CN115851753B - Application of corn ZmBES1/BZR1-1 gene in improving plant yield - Google Patents
Application of corn ZmBES1/BZR1-1 gene in improving plant yield Download PDFInfo
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Abstract
The invention discloses application of a corn ZmBES1/BZR1-1 gene in improving plant yield, relates to the field of genetic engineering, and specifically relates to a relationship between the corn ZmBES1/BZR1-1 gene and plant grain development, and verifies the effect of the ZmBES1/BZR1-1 gene in improving the plant grain size. The ZmBES1/BZR1-1 gene is separated from corn seedling leaves, recombinant plasmid containing the gene is constructed and transformed into plant over-expression, the seed yield of the transgenic plant is obviously improved, and the invention shows that the over-expression of the ZmBES1/BZR1-1 gene can obviously improve the yield of the transgenic plant and has wide application prospect in the field of cultivating high-yield plants.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to application of corn ZmBES1/BZR1-1 genes in improving plant yield.
Background
Corn, rice and the like are important grain crops in China, and high yield is the most important target of researchers in the process of cultivating crop varieties. In recent years, the improvement of agronomic traits of crops by using genetic engineering means is a common technical means, and a new solution is provided for the improvement of crop yield. The application of ZmBES1/BZR1-5 gene of ZL202010362305.2 corn in cultivating large seed plant discloses the ZmBES1/BZR1-5 gene sequence and its function in regulating seed development. For many years, with the deep understanding of genes, the combination of the genes and traditional breeding for cultivating high-yield seeds is a problem to be solved urgently, and although various related reports on the research of gene level yield increase of crops exist, no related report on the effective separation and application of the high-efficiency yield increase genes in the cultivation and planting of crops such as corn, rice and the like exists.
However, functional analysis of the ZmBES1/BZR1-1 gene for maize has not been reported in the prior studies, nor has there been a method for improving crop yield using the gene.
Disclosure of Invention
The invention aims to provide the application of the ZmBES1/BZR1-1 gene of corn in improving the plant yield, so as to solve the problems in the prior art, and the gene can obviously improve the yield of transgenic plants.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides application of a corn ZmBES1/BZR1-1 gene in improving plant yield, wherein the nucleotide sequence of the corn ZmBES1/BZR1-1 gene is shown as SEQ ID NO. 1.
Further, it includes promoting grain length, grain width and thousand grain weight increase of plant seeds.
The invention also provides a method for cultivating the high-yield plant, which is to transform the ZmBES1/BZR1-1 gene of the corn into the plant to be expressed excessively to obtain a homozygous transgenic plant, namely the high-yield plant.
Further, the transformation of the maize ZmBES1/BZR1-1 gene into plants for overexpression specifically comprises: designing an amplification primer of the ZmBES1/BZR1-1 gene to carry out PCR amplification of the gene, constructing a recombinant plasmid containing the ZmBES1/BZR1-1 gene by using an amplification product and a vector, and then transforming the recombinant plasmid into a plant to carry out over-expression of the ZmBES1/BZR1-1 gene.
Further, the nucleotide sequence of the amplification primer is shown as SEQ ID NO. 2-3.
Further, the recombinant plasmid is pCAMBIA2300-35S-ZmBES1/BZR1-1-eGFP or pCAMBIA1300-35S-ZmBES1/BZR1-1-eGFP.
Further, the nucleotide sequence of pCAMBIA2300-35S-ZmBES1/BZR1-1-eGFP is shown as SEQ ID NO. 6; the nucleotide sequence of pCAMBIA1300-35S-ZmBES1/BZR1-1-eGFP is shown as SEQ ID NO 9.
Further, the plants include Arabidopsis thaliana and rice.
