CN116120415B

CN116120415B - Seed weight and yield related protein GmPHD6, related biological material and application thereof

Info

Publication number: CN116120415B
Application number: CN202310155750.5A
Authority: CN
Inventors: 张劲松; 韦伟; 张万科; 陶建军; 阴翠翠; 陈受宜
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2023-02-23
Filing date: 2023-02-23
Publication date: 2023-11-14
Anticipated expiration: 2043-02-23
Also published as: CN116120415A

Abstract

The application discloses a protein GmPHD6 related to seed weight and volume and related biological materials and application thereof. The sequence of soybean protein GmPHD6 is shown in sequence 2. The technical problem to be solved is how to increase the weight and/or volume of plant seeds. Specifically disclosed is the use of a protein, a substance that modulates the expression of a gene encoding said protein, or a substance that modulates the activity or content of said protein, in: a1 Increasing plant seed weight and/or the use of the product to increase plant seed weight; a2 Increasing plant seed volume and/or preparing a product that increases plant seed volume; a3 Plant breeding and/or the preparation of plant breeding products. The weight or volume of the seeds of the plant with the expression of the GmPHD6 protein coding gene knocked out/reduced is obviously higher than that of the wild plant. Substances which inhibit or reduce or down-regulate the expression of the GmPHD6 gene can be used for increasing the weight or volume of plant seeds and can also be used for plant breeding.

Description

Seed weight and yield related protein GmPHD6, related biological material and application thereof

Technical Field

The application relates to a protein GmPHD6 related to seed weight and yield, and related biological materials and applications thereof.

Background

Soybean is an important traditional crop, contains rich nutritional value, is an important economic crop for providing grain and oil and feed, and has many applications in industrial production, such as biofuel, surfactant, softener and the like. China was the largest soybean producing country in the world, however, in recent years, china only accounts for one fourth of the demand for soybean production, so that China becomes the largest soybean import country worldwide, and the total import amount is half of the total export amount of soybean worldwide. The soybean production is far behind the development pace of other domestic grain crops, and the national requirements cannot be met. In recent 50 years, soybean production has increased by only 62%, while other food crops have increased by more than 5 times. Therefore, the improvement of the soybean yield per unit has become a problem to be solved urgently.

The weight (grain weight) of seeds is an important index in seed crop production and is one of the important agronomic traits affecting crop yield. The plant can achieve the aim of increasing yield by increasing the grain weight.

The soybean yield is composed of plant type, pod bearing rate, pod number, seed hundred grain weight and other factors, wherein grain weight is the highest genetic power factor. The influence of grain weight on yield is not limited to leguminous plants, but is also an important factor for yield potential of other monocotyledonous plants, and therefore becomes an important selection trait to be considered in the process of crop variety breeding. Previous studies have shown that grain weight is affected by cultivation environment and genetics. Under normal cultivation conditions, inheritance, i.e. the relevant genes, plays an important role. Thus, research on molecular mechanisms related to particle weight has become a hotspot.

Disclosure of Invention

The application aims to solve the technical problem of how to increase the weight of plant seeds, in particular to soybeans.

In order to solve the above-described problems, the present application provides the following applications.

Use of a protein, a substance that modulates the expression of a gene encoding said protein, or a substance that modulates the activity or content of said protein in any of the following:

a1 For increasing the weight of plant seeds and/or for the preparation of a product for increasing the weight of plant seeds;

a2 For increasing plant seed yield and/or for the preparation of a product for increasing plant seed yield;

a3 Plant breeding and/or the preparation of plant breeding products;

the protein is any one of the following:

b1 Amino acid sequence is a protein shown in sequence 2;

b2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues of the protein B1), has more than 80 percent of identity with the protein B1) and has the function of regulating the weight and/or seed yield of plant seeds;

b3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) or B2) with a protein tag.

Among the above proteins, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

In the above proteins, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

In the above protein, the 80% or more identity may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above protein, sequence 2 (SEQ ID No. 2) consists of 248 amino acid residues. This was designated as GmPHD6 protein or protein GmPHD6. The coding gene is GmPHD6 gene.

Sequence 2 (SEQ ID No. 2) is specifically as follows:

MetAspSerGlyGlyHisTyrAsnProArgThrValGluGluValPheArgAspPheLysGlyArgArgAlaGlyMetIleLysAlaLeuThrThrAspValGluGluPheTyrGlnGlnCysAspProGluLysGluAsnLeuCysLeuTyrGlyPheProThrGluGlnTrpGluValAsnLeuProAlaGluGluValProProGluLeuProGluProAlaLeuGlyIleAsnPheAlaArgAspGlyMetGlnGluLysAspTrpLeuSerLeuValAlaValHisSerAspAlaTrpLeuGlnSerValAlaPheTyrPheGlyAlaArgPheGlyPheAspLysAlaAspArgLysArgLeuPheThrMetIleAsnAspLeuProThrIlePheGluValValThrGlySerAlaLysLysGlnThrLysGluLysSerSerGluAsnAsnGlyAsnLysSerLysSerSerSerLysGlyArgGlySerGluProProLysTyrSerLysGlnValLysAspGluGluGluGlyLeuAspGluGluAspAspAspGluHisGlyGluThrLeuCysGlyAlaCysGlyGluAsnTyrAlaSerAspGluPheTrpIleCysCysAspIleArgGluLysTrpPheHisGlyLysCysValLysIleThrProAlaArgAlaGluHisIleLysHisTyrLysCysProSerCysSerAsnLysArgProArgPro。

in the present application, the modulation may inhibit or reduce or down-regulate.

