CN112694523B - Application of Brassica campestris Bra014815 gene in regulating and controlling branch number and plant type - Google Patents
Application of Brassica campestris Bra014815 gene in regulating and controlling branch number and plant type Download PDFInfo
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Abstract
The invention discloses an application of a Brassica campestris Bra014815 gene in regulation and control of branch number and plant type, which belongs to the technical field of plant genetic engineering, wherein a 196bp fragment on a second exon of the Brassica campestris Bra014815 gene and a pHELLSGATE 12 vector are utilized to construct an RNAi vector of the Bra014815 gene, and an Agrobacterium tumefaciens-mediated flower dipping method (Floral-dip) is utilized to transform Brassica campestris plants, so that the branch number of the Brassica campestris is remarkably increased, the plant height is correspondingly reduced, the apical dominance is weakened, and the pitch spacing of branch parts is shortened. The application of the Bra014815 gene of the cabbage type rape in regulating and controlling the branch number and the plant type is realized by providing the Bra014815 gene for regulating and controlling the plant branches in the cabbage type rape, constructing an RNAi vector containing the gene segment, transferring the gene segment into the cabbage type rape through a transgenic technology, effectively increasing the branches of the cabbage type rape, showing that the Bra014815 gene of the cabbage type rape can be used as a regulating and controlling factor for the branch development of the cabbage type rape, providing reference for researching a plant branch regulating and controlling mechanism, and simultaneously obtaining the multi-branch transgenic cabbage type rape which is a good plant type variety with multiple branches, lodging resistance and suitability for mechanical production.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to an application of a Brassica campestris Bra014815 gene in regulation and control of branch number and plant type.
Background
Brassica is a brassicaceae plant and comprises a plurality of important vegetables and oil crops, such as cabbage type rape, cabbage, rape, cauliflower and the like, wherein the cabbage type rape (Brassica campestris L.) is a cabbage type rape subspecies of Brassica of China, is a raw vegetable of China, has long cultivation history, various varieties and rich nutrition, and is a main vegetable in daily life.
RNA interference is a gene silencing phenomenon induced by double-stranded RNA, can play a role at a transcription level and a post-transcription level, interferes with small segments of RNA which are homologous with a target gene sequence, and efficiently and specifically degrades mRNA, so that an endogenous target gene is silenced, and finally phenotype deletion with corresponding functions is generated. Since the discovery of the phenomenon of RNAi, RNAi technology has been widely used in plant genetic improvement and crop breeding, and has made significant progress.
The apical dominance and lateral branch development of plants play an important role in plant morphogenesis, determine morphological indexes such as height and extension degree of plants, and influence economic indexes such as biomass and fruiting amount. Researches show that high-stalk materials are easy to fall down, while low-stalk materials are reduced in yield, and the key for solving the problems is to culture a new variety which is resistant to fall down and suitable for mechanical production.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the application of a Brassica campestris Bra014815 gene in regulating and controlling the branch number and plant type, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: the application of the Brassica campestris Bra014815 gene in regulating and controlling the branch number and plant type comprises the following specific steps:
s1, selecting plant materials, carriers and strains;
s2, immediately putting fresh cabbage type rape buds of plant materials into liquid nitrogen, extracting the total RNA of the cabbage type rape according to a crude Sangon Biotech total RNA extraction kit, and synthesizing a cDNA first chain according to a Novozan reverse transcription kit;
s3, obtaining a reference sequence of Bra014815 at NCBI, designing a primer with a product of 196bp on a second exon, adding a joint sequence of a pHELLSGATE 12RNAi vector at the 5' end of the primer, amplifying a target fragment by using the primer, and performing agarose gel electrophoresis;
