CN118109509A

CN118109509A - Application of GhBCCP gene in regulating and controlling plant seed size

Info

Publication number: CN118109509A
Application number: CN202410361977.XA
Authority: CN
Inventors: 崔宇鹏; 郭可栋; 张北北; 彭星雨
Original assignee: Anyang Institute of Technology
Current assignee: Anyang Institute of Technology
Priority date: 2024-03-27
Filing date: 2024-03-27
Publication date: 2024-05-31

Abstract

The invention provides an application of GhBCCP gene in regulating and controlling plant seed size, belonging to the technical field of genes. The invention clones cotton GhBCCP1 (GH_A05G 3915) gene from the cotton institute 24 material, and the nucleotide sequence of the GhBCCP1 (GH_A05G 3915) gene is shown as SEQ ID NO:1, successfully constructing an over-expression vector driven by a seed specific promoter, transforming Arabidopsis, planting, culturing and harvesting mature Arabidopsis seeds, and finding that the gene expression quantity of GhBCCP < 1 > is obviously increased compared with a wild control plant and the length and width of GhBCCP < 1 > transgenic Arabidopsis seeds are obviously increased and reach a remarkable level compared with a wild control plant.

Description

Application of GhBCCP gene in regulating and controlling plant seed size

Technical Field

The invention relates to the technical field of genes, in particular to an application of GhBCCP gene in regulating and controlling the size of plant seeds.

Background

Cotton is an important commercial crop and oil crop. The cotton seeds contain rich fatty acid and protein, and are widely applied to processing edible oil, industrial raw materials and biodiesel. Cotton breeding studies have been focused mainly on fiber yield and quality studies, with serious delays in cotton seed yield. Among the cotton seed yield contributors, seed size is an important component that contributes to cotton seed yield, and therefore, studying cotton seed size is of great importance to improving cotton seed byproduct processing.

The seed size of plants is one of the most important features for marking yield, and although some progress has been made in understanding the molecular mechanisms controlling seed size, there are few key genes found to regulate seed size. In the prior art, the identification and functional analysis of the cotton heterogeneous ACCase gene family discloses a gene named GhBCCP (Gh_D06G 1228) and researches on the regulation and control of the shape and the size of cotton seeds, but the sequence of the gene is different from that of the gene GhBCCP1 (GH_A05G3915) of the application, and the gene is not the same. At present, no study on GhBCCP (GH_A05G3915) genes and seed size has been reported.

Disclosure of Invention

The invention aims to provide an application of GhBCCP gene capable of remarkably increasing the length and width of plant seeds.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides an application of GhBCCP gene in regulating and controlling plant seed size, wherein the nucleotide sequence of GhBCCP gene is shown as SEQ ID NO: 1.

Preferably, the amino acid sequence of the protein encoded by the GhBCCP gene is shown in SEQ ID NO: 2.

The invention provides an application of GhBCCP gene in regulating and controlling plant seed size, by cloning cotton GhBCCP (GH_A05G3915) gene from cotton 24 material, successfully constructing a seed specific promoter-driven over-expression vector and transforming Arabidopsis thaliana, planting, culturing and harvesting mature Arabidopsis thaliana seeds, finding that the expression quantity of GhBCCP1 gene is obviously increased compared with wild control plants, and compared with wild control plants, the length and width of GhBCCP1 transgenic Arabidopsis thaliana seeds are obviously increased to a significant level.

Drawings

FIG. 1 is an agarose electrophoresis diagram of GhBCCP gene clone products.

FIG. 2 is a screen of T1 generation Arabidopsis resistant seeds on MS+Kan medium.

FIG. 3 is a PCR identification electrophoretogram of T1 generation Arabidopsis resistant plants.

FIG. 4 is a graph showing the analysis of the expression level of GhBCCP gene in different tissues of T1-generation Arabidopsis thaliana resistant plants.

FIG. 5 is a graph showing the amount of GhBCCP expression in the T3 generation plant Hordeum vulgare of 2 transgenic Arabidopsis thaliana.

FIG. 6 is a plot of GhBCCP transgenic Arabidopsis seed length and width.

FIG. 7 is a diagram showing sizes of GhBCCP transgenic Arabidopsis wild type and 2T 3 generation inbred seeds, with a 1:1 scale, and the size on the diagram is the actual size.

