CN110904117B

CN110904117B - Application of plant PHL2 gene in regulation of plant seed size, dry weight and fatty acid accumulation

Info

Publication number: CN110904117B
Application number: CN201911018665.4A
Authority: CN
Inventors: 胡赞民; 殷祥贞; 范成明; 陈宇红
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2019-10-24
Filing date: 2019-10-24
Publication date: 2022-12-06
Anticipated expiration: 2039-10-24
Also published as: CN110904117A

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to application of plant PHL2 gene in regulating and controlling plant seed size, dry weight and fatty acid accumulation. The PHL2 gene is found to have the function of regulating and controlling the size, dry weight and fatty acid accumulation of plant seeds. Compared with wild type, the size, dry weight and total fatty acid content of seeds of transgenic materials obtained by over-expressing plant PHL2 genes are obviously increased, and the over-expressing plant PHL2 genes do not have adverse effects on plant growth and development and agronomic traits. The new function of PHL2 discovered by the invention has better application potential, and provides a new idea for improving the grease of crops.

Description

Application of plant PHL2 gene in regulation of plant seed size, dry weight and fatty acid accumulation

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to application of plant PHL2 gene in regulating and controlling plant seed size, dry weight and fatty acid accumulation.

Background

PHL2 (PHR 1-like 2) belongs to MYB-CC family, and is a proximal gene of PHR1 (HOSPHORUS STARVATION RESPONSE 1). PHR1 and its homologous gene PSR1 (PHOSPHORUS STARVATION RESPONSE 1) can be involved in PHOSPHORUS metabolism by regulating the expression of some PHOSPHORUS STARVATION-inducing genes. In Arabidopsis, atPHL2 can also be involved in the regulation of phosphorus metabolism by regulating the expression of some phosphorus starvation-induced genes. In addition, PHR1 is critical for arabidopsis thaliana adaptation to high light and maintenance of functional photosynthesis during phosphorus starvation, and PHR1 is also the junction of phosphorus with other essential nutrients cross-talk, such as sulfate, zinc and iron.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide the application of the plant PHL2 gene in regulating and controlling the size, dry weight and fatty acid accumulation of plant seeds.

In order to realize the purpose, the technical scheme of the invention is as follows:

the invention discovers that the plant PHL2 gene has the function of regulating and controlling the size, the dry weight and the fatty acid accumulation of plant seeds, improves the expression quantity of the PHL2 gene in plants, and can obviously improve the size, the dry weight and the fatty acid accumulation of the plant seeds.

In a first aspect, the present invention provides the use of a plant PHL2 protein or a gene encoding the same for regulating the size of a plant seed.

Preferably, the size of the seed is in particular the length, width or projected area of the seed.

In a second aspect, the present invention provides the use of a plant PHL2 protein or a gene encoding the same for regulating the dry weight of plant seeds.

In a third aspect, the present invention provides the use of a plant PHL2 protein or a gene encoding the same for modulating seed development in a plant.

In a fourth aspect, the present invention provides the use of a plant PHL2 protein or a gene encoding the same for modulating fatty acid metabolism in plants.

In a fifth aspect, the present invention provides the use of a plant PHL2 protein or a gene encoding the same for modulating fatty acid accumulation in plant seeds.

Preferably, the fatty acids are total fatty acids or are one or more selected from C16:0, C18:1, C18:2, C18:3, C20:0, C20:1, C20:2 and C22: 1.

In the above applications, the size of the plant seed, the dry weight of the seed, or the fatty acid accumulation of the seed can be increased by increasing the expression level of the plant PHL2 protein or its encoding gene in the plant.

In a sixth aspect, the present invention provides the use of a plant PHL2 protein or a gene encoding the same in plant genetic breeding or in the preparation of transgenic plants.

The application of the PHL2 protein or the coding gene thereof can be applied in the form of the PHL2 protein or the coding gene thereof, or in the form of an expression cassette and a vector containing the coding gene of the PHL2 protein, a host cell containing the expression cassette or the vector, or a transgenic plant cell line containing the coding gene of the PHL2 protein.

The plant PHL2 protein has any one of the following amino acid sequences:

(1) An amino acid sequence shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3;

(2) The amino acid sequence of the protein with the same function is obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3;

(3) An amino acid sequence having at least 80% homology with the amino acid sequence as shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3. Preferably, the homology is at least 90%; more preferably 95%.

The amino acid sequence shown as SEQ ID NO.1 is PHL2 protein AtPHL2 of arabidopsis thaliana; the amino acid sequence shown in SEQ ID No.2 is PHL2 protein BnaC. PHL2.A of Brassica napus; the amino acid sequence shown in SEQ ID No.3 is PHL2 protein BnaC. PHL2.B of Brassica napus. According to the amino acid sequence disclosed by the invention and conservative substitution of amino acids and other conventional technical means in the field, one or more amino acids can be substituted, deleted and/or added by the technical means in the field without influencing the activity of the technical means, so that the mutant of the PHL2 protein with the same activity as the PHL2 protein disclosed by the invention can be obtained.