s4, recovering the target fragment by glue, connecting the pHELLSGATE 12 carrier, adding 1 mu l of K protein after 2 hours at the environment temperature of 25 ℃, and reacting for 10 minutes at the environment temperature of 37 ℃;
s5, transforming the ligation product into DH5 alpha escherichia coli, performing inverted culture at 37 ℃ for 12 hours, and performing sequencing verification;
s6, transforming the recombinant plasmid with correct sequencing into agrobacterium GV3101, picking a single clone, shaking the single clone in a test tube for 24h, then carrying out large shaking, and carrying out enlarged culture: adding 100ml YEB culture medium containing SPE antibiotic into a sterile conical flask, transferring the bacterial liquid in the test tube into the conical flask, and continuously shaking the bacteria to OD600＝0.8～1.0；
S7, subpackaging the cultured bacterial liquid in a 50ml centrifuge tube, centrifuging for 10 minutes at the room temperature at 4000rpm, discarding the supernatant, resuspending the bacterial body precipitated in the 50ml centrifuge tube by using the prepared resuspension liquid, and adjusting the bacterial liquid concentration to OD by using the resuspension liquid600＝0.8～1.0；
S8, culturing the cabbage type rape for 4-5 weeks, removing completely opened flowers in the early stage of flowering, leaving buds to be opened, and poking each bud to open one opening by using forceps so that bacterial liquid can enter in the subsequent dip dyeing process;
s9, screening and identifying the RNAi Bra014815 transgenic cabbage type rape;
s10, phenotype analysis of RNAi Bra014815 transgenic brassica napus.
In the step S1, the plant material is brassica campestris in a laboratory climate room, the brassica campestris is selected as the line of choice of brassica campestris 45, the vector is selected as the phelsgate 12RNAi vector, and the strain is selected as DH5 α escherichia coli and GV3101 agrobacterium.
Further optimizing the technical scheme, in the S5, the sequencing verification method comprises the steps of selecting monoclonal colony shake bacteria and quality-improving particles, designing primers at two ends of the insertion position of a target fragment on a vector, verifying whether a target gene is successfully connected into the vector through PCR, and carrying out sequencing verification on recombinant plasmids.
In the S7 technical scheme, the preparation method of the resuspension is to use 1/2MS liquid culture medium containing 5% sucrose, add 50mg/L acetosyringone AS, and add 0.02% surfactant Silwet L-77.
Further optimizing the technical scheme, in the step S8, plants are inclined, inflorescences are sequentially immersed into the bacterial liquid for 20 seconds, the inflorescences in the bacterial liquid are shaken during the period, and after the dip dyeing is completed, the inflorescences are sprayed with clear water and bagging is completed.
Further optimizing the technical scheme, after bagging is finished, the impregnated plant is protected from light for 24 hours, normal culture conditions are recovered after 24 hours, and after five days, a small opening is opened for a newly emerged bud to be bloomed, and the bud which is not suitable for impregnation is removed.
Further optimizing the technical scheme, and after the buds which are not suitable for dip dyeing are removed, carrying out secondary infection according to the same method.
Compared with the prior art, the invention provides the application of the Brassica campestris Bra014815 gene in regulating and controlling the branch number and the plant type, and the invention has the following beneficial effects:
1. the Bra014815 gene of the cabbage type rape is applied to regulation of the branch number and the plant type, an RNAi vector containing the gene segment is constructed by providing the Bra014815 gene for regulating the plant branch in the cabbage type rape, and the gene segment is transferred into the cabbage type rape by a transgenic technology, so that the branch of the cabbage type rape can be effectively increased.
2. The application of the Brassica campestris Bra014815 gene in regulating and controlling the branch number and the plant type shows that the Brassica campestris Bra014815 gene can be used as a regulating and controlling factor for the branch development of Brassica campestris, provides reference for researching a plant branch regulating and controlling mechanism, and the obtained multi-branch transgenic Brassica campestris is a good plant type variety which has multiple branches, is lodging-resistant and is suitable for mechanical production.