The invention discloses the following technical effects:
according to the invention, zmBES1/BZR1-1 genes are separated from corn seedling leaves, and are transferred into arabidopsis and rice to cultivate transgenic plants, and experiments prove that the transgenic plants can obviously promote the grain length, grain width and thousand grain weight of seeds, so that the gene can improve the yield of the transgenic plants, and has wide application prospects in the field of cultivating high-yield plants.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a homozygous transgenic Arabidopsis seed;
fig. 2 is a transgenic arabidopsis seed grain length and grain width, wherein p <0.05 is represented by x and p <0.01 is represented by x;
FIG. 3 shows a positive PCR assay for transgenic rice;
FIG. 4 is a representation of transgenic rice seed phenotypes;
fig. 5 is a transgenic rice seed grain length and grain width, wherein p <0.05 and p <0.01;
fig. 6 shows thousand grain weight of homozygous transgenic rice, where p <0.05 and p <0.01.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1 Gene cloning and vector construction
Leaves of pentaleaf stage maize B73 seedlings were taken, flash ground with liquid nitrogen, and total RNA was extracted using total RNA extraction kit Trizol (TaKaRa) and according to the instructions thereof. By PrimeScript TM RTregenant kit (Takara Co.) cDNA synthesis was performed using total RNA as a template. The ZmBES1/BZR1-1 gene (ID: zm00001d 046305) cloning primer was designed using Primer5.0, and the specific PCR primers were as shown in Table 1, using the B73cDNA as a template. Amplification was performed using PrimerStar (Takara Co.) high fidelity enzyme, and the PCR amplification reaction system was as shown in Table 2, and the temperature cycling procedure was: 94 ℃ for 3min;98 ℃,10s, and 10s of optimal annealing temperature; 72 ℃,20s;30 cycles; 72 ℃ for 5min; maintained at 4 ℃. Sequencing the amplified product, wherein the sequence of the amplified product is shown as SEQ ID NO: 1.
TABLE 1ZmBES1/BZR1-1 cloning primers
TABLE 2PCR amplification reaction System
1. Construction of transformation Arabidopsis thaliana vector pCAMBIA2300-35S-ZmBES1/BZR1-1-eGFP
According to the dicotyledon expression vector pCAMBIA2300-35S-eGFP multiple cloning site, the upstream primer introduces BamHI cleavage site, the downstream primer introduces SalI site, the specific sequence is as follows:
TABLE 3 construction of pCAMBIA2300-35S-ZmBES1/BZR1-1-eGFP vector primers
The reaction system shown in Table 2 was loaded and amplified. The temperature cycling program is as follows: 94 ℃ for 3min; 10s at 98 ℃,10s at the optimal annealing temperature, 20s at 72 ℃ and 30 cycles; 72 ℃ for 5min; maintained at 4 ℃.
And (5) recovering the PCR product. The recovered pCAMBIA2300-35S-eGFP plasmid was subjected to double digestion and loaded according to the reaction system of Table 4. Mixing the enzyme cutting system uniformly, placing the mixture in a water bath kettle at 30 ℃ for enzyme cutting for 6-8 hours, directly electrophoresis after the enzyme cutting product is added into a loading buffer solution, recovering the enzyme cutting product, connecting the enzyme cutting carrier and the target fragment by using ClonExpress II One Step Cloning Kit (Noruzhan), and reacting the reaction system as shown in table 5 under the reaction conditions: 30mins at 37 ℃.
Table 4 double cleavage reaction System
Table 5 connection system
The ligation product was transformed into E.coli and subjected to colony PCR detection. After the extracted recombinant plasmid is identified by restriction enzyme digestion, the recombinant plasmid bacterial liquid is sent to a sequencing company for DNA sequencing identification, and the plasmid pCAMBIA2300-35S-ZmBES1/BZR1-1-eGFP (SEQ ID NO: 6) with correct sequencing is transformed in the next step.
2. Construction of transformation Rice vector pCAMBIA1300-35S-ZmBES1/BZR1-1-eGFP
The upstream primer was introduced into HindIII cleavage site and the downstream primer was introduced into BamHI site according to the monocot expression vector pCAMBIA1300-35S-eGFP multiple cloning site, the specific sequences of which are shown in Table 6. Amplification was performed using PrimerStar (Takara Co.) high fidelity enzyme and the PCR amplification reaction system was as shown in Table 2. The PCR product was then recovered.
