CN117431262A - Application of BjuPP2C52 gene in resistance breeding of stem tumor mustard - Google Patents
Application of BjuPP2C52 gene in resistance breeding of stem tumor mustard Download PDFInfo
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Abstract
The invention belongs to the technical field of plant molecular breeding, and particularly relates to application of BjuPP2C52 gene in resistance breeding of stem nodule. The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2. The over-expression vector of the gene is transferred into stem tumor mustard, and a large amount of fur appears on leaves and stem segments of the obtained transgenic plant. The plant epidermal hair is the apophysis of pointed protrusion or epidermal tissue caused by glandular hair and cortical cell deformation, and has the defensive effects of increasing plant strength, avoiding plant being gnawed by various animals, reducing water evaporation and the like. The research result also provides a molecular genetic improvement target for brassica plant resistance breeding including stem tumor mustard. Aphids and viral disease transmission caused by the aphids are one of the main factors influencing the yield of the stem nodule mustard, and the cultivation of the transgenic plants of the scheme is favorable for improving the disease and pest resistance of the stem nodule mustard, and has a great popularization and application prospect.
Description
Technical Field
The invention belongs to the technical field of plant molecular breeding, and particularly relates to application of BjuPP2C52 gene in resistance breeding of stem nodule.
Background
Stem mustard (Brassica junsea var. Tumida Tsen et Lee), commonly known as green mustard, is a variety of Brassica species of brassicaceae, a characteristic vegetable in China, and has a cultivation history of nearly 300 years. Chongqing Fuling is the largest planting and processing base of the stem mustard, and the tumor stem which is deformed and developed is the main economic harvest of the stem mustard, is the main raw material for preparing Fuling hot pickled mustard tuber, and can be eaten fresh. The Fuling green vegetable head is identified as the first brand of vegetables in Chongqing, and the Fuling area is the country of the Chinese green ecological green vegetable head. Although the planting of the stem mustard brings ideal economic benefit, in the planting process, aphids and viral disease transmission caused by the aphids are one of main factors influencing the yield, and how to control the damage of the aphids with high efficiency and safety is a technical problem which is solved by the technicians in the field for a long time.
Plant epidermal hair is the apoplectic production of pointed projections or epidermal tissue caused by the deformation of glandular hair and cortical cells, and is widely distributed over many plant leaves, fruits, stems and other organs. The leaf coat has various defensive properties, and can be classified into protective, secretory and absorptive functions according to their functions, so that external mechanical damage and insect diseases can be avoided (Kenzo et al, 2008;Balkunde et al, 2010;Zalucki et al, 2012;Hamaoka et al, 2017). Therefore, by promoting the formation and growth of the epidermal hair of the stem nodule (plant), the defensive power of the stem nodule can be enhanced to a certain extent, and the damage of diseases, insects and the like can be reduced.
The person skilled in the art researches on genes and action mechanisms affecting the epidermal hair growth of the stem tumor mustard, and discovers that the epidermal hair character of the leaf of the stem tumor mustard is controlled by two pairs of nuclear genes, and the leaf with the epidermal hair is incompletely dominant to the no-epidermal hair and has additive effects (Liu Yihua, etc. 2006). About 40-70 genes related to epidermal hair are found in Arabidopsis thaliana, wherein the major gene GL1 (Glabrous 1) inhibits epidermal hair formation (Kim, et al 2010). Genes GL1, GL3 (Glabrous 3) and TTG1 (Transparent testa glabra 1) are all involved in transcriptional regulation of plant coat hair, thereby indirectly affecting coat hair morphology and formation (Payne, et al, 2000). GL1 is highly expressed in development, and is involved in regulating early development of epidermal cell trichomes, GL3 is mainly involved in controlling the number of epidermal hairs, and TTG1 is involved in forming epidermal hairs. GL1, GL3 and TTG1 encode R2R3 class MYB transcription factor, bHLH transcription factor and WD40 protein, respectively, which together form a WD40-bHLH-MYB protein complex, which regulates expression of downstream genes and ultimately forms the coat (Payne et al.,2000;Zhao et al,2008;Pesch et al,2009). However, no gene capable of directly regulating the epidermal hair development of the stem nodule mustard has been found at present, and a certain obstacle is caused to the genetic breeding of the stem nodule mustard due to the lack of molecular genetic improvement targets for resisting insect attack.