Drawings
FIG. 1 is a PCR verification diagram of a target fragment of an RNAi Bra014815 recombinant plasmid for regulating and controlling the branch number and the plant type of a Brassica campestris Bra014815 gene provided by the invention;
FIG. 2 is a sequencing verification diagram of a target fragment of an RNAi Bra014815 recombinant plasmid for regulating and controlling the branch number and the plant type of a Brassica campestris Bra014815 gene provided by the invention;
FIG. 3 is a screening diagram of RNAi Bra014815 transgenic brassica napus on a resistance medium for regulating the branch number and the plant type of the brassica napus Bra014815 gene;
FIG. 4 is a PCR verification diagram of RNAi Bra014815 transgenic brassica rapa of the invention for regulating the branch number and plant type by using Bra014815 gene;
FIG. 5 is a diagram showing the verification of the expression level of Bra014815 gene in RNAi Bra014815 transgenic brassica rapa, which is an application of the brassica rapa Bra014815 gene in the regulation of the number of branches and plant types;
FIG. 6 is a phenotypic graph of RNAi Bra014815 transgenic brassica napus for regulating the branch number and plant type of brassica napus Bra014815 gene;
FIG. 7 is a statistical diagram of plant height of 5 weeks of RNAi Bra014815 transgenic brassica rapa of the invention in the application of Bra014815 gene in controlling the number of branches and plant type;
FIG. 8 is a statistical chart of the number of branches of a transgenic brassica rapa type 5 weeks after RNAi Bra014815 gene is applied to control the number and plant type of branches;
FIG. 9 is a proportion diagram of the length of the main inflorescence of the Bra014815 gene of brassica rapa pekinensis in 5 weeks accounting for the plant height when the RNAi Bra014815 gene regulates the number of branches and the plant type;
FIG. 10 is a statistical chart of the first three internode distances from top to bottom at 5 weeks for RNAi Bra014815 transgenic brassica rapa, which is applied to control the number of branches and plant types of brassica napus Bra014815 gene.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment is as follows:
the application of the Brassica campestris Bra014815 gene in regulating and controlling the branch number and plant type comprises the following specific steps:
s1, selecting plant materials, carriers and strains;
s2, immediately putting fresh cabbage type rape buds of plant materials into liquid nitrogen, extracting the total RNA of the cabbage type rape according to a crude Sangon Biotech total RNA extraction kit, and synthesizing a cDNA first chain according to a Novozan reverse transcription kit;
s3, obtaining a reference sequence of Bra014815 at NCBI, designing a primer with a product of 196bp on a second exon, adding a joint sequence of pHELLSGATE 12RNAi vector at the 5' end of the primer, amplifying a target fragment by using the primer and carrying out agarose gel electrophoresis;
wherein the primer sequences are as follows:
s4, recovering the target fragment by glue, connecting the pHELLSGATE 12 carrier, adding 1 mu l of K protein after 2 hours at the environment temperature of 25 ℃, and reacting for 10 minutes at the environment temperature of 37 ℃;
wherein the connecting system is as follows:
s5, transforming the ligation product into DH5 alpha escherichia coli, performing inverted culture at 37 ℃ for 12 hours, and performing sequencing verification to obtain a result shown in figure 1, thereby completing the construction of the Bra014815RNAi vector;
s6, transforming the cabbage rape by using an agrobacterium-mediated flower dipping method, transforming the recombinant plasmid with correct sequencing into agrobacterium GV3101, selecting a single clone, shaking the single clone in a test tube for 24h, then greatly shaking: adding 100ml YEB culture medium containing SPE antibiotic into sterile conical flask, transferring the bacterial liquid in the test tube into the conical flask, and continuing to shake bacteria to OD600＝0.8～1.0；
S7, subpackaging the cultured bacterial liquid in a 50ml centrifuge tube, centrifuging for 10 minutes at the room temperature at 4000rpm, discarding the supernatant, resuspending the bacterial body precipitated in the 50ml centrifuge tube by using the prepared resuspension liquid, and adjusting the bacterial liquid concentration to OD by using the resuspension liquid600＝0.8～1.0；
S8, culturing the cabbage type rape for 4-5 weeks, removing completely opened flowers in the early flowering stage, leaving buds to be opened, and poking each bud with a pair of tweezers to allow bacterial liquid to enter during subsequent dip dyeing;
s9, screening and identifying the transgenic rapes of the RNAiBra014815 Chinese cabbage type;
wherein, the T0 generation seeds obtained by dip dyeing are sterilized and planted in a KN-containing 1/2MS culture medium according to an aseptic operation, true leaves grown from wild type brassica napus seedlings are whitened, while green true leaves can be grown from transgenic brassica napus seedlings, and the result is shown in figure 3. And (3) transplanting the preliminarily screened transgenic seedlings into soil culture, and cleaning a culture medium at the roots of the seedlings when the seedlings are transplanted. DNA of the soil-cultured seedlings was extracted, and PCR detection was performed using primers designed from pHELLSGATE 12RNAi vector backbone sequences, the results of which are shown in FIG. 4. Then, RNA was extracted from the 3 selected transgenic plants, and the expression level of the Bra014815 gene was detected by fluorescent quantitative PCR, and the results are shown in fig. 5.