TABLE 6 construction of pCAMBIA1300-35S-ZmBES1/BZR1-1-eGFP vector primers
The expression vector pCAMBIA1300-35S-eGFP plasmid was subjected to double digestion and loaded according to the reaction system of Table 4. Mixing the enzyme digestion system uniformly, placing the mixture in a water bath kettle at 30 ℃ for enzyme digestion for 6-8 hours, directly electrophoresis after adding a sample buffer solution into enzyme digestion products, recovering enzyme digestion products, connecting target genes and carriers by using ClonExpress II One Step Cloning Kit (Noruzhan), and reacting under the reaction conditions shown in table 5: 30mins at 37 ℃.
The ligation product was transformed into E.coli and subjected to colony PCR detection. After the recombinant plasmid is extracted and identified by restriction enzyme digestion, the recombinant plasmid bacterial liquid is sent to a sequencing company for DNA sequencing identification, and the plasmid pCAMBIA1300-35S-ZmBES1/BZR1-1-eGFP (SEQ ID NO: 9) with correct sequencing is transformed in the next step of rice.
EXAMPLE 2 Arabidopsis, rice transformation and Positive identification
1. Transformation of Arabidopsis thaliana by flower-borne infection
Exploring the function of corn ZmBES1/BZR1-1, we transformed the pCAMBIA2300-35S-ZmBES1/BZR1-1-eGFP vector into Arabidopsis thaliana Col (WT) wild-type using Agrobacterium-mediated batting.
1.1 inflorescence dip-dyeing transformation
(1) The agrobacterium containing the recombinant plasmid is streaked on a YEP plate containing Kana and Rif, and cultured for 2-3 d at 28 ℃.
(2) Single colonies of Agrobacterium were picked and inoculated in 3mL of YEP liquid medium containing Kana and Rif, at 28℃at 200r/min, and cultured overnight with shaking.
(3) Inoculating 1mL of overnight-cultured initial Agrobacterium solution in 100mL of liquid YEP medium (containing Kana and Rif), and shake culturing at 28deg.C to bacterial solution OD 600 The value is 1.2-1.5.
(4) Centrifuging at 4deg.C at 5000r/min for 10min to collect cells, suspending thallus with 5% sucrose solution, and adjusting OD 600 The value is 0.8-1.0, and the surfactant silwet L-77 is added according to the proportion of 1-2 percent.
(5) Culturing wild Col of Arabidopsis thaliana by using nutrient soil for potting, pruning after flowering to form pods and flowers, soaking inflorescences for 1.5-2.0 min by using the infection liquid, and culturing in the dark for 10h.
(6) Culturing for 3-4 weeks under proper conditions, collecting seeds, and performing the next screening treatment.
1.2 selection of resistant Medium
(1) The harvested infected seeds were aliquoted into 1.5mL EP tubes and immersed in 500. Mu.L of 70% alcohol for 30s.
(2) After the alcohol is sucked off, the centrifuge tube is continuously shaken during the soaking with 500 mu L of 10% sodium hypochlorite for 5-10 min until the seeds are fully contacted with the sodium hypochlorite.
(3) Washing the seeds with sterilized water for 4-6 times, and discarding the upper ddH layer after the seeds subside 2 O; 200. Mu.L of 0.1% agar water suspended seeds.
(4) Seeds were sown on demand in 1/2MS medium containing 40mg/L kanamycin (Kana) for cultivation.
(5) Repeating the steps until the T3 generation homozygous positive seedlings are harvested.
1.3 genomic PCR identification of the obtained positive lines
(1) 100mg of fresh plant tissue is taken and added with liquid nitrogen for full grinding. 400. Mu.L of buffer FP1 and 6. Mu.L of RNaseA (10 mg/mL) were added thereto, vortexed for 1min, and left at room temperature for 10min.