Disclosure of Invention
The invention aims to provide an application of BjuPP2C52 gene in resistance breeding of stem mustard, so as to solve the technical problem that a molecular genetic improvement target spot with an insect-resistant effect on stem mustard is lacking in the prior art.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the BjuPP2C52 gene is applied to resistance breeding of stem tumor mustard, and the nucleotide sequence of the BjuPP2C52 gene is shown in SEQ ID NO. 1.
Further, the protein sequence encoded by the BjuPP2C52 gene is shown as SEQ ID NO. 2.
Further, the BjuPP2C52 gene is overexpressed in the stem tumor mustard plant.
Further, the application of BjuPP2C52 gene in the resistance breeding of the stem nodule comprises the following steps in sequence:
s1: constructing an over-expression vector integrated with BjuPP2C52 gene and transferring into agrobacterium;
s2: using agrobacterium to dip the cotyledon of the stem nodule mustard seedling, and then obtaining a T0 generation plant through induction culture; and (3) carrying out selfing for multiple generations after screening to obtain the stem nodule mustard transgenic plant with stable characters.
Further, a large amount of coat appears on the leaves and stem segments of the stem nodule transgenic plants.
Further, in S1, extracting total RNA in the bud of the stem tumor mustard in the bud stage, reversely transcribing the total RNA into cDNA, and carrying out PCR amplification on the cDNA by using upstream and downstream primers shown as SEQ ID NO.3 and SEQ ID NO.4 to obtain BjuPP2C52 gene fragments; the BjuPP2C52 gene fragment was integrated into the p1300-GFP vector to obtain an over-expression vector.
Further, in S1, the expression vector is transferred into agrobacterium GV3101 competent cells using liquid nitrogen freeze-thawing.
Further, in S2, using agrobacterium to dip the cotyledon of the stem nodule mustard seedling, sequentially inducing to form a callus, inducing to form an adventitious bud, and inducing to root, and finally obtaining a T0 generation plant; and (3) carrying out selfing for multiple generations after screening to obtain the stem nodule mustard transgenic plant with stable characters.
The technical scheme also provides application of the BjuPP2C52 gene in promoting the occurrence of a large number of fur on stems and leaves of stem tumor mustard, and the nucleotide sequence of the BjuPP2C52 gene is shown as SEQ ID NO. 1; the protein sequence is shown as SEQ ID NO. 2.
The technical scheme also provides application of the BjuPP2C52 gene in improving the resistance of the stem tumor mustard pests, and is characterized in that: the BjuPP2C52 gene is overexpressed in the stem nodule mustard plant, so that a large number of fur appears on the leaves and stem segments of the stem nodule mustard, and the capability of resisting harmful organisms of the stem nodule mustard is improved; the pest includes aphids;
the nucleotide sequence of the BjuPP2C52 gene is shown as SEQ ID NO. 1; the protein sequence is shown as SEQ ID NO. 2.
Wherein, the pest infection comprises the damage of pests (such as aphids) and the like to plants and the damage of various harmful microorganisms to the plants.
The principle of the scheme is as follows:
the inventor researches and discovers that BjuPP2C52 gene promotes the growth of a large amount of coat on leaves and stem segments of plants by over-expressing BjuPP2C52 gene in the stem mustard, improves the defense mechanism of the stem mustard, reduces the harm of diseases and insects and reduces pesticide application. The coat hair itself is a defense mechanism for common plants, and it is widely recognized by those skilled in the art of plant breeding that plants with high densities of surface hairs have significantly reduced insect pests because the presence of coat hair prevents the plant from being gnawed by various types of animals (e.g., insect pests). Meanwhile, the existence of the surface fur can increase the plant strength, and the occurrence of mechanical damage is avoided. In addition, a certain amount of epidermis hair grows on the plant stem and leaf, so that the moisture can be prevented from being lost through the air holes, the moisture waste is reduced, and meanwhile, the direct irradiation of sunlight is reduced, so that the temperature of the leaf is not too high, and the plant growth is facilitated. The technical proposal provides a regulatory gene for the genetic improvement of the stem nodule, provides more selection possibilities for plant genetic engineering, and simultaneously provides support for the improvement of the stem nodule by a molecular means.