Detailed Description

The sources of enzymes, reagents, medicines, vectors and competent cells used in the invention are as follows:

Enzyme and kit: the total plant RNA extraction kit (DP 432) was purchased from Tiangen Biochemical (technology) Beijing Co., ltd; RNA reverse transcription Kit TRANSSCRIPT ONE-Step gDNARemoval AND CDNA SYNTHESIS Supermix (AT 311-02), fluorescence quantitative enzyme TRANSSTART TOP GREEN QPCR Supermix (AQ 131-04), cloning vector pEASY-T5 Zero Cloning Kit (CT 501-01) and plasmid extraction Kit EasyPure HiPure PLASMID MINIPREP KIT (EM 111-01) were purchased from Beijing full gold Biotechnology Co., ltd; the high-fidelity PCR amplification enzyme KOD-Plus-Neo (KOD-401) was purchased from TOYOBO BioCo; DNA FRAGMENT Purification Kit ver 4.0.0 gel recovery kit and DNAPurification Kit PCR product purification kit were purchased from TaKaRa biosystems; an infusion ligase was purchased from nuprandial biotechnology limited; coli competent cells DH 5. Alpha. Were purchased from Shanghai; agrobacterium competent LBA4404 was purchased from Beijing Bomaide Gene technologies Co., ltd; both restriction enzymes XbaI and SacI were available from NEB company.

Other drugs: agarose was purchased from Beijing full gold biotechnology Co., ltd; peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride, etc. are domestic analytical purities; ampicillin (IA 0340), kanamycin (YZ-130556), rifampicin (IR 0110), streptomycin (IS 0360) and the like were purchased from Beijing Soy Bao technology Co.

Preparing a solution: the various reagents mentioned herein but not listed are formulated as described in third edition of the guidelines for molecular cloning experiments, with biochemical reagents being analytically pure or superior.

LB medium: yeast extract (Yeast extract) 5g/L, tryptone (Tryptone) 10g/L and sodium chloride (NaCl) 10g/L, and finally, ddH ₂ O is used for fixing volume to 1L; LB solid medium: yeast extract (Yeast extract) 5g/L, sodium chloride (NaCl) 10g/L, agar powder 15g/L, tryptone (Tryptone) 10g/L, and ddH ₂ O to 1L.

The main instrument is as follows: PCR amplification apparatus (BIO-RAD), electrophoresis apparatus (BIO-RAD), fluorescence quantitative PCR apparatus (ABI 7500), gel imaging system (BIO-RAD), high-speed centrifuge (Hettich MIKRO R), enzyme-labeled apparatus (BIO-TEC KC 4), and vacuum freeze-dryer (ALPHAI-5).

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Cloning of example 1GhBCCP1

(1) Cotton stations 24 in the test materials were planted in the Anyang institute of technology test base and managed as a general field. The obtained tissue is mixed ovule of 3 days, 5 days, 10 days and 20 days after flowering, and the obtained material is rapidly frozen in liquid nitrogen and stored in a refrigerator at-80deg.C for use. The total RNA of the plants is extracted by using a Tiangen company kit.

(2) 1000Ng of RNA was reverse transcribed into cDNA, and the reverse transcription product cDNA solution was diluted 4-fold as a template for PCR reaction. The total RNA of the cotton ovule mixture extracted in each period is used as a template, and is reversely transcribed into cDNA by using a full-gold reverse transcription kit, and the cDNA synthesis system is as follows.

Remarks: incubation was performed in a PCR apparatus at 42℃for 30min, and finally at 85℃for 5s. Preserving at 4 ℃ for standby.

(3) Amplifying target gene by PCR reaction

The CDS full-length sequence of the GH_A05G3915 (GhBCCP) gene was retrieved from the cotton functional genome database (cotton functional genomic database (https:// cottonfgd /)) using the local Blast method, and the primer sequence was designed using primer 5.0 software and the primer sequence was as follows:

GhBCCP1-F(SEQ ID NO：3)：5′-ATGGCTTCTTCACTCTCTTTTC-3’

GhBCCP1-R(SEQ ID NO：4)：5′-TCACCCCACTATTACAAAT-3’

the cotton GhBCCP gene was PCR amplified using the synthesized primers GhBCCP-F and GhBCCP-R and the cDNA obtained by reverse transcription as a template. The target gene amplification system is as follows.