In the above, the knockout or inhibition or reduction or downregulation of the expression of the gene encoding the above protein or the activity or content of the above protein may increase the seed weight and/or seed volume of the plant.

In the above application, the protein is derived from soybean.

In the above, the soybean may be Jinma 23.

In the above, the substance that regulates gene expression may be a substance that performs at least one of the following 6 regulation: 1) Regulation at the level of transcription of said gene; 2) Regulation after transcription of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated by the gene).

In the above application, the substance regulating the expression of the coding gene of the protein is any one of the following:

d1 A nucleic acid molecule which inhibits or reduces or down-regulates the expression of a gene encoding the above protein;

d2 Expression of D1) the gene encoding the nucleic acid molecule;

d3 An expression cassette comprising D2) said gene;

d4 A recombinant vector comprising the gene of D2) or a recombinant vector comprising the expression cassette of D3);

d5 A recombinant microorganism comprising the gene of D2), or a recombinant microorganism comprising the expression cassette of D3), or a recombinant microorganism comprising the recombinant vector of D4);

d6 A transgenic plant cell line containing the gene of D2), or a transgenic plant cell line containing the expression cassette of D3), or a transgenic plant cell line containing the recombinant vector of D4);

d7 A transgenic plant tissue containing the gene of D2), or a transgenic plant tissue containing the expression cassette of D3), or a transgenic plant tissue containing the recombinant vector of D4);

d8 A transgenic plant organ containing the gene of D2), or a transgenic plant organ containing the expression cassette of D3), or a transgenic plant organ containing the recombinant vector of D4).

D2 In said nucleic acid molecules, the person skilled in the art can easily mutate the nucleotide sequence of the application which inhibits or reduces or down-regulates the expression of the gene encoding the protein GmPHD6 by means of known methods, for example directed evolution or point mutation. Those artificially modified nucleotides having 80% or more identity with the nucleotide sequence isolated according to the present application that inhibits or reduces or down-regulates the expression of the gene encoding the protein GmPHD6 and having the function of inhibiting or reducing or down-regulating the expression of the gene encoding the protein GmPHD6 are all derived from the nucleotide sequence according to the present application and are identical to the sequence according to the present application.

The 80% or more identity may be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and search is performed to calculate the identity of amino acid sequences, and then the value (%) of identity can be obtained.

In the above D4), the recombinant vector may be a vector edited with a plant gene. The plant gene editing vector may be a pCBSG015 vector.

As a specific example, the recombinant vector is recombinant vector pCBSG015-sgRNA. The recombinant vector pCBSG015-sgRNA is a recombinant plasmid obtained by inserting a DNA fragment with a sequence of CCGCACCGTCGAAGAGGTT between enzyme cutting sites of pCBSG015 vector restriction enzyme Bsa I and keeping other sequences of the pCBSG015 vector unchanged.

The microorganism of the above D5) may be Agrobacterium. The agrobacterium is EHA105.

In the above application, the nucleic acid molecule of D1) is a gRNA targeting the protein coding gene of claim 1, and the target sequence of the gRNA is sequence 3 in the sequence table.

In the above, the target sequence of the gRNA may be 5 '-CCGCACCGTCGAAGAGGTT-3'.

In the above-mentioned use, the plant is any one of the following:

g1 Dicotyledonous plants, G2) rosaceous plants, G3) leguminous plants, G4) soyabean plants, G5) soyabean plants.

In order to solve the above problems, the present application provides a method for increasing the weight and/or seed yield of plant seeds.

The method comprises increasing plant seed weight and/or seed yield by knocking out or inhibiting or reducing or down-regulating expression of genes encoding the above proteins in plants, and/or activity and/or content of the above proteins.

In order to solve the above problems, the present application provides a method of growing plants with high seed weight and/or seed yield.

The method comprises inhibiting or reducing or down-regulating the expression level of a gene encoding the above protein in a plant of interest, and/or the activity and/or content of the protein gives a high seed weight and/or seed yield plant having a higher seed weight and/or seed yield than the plant of interest.

In the above, the seed weight may be a seed dry weight. The seed weight may be hundred grains. The seed weight may be individually heavy.

In the above method, the knocking out or suppressing or reducing or down-regulating the expression of the gene encoding the above protein in the plant comprises introducing a gene knockout vector having the sequence 3 as a target sequence into the target plant.

In the above, the gene knockout system may be a pCBSG015 vector. The gene knockout system with the sequence 3 as the handle sequence can be a recombinant vector pCBSG015-sgRNA. The recombinant vector pCBSG015-sgRNA is a recombinant plasmid obtained by inserting a DNA fragment with a sequence of CCGCACCGTCGAAGAGGTT between enzyme cutting sites of pCBSG015 vector restriction enzyme Bsa I and keeping other sequences of the pCBSG015 vector unchanged. The recombinant plasmid was designated as recombinant vector pCBSG015-sgRNA.

In order to solve the above problems, the present application provides a method for cultivating soybeans with high seed weight and/or seed yield,

the method comprises the following steps of carrying out any one of the following operations on the genome of the target soybean:

k1 1 adenine deoxyribonucleotide is inserted between the 29 th position and the 30 th position of the sequence 3 in the sequence table in the target soybean genome;

k2 1 cytosine deoxyribonucleotide is inserted between the 29 th position and the 30 th position of the sequence 3 in the target soybean genome sequence table.

In the above application or method, the plant is any one of the following:

j1 Dicotyledonous plants, J2) Rosales, J3) Leguminosae, J4) Glycine plants, J5) Glycine max.