Protein Phosphatases (PPs) are involved in regulating protein phosphorylation by reversing the action of protein kinases, including the general classes tyrosine phosphatases and serine/threonine phosphatases 2, where serine/threonine phosphatases can be classified into the general classes protein phosphatase 1 (PP 1) and protein phosphatase 2 (PP 2) 2, and PP2 can be classified into the general classes PP2A, PP B and PP2C 3. PP2Cs are mainly involved in regulating the protein kinase pathway, are negative regulators activated by different environmental stresses and developmental signals, and are divided into different subfamilies according to their sequence similarity (Kerk et al, 2002; liu et al, 2013). The C-terminal of the PP2Cs family protein contains a highly conserved catalytic domain; the N-terminus is a hypervariable domain containing protein interactions, localization and autorepression of phospholipase activity sequences, which play an important role in plant signaling. The research shows that the PP2Cs A family genes in plants are negative regulators of ABA reaction; the B family genes mainly regulate plant stomatal opening, seed germination, abscisic acid-induced gene expression, and in addition, the B family genes can also bind to MAPK to activate their activity (ubrasaite et al, 2010). In arabidopsis, the AtPP2C52 gene belongs to the PP2Cs E family, and AtPP2C52 can interact with AtAGB1 to regulate the GA signaling pathway of arabidopsis (Tsugama et al 2012;Umbrasaite et al, 2010). It can be seen that in arabidopsis, the AtPP2C52 gene function is not directly related to plant defense mechanisms such as the formation of plant surface coat. In the prior art, although the PP2C52 gene of Arabidopsis thaliana is studied, no study on the function of the BjuPP2C52 gene of the stem tumor mustard is reported, and the specific function is still unclear. Prior to this study, the inventors have not contemplated that the PP2C52 gene (BjuPP 2C52 gene) in the stem mustard is associated with the formation of plant coat hair. The technology fills the blank of the prior art, creates conditions for molecular genetic breeding of brassica plants such as stem tumor mustard and the like, and can improve the mechanical injury resistance and insect-repellent capacity of crops through a mode of over-expressing BjuPP2C52 genes.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention clones the full-length sequence of the BjuPP2C52 gene of the stem tumor mustard for the first time, obtains the coded protein sequence, and overexpresses the gene in the stem tumor mustard, and the test result shows that the overexpression of the gene can enable leaves and stem segments to grow a large number of fur. The invention provides an important target point for genetic improvement of the anti-insect molecule of brassica plants, and has important value and significance for cultivation and production of new varieties of stem mustard.
(2) The invention discovers through observing the phenotype of over-expression and wild plants that the over-expression BjuPP2C52 can lead leaves and stem segments to generate a large amount of fur, and provides an effective gene for the genetic engineering of stem nodule and other plants. The method lays a foundation for cultivating new insect-resistant germplasm of the stem nodule mustard in future. Therefore, the invention has strong application prospect and popularization value.
Drawings
FIG. 1 is a diagram showing the amplification of the BjuPP2C52 gene of the stem tumor mustard of example 1 (M: marker) according to the present invention.
FIG. 2 is an electrophoresis chart of the PCR detection result of the genomic DNA of the transgenic stem tumor mustard of example 2 of the present invention (lanes W1-W3 are wild type controls, M is marker, and 1-20 are 20T 0 transgenic plants obtained).
FIG. 3 is a graph showing the results of detecting the expression level of BjuPP2C52 mRNA in transgenic phoma mustard according to example 2 of the present invention (WT is a wild-type control, OE1, OE2 and OE3 are 3 BjuPP2C52 overexpressing plants obtained by screening).
FIG. 4 is a graph showing the leaf and stem segment patterns of transgenic nodulizer plants and wild type plants in example 2 of the present invention (WT is wild type control, OE1, OE2 and OE3 are 3 BjuPP2C52 overexpressing plants obtained by screening).
FIG. 5 is a photograph of an outdoor grown transgenic phoma mustard of example 2 of the present invention (example plant one).
FIG. 6 is a photograph of an outdoor grown transgenic phoma mustard of example 2 of the present invention (example plant two).