(2) 130. Mu.L of buffer FP2 was added, thoroughly mixed, centrifuged at 12000r/min (13400 Xg) for 5min with vortexing, and the supernatant was transferred to a new centrifuge tube.
(3) The optional steps are as follows: the supernatant was centrifuged again at 12000r/min (13400 Xg) for 5min and the supernatant was transferred to a new centrifuge tube.
(4) 0.7 volumes of isopropanol was added to the supernatant and mixed well, at which point flocculent genomic DNA appeared. Centrifuge at 12000r/min (13400 Xg) for 2min, discard supernatant and leave pellet.
(5) 600. Mu.L of 70% ethanol was added thereto, vortexed and shaken for 5s, centrifuged at 12000r/min (13400 Xg) for 2min, and the supernatant was discarded.
(6) Repeating the step (5).
(7) And (5) uncovering and inverting, and airing residual ethanol thoroughly at room temperature for 5-10 min.
(8) Adding proper amount of elution buffer TE, dissolving DNA in water bath at 65 deg.c for 10-60 min, and mixing to obtain DNA solution.
(9) PCR detection was performed using the extracted genomic DNA as a template using PCR primers as shown in Table 8, with a procedure of 94℃for 3min;94℃for 30s, an optimum annealing temperature of 30s,72℃for 60s/kb,35 cycles; 72 ℃ for 5min; preserving at 4 ℃.
Table 8 transgenic Arabidopsis thaliana detection primers
(10) After T2 generation seeds of the transformed Arabidopsis thaliana are harvested and sterilized, the T2 generation seeds are sown on a 1/2MS solid medium containing kanamycin, and after 7-8 days, the T2 generation transgenic lines W1-2 and W1-4 are all green and strong plants, and are identified as homozygous transformants, and non-homozygous transformants show 3:1 segregation (the ratio of normal growth to yellowing death plants). The method lays a homozygous transgenic strain W1-2 and W1-4, and the T3 generation seeds are used for further analysis.
2. Rice transformation
The constructed pCAMBIA1300-35S-ZmBES1/BZR1-1-eGFP vector was transferred to the Semi BioCo (Nanjing) for transformation of rice wild type "Nip".
2.1 genomic PCR identification of the positive lines obtained:
(1) 100mg of fresh plant tissue is taken and added with liquid nitrogen for full grinding. 400. Mu.L of buffer FP1 and 6. Mu.L of RNaseA (10 mg/mL) were added thereto, vortexed for 1min, and left at room temperature for 10min.
(2) 130. Mu.L of buffer FP2 was added, thoroughly mixed, centrifuged at 12000r/min (13400 Xg) for 5min with vortexing, and the supernatant was transferred to a new centrifuge tube.
(3) The optional steps are as follows: the supernatant was centrifuged again at 12000r/min (13400 Xg) for 5min and the supernatant was transferred to a new centrifuge tube.
(4) 0.7 volumes of isopropanol was added to the supernatant and mixed well, at which point flocculent genomic DNA appeared. Centrifuge at 12000r/min (13400 Xg) for 2min, discard supernatant and leave pellet.
(5) 600. Mu.L of 70% ethanol was added thereto, vortexed and shaken for 5s, centrifuged at 12000r/min (13400 Xg) for 2min, and the supernatant was discarded.
(6) Repeating the step (5).
(7) And (5) uncovering and inverting, and airing residual ethanol thoroughly at room temperature for 5-10 min.
(8) Adding proper amount of elution buffer TE, dissolving DNA in water bath at 65 deg.c for 10-60 min, and mixing to obtain DNA solution.
(9) PCR detection was performed using the extracted genomic DNA as a template using PCR primers as shown in Table 9, with a procedure of 94℃for 3min;94℃for 30s, an optimum annealing temperature of 30s,72℃for 60s/kb,35 cycles; 72 ℃ for 5min; preserving at 4 ℃.