Note that 28 cycles of amplification were performed from Step2 to Step4

(4) Preserving at 4 ℃ after the reaction is finished, and detecting by using 0.8% agarose electrophoresis to obtain a PCR amplification product (figure 1) with the size of 873bp, wherein the amplification product is GhBCCP gene, and the nucleotide sequence of the amplification product is shown as SEQ ID NO:1, which encodes 290 amino acid residues, the amino acid sequence of which is shown in SEQ ID NO:2 is shown in the figure

Nucleotide sequence SEQ ID NO:1:

ATGGCTTCTTCACTCTCTTTTCCATGCCCCAAGATCTCTTCTTTTTTGAAAACAAACCAGCAAACCCAAACTCACAAGGGTTCTTTGTCTCTTCCTCCAACTTCAAATTTCAACTCCAAATCTTGCTTGTCATTTGGATCTTCAATCCGCGTACCTGCATCTTCAGCACCTCAGTGGCCTAATGATGGGAAACAAACTACAGTCTTCGCAAGCCATACGCAGCTTAACGAGGTTGCTGCAGAGAAATCATCGAATTCTGTAGCGGTTGTTGACATGAAACCTAAAGTTGCATTGCCTGAGGAAGATGATAAAAAATCCGCAGAGAAGGCTATTCCAGATGCTGCGGCAATTTCAGAATTCATGGCACAAGTTTCAGACCTTGTAAAACTTGTTGATTCACGAGATATTACGGAGCTGCAACTGAAGCAATCGGATTGTGAGCTTGTCATAAGAAAGAAGGAAGCTTTGCAGCAGCTGGAATCAGCATCTCCAATTGTCATGCAGCAGTACATGCCTCAACCGACGTTTCAAACCCCAGCTCCAGCAGCCCCAGTAGCAGCTCCTGCTCCTGCTAACCCAGCCCCTCCAGCAGCAGCACCTTCGTCACCTCCCCCTGCTAAAGCGGTTGGTTCTTCTCATCCGCCCCTTAAATGCCCCATGGCTGGAACGTTCTACAGGAGCCCTGCACCGGGTGAACCACCATTTGTTAAGGTGGGAGATAAAGTACAGAAAGGCCAAGTTGTGTGCATCATCGAGGCAATGAAACTAATGAACGAAATTGAAGCTGATCAATCCGGAACCGTTACAGAGATATTGGTAGAGGATGGAAAACCAGTTAGTGTAGACATGCCTCTATTTGTAATAGTGGGGTGA

Amino acid sequence SEQ ID NO:2:

MASSLSFPCPKISSFLKTNQQTQTHKGSLSLPPTSNFNSKSCLSFGSSIRVPASSAPQWPNDGKQTTVFASHTQLNEVAAEKSSNSVAVVDMKPKVALPEEDDKKSAEKAIPDAAAISEFMAQVSDLVKLVDSRDITELQLKQSDCELVIRKKEALQQLESASPIVMQQYMPQPTFQTPAPAAPVAAPAPANPAPPAAAPSSPPPAKAVGSSHPPLKCPMAGTFYRSPAPGEPPFVKVGDKVQKGQVVCIIEAMKLMNEIEADQSGTVTEILVEDGKPVSVDMPLFVIVG

(5) The reaction product of the desired fragment was purified and recovered by TaKaRa MiniBEST DNA FRAGMENT PurificationKit ver 4.0.

(6) The products recovered from the above gel were ligated with pEASY-T5 cloning vector and transformed into E.coli competent DH 5. Alpha.

(7) Heat shock in a 42℃water bath for 45s, resuscitating for 90s on ice, then adding 600. Mu.l of the non-resistant LB liquid medium, and shaking culturing at 160rpm/min at 37℃for 1h. The whole broth was then plated onto LB solid medium (containing kan resistance). The culture was carried out overnight at 37℃and the single clone was picked from kan-resistant LB medium and cultured overnight at 37℃in 500. Mu lLB medium containing kan.