Advantageous effects

The application constructs a GmPHD6 gene knockout vector: recombinant vector pCBSG015-sgRNA. The group vector pCBSG 0-sgRNA is a recombinant plasmid obtained by inserting a DNA fragment with a sequence of CCGCAC CGTCGAAGAGGTT between restriction sites of a restriction enzyme Bsa I of a pCBSG015 vector and keeping other sequences of the pCBSG015 vector unchanged, and the recombinant plasmid is named as a recombinant vector pCBSG015-sgRNA.

The pCBSG015-sgRNA was introduced into Agrobacterium EHA105 competent cells. The agrobacteria EHA105-pCBSG015-sgRNA is obtained. Two homozygous plants phd-58 and phd-6-89, from which the GmPHD6 gene was knocked out, were obtained by infecting soybean with Agrobacterium EHA105-pCBSG015-sgRNA. The hundred weight/volume and individual weight/volume of greenhouse potting phd6-58 and phd6-89 were both found to be significantly higher than the recipient control Jack by manual culture of phd-58, phd-89 with control Jack. The field trials showed that the hundred grain weights/volumes of phd6-58 and phd6-89 were also significantly higher than the control Jack, whereas the total grain weight/volume of 20 individual plants, mutants phd6-58 and phd6-89 were all higher than the control, with phd6-89 reaching significant levels. In a word, both greenhouse potting and field results show that the GmPHD6 can negatively regulate the grain weight of soybean seeds, and the reduction of the expression level of GmPHD6 can remarkably improve the grain weight of soybean seeds and the yield/volume of single plants.

Drawings

FIG. 1 is the GmPHD6 gene editing site. The 1 target sequence (T1) of GmPHD6 and its specific position on the GmPHD6 gene are shown.

FIG. 2 shows the phd mutant gene editing sequencing verification. A is phd-58; b is phd-89

FIG. 3 shows the molecular detection of homozygous GmPHD6 mutants phd-58 and phd-89.

FIG. 4 shows that GmPHD6 gene CRISPR material increases soybean seed grain weight; a shows control Jack and mutant phd6-58, phd6-89 mature plants; b is the phenotype of the control Jack, mutants phd6-58 and phd6-89 kernels

FIG. 5 is a graph showing hundred and individual plant weight statistics for potting controls Jack and phd6-58 and phd 6-89; hundred weight statistics of A control Jack and two mutants phd-58, phd6-89 SD (n=20), P.ltoreq.0.001, T test; b control Jack and individual plant weight statistics of two mutants phd-58, phd6-89. SD (n=20), p.ltoreq.0.001, t test.

FIG. 6 is a field trial of control Jack and two mutants phd-58, and phd-89; SD (n=20), p.ltoreq.0.001, t test; b, 20 individual plant weight statistics SD (n=20), p.ltoreq.0.01, t test for control Jack and two mutants phd-58, phd 6-89; c, comparison of yields per hectare for control Jack and two mutants phd-58, phd6-89

Detailed Description

The soybean variety jin bean 23 (Glycine max l. Merr. Jd 23) in the following examples is described in the following literature: wei W, huang J, hao YJ, zou HF, wang HW, zhao JY, liu XY, zhang WK, ma B, zhang JS and Chen SY (2009) Soybean GmPHD-Type Transcription Regulators Improve Stress Tolerance in Transgenic Arabidopsis plants.PLoS ONE,4 (9), e7209.

Soybean variety Jack (Glycine max l. Merr. Jack) is described in the following documents: jiana Han, bingfu Guo, yong Guo, bo Zhang, xiaobo Wang, and Li-Juan Qiu, creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology, front Plant sci.2019;10:1446Published online 2019 Nov 22.doi:10.3389/fpls.2019.01446.

Agrobacterium tumefaciens EHA105 is described in the following documents: rodrigues SD, karimi M, impens L, van Lerberrge E, coussens G, aessaert S, rombut D, holterpels D, ibrahim HMM, van Montagu M, wagemans J, jacobs TB, de Coninck B, pauwels L., effect CRISPR-mediated base editing in Agrobacterium spp, # Proc Natl Acad Sci U S A.2021 Jan,12;118 (2) e2013338318. Doi: 10.1073/pnas.2013338318.

The gene editing was performed by unmixed biosciences, inc. (original name hundred biosciences), and the pCBSG015 vector was supplied by unmixed biosciences, inc., under the product number wimi-pCXB053.

The above biological materials are obtained from research of genetics and developmental biology of national academy of sciences after the public agrees with the economic crops of Shanxi academy of sciences, and the biological materials are only used for repeated experiments related to the application and cannot be used for other purposes.

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The following examples were run using SPSS11.5 statistical software and the experimental results were expressed as mean ± standard deviation, using One-way ANOVA test, P < 0.05 (x) indicated significant differences, P < 0.01 (x) indicated very significant differences, and P < 0.001 (x) indicated very significant differences.

Example 1 acquisition of the Soybean GmPDH6 Gene

Early studies found that GmPHD6 overexpression resulted in a decrease in soybean seed grain weight. It is presumed that GmPHD6 negatively regulates soybean seed grain weight.

Primers were designed based on the Williams 82 (W82) reference genomic sequence:

GmPHD6 sense:5‘-ATGGACAGTGGAGGACACTA-3’，

GmPHD6 antisense:5‘-TCAAGGCCTTGGTCTCTTATT-3’

using RT-PCR method, amplifying GmPHD6 gene from soybean total RNA: leaves of Jinma 23 (JD 23) were taken, ground in liquid nitrogen, suspended in 4mol/L guanidine thioglycolate, extracted with acidic phenol and chloroform, precipitated by adding absolute ethanol to the supernatant, and finally the precipitate was dissolved in water to obtain total RNA. A PCR amplification reaction was performed using 5. Mu.g of total RNA as a template by reverse transcription using a reverse transcription kit (Invitrogen) according to the method of the kit.