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art unless otherwise indicated; the experimental methods used were all conventional and can be carried out according to the recombinant techniques described (see molecular cloning, laboratory manual, 2 nd edition, cold spring harbor laboratory Press, cold spring harbor, N.Y.; maXetal, arobast CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants.molecular plant.2015,8 (8): 1274-1284.); the materials, reagents, and the like used are all commercially available.
Example 1: full-length gene clone of stem tumor mustard BjuPP2C52 and construction of over-expression vector
The bud of the stem tumor mustard in the bud stage preserved at the temperature of minus 80 ℃ is taken as a material, a total RNA is extracted according to the specification steps by adopting a total RNA extraction kit (RNAprep pure Plant Kit) of a root plant, 2 mu L of total RNA of the bud of the stem tumor mustard in the bud stage is taken, and the first strand cDNA is synthesized by reverse transcription according to the specification steps of All-in-One 5 xRT Master mix (G592, abm).
The reverse transcription procedure is: 37 ℃ for 15min;60 ℃ for 10min;95℃for 3min.
Using the reverse transcription synthesized cDNA as a template, the BjuPP2C52 coding sequence was amplified using high fidelity enzyme (MegaFiTM Fidelity x PCR MasterMix), PCR amplification system: primix 25. Mu.L, forward primer (BjuPP 2C52-F, 10. Mu.M) 2. Mu.L, reverse primer (BjuPP 2C52-R, 10. Mu.M) 2. Mu.L, template(cDNA) 2. Mu.L, sterile ddH 2 O was made up to 50. Mu.L.
The forward primer and the reverse primer are as follows:
BjuPP2C52-F：5’-GAGAACACGGGGGACTCTAGAATGGGAGGTTGTGTGTCGAC-3’，(SEQ ID NO.3)；
BjuPP2C52-R：5’-GCCCTTGCTCACCATTCTAGATCAAGTCTTGGACTTTTCTTC-3’，(SEQ ID NO.4)。
the PCR amplified products were analyzed by agarose gel electrophoresis, and a specific amplification band was observed at 1kb to 2kb under ultraviolet irradiation, as shown in FIG. 1. The agarose gel was used for recovery using the agarose gel recovery kit (D1200, solarbio) for later use.
The BjuPP2C52 gene fragment obtained by amplification is subjected to recombination connection with a p1300-GFP vector linearized by using XbaI (Thermo Scienfic FastDigest, 91252720) restriction enzyme by using recombination ligase (2 xPro Ligation-Free Cloning MasterMix, E086-1, abm), the product is transformed into escherichia coli DH5a, 2-3 positive clones are picked from an LB culture plate containing calicheamicin sulfate (50 mg/L) and subjected to colony PCR detection, and bacterial liquid with the size of a target strip obtained by amplification is sent to a large gene for sequencing. The result shows that the full-length sequence of the stem tumor mustard BjuPP2C52 gene is shown as SEQ ID NO.1, and the stem tumor mustard BjuPP2C52 gene contains an open reading frame (containing a termination code) of 1398 bp. According to SEQ ID NO.1, the protein sequence of the obtained stem tumor mustard BjuPP2C52 is shown as SEQ ID NO.2 and contains 466 amino acids (representing termination signals) through translation by Primer premier software.
The nucleotide sequence of BjuPP2C52 gene is shown as SEQ ID NO.1, and the protein sequence is shown as SEQ ID NO. 2.
