression and response in chronic myeloid leukemia and uses thereof

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1419541A (en) * 2000-03-27 2003-05-21 应用研究系统Ars股份公司 Pharmaceutically active pyrrolidine derivatives as BAX inhibitors
BRPI0408286A (en) * 2003-03-12 2006-03-07 Basf Plant Science Gmbh process for generating or increasing plant resistance to at least one biotic or abiotic stress factor, polypeptide sequence encoding a bi1 protein, nucleic acid sequence, recombinant expression cassette, recombinant vector and organism
DE102004024184A1 (en) * 2004-05-13 2006-01-26 Basf Plant Science Gmbh Novel nucleic acid sequences and their use in methods for achieving pathogen resistance in plants
WO2011084718A1 (en) * 2009-12-17 2011-07-14 The Curators Of The University Of Missouri Plant genes associated with seed oil content and methods of their use
CN102939383A (en) * 2009-12-30 2013-02-20 先锋国际良种公司 Methods and compositions for targeted polynucleotide modification
CN102892890A (en) * 2010-03-18 2013-01-23 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and method for making the same
CN102660555A (en) * 2012-05-03 2012-09-12 广西大学 Peanut senescence gene for regulating programmed cell death, coding sequence and application
CN103740753A (en) * 2013-12-28 2014-04-23 杭州师范大学 Application of BI-1 gene in promoting growth of catharanthus roseus cells and method
CN111328287A (en) * 2017-07-04 2020-06-23 库瑞瓦格股份公司 Novel nucleic acid molecules
CN113736819A (en) * 2021-09-02 2021-12-03 浙江大学 Method for constructing butterfly flower gene silencing system

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
A novel VIGS methoed by agroinoculation of cotton seeds and application for elucidating functions of GhBI-1 in salt-stress response;Jingxia Zhang等;Plant Cell Rep;全文 *
An efficient method for the production of transgenic peanut plants by pollen tube transformation mediated by Agrobacterium tumefaciens;Zhou Min;Plant Cell;20230327;全文 *
Predicted:Arachis hypogaea bax inhibitor 1(LOC112802210），mRNA;NCBI;Genbank Database;Accession No.XM_025845315.2 *
花生AhBI-1的克隆、生物信息学分析、亚细胞定位及其在铝胁迫下的表达分析;罗锦等;分子植物育种;第4页倒数第二段 *

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