阅读说明：本技术 栽培花生AhGPAT9A基因及其在改良种子含油量中的应用 (Cultivated peanut AhGPAT9A gene and application thereof in improving oil content of seeds ) 是由 刘风珍 万勇善 吕玉英 张秀荣 张昆 骆璐 杨会 朱素青 华方静 于 2020-07-02 设计创作，主要内容包括：本发明公开了一种栽培花生AhGPAT9A基因及其在改良种子含油量中的应用。本发明克隆得到了来源于栽培花生A染色体组的AhGPAT9A基因；并通过构建转基因花生株系,对AhGPAT9A基因的功能进行了考察,发现AhGPAT9A基因对花生种子的含油量有显著影响。因此,本发明对解析花生高油分子机制,提升高油分子育种技术水平,培育高油新品种都具有重要的理论指导意义和实践应用价值。(The invention discloses a cultivated peanut AhGPAT9A gene and application thereof in improving oil content of seeds. The AhGPAT9A gene derived from the peanut A genome is cloned; and by constructing a transgenic peanut strain, the function of the AhGPAT9A gene is investigated, and the AhGPAT9A gene is found to have a remarkable influence on the oil content of peanut seeds. Therefore, the method has important theoretical guidance significance and practical application value for analyzing the mechanism of the peanut high-oil molecules, improving the breeding technical level of the high-oil molecules and cultivating new high-oil species.)

1. An AhGPAT9A gene derived from the chromosome group of cultivated peanut A, wherein the nucleotide sequence of the AhGPAT9A gene is shown as any one of the following (1) to (3):

(1) a nucleotide sequence shown as SEQ ID NO. 1;

(2) a nucleotide sequence shown as SEQ ID NO. 2;

(3) a nucleotide sequence other than (2) encoding the amino acid composition shown in SEQ ID NO. 3.

2. A recombinant expression vector, a genetically engineered bacterium or a transgenic strain carrying the AhGPAT9A gene of claim 1.

3. The AhGPAT9A gene as shown in any one of the following (1) to (3) is applied to regulating the oil content of peanut seeds;

(1) the AhGPAT9A gene has a DNA sequence shown in SEQ ID NO. 1;

(2) the cDNA sequence of the AhGPAT9A gene is shown in SEQ ID NO. 2;

(3) the AhGPAT9A gene, which has a nucleotide sequence coding for the amino acid composition shown in SEQ ID NO.3 except for (2).

4. Use of a recombinant expression vector, a genetically engineered bacterium or a transgenic strain carrying the AhGPAT9A gene of claim 1 for regulating the oil content of peanut seeds.

5. Use of a protein according to any one of (1) to (3) below for modulating oil content in a peanut seed;

1) the amino acid sequence is a protein shown as SEQ ID NO. 3;

2) the protein which has the same function with the protein shown in SEQ ID NO.3 is obtained by replacing, deleting or inserting one, a plurality of or dozens of amino acids in the amino acid sequence shown in SEQ ID NO. 3;

3) and (3) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in SEQ ID NO. 3.

6. A method for improving the oil content of peanut seeds by using an AhGPAT9A gene is characterized by comprising the following steps: a step of overexpressing the AhGPAT9A gene shown in any one of the following (1) to (3) in a peanut plant;

(1) the AhGPAT9A gene has a DNA sequence shown in SEQ ID NO. 1;

(2) the cDNA sequence of the AhGPAT9A gene is shown in SEQ ID NO. 2;

(3) the AhGPAT9A gene, which has a nucleotide sequence coding for the amino acid composition shown in SEQ ID NO.3 except for (2).

7. A method for cultivating a high-oil transgenic peanut strain is characterized by comprising the following steps:

(1) constructing a overexpression vector pGB-GPAT9A-OE of the peanut AhGPAT9A gene, transferring the overexpression vector pGB-GPAT9A-OE into agrobacterium LBA4404 competent cells by a freeze-thaw method, and screening to obtain a recombinant agrobacterium strain carrying a plasmid pGB-GPAT 9A-OE;

(2) selecting a single colony of the recombinant agrobacterium strain obtained in the step (1), performing activated culture in a YEB liquid culture medium, centrifuging, collecting thalli, and suspending the thalli by using an MS liquid culture medium until OD600 is 0.6-0.7 to obtain a bacterial suspension; taking young leaves of seeds of a peanut cultivar Fenghua No.2 as transgenic infection explants, inoculating the transgenic infection explants in a bud cluster induction culture medium under an aseptic condition for pre-culture, placing the pre-cultured explants in a bacterial suspension for oscillation infection for 10-20min, sucking a bacterial solution on the surfaces of the explants by using filter paper, transferring the bacterial solution to the bud cluster induction culture medium, performing dark culture for 3-5d, transferring the bacterial solution to a bud cluster induction culture medium added with cephalo, performing culture for 28-32d, transferring the bacterial solution to a differentiation culture medium, and continuing subculture until seedlings grow out to obtain regenerated seedlings; transferring the regenerated seedlings into a screening culture medium containing a herbicide, and screening to obtain resistant seedlings; transferring the resistant seedlings to an MS culture medium for rapid propagation;

(3) and carrying out Bar gene detection on the fast propagated resistant seedlings, transplanting the T0 transgenic plants with positive gene detection into a field, continuously planting the harvested seeds in the second year, continuously carrying out Bar gene detection on DNA of the T1 transgenic plants for three or more generations, and obtaining the transgenic peanut lines with stable high-oiliness.