The nucleotide sequence shown as SEQ ID NO.4 is the CDS sequence of the PHL2 protein AtPHL2 of Arabidopsis; the nucleotide sequence shown as SEQ ID NO.5 is CDS sequence of PHL2 protein BnaC. PHL2.A of Brassica napus; the nucleotide sequence shown as SEQ ID NO.6 is CDS sequence of PHL2 protein BnaC. PHL2.B of Brassica napus. The coding gene of the PHL2 protein can be any nucleotide sequence capable of coding the PHL2 protein. In view of the degeneracy of the codons and the preference of codons for different species, one skilled in the art can use codons suitable for the expression of a particular species as needed.

The CDS of the plant PHL2 protein has any one of the following nucleotide sequences:

(1) A nucleotide sequence shown as SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6;

(2) The nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6 is obtained by replacing, inserting or deleting one or more nucleotides to obtain the nucleotide sequence which codes the same functional protein.

In a seventh aspect, the present invention provides a method of modulating seed size, dry weight or fatty acid accumulation in a plant comprising: regulating the expression level of the PHL2 protein in the plant;

the PHL2 protein has any one of the following amino acid sequences:

(1) An amino acid sequence shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3;

(3) An amino acid sequence having at least 80% homology with the amino acid sequence shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3; preferably, the homology is at least 90%; more preferably 95%.

Specifically, the method is to increase the seed size, dry weight or fatty acid accumulation of a plant by overexpressing a gene encoding a PHL2 protein in the plant;

(1) A nucleotide sequence shown as SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6;

(2) The nucleotide sequence shown as SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6 is obtained by replacing, inserting or deleting one or more nucleotides to obtain the nucleotide sequence which codes the same functional protein.

The overexpression of the gene encoding the PHL2 protein can be achieved by means of conventional techniques in the art, such as: an expression vector carrying a gene encoding the PHL2 protein is introduced into the plant.

The gene encoding the over-expressed PHL2 protein may be transcribed using a commonly used promoter to promote the gene encoding the PHL2 protein, for example: the CaMV 35S promoter.

In the present invention, the plant is a monocotyledon or a dicotyledon. Such plants include, but are not limited to, arabidopsis, oilseed rape, soybean, cotton, peanut, palm and the like oilseed plants, and wheat, rice, corn and the like.

The invention has the beneficial effects that:

the PHL2 gene is found to have the function of regulating and controlling the size, dry weight and fatty acid accumulation of plant seeds. Experiments prove that compared with wild type, the transgenic material obtained by over-expressing AtPHL2 of Arabidopsis thaliana and BnaC.PHL2.a and BnaC.PHL2.b of Brassica napus in Arabidopsis thaliana has the length, width and projection area of the Arabidopsis thaliana seeds transformed with AtPHL2, bnaC.PHL2.a and BnaC.PHL2.b increased by 1.36-19.38%, 1.96-12.89% and 3.17-34.93% respectively; the dry weight of the arabidopsis seeds transformed with AtPHL2 and BnaC. PHL2.B is increased by 12.96-47.06%, and the content of total fatty acid is increased by 14.99-31.11%. Meanwhile, atPHL2, bnaC. PHL2.A and BnaC. PHL2.B are over-expressed in Arabidopsis, and unfavorable agronomic traits such as plant growth and development inhibition are not found except that the size and dry weight of Arabidopsis seeds and the accumulation of fatty acid in the seeds are obviously improved. The functions and the application of the PHL2 discovered by the invention have better application potential, and a new idea is provided for improving the grease of crops.

Drawings

FIGS. 1A, 1B and 1C are schematic structural views of the entry vectors pGWC-AtPHL2, pGWC-BnaC. PHL2.A and pGWC-BnaC. PHL2.B, respectively, in example 1 of the present invention.

Fig. 2A, fig. 2B and fig. 2C are schematic structural diagrams of patpol 2, pbnac. Phl2.A and pbnac. Phl2.B plant expression vectors in example 1 of the present invention, respectively.

FIG. 3 is an analysis of seed sizes of different transgenic Arabidopsis lines in example 3 of the present invention; wherein WT represents a wild type; 35S, atPHL2, eGFP WT represents a wild-type AtPHL2 gene-transferred strain; 35S, bnaC, PHL2.A, eGFP WT represents a wild-type BnaC, PHL2.A gene-transferred strain; 35S, bnaC.PHL2.B, eGFP WT represents a wild-type BnaC.PHL2b gene-transferred strain; 35S, bnaC.PHL2.B, eGFP phl2-1 represents a phl2-1 mutant strain transformed into BnaC.PHL2b gene; the #1 and #2 represent different transgenic lines respectively; * Indicating that the difference was significant at the level of 0.01-straw-over-p-straw-over 0.05; * Indicates significant differences at p <0.01 levels.

FIG. 4 is an analysis of the dry weight of seeds of different transgenic Arabidopsis lines in example 4 of the present invention; wherein WT represents a wild type; atPHL2 WT, bnac. Phl2.A WT and bnac. Phl2.B WT indicate wild type transgenic lines AtPHL2, bnac. Phl2.A and bnac. Phl2.B, respectively; bnaC.PHL2.B phl2-1 shows that phl2-1 mutant is transformed into BnaC.PHL2.B gene strain; * Indicating that the difference was significant at the level of 0.01-straw-over-p-straw-over 0.05; * Indicates significant differences at p <0.01 levels.