(8) And (3) performing PCR (polymerase chain reaction) verification on bacterial liquid, sequencing the bacterial liquid containing the size band of the target gene fragment, and designating the clone with correct sequencing as pEASY-GhBCCP1.

Example 2 construction of p2301M alpha-GhBCCP plant seed-specific expression vectors

1. Plasmid extraction and enzyme digestion

The p2301M alpha plasmid vector is stored in the laboratory, and the construction method is the same as that of the p2301M alpha plasmid vector in the 'Liu Zhengjie et al' seed specific expression vector construction and genetic transformation of upland cotton heterogeneous ACCase gene 'molecular plant breeding 9.3 (2011): 8, see 3.2 strain and plasmid part'. The plasmid extraction adopts a full-formula gold company plasmid extraction kit, the plasmid concentration is detected to be 120 ng/. Mu.l, the agarose gel electrophoresis detection shows that the plasmid has no protein pollution, the test requirement is met, and the incision enzyme is subjected to double enzyme digestion by using XbaI and SacI.

2. Construction of p2301M alpha-GhBCCP 1 expression vector

(1) Amplification of ORF sequence of the target gene: xbaI-SacI was designed as an insertion site according to the multiple cloning site on the final vector p2301 M.alpha.and primers were synthesized.

The primer sequences were as follows:

GhBCCP1-XbaI-F (SEQ ID NO: 5): 5'-GCTCTAGAATGGCTTCTTCACTCTCTTTTC-3' (the underlined part contains the recognition sequence for the restriction enzyme XbaI);

GhBCCP1-SacI-R (SEQ ID NO: 6): 5'-CGAGCTCTCACCCCACTATTACAAAT-3' (the underlined part contains the recognition sequence for the restriction enzyme SacI).

The GhBCCP-mesh gene sequence containing XbaI and SacI cleavage sites was amplified using KOD-Plus-Neo high-fidelity enzyme, and the reaction system was as follows.

The conditions required for the PCR reaction were set as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 30s, extension at 68 ℃ for 30s,30 cycles; extending at 68℃for 5min.

(2) Fusion expression vector construction:

1) The vector p2301Mα was expressed by restriction enzymes XbaI and SacI for cleavage of the plant specific promoter, and the vector backbone was recovered.

2) The GhBCCP1 amplified product containing XbaI and SacI cleavage sites was recovered and purified, and the recovered product was digested with restriction enzymes XbaI and SacI.

3) And (3) connecting the carrier framework obtained in the step (1) with the recovered product obtained in the step (2) to obtain a connecting product.

4) Performing heat shock transformation on the connection product obtained in the step 3to obtain escherichia coli DH5 alpha, performing inversion overnight culture in a 37 ℃ incubator, and performing sequencing on positive clones; the correct strain was designated as p2301 M.alpha. -GhBCCP/DH 5. Alpha. As a result of the sequencing, and the plasmid extracted from the correct strain was designated as p2301 M.alpha. -GhBCCP1 plasmid.

Example 3 transformation of LBA4404 competent cells with vector plasmid

A. Taking 100 μl of LBA4404 competent cells melted on ice, adding 2 μl (about 500 ug) of the constructed p2301M alpha-GhBCCP plasmid into a centrifuge tube containing competent cells under aseptic conditions, gently mixing, and standing in an ice water bath for 5 min;

b. Placing the centrifuge tube in liquid nitrogen for quick freezing for 5 minutes;

c. then, rapidly placing the centrifuge tube in a water bath at 37 ℃ for incubation for 5 minutes, and placing the centrifuge tube back in an ice water bath for 5 minutes;

d. And (3) coating bacterial liquid in a 100 mu L centrifuge tube on LB screening medium containing kanamycin, streptomycin and rifampicin, culturing for 3d at 28 ℃, selecting positive clones (p 2301M alpha-GhBCCP 1/LBA 4404), culturing for 48h on LB liquid medium at 28 ℃ until the final concentration of glycerol is about 15%, and preserving at-20 ℃ for later use.