The 20. Mu.l PCR reaction system was: mu.l of one-stranded cDNA (0.05. Mu.g), 1. Mu.l of the above primer (20. Mu.M), 2. Mu.l of 10 XPCR buffer, 0.4. Mu.l of dNTP (10 mM) and 1U of Taq DNA polymerase were made up to 20. Mu.l with ultrapure water, and the liquid paraffin was covered with a liquid surface. The reaction was performed on a PE9600 PCR apparatus, the procedure of which was denaturation at 94℃for 5min; then, the temperature is 94 ℃ for 1min, the temperature is 56 ℃ for 1min and the temperature is 72 ℃ for 1min, and 30-32 cycles are performed; then extending for 10min at 72 ℃; preserving at 4 ℃. The PCR product of about 750bp is obtained, and after the PCR product is recovered, the sequence analysis shows that the PCR product is 747bp, the nucleotide sequence with the sequence 1 in the sequence table is GmPHD6 gene, and the encoding protein is named GmPHD6. The amino acid sequence of GmPHD6 is sequence 2 in the sequence table.

Sequence 1 (SEQ ID No. 1) is specifically as follows:

Atggacagtggaggacactacaacccccgcaccgtcgaagaggttttccgggacttcaagggccgccgggctggcatgatcaaagccctcaccactgatgttgaggaattttaccagcagtgtgatcctgaaaaggagaatttatgtctgtatggattccccactgaacaatgggaagtaaatttgcctgctgaagaagttcccccagaacttcccgagccagcactgggcataaattttgctagggatgggatgcaagaaaaggactggctatctttagttgctgtacacagtgatgcatggctacaatcagtggctttctactttggggcaagatttggttttgataaagctgacagaaagcgcctgttcactatgattaatgatttgcctacaatatttgaggttgtaactggatctgcaaagaaacagacaaaggagaagtcatcagagaacaatggcaacaaatcaaagtccagttcaaaagggcgaggatctgaacccccaaagtattcaaagcaagtgaaggatgaggaggagggattggacgaagaagacgatgacgagcatggggagaccttgtgtggagcatgtggggagaactatgcatccgatgagttctggatttgttgcgacatacgtgagaagtggtttcatggcaagtgtgtgaagatcactcctgcccgggctgagcacatcaaacactacaagtgcccctcatgcagcaataagagaccaaggccttga。

example 2: mutation of GmPHD6 gene results in increased seed weight of soybean seeds

In the embodiment, the GmPHD6 gene in the soybean is mutated by CRISPR/Cas9 technology, and the grain weight of the soybean seeds is obviously increased compared with that of a control after the gene mutation. The DNA sequence of the GmPHD6 gene is SEQ ID No.3 in the sequence table, and encodes a protein shown in SEQ ID No.2 in the sequence table.

Sequence 2 (SEQ ID No. 2) is specifically as follows:

MDSGGHYNPRTVEEVFRDFKGRRAGMIKALTTDVEEFYQQCDPEKENLCLYGFPTEQWEVNLPAEEVPPELPEPALGINFARDGMQEKDWLSLVAVHSDAWLQSVAFYFGARFGFDKADRKRLFTMINDLPTIFEVVTGSAKKQTKEKSSENNGNKSKSSSKGRGSEPPKYSKQVKDEEEGLDEEDDDEHGETLCGACGENYASDEFWICCDIREKWFHGKCVKITPARAEHIKHYKCPSCSNKRPRP。

sequence 3 (SEQ ID No. 3) is specifically as follows:

ATGGACAGTGGAGGACACTACAACCCCCGCACCGTCGAAGAGGTTTTCCGGGACTTCAAGGGCCGCCGGGCTGGCATGATCAAAGCCCTCACCACTGGTTACTCTCTCTTTCTCTCTAAATTTTCTTCTTTCTTTTTCTTTTTTTTTTAAATTCTACTCACTAAAGTTTATTTATTTATAATTTTTTTTTTTTGCAGATGTTGAGGAATTTTACCAGCAGTGTGATCCTGGTTAGGGTTTGGCCCTTTCTTCTTTTTTTTTTTTTTGTGTTTTGACTCTCCAATTGTGATTCTGTTTTATCGTTGTTTTTTTGTTTTGTTTGTTTCAAGTGTTCGGTGTCTGTGATTTCTTCTGTGACAGTTGCTTACTCATTTTACTGCATTGTGTGTGAGTCATGAAAATTGGCCCTTTCTTTTTCTTTTTTTTTTTAAATTTTAATGACCATGAAGCGTGAAATAGCAAATTGAGCCGTTGAAGAAATTTTTTTAGCGCTTTTGTGATTTTGATTAACCTTGCTGTTGGGGATGGTGCGTTGTAGCTTTGTTGGGTGGACTTGTTATGCATTTTTCTTTGGTCTTTATGGATGAGGTTCTTGTGATTTTGAATAAATGGTTTGCTTCTTCTGATCTATGGTCTTAGATATATTTTGTGTTCTGAATTCTGATTGTATTAAACTATTTTGATTACCAAGGGGTCTAGATTATTTGGGCTTTTTTGGGGAGTCTGATTACAACCTCTTTTTTTTTTTTTTCTTTTTTCTCTTAGCAACCCCTTTCGCGTTTTTGTAATTTTTTCTGACAAAAATCCTCTTCCTGGGACAATTTTTGGAATCTATAGAAATTCCAGAATTTAATTTTCCAGGTTCATCAAACTAATTTCCGGTTAACTTTTGTTGTTGTTGCTGCTCCTTTTTCCAACCATAGATGGGATCTTACTTCTTAGTTTGAGTGACATCCTTGTTTTATGACTTTTGTCACTTACAATACATATTCTTTTTGGTAGTATACATGGGTTTGTCTAGAAGTTCCCCATTCTATGATATTTTACAGATTTTGAATCAACCATATGTGGTGAATGAAGATTTTGGTTTGGGATTTTGAAAGTTTTGAAGAACTGGATTTTGTATTTCTCTTGTTTGCTTTCAATAAGTGAGACTAATTCCTTAACATGTGTTGTATTATTTGTTTTAATAGCTAGAGCCTACAGCTAATTATATTCTTTGTTGTTTTCTTTCTTACTTAGTTAGCAGCATCGTTGTCTTGGTAATATTCAAGTTAAAATTAGATGTTGAATTTCTTTTCTTTTCTTTTCATTATAGTATTTAGTATTTTTGCATGTATCTCAAAAATCTAATTTGCAATTTGTGGTATATTAAACAAGTTTACATTGTAAAATTTATTGATGCTTTATTTGTTTACCTGTTGTACATCAGAAAAGGAGAATTTATGTCTGTATGGATTCCCCACTGAACAATGGGAAGTAAATTTGCCTGCTGAAGAAGTTCCCCCAGAACTTCCCGAGCCAGCACTGGGCATAAATTTTGCTAGGGATGGGATGCAAGAAAAGGACTGGCTATCTTTAGTTGCTGTACACAGTGATGCATGGCTACAATCAGTGGCTTTCTACTTTGGGGCAAGATTTGGTTTTGATAAAGCTGACAGGTATAATAGTTCTTTTCTCCAATCCTTTATTATCTTATACTCTCTGGAATTGTTTCAGGTGTTCAATGAGAACATGTTTTGAATGCATGCCATTGCCTTTTTCTTCTGAACTCATGTTTTTTATGTGGCAGTATTGATTTATGTGTCTGCATAGTGTTTTCCGTAGAGATTTAGTTAATGGAAAAATATTTTAACATGGACTCTTCATTTGTATCAATGGAGTATGTCTTCTGATTGTTTTTTTGACTTAAATTATGGTGCCTAAATAAGCATATACTCCCTCCGTTCCTAATTATAAGATCGAAGTTTGAAATGATGTTTGTTCCTTTTTATAAGACTCAATCTATAATGTTCACTACATTAATTATTTATAGACTAGAAATACCCCTAATTAAAAGAAGAGAGAAATAATAATGACAAGGATAATTTTGGAAAAAATTATAAACTAAGACAAATTTATTACTATCAACTAATTTAACCACTTTTCTTAATCTTGATGAATTTGGTAATTGAGTCTTATAAATAGGAACAGAGAGAGTATTTTATATGGAAAAAGTTTCAGTCAACACAAACCATCATGAGATGTCTACCACTTGTAGGTTAGAGCTTTGCTTTATTTATGCAATTTTACCAATTGCTAAAGGAAAAGCACAACTGAAAAAAGCATAAGCAAGTCGCTTTTCTTATGCCCTGTGTCATTCATTATCTTTCCTTTGCTTATACTGGATCAAAATGGTTTTTGCAATTGATCTATTTCATCAACAATTCAATGTTTTTTGCATACTTGTCTGTCACGTGTACTCATCAGGAAAGAATGATATCTCCTTAAGAATTTACATTTAAGATTCCAAGCTTTTCTCTTTATGGAAAAAGGGGGTTTGGCACTTTGCATTGTTTACATTGGTCTACAACAAGCTGCTGACAGTAAAAATTCCGTATACATGCCCCTCATGACTAGTTAGCAGTGAAATATATGTATGTGTGTTGGAATACAGAATTGTATGTGATGATGCATGTAAAAATTAGCATGATGAAAAAAAATGTTCATTCAAAGCAAATATACTTGTGATATCCTGATCAGTGACAACATGGAATTCTTTGTGCAGAAAGCGCCTGTTCACTATGATTAATGATTTGCCTACAATATTTGAGGTTGTAACTGGATCTGCAAAGAAACAGACAAAGGAGAAGTCATCAGAGAACAATGGCAACAAATCAAAGTCCAGTTCAAAAGGGGTAAGAAAAACATCTTGTTACATCTGCACATCCTTGTATTTTTCTGGGTTTTTTTAAAATCATTTTTAATATCATAATTGTACTATTTATTGCTCATGCTCTTGTCACTATTGAAGGTCCATATATTTGGTCTGAGTAGAAGTGGGAAATGTCTTATTATCATAATATGCTAAATAGGGTTGACATGCTGTTTACTTGTTAATTTATTTCCTTCATGAAATGTCATTTTTACATAATTGTCTGCTCTATGGAACAAAATAAAAATAGCGAGGATCTGAACCCCCAAAGTATTCAAAGCAAGTGAAGGATGAGGAGGAGGGATTGGACGAAGAAGACGATGACGAGCATGGGGAGACCTTGTGTGGAGCATGTGGGGAGAACTATGCATCTGATGAGTTCTGGATTTGTTGCGACATATGTGAGAAGTGGTTTCATGGCAAGTGTGTGAAGATCACTCCTGCCCGGGCTGAGCACATCAAACACTACAAGTGCCCCTCATGCAGCAATAAGAGACCAAGGCCTTGA。

the method comprises the following specific steps:

construction of pCBSG015 Gene editing vector

Selection of target sequences: the target site sequence is T1 (nucleotide 27-45 of SEQ ID NO.1, namely SEQ ID NO. 4), and the position of T1 in SEQ ID NO.1 is shown in figure 1.