S10, phenotype analysis of RNAi Bra014815 transgenic brassica napus.
Wherein, three strains of wild type flowering cabbage 45 and RNAi Bra014815 transgenic flowering cabbage 45 are simultaneously planted and cultured for 5 weeks under the same conditions, as shown in FIGS. 6 to 10, and the plant height, the number of branches, the length of the main inflorescence and the first three internode distances counted from top to bottom of each strain are counted. The results show that the transgenic brassica napus of RNAiBra014815 has reduced plant height, significantly increased number of branches, reduced apical dominance and shortened internode distance of the growing branch part compared with the wild type.
Specifically, in the S1, the plant material is brassica campestris in a laboratory climate room, the brassica campestris is selected as the line of choice of brassica campestris 45, the vector is selected as the phelsgate 12RNAi vector, and the strain is selected as DH5 α escherichia coli and GV3101 agrobacterium.
Specifically, in S5, the sequencing verification method includes selecting monoclonal colony shake bacteria and quality-improved particles, designing primers at two ends of the insertion site of the target fragment on the vector, verifying whether the target gene is successfully linked to the vector by PCR, and performing sequencing verification on the recombinant plasmid, with the result shown in fig. 2.
Specifically, in the S7, the preparation method of the resuspension comprises the steps of adding 50mg/L acetosyringone AS into 1/2MS liquid culture medium containing 5% of sucrose, and adding 0.02% of surfactant Silwet L-77.
Specifically, in S8, the plant is tilted, the inflorescences are immersed into the bacterial liquid for 20 seconds, the inflorescences in the bacterial liquid are shaken during the immersion, and after the immersion dyeing is completed, the inflorescences are sprayed with clear water to complete bagging.
Specifically, the impregnated plant is protected from light for 24 hours after bagging is completed, normal culture conditions are recovered after 24 hours, a small opening is opened for a newly emerged bud to be bloomed after five days, and the bud which is not suitable for impregnation is removed.
Specifically, after the buds which are not suitable for padding are removed, the second infection is carried out in the same manner.
The invention has the beneficial effects that:
1. the Bra014815 gene of the cabbage type rape is applied to regulation of the branch number and the plant type, an RNAi vector containing the gene segment is constructed by providing the Bra014815 gene for regulating the plant branch in the cabbage type rape, and the gene segment is transferred into the cabbage type rape by a transgenic technology, so that the branch of the cabbage type rape can be effectively increased.
2. The application of the Brassica campestris Bra014815 gene in regulating and controlling the branch number and the plant type shows that the Brassica campestris Bra014815 gene can be used as a regulating and controlling factor for the branch development of Brassica campestris, provides reference for researching a plant branch regulating and controlling mechanism, and the obtained multi-branch transgenic Brassica campestris is a good plant type variety which has multiple branches, is lodging-resistant and is suitable for mechanical production.