FIG. 5 is the compositional analysis of seed fatty acids in different transgenic Arabidopsis lines according to the invention in example 5; wherein denotes that the differences at the 0.01-p-t-0.05 level were significant; * Indicates significant differences at the 0.001-but-p-but 0.01 level; * Indicates significant differences at p <0.001 levels.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 cloning of Arabidopsis thaliana and Brassica napus PHL2 genes and construction of expression vectors

1. Extraction of plant Total RNA

The method comprises the following steps of extracting the total plant RNA according to an RNAprep Pure plant total RNA extraction kit (catalog number: DP432, tiangen Biochemical technology (Beijing) Co., ltd.), and comprises the following steps:

(1) Homogenizing 50-100 mg of arabidopsis thaliana or brassica napus leaves, quickly grinding the leaves into powder in liquid nitrogen, adding 450 mu L of RL (adding 10 mu L of beta-mercaptoethanol), and violently shaking and uniformly mixing the leaves by vortex;

(2) Transferring all the solution to a filter column CS (the filter column CS is placed in a collecting tube), centrifuging for 2min at 12,000rpm, carefully sucking the supernatant in the collecting tube to an RNase-Free centrifuge tube, and preventing the suction head from contacting with cell debris in the collecting tube to precipitate as much as possible;

(3) Slowly adding 0.5 times of the volume of the supernatant of absolute ethyl alcohol (225 μ L), mixing, transferring the obtained solution and precipitate into an adsorption column CR3, centrifuging at 12000rpm for 60sec, pouring off the waste liquid in the collection tube, and returning the adsorption column CR3 to the collection tube;

(4) Adding 350 μ L deproteinized solution RW1 into adsorption column CR3, centrifuging at 12000rpm for 60sec, pouring off waste liquid in the collection tube, and returning adsorption column CR3 into the collection tube;

(5) Preparing DNase I working solution: putting 10 mu L of DNase I stock solution into a new RNase-Free centrifuge tube, adding 70 mu L of RDD buffer solution, and gently and uniformly mixing;

(6) Adding 80 μ L of DNase I working solution into the center of the adsorption column CR3, and standing at room temperature for 15min;

(7) Adding 350 μ L deproteinized solution RW1 into adsorption column CR3, centrifuging at 12,000rpm for 60sec, pouring off waste liquid in the collection tube, and returning adsorption column CR3 to the collection tube;

(8) Adding 500 μ L of rinsing solution RW into adsorption column CR3, standing at room temperature for 2min, centrifuging at 12000rpm for 60sec, pouring off waste liquid in the collection tube, and placing adsorption column CR3 back into the collection tube;

(9) Repeating the step 8;

(10) Centrifuging at 12,000rpm for 2min, and discarding the waste liquid. Placing the adsorption column CR3 at room temperature for several minutes to thoroughly air-dry the residual rinsing liquid in the adsorption material;

(11) Placing the adsorption column CR3 into a new RNase-Free centrifuge tube, suspending 60 μ L RNase-Free ddH2O dropwise into the middle part of the adsorption membrane, standing at room temperature for 2min, and centrifuging at 12,000rpm for 2min to obtain RNA solution. The RNA samples were stored at-80 ℃.

2. Synthesis of cDNA

The total plant RNA extracted in 1 above was subjected to reverse transcription based on TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (catalog No.: AE311-03, kyoto Kogyo gold Biotechnology Co., ltd.) as follows:

(1) A reverse transcription reaction system (20. Mu.L) was prepared as follows:

(2) The reaction was gently mixed and incubated at 42 ℃ for 30min.

(3) TransScript RT/RI Enzyme and gDNA removal solution were inactivated by heating at 85 ℃ for 5sec and stored at-20 ℃ until use.

3. Acquisition of AtPHL2, bnaC, PHL2.A and BnaC, PHL2.B genes and construction of expression vector

Searching AtPHL2 (the protein sequence of AtPHL2 is shown as SEQ ID NO.1 and the CDS sequence is shown as SEQ ID NO. 4) in an Arabidopsis genome database (TAIR, http:// www.arabidopsis.org /), searching BnaC.PHL2.A (the protein sequence of BnaC.PHL2.A is shown as SEQ ID NO.2 and the CDS sequence is shown as SEQ ID NO. 5) and BnaC.PHL2.B (the protein sequence of BnaC.PHL2.B is shown as SEQ ID NO.3 and the CDS sequence is shown as SEQ ID NO. 6) in a Brassica napus genome database (http:// www.genoscope.cns.fr/brassicana), and designing amplification specific primers of AtPHL2, bnaC.PHL2.A and BnaC.PHL2.B according to the gene sequences, wherein the amplification specific primers are as follows:

AtPHL2_F:5′-agcaggctttgactttatgtactcagcgattcgctcg-3′；

AtPHL2_R:5′-tgggtctagagactttcctccaatggtgctactaggcataatc-3′；

BnaC.PHL2.a_F:5′-agcaggctttgactttatgtactcggcgattcgctcg-3′；

BnaC.PHL2.a_R:5′-tgggtctagagactttcctccaatggtactactactaggcacg-3′；

BnaC.PHL2.b_F:5′-agcaggctttgactttatgtactcggcgattcgctcc-3′；

BnaC.PHL2.b_R:5′-tgggtctagagactttcctccaatggtactactaggcacagtc-3′。

the above primers were used for gene cloning. The detailed procedures for gene cloning are as follows:

(1) A PCR reaction system (total volume: 50. Mu.L) was prepared from the following components:

(2) The PCR reaction conditions were as follows:

and respectively connecting the PCR products obtained by cloning to an entry vector pGWC by using an In-fusion system, wherein the specific method comprises the following steps:

reaction system: mu.L of digested pGWCm (100 ng/. Mu.L, digested with AhdI), 1. Mu.L of PCR product (80 ng/. Mu.L), 2. Mu.L of In-fusion Mix. The reaction conditions are as follows: 50-60 min at 50 ℃. The ligation product is transformed into Escherichia coli DH5 alpha, positive clones are identified and screened by PCR, and after sequencing identification, recombinant plasmids are obtained and are named as pGWC-AtPHL2, pGWC-BnaC.PHL2.A and pGWC-BnaC.PHL2.B in sequence (as shown in figure 1A, figure 1B and figure 1C).

AtPHL2, bnaC. PHL2.A and BnaC. PHL2.B were each constructed into the plant expression vector pHZM27 by the Gateway system (pHZM 27 is by-P in pEarley gate 103) _35S ::attR1::ccdB::Cm::attR2::mGFP::T _OCS Introducing gypsy on both sides to construct gypsy, namely gypsy P _35S ::attR1::ccdB::Cm::attR2::mGFP::T _OCS Gypsy-, pEarleyGate 103 was purchased from Biovector NTCC Inc., and designated pAtPHHL 2, pBnaC. PHL2.A, and pBnaC. PHL2.B, respectively (as shown in FIGS. 2A, 2B, and 2C). The recombinant plasmids pAtPHHL 2, pBnaC.PHL2.A and pBnaC.PHL2.B are respectively transformed into agrobacterium GV3101 and are identified by PCR for later use.

Example 2 genetic transformation of AtPHL2, bnaC. PHL2.A and BnaC. PHL2.B genes and screening of Positive transgenic lines

The expression vectors patph 2, pbnac. Phl2.A and pbnac. Phl2.B constructed in example 1 were transformed into arabidopsis thaliana using dip-flower method, respectively, as follows:

agrobacterium harboring the plasmids pAtPHHL 2, pBnaC. PHL2.A and pBnaC. PHL2.B constructed in example 1 were cultured in LB liquid medium (with 50mg/L kanamycin, 50mg/L gentamicin and 50mg/L rifampicin added) to OD =0.8, centrifuged at 5000rpm for 5min to collect the cells, and an equal volume of suspension (10 mM MgCl. Sub.L. Sub.MgCl. Sub.2. A) was used ₂ 0.005% Silwet L-77,5% sucrose) and transformed into Arabidopsis at the early stage of flowering once and dipped into flowers once more at intervals of 7 days. After the seeds are mature, collecting T0 generation seeds, planting the seeds in a culture dish, spraying Barsta (0.3%) for screening 10 days after seedling emergence, and spraying once again at an interval of 5 days; and after PCR identification, transplanting the T1 generation positive seedlings into a nutrition pot for culture, and harvesting seeds after the seedlings are mature.

Example 3 analysis of seed size of plants transgenic for AtPHL2, bnaC. PHL2.A and BnaC. PHL2.B genes

Seeds from 10 mature Arabidopsis shoots were collected for each transgenic line and dried at 37 ℃. Observing and photographing the seeds by using a stereoscope, and analyzing the photographed images of the seeds by using Image-Pro Plus software to obtain the length, width and projection area of the seeds.

The results of the experiment are shown in fig. 3, and the results show that: the over-expression of the AtPHL2, bnaC. PHL2.A and BnaC. PHL2.B genes can promote the length, width and projection area of the seeds to be remarkably increased: compared with wild type, the length, width and projection area of 9 strain seeds over-expressing AtPHL2 gene are respectively increased by 1.36-13.49%, 1.96-8.11% and 3.17-23.40%; compared with wild type, the length, width and projection area of 9 lines of seeds of BnaC.PHL2.a transgenic lines are respectively increased by 2.40-19.38%, 3.66-12.89% and 3.74-34.93%; compared with wild type, the length, width and projection area of 9 strain seeds of BnaC.PHL2.B gene are respectively increased by 3.46-14.49%, 4.90-9.91% and 7.21-26.93%.

Example 4 seed Dry weight analysis of AtPHL2 and BnaC. PHL2.B Gene lines

Seeds of AtPHL2 and BnaC. PHL2.B gene-transferred Arabidopsis thaliana are collected respectively, dried at 37 ℃, and then the dry weight of 500 seeds is counted, and 10 plants of each transgenic line are taken as 10 biological repeats.