Selection of promoters: the T1 target site was initiated using AtU6 from arabidopsis thaliana.

Preparation of sgRNA expression cassette containing target site: the pCBSG015 vector was subjected to restriction enzyme digestion with Bsa I, a T1 sequence was directly synthesized by a primer synthesis method, and a 16bp vector sequence was added to each of both ends as a homology arm (Sense-U6-T1), a reverse complement sequence (Anti-U6-T1) was synthesized, and the Sense-U6-T1 and Anti-U6-T1 were annealed to form a double strand, and homologous recombination was performed with the linearized pCBSG015 vector according to the instructions of the homologous recombination kit. The operation is specifically as follows:

Sense-U6-T1：5｀-ggcaccgagtcggtgcCCGCACCGTCGAAGAGGTTgttgaacaacggaaac-3｀；

Anti-U6-T1:5 '-gtttccgttgttcaacAACCTCTTCGACGGTGCGGgcaccgactcggtgcc-3'; lower case letters are homology arms and upper case letters are T1 sequences.

Preparation of annealed AtU6-T1-gRNA fragment: the synthesized Sense-U6-T1 is annealed to the Anti-U6-T1 sequence to form a double strand. The reaction system is as follows: the synthesized sequence was dissolved in 75mM NaCl solution at a final concentration of 0.2 nM/. Mu.l, and the forward and reverse strand solutions (Sense-U6-T1 and Anti-U6-T1) were mixed in equal amounts. Heating in 95 ℃ water bath for 5-10min, and slowly cooling to obtain annealed AtU-T1-gRNA fragments.

Tangential enzyme-carrying: pCBSG015 plasmid 1-2. Mu.g, 10 XCutSmart ^TM Buffer (NEB) 5. Mu.l, bsa I endonuclease 1. Mu.l, sterile double distilled water make up 50. Mu.l. The mixture was reacted in a water bath at 37℃for 30 minutes, and then recovered and purified by EZ-10Column DNA Purification Kit (Shanghai chemical industry), and dissolved in water to obtain a linearized pCBSG015 vector.

The homologous recombination reaction of the target sgRNA expression cassette and the pCBSG015 vector is completed by adopting a homologous recombination method (an easy Geno rapid recombination cloning kit of the company TianGen). The reaction system is as follows: 2X EasyGeno Assembly mix Buffer. Mu.L, 0.5. Mu.L of linearized pCBSG015 vector, 4.5. Mu.L of annealed AtU-T1-gRNA fragment. The reaction conditions are as follows: 15min at 50 ℃. Transferring the connection product into E.coli DH5 alpha competent cells, picking positive colony to extract plasmid, and obtaining recombinant vector pCBSG015-sgRNA after sequencing correctly.

The recombinant vector pCBSG015-sgRNA is obtained by replacing the segment between 5 '-ggcaccgagtcggtgc-3' and 5 '-gttgaacaacggaaac-3' of the pCBSG015 vector with the DNA segment with the nucleotide sequence of 5 '-CCGCACCGTCGAAGAGGTT-3', and keeping the other sequences of the pCBSG015 vector unchanged, and the recombinant plasmid is named as the recombinant vector pCBSG015-sgRNA.

The recombinant vector pCBSG015-sgRNA was transferred into competent cells of Agrobacterium EHA105. The agrobacteria EHA105-pCBSG015-sgRNA is obtained.

Genetic transformation of soybean

The prepared agrobacterium EHA105-pCBSG015-sgRNA is used for infecting soybean varieties Jack to obtain soybean plants edited by GmPHD6 genes, which are completed by the hundred grid company. The T0 generation seeds are harvested, the T1 generation seeds are obtained by selfing the T1 generation seeds, the T1 generation seeds are planted to obtain T2 generation seedlings, and the following detection is carried out.

Screening of GmPHD6 Gene mutant homozygote

And (3) using the genomic DNA of the T2 generation seedling obtained in the step (II) as a template, amplifying a DNA sequence of about 560bp nearby the target sequence by using PCR primers designed nearby the target sequence at the upstream of about 120bp and nearby the target sequence at the downstream of about 420bp, and sequencing to detect the GmPHD6 gene editing mode, wherein the target PCR primers and the sequencing primers are shown in Table 2.

TABLE 2 target detection primer sequences

Target PCR primer:	G6-5’:GGGGTATTACGGATTACGGAG
			G6-3’:TTGCTATTTCACGCTTCATGG

after sequencing is successful, analyzing a website DSDecode (http:// dsDecode. Scgene. Com /) in a CRISPR target editing mode, comparing the website DSDecode with a gene standard sequence in a mode of combining a manual read peak diagram, and analyzing and subculturing the editing modes of each target sequence and upstream and downstream sequences thereof.

The gene editing modes of the GmPHD6 gene homozygous mutants selected in the above manner, selected by the selection method, are shown in FIG. 2. In the select 12#58 mutant, compared to the wild soybean variety Jack, the GmPHD6 gene in the genome was mutated: in the two homologous chromosomes, 1 nucleotide A is inserted between 29 th and 30 th positions of a sequence 3 in a sequence table, namely 1 adenine deoxyribonucleotide (A) is inserted between 29 th and 30 th positions of SEQ ID NO.1, so that encoded protein is terminated in advance at 39 th amino acid positions, the encoded amino acid sequence is mutated into MDSGGHYNPRHRRRGFPGLQGPPGWHDQSPHHWLLSLSL, the GmPHD6 gene is subjected to frame shift mutation, and protein translation is terminated in advance, so that the GmPHD6 gene is knocked out. The slect12#58 homozygote was designated phd6-58.