The research of the invention shows that: the Brassica napus chromatin remodeling factor Bra014815 gene can regulate and control the branch number of Brassica napus, provides a reference value for improving the plant type and lodging resistance of crops by using multi-branch mutants so as to improve the yield of the crops, and has important significance for understanding the mechanism of regulating and controlling the plant growth and development by the chromatin remodeling factor.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. The application of the Brassica campestris Bra014815 gene in regulating and controlling the branch number and plant type of Brassica campestris is characterized by comprising the following specific steps:
s1, selecting plant materials, carriers and strains;
s2, immediately putting fresh cabbage type rape buds of plant materials into liquid nitrogen, extracting the total RNA of the cabbage type rape according to a raw Sangon Biotech total RNA extraction kit, and synthesizing a cDNA first chain according to a Novozan reverse transcription kit;
s3, obtaining a reference sequence of Bra014815 at NCBI, designing a primer with a product of 196bp on a second exon, adding a joint sequence of pHELLSGATE 12RNAi vector at the 5' end of the primer, amplifying a target fragment by using the primer and carrying out agarose gel electrophoresis;
s4, recovering the target fragment by glue, connecting the pHELLSGATE 12 carrier, adding 1 mu l of K protein after 2 hours at the environment temperature of 25 ℃, and reacting for 10 minutes at the environment temperature of 37 ℃;
s5, transforming the ligation product into DH5 alpha escherichia coli, performing inverted culture at 37 ℃ for 12 hours, and performing sequencing verification;
s6, transforming the recombinant plasmid with correct sequencing into agrobacterium GV3101, picking a single clone, shaking the single clone in a test tube for 24h, then turning to big shake: adding 100ml of YEB culture medium containing SPE antibiotics into a sterile conical flask, transferring the bacterial liquid in the test tube into the conical flask, and continuously shaking the bacteria until OD600nm = 0.8-1.0;
s7, subpackaging the cultured bacterial liquid in a 50ml centrifugal tube, centrifuging at 4000rpm at room temperature for 10 minutes, removing supernatant, resuspending the bacterial body precipitated in the 50ml centrifugal tube by using prepared resuspension liquid, and adjusting the concentration of the bacterial liquid to OD600nm = 0.8-1.0 by using resuspension liquid;
s8, culturing the cabbage type rape for 4-5 weeks, removing completely opened flowers in the early flowering stage, leaving buds to be opened, and poking each bud with a pair of tweezers to allow bacterial liquid to enter during subsequent dip dyeing;
s9, screening and identifying the RNAi Bra014815 transgenic cabbage type rape;
s10, phenotype analysis of RNAi Bra014815 transgenic brassica napus.
2. The application of the Brassica napus Bra014815 gene in regulating and controlling the branch number and plant type of Brassica napus as claimed in claim 1, wherein in S5, the sequencing verification method comprises selecting monoclonal bacterial colony shake bacteria and quality-improving particles, designing primers at two ends of the insertion position of a target fragment on a vector, verifying whether the target gene is successfully connected to the vector by PCR, and carrying out sequencing verification on recombinant plasmids.
3. The use of the Brassica napus Bra014815 gene AS claimed in claim 1 for regulating the branch number and plant type of Brassica napus, wherein in S7, the resuspension is prepared by adding 50mg/L acetosyringone AS to 1/2MS liquid medium containing 5% sucrose, and adding 0.02% surfactant Silwet L-77.
4. The use of the brassica napus Bra014815 gene as claimed in claim 1 for regulating the number and plant type of branches of brassica napus, wherein in S8, the plant is tilted, inflorescences are immersed in the bacterial solution one by one for 20 seconds while shaking the inflorescences in the bacterial solution, and after the dip dyeing, the inflorescences are sprayed with clear water and bagged.
5. The application of the Brassica napus Bra014815 gene in regulating and controlling the branch number and plant type of Brassica napus as claimed in claim 4, wherein the bag is covered, the soaked plant is protected from light for 24h, the normal culture conditions are recovered after 24h, five days later, a small opening is opened for a newly emerged bud to be bloomed, and the bud which is not suitable for soaking is removed.
6. The use of Brassica napus Bra014815 gene in regulating the number and plant type of branches of Brassica napus according to claim 5, wherein the buds unsuitable for soaking are removed and a second infection is carried out in the same manner.
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