The results are shown in FIG. 4, which shows: overexpression of both AtPHL2 and bnac. Phl2.B genes can significantly increase the dry weight of seeds: the dry weight of seeds transformed into AtPHL2 gene lines 1, #3, #5 and #7 in WT increased by 35.98%, 14.50%, 12.96% and 32.87%, respectively, compared to Wild Type (WT); the dry weight of seeds transformed in WT of bnac. Phl2.B gene lines #1, #3, #5 and #10 increased by 28.06%, 28.25%, 47.06% and 27.51%, respectively, compared to wild type; the dry weight of seeds transformed into BnaC. PHL2.B gene lines #3, #5, #8 and #9 in mutant PHL2-1 (SALK _114420C, see Arabidopsis database www.arabidopsis.org, for the mutant obtained by T-DNA insertion in Arabidopsis, the insertion site is located at the 3' end of the first intron of PHL 2) was increased by 17.97%, 18.38%, 29.34% and 17.27%, respectively, compared to the wild type.

Example 5 analysis of fatty acid composition in seeds transformed with AtPHL2 and BnaC. PHL2.B genes

Respectively collecting seeds of AtPHL2 and BnaC. PHL2.B transgenic Arabidopsis thaliana, drying at 37 deg.C, grinding thoroughly, weighing 0.01g, adding 3mL 7.5% ₃ OH (C17: 0 standard substance is added as an internal reference), water bath is carried out for 4-5h at 70 ℃, and the mixture is inverted and mixed for several times. 2mL of HCl-CH was added ₃ OH (V/V, 1:1) solution, 2mL 14% BF3-CH ₃ OH solution, water bath at 70 ℃ for 1.5h. Add 1mL of 0.9% NaCl and 4mL of n-hexane, mix well with shaking, centrifuge at 4,000rpm for 8min, and transfer the upper organic phase to a new tube. Nitrogen was blown dry and 300. Mu.L of ethyl acetate was dissolved. Each transgenic line was randomly selected 4 plants as 4 biological replicates.

The samples to be tested were analyzed by gas chromatography-triple quadrupole tandem mass spectrometer (GC-QQQ, agilent 7890A-7001B). The column was HP-FFAP (30 mm. Times.0.25mm ID,0.25 μm; agilent). The carrier gas is high purity helium. The specific parameters are set as follows: the flow rate is 1mL/min; the temperature of a sample inlet is 220 ℃; the transmission line temperature is 230 ℃; the sample introduction mode is non-shunting; the sample injection volume is 1 mu L; the ion source is EI (70 eV); the ion source temperature is 230 ℃; the scanning mode is full scanning (50-550 m/z); the initial temperature of the column box is 60 ℃, the temperature is firstly increased to 180 ℃ at the speed of 10 ℃/min, then is continuously increased to 210 ℃ at the speed of 3 ℃/min, and finally is continuously increased to 220 ℃ at the speed of 5 ℃/min, and is kept for 15min. And (3) performing qualitative and quantitative determination on the sample by using a self-contained software Masshunter works of a gas workstation. The database used for qualitative identification was NIST. Then according to the gas chromatography analysis result, the peak areas corresponding to different fatty acids are compared with the peak area of the C17:0 internal standard to calculate the content of each fatty acid component and the content of total fatty acids.

The results are shown in FIG. 5, which shows: no change in fatty acid composition occurred in the seeds of arabidopsis thaliana (all containing C16:0, C18:1, C18:2, C18:3, C20:0, C20:1, C20:2, C20:3, and C22: 1) regardless of whether AtPHL2 and bnac. Phl2.B were overexpressed in WT or mutant phl2-1, but the total fatty acid content increased by 14.99% to 31.11% in arabidopsis thaliana seeds. In line #1 overexpressing AtPHL2 in WT (WT-AtPHL 2# 1), the contents of C18:1 and C20:1 increased significantly, by 13.64% and 27.50%, respectively. In line #3 overexpressing AtPHL2 in WT (WT-AtPHL 2# 3), the contents of C16:0, C18:3, C20:0, C20:1, C20:2, and C22:1 were all significantly increased by 7.11%, 12.96%, 24.45%, 20.02%, 35.25%, 25.15%, and 30.50%, respectively. In line #1 overexpressing bnac. Phl2.B in WT (WT-bnac. Phl2.B # 1), the contents of C16:0, C18:2, C18:3, C20:1 and C20:2 were all significantly increased by 9.70%, 12.74%, 25.33%, 33.50% and 8.22%, respectively. In line #3 overexpressing bnac. Phl2.B in WT (WT-bnac. Phl2.B # 3), the content of both C18:3 and C20:1 were significantly increased, by 20.48% and 28.50%, respectively. In line #3 overexpressing BnaC. PHL2.B in phl2-1 (phl 2-1-BnaC. PHL2.B # 3), the contents of C16:0, C18:2, C18:3, C20:0 and C20:1 were all significantly increased by 18.41%, 15.09%, 19.90%, 42.27%, 10.93% and 37.78%, respectively. In line #5 overexpressing BnaC. PHL2.B in phl2-1 (phl 2-1-BnaC. PHL2.B # 5), the contents of C16:0, C18:2, C18:3, C20:0, C20:1 and C20:2 were all significantly increased by 20.02%, 24.83%, 20.13%, 41.24%, 28.74%, 48.65% and 23.18%, respectively.