In the select 12#89 mutant, 1 nucleotide insertion occurred at the T1 site, i.e., a C was inserted at nucleotide 30 of SEQ ID NO. 1.

Compared with soybean variety Jack, the select 12#89 homozygote has mutation in the gene of GmPHD6 in soybean genome: in the two homologous chromosomes, 1 cytosine deoxyribonucleotide (C) is inserted between 29 th position and 30 th position of a sequence 3 in a sequence table, so that the encoded protein is terminated in advance at 32-position amino acid, and the encoded amino acid sequence is mutated into MDSGGHYNPRHRRRGFPGLQGPPGWHDQSPHH, so that the GmPHD6 gene is knocked out. The slect12#89 homozygote was designated phd6-89. Seeds from the T2 generation plants (T3 generation) were harvested for subsequent experiments.

Example 3 phenotypic analysis of GmPHD6 Gene CRISPR materials phd-58 and phd-6-89 (one) detection of GmPHD6 Gene expression levels of phd6-58 and phd6-89

Extracting total RNA of soybean varieties Jack and phd6-58 and phd6-89 seedlings, carrying out reverse transcription, and respectively taking cDNA obtained by reverse transcription as a template, wherein the primers are as follows: gmPHD6-up 5' -ATGGACAGTGGAGGACACTA-3 and GmPHD6-dp 5'-TCAAGGCCTTGGTCTCTTATT-3' were subjected to Real Time-PCR to determine the expression level of the GmPHD6 gene.

The soybean Tublin gene is used as an internal standard, and the internal standard Primer is Primer-TF:5'-AACCTCCTCCTCATCGTACT-3' Primer-TR:5'-GACAGCATCAGCCATGTTCA-3'. As a result, as shown in FIG. 3, when the relative expression level of GmPHD6 in Jack was 1, the expression levels of GmPHD6 in phd6-58 and phd6-89 were about 0.2 and 0.7, respectively. The expression level of GmPHD6 in phd-58 and phd-89 was shown to be significantly reduced (FIG. 3).

(II) Down-regulating GmPHD6 Gene to increase seed weight of Soybean seed

First generation seeds of phd6-58 and phd6-89 described above (seeds of T2 generation plants of the transgenic event in step (two) of example 2) and control soybean variety Jack seeds were planted in greenhouse pots, greenhouse conditions: illumination time 16h:8h (day: right) temperature: seed is collected in the daytime at 30-37 ℃ and at night at 25-28 ℃ for 130 days. The Jack phenotypes of phd6-58 and phd6-89 and control soybean varieties were observed after seed maturation, as shown in fig. 4 a, with phd6-58 and phd6-89 plant heights greater than control Jack.

Ten phd-58, phd-6-89 and control soybean variety Jack seeds were compared side-by-side, and thousand phd-58, phd6-89 and control soybean variety Jack seeds were tiled for control, resulting in seeds of phd6-58 and phd-89 significantly larger than the recipient control Jack, as shown in fig. 4B.

Hundred weight statistics: after the potted soybean seeds described above were matured, the individual harvested seeds were dried at 37℃for one week and the seed hundred weight (i.e., the hundred weight of thoroughly dried seeds) of the recipient controls Jack, phd6-58 and phd6-89 were measured. Each strain was obtained by taking 20 seeds, weighing 50 seeds for each strain, repeating the biological experiment three times, converting the biological experiment into hundred weight, and taking the average value of + -standard deviation to represent the average value, wherein the average value of + -standard deviation is tested by using One-way ANOVA, P < 0.05 (x) represents a significant difference, P < 0.01 (x) represents a very significant difference, and P < 0.001 (x) represents a very significant difference.

The results are shown in FIG. 5A, with the receptor controls Jack, phd6-58 and phd6-89 hundred grams at 16.5+ -2.1,18.2 + -4.2 and 19.0+ -3.8 grams, respectively. The weight of phd-58 and phd-89 hundred grains was increased by 10.3-15.1% compared to control Jack.

Seeds of the phd6-58 and phd6-89T3 generation and seeds of the recipient control Jack were planted in the field, all seeds were planted in 5 pieces of 4m, respectively ² After the seeds are mature, the individual harvested seeds are dried at 37℃for one week and the seed hundred weight (i.e., the hundred weight of thoroughly dried seeds) of the recipient controls Jack, phd6-58 and phd6-89 is measured. Each strain is respectively from 5 fieldsThe method comprises the steps of taking 20 single plants, weighing 100 seeds for each plant line, repeating biological experiments three times, taking an average value of +/-standard deviation, and using One-way ANOVA test, wherein P < 0.05 (in the x) represents a significant difference, P < 0.01 (in the x) represents a very significant difference, and P < 0.001 (in the x) represents a very significant difference.

As a result, as shown in FIG. 6A, the hundred-grain weights of the recipient controls Jack, phd6-58 and phd6-89 were 14.7.+ -. 0.2, 15.6.+ -. 0.7 and 15.8.+ -. 0.3 g, respectively. The weight of phd-58 and phd-89 hundred grains was increased by 6.1-7.5% compared to control Jack.

Comparison of yields of the three receptor control Jack, mutants phd-58 and phd6-89 individuals

The first generation seeds of phd6-58 and phd6-89 described above (seeds of T2 generation plants of the transgenic event in step (II) of example 2) and control Jack seeds were planted in greenhouse pots and cultured using the culture conditions of step (II) of example 3, and the individual yield of the recipient control Jack, mutants phd6-58 and phd6-89 was examined after seed maturation. Mature seeds harvested by a single plant are dried for one week at 37 ℃,10 plants are taken for each plant, all the seeds of each plant are collected respectively, and the total weight of each plant of thoroughly dried seeds is weighed. The biological experiments were repeated three times, and the results were expressed as mean ± standard deviation, using One-way ANOVA test, P < 0.05 (x) indicated significant differences, P < 0.01 (x) indicated very significant differences, and P < 0.001 (x) indicated very significant differences.