Example 4 genetic transformation, identification and Gene expression analysis of p2301M alpha-GhBCCP 1 seed-specific expression vectors in Arabidopsis

The agrobacterium solution transformed with p2301M alpha-GhBCCP 1/LBA4404 was first cultured with 1ml of an LB medium containing kan, then 100. Mu.l of the cultured overnight bacterial solution was aspirated and added to 100ml of fresh liquid LB medium containing kan, and shake-cultured at 28℃for 16h. The supernatant was discarded after centrifugation at 13000rpm/min for 5min, and the bacteria were resuspended in 100ml of permeate (10% sucrose+400. Mu.l/L Silwet-77) to an OD ₆₀₀ = 0.8 or so. Genetic transformation is carried out by using Columbia type Arabidopsis thaliana as a receptor and using an Arabidopsis thaliana flower dipping infection method, T0 generation transgenic seeds are harvested, positive transformants are screened on MS culture medium containing kanamycin (50 mg/L) (figure 2), T1 generation resistant plants are obtained, and seeds are collected after flowers are developed. After T ₁ generation of resistant plants are transplanted for 5 weeks, taking leaves of the arabidopsis plants to extract DNA, and carrying out PCR amplification on the resistance marker NPTII to further screen positive arabidopsis plants. The results are shown in FIG. 3. The result shows that the target band can be obtained near 795bp of the agarose gel electrophoresis pattern, and the success of the transformation of the seed-specific expression vector of the gene is preliminarily proved. Meanwhile, after 7 weeks of T1 generation resistant plants are transplanted, RNA of the horn fruits, leaves and stems of the arabidopsis plants is taken, cDNA is synthesized, specific primers (GhBCCP-QRT-F (SEQ ID NO: 7): TTGGTTCTTCTCATCCGCCC and GhBCCP-QRT-R (SEQ ID NO: 8): GCCTCGATGATGCACACAAC) are designed, and cDNA of three different tissue parts (horn fruits, leaves and stems) of the transformed plants of the vector are used as templates, wherein the internal reference primers are ATACTINQF (SEQ ID NO: 9) respectively: CCATGAACCCACCTATAACTCC and ATACTINQR (SEQ ID NO: 10): TACTCTGCCTTTGCGATCCAC, performing fluorescent quantitative PCR. As a result, as shown in FIG. 4, ghBCCP gene in T1 transformed plants had a higher expression level only in the Horn fruit, while GhBC gene was expressed in leaves and stems very little or no. It was demonstrated that the GhBCCP gene transformed in Arabidopsis achieved seed-specific expression.

And respectively harvesting T1 generation single plant seeds, taking about 50 seeds to be distributed on an MS+Kan (50 mg/L) culture medium, counting the separation ratio of yellow-green seedlings after the cotyledons of the seedlings are completely unfolded (about 10 days), and carrying out chi-square test to ensure that the seedlings meet the requirements of green seedlings: the primary prediction of yellow seedlings = 3:1 isolated plants was single copy insert lines, from which seeds were individually harvested for subsequent analysis.

Example 5 measurement of seed Length and seed Width

2 Single-copy T3 generation pure lines with high expression level of GhBCCP gene in fructus corni are selected and grown on MS+Kan (50 mg/L) culture medium (figure 5), the two lines are named GhBCCP1OE6 and OE13 respectively, and 25 plants are transplanted in each line. To measure seed size, plants were grown simultaneously under the same conditions. After harvesting the mature arabidopsis seeds of the corresponding strain, they were dried at 24 ℃.

The length and width of the seeds were photographed with a zeiss Axio ImagerA microscope and then measured with ImageJ software. Data were measured as at least 7 independent seeds, and the mean ± SD of the data. The aspect ratio of the seeds was calculated. As a result, as shown in FIG. 6, ghBCCP.sup.1 OE mature dry seeds were longer in length and wider in width than the wild-type control, and GhBCCP.sup.1 OE seeds were larger than the wild-type seeds (WT) (FIG. 7).

As can be seen from the above examples, the GhBCCP gene provided by the invention is applied to the regulation of the size of plant seeds, and the length and width of the seeds are remarkably improved.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

The application of GhBCCP1 gene in regulating the size of plant seeds is characterized in that: the nucleotide sequence of GhBCCP gene is shown as SEQ ID NO: 1.
2. The use according to claim 1, wherein the GhBCCP gene encodes a protein having the amino acid sequence set forth in SEQ ID NO: 2.