The invention takes model plant arabidopsis thaliana as an example and experiments prove the functions of the genes AtPHL2, bnaC. PHL2.A and BnaC. PHL2.B, and considering that arabidopsis thaliana is a model plant, the genes which can play a role in arabidopsis thaliana have similar effects in various crops, so the genes AtPHL2, bnaC. PHL2.A and BnaC. PHL2.B can be used for regulating and controlling the size, the dry weight or the fatty acid accumulation of plant seeds in oilseed rape, soybean, cotton, peanut, palm and other oil plants and wheat, rice, corn and other crops.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto without departing from the scope of the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Tyr Ser Ala Ile Arg Ser Leu Pro Leu Asp Gly Gly Asp Tyr His

1 5 10 15

Gly Pro Leu Asp Gly Thr Asn Leu Pro Gly Asp Ala Cys Leu Val Leu

20 25 30

Thr Thr Asp Pro Lys Pro Arg Leu Arg Trp Thr Ala Glu Leu His Glu

35 40 45

Arg Phe Val Asp Ala Val Thr Gln Leu Gly Gly Pro Asp Lys Ala Thr

50 55 60

Pro Lys Thr Ile Met Arg Thr Met Gly Val Lys Gly Leu Thr Leu Tyr

65 70 75 80

His Leu Lys Ser His Leu Gln Lys Phe Arg Leu Gly Arg Gln Ala Cys

85 90 95

Lys Asp Ser Thr Asp Asn Ser Lys Asp Ala Ser Cys Ala Gly Glu Ser

100 105 110

Gln Asp Thr Gly Ser Ser Ser Ser Ser Ser Leu Arg Met Ala Ala Gln

115 120 125

Glu Gln Asn Glu Gly Tyr Gln Val Thr Glu Ala Leu Arg Ala Gln Met

130 135 140

Glu Val Gln Arg Arg Leu His Glu Gln Leu Glu Val Gln Arg Arg Leu

145 150 155 160

Gln Leu Arg Ile Glu Ala Gln Gly Lys Tyr Leu Gln Ser Ile Leu Glu

165 170 175

Lys Ala Cys Lys Ala Phe Asp Asp Gln Ala Ala Ala Phe Val Gly Leu

180 185 190

Glu Ala Ala Arg Glu Glu Leu Ser Glu Leu Ala Ile Lys Val Ser Asn

195 200 205

Ser Ser Gln Gly Thr Ala Val Pro Phe Phe Asp Thr Thr Lys Met Met

210 215 220

Met Met Pro Ser Val Ser Glu Leu Ala Val Ala Val Asp Thr Lys Asn

225 230 235 240

Asn Thr Thr Thr Asn Cys Ser Val Glu Ser Ser Leu Thr Ser Asn Thr

245 250 255

Asn Gly Ser Ser Leu Ser Ala Ala Ser Met Lys Lys Arg Leu Arg Gly

260 265 270

Asp Asp Val Gly Leu Gly Tyr Glu Ala Gly Trp Ile Met Pro Ser Ser

275 280 285

Ser Thr Ile Gly

290

<210> 3

<211> 294

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Tyr Ser Ala Ile Arg Ser Leu Pro Leu Asp Gly Gly Glu Tyr His

1 5 10 15

Gly Pro Leu Asp Gly Thr Asn Leu Pro Gly Asp Ala Cys Leu Val Leu

20 25 30

Thr Thr Asp Pro Lys Pro Arg Leu Arg Trp Thr Ala Glu Leu His Glu

35 40 45

Arg Phe Val Glu Ala Val Thr Glu Leu Gly Gly Pro Glu Lys Ala Thr

50 55 60

Pro Lys Thr Leu Met Arg Thr Met Gly Val Lys Gly Leu Thr Leu Tyr

65 70 75 80

His Leu Lys Ser His Leu Gln Lys Phe Arg Gln Gly Arg Gln Ala Cys

85 90 95

Lys Asp Ser Thr Asp Asn Ser Asn Lys Asp Ala Ser Cys Val Gly Glu

100 105 110

Ser Gln Asp Thr Gly Ser Ser Ser Pro Ser Ser Leu Lys Leu Ala Ala

115 120 125

Gln Glu Gln Asn Glu Ser Tyr Gln Val Thr Glu Ala Leu Arg Ala Gln

130 135 140

Met Glu Val Gln Arg Arg Leu His Glu Gln Leu Glu Val Gln Arg Arg

145 150 155 160

Leu Gln Val Arg Ile Glu Ala Gln Gly Lys Tyr Leu Gln Thr Ile Leu

165 170 175

Glu Lys Ala Cys Lys Ala Phe Asp Glu Gln Ala Ala Met Phe Thr Gly

180 185 190

Leu Glu Thr Ala Arg Glu Glu Leu Ser Glu Leu Ala Ile Lys Val Ser

195 200 205

Asn Asn Ser Gln Gly Thr Thr Val Pro Tyr Phe Asp Ala Thr Lys Met

210 215 220

Met Met Met Met Pro Ser Leu Ser Glu Leu Glu Val Ala Ala Ile Asp

225 230 235 240

His Lys Ser Asn Ile Thr Thr Thr Asn Cys Ser Val Glu Ser Ser Leu

245 250 255

Thr Ser Asn Thr Asn Gly Ser Ser Val Ser Ala Ala Ser Met Lys Lys

260 265 270

Arg His Arg Gly Gly Asn Asn Val Gly Tyr Glu Gly Ser Trp Thr Val

275 280 285

Pro Ser Ser Thr Ile Gly

290

<210> 4

<211> 888

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgtactcag cgattcgctc gcttccactc gatggtggac acgttggtgg tgactaccat 60