Table 9 transgenic rice detection primers
(10) The transformed rice seeds were harvested for the T2 generation and seeds of all positive lines were detected for the progeny after sowing for further analysis. The identification method of the transgenic lines is the same as that of the step (10) of 1.2, and the pure transgenic lines are R1-8 and R1-13 after kanamycin screening.
Example 3 purity and Strain yield analysis
1. Arabidopsis seed phenotype identification
Homozygous transgenic Arabidopsis lines W1-2 and W1-4 and Wild Type (WT) were sterilized with 5% sodium hypochlorite solution, sown on 1/2MS medium, 15d later transplanted to nutrient-containing soil: vermiculite = 3:1 soil in a flowerpot of 50mm x 50mm, 4 plants were sown per pot, cultivated under the same conditions for direct harvest, and seed grain length and grain width were measured.
2. Phenotypic identification of rice seeds
Homozygous transgenic rice lines R1-8, R1-13 and wild type (NIP) were sown in paddy fields, and the seeds were cultivated under the same conditions for direct harvest, and the seed grain length, grain width and thousand grain weight were measured.
3. Analysis of results
3.1 analysis of transgenic Arabidopsis yield
Through resistance screening, the stable expression of the target gene ZmBES1/BZR1-1 in arabidopsis is determined, and the grain length and grain width analysis of homozygous transgenic arabidopsis (figure 1) shows that the grain length of the over-expression lines W1-2 and W1-4 seeds is obviously increased, and the grain widths of the W1-2 and W1-4 seeds are obviously increased (figure 2).
3.2 analysis of transgenic Rice yield
The target gene was detected by PCR to determine a positive strain (FIG. 3). The grain length, grain width and thousand grain weight analysis of the positive lines (FIG. 4) showed that the grain length of the overexpressing lines R1-8 and R1-13 was significantly increased with no significant change in grain width (FIG. 5). Thousand kernel weight was significantly increased compared to control NIP (fig. 6).
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (7)
1. The application of the corn ZmBES1/BZR1-1 gene in increasing the grain length and grain width of arabidopsis seeds is characterized in that the nucleotide sequence of the corn ZmBES1/BZR1-1 gene is shown as SEQ ID NO. 1.
2. The application of the corn ZmBES1/BZR1-1 gene in increasing the grain length and thousand grain weight of rice seeds is characterized in that the nucleotide sequence of the corn ZmBES1/BZR1-1 gene is shown as SEQ ID NO. 1.
3. A method for cultivating high-yield plants is characterized in that corn ZmBES1/BZR1-1 genes are transformed into plants to be over-expressed, and homozygous transgenic plants are obtained;
the plant is Arabidopsis thaliana and rice;
when the plant is arabidopsis thaliana, the grain length and grain width of the obtained transgenic plant seeds are increased;
when the plant is rice, both the grain length and thousand seed weight of the obtained transgenic plant seed are increased.
4. The method according to claim 3, wherein said transforming the maize ZmBES1/BZR1-1 gene into plant overexpression comprises in particular: designing an amplification primer of the ZmBES1/BZR1-1 gene to carry out PCR amplification of the gene, constructing a recombinant plasmid containing the ZmBES1/BZR1-1 gene by using an amplification product and a vector, and then transforming the recombinant plasmid into a plant to carry out over-expression of the ZmBES1/BZR1-1 gene.
5. The method of claim 4, wherein the amplification primer has a nucleotide sequence set forth in SEQ ID NO. 2-3.
6. The method according to claim 4, wherein the recombinant plasmid is pCAMBIA2300-35S-ZmBES1/BZR1-1-eGFP or pCAMBIA1300-35S-ZmBES1/BZR1-1-eGFP.
7. The method according to claim 6, wherein the nucleotide sequence of pCAMBIA2300-35S-ZmBES1/BZR1-1-eGFP is shown in SEQ ID NO. 6; the nucleotide sequence of pCAMBIA1300-35S-ZmBES1/BZR1-1-eGFP is shown as SEQ ID NO 9.
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