SEQ ID NO.1(5’→3’)：
ATGGGAGGTTGTGTGTCGACTAGTAGTAAGAGTACGTGTAGCAGCTGGAGCAATGGAGAGAAGCCAATGCCTCGTCCATATCTTGGGATCGGTTGTTGTGTGAGCAAAAGGGCGAAGAGAACGTTTTCAGACCATATCCTCTCACTCCAGAACTTGGCCTCTATACCAAACCGTATCATCACCAGCGGCAAGAGCAGGAGCTCTTGCATTTTCACTCAACAAGGACGCAAGGGTATTAACCAAGACGCCATGATTGTGTGGGAAGATTTTATGTCTGAGGATGTGACGTTTTGTGGTGTATTTGATGGTCATGGTCCTTTTGGTCATCTTGTTGCTCGTAAAGTGAGAGATACGTTGCCTGTGAAGTTGCAGTCTTTCTTTCATGCGCTTCAGTCAAAGCAAAACGGTCGGTTCAGAAGAAGTTCAAGCAAATCAGCTGTCCAGGAAGCTGTTAAAGAAGGAACCGATGAAGATAAACTAAAAGGCTTGTGGGGAGAAGCTTTCTTTAAATCATTTAAGGCCATGGACAAGGAACTGCGGTCTCACCCTAATGTGGATTGTTTCTGTAGTGGTAGCACTGCTGTTACAATTCTCAAACAGGGGTCTAATCTCTTCATGGGGAACATTGGTGATTCTCGAGCCATTCTTGGATCAAAAGACAGTAATGATACCATGGTAGCAACTCAACTAACTGTTGATCTGAAACCTGATTTACCGAGGGAAGCTGAGAGGATTAAACGGTGCAAAGGCCGTGTCTTTGCTCTGCAAGATGAGCCAGAGGTGTCACGAGTTTGGCTACCTTTTGATGACGCTCCTGGACTGGCCATGGCTAGGGCGTTTGGTGACTTCTGTCTGAAAGAGTATGGAGTCATTTCGATACCTGAGTTCACTCACCGTGTCCTTACAGACAAAGACCAGTTCATTGTTCTTGCCTCTGATGGAATATGGGACGTGCTAAGCAACGAAGAAGTGGTCGATATTGTAGCTTCATCTTCAAGCCGGGCATCAGCAGCTAGGATCTTGGTGAACTCGGCTGCACGTGAGTGGAAACTGAAGTATCCAACTTCAAAAATGGACGACTGTGCAGTTGTCTGTTTGTTTCTTGATGGGAAAATGGATTCTGAGTCGGATTACGATGAGCAGGGCTTCTCTTCAGCCACAAATGCTGTGGAATCAGATGATGGACAAAGATCAGAACCGTGTCTACAAAGAAACTTCACAGTTAGATCATCATCAGATCAAGAAAACGAGACGTATGGTAATAATGTGAATGCAGATACTGAGGGAGAGGATGAGAAAACTGTGGGAGATCAAAACTGGTTGGGGCTGGAAGGTGTTACAAGAGTGAACTCACTCGTTCAGCTCCCGAGATTCTCTGAAGAAAAGTCCAAGACTTGA；
SEQ ID NO.2(N→C)：
MGGCVSTSSKSTCSSWSNGEKPMPRPYLGIGCCVSKRAKRTFSDHILSLQNLASIPNRIITSGKSRSSCIFTQQGRKGINQDAMIVWEDFMSEDVTFCGVFDGHGPFGHLVARKVRDTLPVKLQSFFHALQSKQNGRFRRSSSKSAVQEAVKEGTDEDKLKGLWGEAFFKSFKAMDKELRSHPNVDCFCSGSTAVTILKQGSNLFMGNIGDSRAILGSKDSNDTMVATQLTVDLKPDLPREAERIKRCKGRVFALQDEPEVSRVWLPFDDAPGLAMARAFGDFCLKEYGVISIPEFTHRVLTDKDQFIVLASDGIWDVLSNEEVVDIVASSSSRASAARILVNSAAREWKLKYPTSKMDDCAVVCLFLDGKMDSESDYDEQGFSSATNAVESDDGQRSEPCLQRNFTVRSSSDQENETYGNNVNADTEGEDEKTVGDQNWLGLEGVTRVNSLVQLPRFSEEKSKT*。
The p1300-BjuPP2C52-GFP plasmid in E.coli described above was extracted and purified using a plasmid extraction kit (D6943, omega). Transferring the p1300-BjuPP2C52-GFP over-expression vector into competent cells of agrobacterium GV3101 (AC 1001, shanghai only) by using a liquid nitrogen freeze thawing method, selecting a monoclonal culture until bacterial liquid is turbid, adding sterilized 50% glycerol into the bacterial liquid in an equal volume, and storing the bacterial liquid at the temperature of-80 ℃ for later use.