ggacctcttg acggaaccaa tcttcccggt gacgcttgtt tggttttaac gactgaccct 120

aaacctcgtc tccggtggac aactgagctt catgagagat tcgttgacgc cgttactcag 180

ctcggtggtc ctgacaaagc gactcccaaa actattatga gaacaatggg agtgaagggt 240

ctcactctct accacctcaa atcacatctt cagaaattcc gcctagggag gcaagctggc 300

aaagaatcaa ctgagaactc taaagatgct tcttgtgtag gggagagtca ggacacaggt 360

tcatcttcga catcatcaat gagaatggcg cagcaggagc agaacgaggg ttaccaagtc 420

accgaagctc tacgtgctca gatggaagtc caaagaagac tacacgatca attggaggtg 480

caacggaggc tccagctgag gatagaggca caaggaaaat acctgcaatc gattcttgaa 540

aaagcttgca aggcctttga cgagcaagct gctacttttg ctggacttga ggctgctagg 600

gaagagctat cagagctagc catcaaagtc tccaatagct ctcaaggaac atcagtcccg 660

tacttcgatg caacaaagat gatgatgatg ccatcgttgt cagagcttgc agtagcaata 720

gacaacaaaa acaacatcac aaccaactgt tcagtagaaa gctctctgac ttccatcaca 780

catgggagct ctatatctgc tgcatcaatg aagaagcgtc aacgtggaga caatttgggc 840

gtagggtatg aatcaggctg gattatgcct agtagcacca ttggataa 888

<210> 5

<211> 879

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgtactcgg cgattcgctc gcttcctctc gacggcggtg actaccatgg accccttgac 60

ggtactaatc ttcccggaga cgcctgtttg gtcttaacca ctgaccccaa accccgtctc 120

cggtggaccg cggagctcca tgagaggttc gttgacgccg tcacgcagct cggaggtccc 180

gacaaagcga cgcccaaaac aatcatgaga acaatgggag tgaaaggcct caccctctac 240

cacctcaaat cacatctcca gaaattccgg ctagggaggc aagcttgcaa agattcaacc 300

gacaactcca aggatgcttc ttgtgctggg gagagtcagg acacaggttc atcttcatcg 360

tcatcactga gaatggcagc gcaggagcag aacgagggtt accaagtcac ggaagctctg 420

cgcgctcaga tggaagtcca aagaaggctg cacgagcaat tggaggtaca gcggagactc 480

cagctgagga tagaggcaca aggaaagtac ctacaatcga ttcttgagaa agcttgcaag 540

gcctttgacg accaagctgc tgcttttgtt gggctcgagg cagctaggga agagctctca 600

gagctagcca tcaaagtgtc caatagctct caaggaacag cagtcccgtt cttcgataca 660

acaaagatga tgatgatgcc atctgtgtcc gagcttgcag tagcagtaga caccaaaaac 720

aacaccacaa ccaactgttc agttgaaagc tctctgactt ccaacaccaa tgggagctcg 780

ctttctgctg catcgatgaa gaagcggctt cgcggagacg atgtaggcct aggctatgaa 840

gcagggtgga ttatgcctag tagtagtacc attggataa 879

<210> 6

<211> 885

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgtactcgg cgattcgctc ccttcctctc gacggcggtg agtaccatgg acctctcgac 60

ggaaccaatc ttcccggaga cgcctgtttg gtcttgacca ctgacccgaa accccgtctc 120

cggtggacgg cggagctcca tgagaggttc gttgaagccg tcacggagct cggtggtccc 180

gaaaaagcga cgcccaaaac tctgatgaga acaatgggag tgaaaggtct caccctctac 240

cacctcaaat ctcatcttca gaaatttcgg caagggaggc aagcttgtaa agactcaact 300

gacaactcca acaaggatgc ttcttgtgtt ggggagagtc aggacacagg ttcctcttca 360

ccgtcatcat tgaaactagc tgcgcaggaa cagaacgaga gttaccaagt cactgaagct 420

ctgcgcgctc aaatggaagt tcaaagaaga ctgcacgagc aattggaggt gcaacggaga 480

ctccaggtaa ggatagaggc ccaagggaaa tacctacaaa cgattctcga gaaagcttgc 540

aaggcctttg acgagcaagc tgctatgttt actgggcttg agacagctag ggaagagctg 600

tcggagctag ccatcaaagt ctctaataac tctcaaggaa caacagtccc atactttgat 660

gcaacaaaga tgatgatgat gatgccgtct ttgtccgagc ttgaagtagc agcaatagac 720

cacaaaagca acatcacaac aaccaactgt tctgttgaaa gctctctgac ttccaacacc 780

aatgggagct cggtttctgc tgcatcgatg aagaagaggc atcgtggagg aaacaatgtc 840

gggtatgaag ggagctggac tgtgcctagt agtaccattg gatag 885

<210> 7

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

agcaggcttt gactttatgt actcagcgat tcgctcg 37

<210> 8

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tgggtctaga gactttcctc caatggtgct actaggcata atc 43

<210> 9

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agcaggcttt gactttatgt actcggcgat tcgctcg 37

<210> 10

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgggtctaga gactttcctc caatggtact actactaggc acg 43

<210> 11

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

agcaggcttt gactttatgt actcggcgat tcgctcc 37

<210> 12

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tgggtctaga gactttcctc caatggtact actaggcaca gtc 43

Claims

1. The application of plant PHL2 protein or coding gene thereof in increasing the size of arabidopsis thaliana seeds;

the amino acid sequence of the PHL2 protein is shown in SEQ ID NO. 1.