As a result, as shown in FIG. 5B, the individual weights of the receptor controls Jack, phd6-58 and phd-89 were 73.0.+ -. 11.3,88.2.+ -. 10.9 and 83.1.+ -. 12.8 g, respectively. Mutants phd-58 and phd-6-89 increased 13.8% -20.8% over control Jack.

The second generation seeds of phd6-58 and phd6-89 (T3 generation seeds of the transgenic event of example 2, step (II)) and control Jack seeds were planted in the field, and all seeds were planted in 5 pieces of 4m, respectively ² After the seeds were matured, the individual harvested seeds were dried at 37℃for one week and the grain weights of 20 individual plants of the recipient controls Jack, phd6-58 and phd6-89 were measured. Randomly selecting 20 individual plants from 5 fields of control Jack, phd6-58 and phd-89, respectively, collecting dried seeds, measuring 20The weights of all seeds of the single plant were repeated three times, and the results were expressed as mean ± standard deviation, using One-way ANOVA test, P < 0.05 (x) indicated significant differences, P < 0.01 (x) indicated significant differences, and P < 0.001 (x) indicated significant differences.

As a result, as shown in FIG. 6B, the individual weights of the receptor controls Jack, phd6-58 and phd-89 were 261.0.+ -.53, 278.+ -.50 and 313.+ -.27 g, respectively. The grain weights of phd-58 and phd-8920 individuals increased by 6.5% -19.9% compared to control Jack.

The above yields (3X 4m per strain ² ) Converted to yield per hectare (1 hectare=10000 m) ² ) The yields per hectare of the controls Jack, phd6-58 and phd-89 were approximately 2578Kg/ha,2782Kg/ha and 3111Kg/ha. The yields per hectare were increased by about 7.9% -20.6% for phd-58 and phd-89 compared to control Jack.

The above statistics show that the hundred weight and individual plant weight of greenhouse potting phd6-58 and phd6-89 are significantly higher than the recipient control Jack. The field trials showed that the hundred grain weights of phd6-58 and phd6-89 were also significantly higher than the control Jack, whereas the total grain weight of 20 individual plants, mutants phd6-58 and phd6-89 were all higher than the control, with phd6-89 reaching significant levels. In a word, both greenhouse potting and field results show that the GmPHD6 can negatively regulate the grain weight of soybean seeds, and the reduction of the expression level of GmPHD6 can remarkably improve the grain weight of soybean seeds, the yield of single plant and the yield per hectare.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

1. Use of a substance that inhibits or reduces or down-regulates expression of a gene encoding a protein or a substance that reduces the content of a protein in any of the following:

the protein is any one of the following:

b1 Amino acid sequence is a protein shown in sequence 2;

b2 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) with protein tags;

the substance which inhibits or reduces or down-regulates the expression of the encoding gene of the protein or the substance which reduces the content of the protein is any one of the following substances:

d1 A nucleic acid molecule that inhibits or reduces or down-regulates expression of a gene encoding the protein;

d2 An expression cassette comprising D1) said nucleic acid molecule;

d3 A recombinant vector comprising D1) said nucleic acid molecule, or a recombinant vector comprising D2) said expression cassette;

d4 A recombinant microorganism comprising D1) said nucleic acid molecule, or a recombinant microorganism comprising D2) said expression cassette, or a recombinant microorganism comprising D3) said recombinant vector;

the plant is leguminous plant.

2. The use according to claim 1, wherein the protein is derived from soybean.

3. The use according to claim 1 or 2, wherein the leguminous plant is a soyabean plant.

4. The use according to claim 3, wherein the soyabean is soybean.

5. The use according to claim 1 or 2, wherein D1) the nucleic acid molecule is a gRNA targeting the gene encoding the protein of claim 1, the target sequence of the gRNA being sequence 3 in the sequence listing.

6. The use according to claim 5, wherein the leguminous plant is a plant of the genus glycine.

7. The use according to claim 6, wherein the soyabean is soybean.

8. A method of growing a high seed weight and/or seed yield plant, comprising inhibiting or reducing or down-regulating the expression of a gene encoding a protein as claimed in claim 1 or 2 in a plant of interest, or reducing the protein content to obtain a high seed weight and/or seed yield plant having a higher seed weight and/or seed yield than the plant of interest; the target plant is leguminous plant.

9. The method of claim 8, wherein said inhibiting or reducing or down-regulating the expression level of a gene encoding the protein of claim 1 or 2 in a plant, or reducing the protein level, comprises introducing into said plant of interest a gene knockout vector comprising a gRNA targeting sequence 3.

10. The method of claim 8 or 9, wherein the leguminous plant is a soyabean plant.

11. The method of claim 10, wherein the soyabean is soybean.

12. The method according to claim 8, wherein the inhibiting or reducing or down-regulating the expression level of a gene encoding the protein according to claim 1 or 2 in the plant of interest, or the reducing the protein content is performed on the genome of the plant of interest by any one of the following operations:

k1 1 adenine deoxyribonucleotide is inserted between the 29 th position and the 30 th position of the sequence 3 in the sequence table in the genome of the target plant;

k2 Inserting 1 cytosine deoxyribonucleotide between the 29 th position and the 30 th position of the sequence 3 in a target plant genome sequence table;

the target plant is soybean.