Example 2: agrobacterium-mediated transformation of phoma mustard p1300-BjuPP2C52-GFP and screening of transgenic positive plants
The stem nodule mustard seeds were sterilized and sown in 1/2MS medium until the two cotyledons were fully expanded. Culturing the stored Agrobacterium GV3101 bacterial liquid containing p1300-BjuPP2C52-GFP plasmid in YEB (50 mg/mL calicheamicin+20 mug/mL rifampicin) liquid culture medium until the bacterial liquid is turbid, and amplifying culturing to OD according to the ratio of 1:1000 600 About 0.5, the cells were collected by centrifugation at 3000rpm for 10min, and then the collected cells were resuspended in 30g/L sucrose solution to adjust OD 600 =0.01. Then cutting the stem nodule bacteria-free seedling cotyledon with a handle, placing the stem nodule bacteria-free seedling cotyledon with a handle into a resuspended agrobacterium bacterial solution, soaking for 10min, sucking the excessive bacterial solution by using a sterile filter paper, spreading the cotyledon in a co-culture medium (MS+2mg/L6-BA+0.15 mg/L NAA+0.8% agar) for dark culture for 2d, and placing the cotyledon into a callus induction culture medium (MS+2mg/L6-BA+0.15 mg/LNAA+20mg/L hygromycin+500 mg/L timetin+0.8% agar) for culture. After observing compact green callus, cutting the callus, continuously placing the callus in an adventitious bud differentiation culture medium (MS+2mg/L6-BA+0.15 mg/L NAA+20mg/L hygromycin+500 mg/L timentin+0.8% agar) for culture, finally placing the grown adventitious bud in a rooting culture medium (MS+0.2mg/L NAA+20mg/L hygromycin+500 mg/L timentin+0.8% agar) for culture until roots grow out, and transferring the plant into nutrient soil for culture, thus obtaining the plant which is the stem tumor mustard p1300-BjuPP2C52-GFP T0 transgenic plant. PCR and electrophoresis detection are carried out on T0 generation transgenic plants, the experimental results are shown in figure 2, the electrophoresis bands of wild plants W1-W3 do not appear, and the electrophoresis bands shown by red arrows appear in plants with successful transgenesis. The object of electrophoresis detection is a tag protein GFP, and the primer for detecting GFP gene is as follows: GFP-F5'-ATGGTGAGCAAGGGCGAGGAG-3' (SEQ ID NO. 5); GFP-R5'-CTTGTACAGCTCGTCCATGC-3' (SEQ ID NO. 6).
And (3) carrying out selfing on the obtained stem tumor mustard p1300-BjuPP2C52-GFP T0 generation plant for multiple generations, extracting leaf genome, and detecting by using a tag protein GFP primer. And finally, detecting BjuPP2C52 expression quantity of the preliminarily screened positive plants by using qRT-PCR. The primer for detecting BjuPP2C52 gene is as follows:
BjuPP2C52-F：5'-ATGGGAGGTTGTGTGTCGAC-3'，(SEQ ID NO.7)；
BjuPP2C52-R：5'-CATCAAATACACCACAAAAC-3'，(SEQ ID NO.8)。
the results are shown in FIG. 3, and FIG. 3 shows a phenotype of BjuPP2C52 transgene and the growth of wild type stem mustard into four true leaves. Wherein, FIG. 3a shows the phenotype observation of BjuPP2C52 overexpression and wild-type plants, and FIG. 3b shows the detection of BjuPP2C52 overexpression and wild-type plant mRNA expression level. The expression of the gene of interest, bjuPP2C52, was up-regulated in all 3 representative transgenic lines (OE 1, OE2 and OE 3) compared to the wild-type plant (WT), and the OE3 expression level was highest, indicating that BjuPP2C52 had been introduced into the stem tumor mustard genome and successfully transcribed.
Leaf phenotype plots for transgenic and wild-type stem nodule mustard grown to four true leaves are shown in FIG. 4. FIGS. 4a, b, f and g are leaf phenotype observations of BjuPP2C52 overexpression versus wild-type plants; FIGS. 4C-e are Table observations of BjuPP2C52 overexpression and wild-type plant stem segments. Experimental results show that BjuPP2C52 over-expressed plants generate a large amount of coat in the leaves and stem segments, and the coat is a plant defense mechanism and can avoid external mechanical damage and damage of diseases and insect pests. Through the genetic transformation of BjuPP2C52 overexpression of the stem mustard, the stem mustard plant with stronger resistance can be obtained, so that the using amount of insect-proof pesticides is reduced, the yield is improved, and conditions are created for the development of green agriculture.