2. The application of plant PHL2 protein or its coding gene in the genetic breeding for increasing the size of Arabidopsis seed;

the amino acid sequence of the PHL2 protein is shown in SEQ ID NO. 1.

3. The use according to claim 1 or 2, wherein the nucleotide sequence of the CDS of the PHL2 protein is as shown in SEQ ID No. 4.

4. The application of plant PHL2 protein or its coding gene in increasing plant seed size;

the amino acid sequence of the PHL2 protein is shown as SEQ ID NO.2 or SEQ ID NO. 3.

5. The application of plant PHL2 protein or its coding gene in the genetic breeding for increasing the size of plant seeds;

6. The use according to claim 4 or 5, wherein the nucleotide sequence of the CDS of the PHL2 protein is as shown in SEQ ID No.5 or SEQ ID No. 6.

7. The application of plant PHL2 protein or coding gene thereof in increasing the dry weight of arabidopsis seeds;

the amino acid sequence of the PHL2 protein is shown in SEQ ID NO. 1.

8. The application of plant PHL2 protein or coding gene thereof in genetic breeding for increasing the dry weight of arabidopsis seeds;

the amino acid sequence of the PHL2 protein is shown in SEQ ID NO. 1.

9. The application of plant PHL2 protein or coding gene thereof in increasing the accumulation of fatty acid in arabidopsis seeds;

the amino acid sequence of the PHL2 protein is shown in SEQ ID NO. 1.

10. The application of plant PHL2 protein or coding gene thereof in genetic breeding for increasing the accumulation of fatty acid in arabidopsis seeds;

the amino acid sequence of the PHL2 protein is shown in SEQ ID NO. 1.

11. The use according to any one of claims 7 to 10, wherein the nucleotide sequence of the CDS of the PHL2 protein is shown as SEQ ID No. 4.

12. The application of plant PHL2 protein or its coding gene in increasing the dry weight of plant seed;

the amino acid sequence of the PHL2 protein is shown in SEQ ID NO. 3.

13. The use of a plant PHL2 protein or a gene encoding the same in genetic breeding to increase the dry weight of plant seeds;

the amino acid sequence of the PHL2 protein is shown in SEQ ID NO. 3.

14. The application of plant PHL2 protein or its coding gene in increasing the fatty acid accumulation of plant seeds;

the amino acid sequence of the PHL2 protein is shown in SEQ ID NO. 3.

15. The application of plant PHL2 protein or its coding gene in genetic breeding for increasing the fatty acid accumulation of plant seeds;

the amino acid sequence of the PHL2 protein is shown in SEQ ID NO. 3.

16. The use according to any one of claims 12 to 15, wherein the nucleotide sequence of the CDS of the PHL2 protein is shown as SEQ ID No. 6.

17. A method of increasing seed size of arabidopsis thaliana, comprising: increasing the expression level of the PHL2 protein in Arabidopsis;

the amino acid sequence of the PHL2 protein is shown in SEQ ID NO. 1.

18. The method of claim 17, wherein a gene encoding the PHL2 protein is overexpressed in the arabidopsis thaliana, increasing the seed size of the arabidopsis thaliana;

the nucleotide sequence of CDS of the PHL2 protein is shown as SEQ ID NO. 4.

19. A method of increasing seed size in a plant comprising: increasing the expression level of the PHL2 protein in the plant;

20. The method of claim 19, wherein overexpressing a gene encoding a PHL2 protein in said plant increases the seed size of said plant;

the nucleotide sequence of CDS of the PHL2 protein is shown as SEQ ID NO.5 or SEQ ID NO. 6.

21. A method of increasing seed dry weight or seed fatty acid accumulation in arabidopsis comprising: increasing the expression level of PHL2 protein in Arabidopsis;

the amino acid sequence of the PHL2 protein is shown in SEQ ID NO. 1.

22. The method of claim 21, wherein a gene encoding the PHL2 protein is overexpressed in the arabidopsis thaliana, increasing seed dry weight or seed fatty acid accumulation of the arabidopsis thaliana;

the nucleotide sequence of CDS of the PHL2 protein is shown as SEQ ID NO. 4.

23. A method of increasing seed dry weight or seed fatty acid accumulation in a plant comprising: increasing the expression level of the PHL2 protein in a plant;

the amino acid sequence of the PHL2 protein is shown in SEQ ID NO. 3.

24. The method of claim 23, wherein overexpressing a gene encoding a PHL2 protein in said plant increases seed dry weight or seed fatty acid accumulation in said plant;

the nucleotide sequence of CDS of the PHL2 protein is shown as SEQ ID NO. 6.