The above experimental results are all experimental results of cultivating the stem nodule mustard in a laboratory, and the inventors also tried to transfer the stem nodule mustard plant transformed with BjuPP2C52 gene to an outdoor field for planting. The outdoor field planting plant has long nutrition growth time, the plant is relatively thick, and the leaves and stem sections of the plant still generate a large amount of fur. FIGS. 5 and 6 show photographs of BjuPP2C52 gene overexpressing stem mustard cultivated outdoors for two months after sowing, and a large amount of fur on the stem segments and leaves thereof can be seen. Whereas common stem mustard without transgenic manipulation does not have epidermal hairs in the leaves and stem segments.
The foregoing is merely exemplary of the present invention, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present invention, and these should also be regarded as the protection scope of the present invention, which does not affect the effect of the implementation of the present invention and the practical applicability of the patent. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (10)
- The application of BjuPP2C52 gene in resistance breeding of stem nodule is characterized in that: the nucleotide sequence of the BjuPP2C52 gene is shown as SEQ ID NO. 1.
- 2. Use of the BjuPP2C52 gene according to claim 1 in the breeding of resistance to stem nodule mustard, characterized in that: the protein sequence coded by the BjuPP2C52 gene is shown as SEQ ID NO. 2.
- 3. Use of the BjuPP2C52 gene according to any one of claims 1 or 2 in the breeding of resistance to stem nodule, characterized in that: the BjuPP2C52 gene is overexpressed in the stem nodule mustard plant.
- 4. Use of the BjuPP2C52 gene according to claim 3 in the breeding of resistance to stem nodule mustard, characterized in that: the method comprises the following steps of:s1: constructing an over-expression vector integrated with BjuPP2C52 gene and transferring into agrobacterium;s2: using agrobacterium to dip the cotyledon of the stem nodule mustard seedling, and then obtaining a T0 generation plant through induction culture; and (3) carrying out selfing for multiple generations after screening to obtain the stem nodule mustard transgenic plant with stable characters.
- 5. The use of the BjuPP2C52 gene according to claim 4 in the breeding of resistance to stem nodule, characterized in that: a large number of coat hairs appear on leaves and stem segments of the stem nodule mustard transgenic plants.
- 6. The use of the BjuPP2C52 gene according to claim 4 in the breeding of resistance to stem nodule, characterized in that: in S1, extracting total RNA in buds of the stem nodule in the bud stage of the stem nodule mustard, reversely transcribing the total RNA into cDNA, and carrying out PCR amplification on the cDNA by using upstream and downstream primers shown as SEQ ID NO.3 and SEQ ID NO.4 to obtain BjuPP2C52 gene fragments; the BjuPP2C52 gene fragment was integrated into the p1300-GFP vector to obtain an over-expression vector.
- 7. The use of the BjuPP2C52 gene according to claim 6 in the breeding of resistance to stem nodule mustard, characterized in that: in S1, the expression vector was transferred into agrobacterium GV3101 competent cells using liquid nitrogen freeze-thawing.
- 8. The use of the BjuPP2C52 gene according to claim 5 in the breeding of resistance to stem nodule, characterized in that: in S2, using transgenic agrobacterium to dip the cotyledon of the stem nodule mustard seedling, sequentially inducing to form a callus, inducing to form an adventitious bud, and inducing to root to finally obtain a T0 generation plant; and (3) carrying out selfing for multiple generations after screening to obtain the stem nodule mustard transgenic plant with stable characters.
- The application of the BjuPP2C52 gene in promoting the appearance of a large number of coat on stem and leaf of stem tumor mustard, which is characterized in that the nucleotide sequence of the BjuPP2C52 gene is shown as SEQ ID NO. 1; the protein sequence is shown as SEQ ID NO. 2.
- The use of the bjupp2c52 gene for increasing the resistance of a stem tumor mustard pest, characterized in that: the BjuPP2C52 gene is overexpressed in the stem nodule mustard plant, so that a large number of fur appears on the leaves and stem segments of the stem nodule mustard, and the capability of resisting harmful organisms of the stem nodule mustard is improved; the pest includes aphids;the nucleotide sequence of the BjuPP2C52 gene is shown as SEQ ID NO. 1; the protein sequence is shown as SEQ ID NO. 2.
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