8. The method as claimed in claim 7, wherein in step (1), the overexpression vector pGB-GPAT9A-OE is constructed by:

specifically amplifying peanut AhGPAT9A gene by using primers shown in SEQ ID NO.4 and SEQ ID NO.5, connecting the amplified product with a cloning vector pEASY-T1 to obtain a recombinant plasmid pEASY-AhGPAT 9A;

and carrying out double digestion on the recombinant plasmids pEASY-AhGPAT9A and pGBVE expression vectors by using restriction enzymes Bam H I and Not I respectively, recovering corresponding target fragments, and connecting the recovered target gene AhGPAT9A to the rear of a 35S promoter in the expression vector pGBVE by using T4DNA ligase to obtain a super-expression vector pGB-GPAT 9A-OE.

9. The method according to claim 7, wherein in step (2), the composition of the sprout inducing medium is: MS culture medium +5.0mg/L6-BA +0.75mg/L NAA;

preferably, in step (2), the composition of the differentiation medium is: MS culture medium +6 mg/L6-BA;

preferably, in step (2), the composition of the screening medium is: MS culture medium +1.2mg/L PPT.

10. The method according to claim 7, wherein in step (3), the Bar gene is detected by: the primers shown in SEQ ID NO.8 and SEQ ID NO.9 are used for PCR amplification, and the positive transgenic plant can generate a 442bp target band.

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a cultivated peanut AhGPAT9A gene and application thereof in improving oil content of seeds.

Background

Peanuts (Arachis Hypogaea L.) are an important oil and economic crop worldwide. About 55% of the total peanut yield in China is used for oil extraction, so the peanuts occupy a great position in the aspects of guaranteeing the safety of grease supply in China and stabilizing the domestic oil source. The oil content is one of the most important quality traits of peanuts, and the high oil content is an important target of peanut breeding research. The oil content difference among different peanut varieties can reach more than 20 percent, which indicates that the oil content of the peanut varieties still has great space for improvement, and the cultivation of high-oil high-quality varieties has great genetic potential. Therefore, the research on the peanut oil content related gene, particularly the mining and utilization of the key gene for oil synthesis has important theoretical guidance significance and practical application value for analyzing the peanut high oil molecule mechanism, improving the high oil molecule breeding technical level and cultivating new high oil varieties.

Triacylglycerols (TAGs) are the major form of vegetable lipids, and in the biosynthetic pathway of vegetable TAGs, triacylglycerol acyltransferases (GPAT) transfer the fatty acyl group on an acyl carrier protein or acyl-CoA to the sn-1 position of glycerol-3-phosphate (G3P) to form lysophosphatidic acid (LPA), the first acyltransferase to catalyze TAG synthesis, and play an important role in seed development and lipid accumulation. The plant GPAT family contains many members. In arabidopsis, GPAT comprises 10 members, is divided into 3 types according to subcellular localization, enzyme activity and substrate selectivity, and is respectively located in mitochondria, chloroplasts and endoplasmic reticulum, wherein GPAT9 is located in the endoplasmic reticulum and belongs to membrane-bound protein, and research shows that the arabidopsis GPAT9 gene down-regulated expression causes the shrinkage of seeds, and the oil content is obviously lower than that of a wild type; over-expression of GPAT9 not only results in significant increase of seed grain weight and oil content, but also greatly increases the content of TAG in leaves. In peanuts, it was studied to clone a GAPT9 gene from the cultivar flower 19, which gene is expressed in stems, flowers and seeds, but its role in the seed oil accumulation process was not studied in depth.

Cultivated peanuts (Arachis hypogaea L.) are heterotetraploids (2n 4x 40, AABB) formed by natural crossing of diploid ancestral wild species a.duranensis (AA) and a.ipaensis (BB) of two peanut blocks followed by a single natural doubling event, with a genome size of 2.7 Gb. The cultivated peanut comprises two chromosome groups A and B, and researches show that homologous genes between the chromosome group A and the chromosome group B of the cultivated peanut can have various complex relationships such as redundancy, accumulation, complementation and the like in function. In the existing research, most of the cloned peanut genes are not distinguished from A and B chromosome sets, and related reports on AhGPAT9A genes derived from a peanut A chromosome set and functions thereof are not found at present.

In addition, as peanuts are one of the crops which are difficult to be genetically transformed, in the process of establishing a peanut genetic transformation system, the problems of low regeneration and transformation rate, limitation of variety genotypes, difficult stable existence of transformed genes in transgenic plants and the like exist, so that the functional verification and transformation application of peanut genes are very difficult.

Disclosure of Invention

Aiming at the prior art, after long-term research and exploration, the AhGPAT9A gene derived from the peanut A genome is cloned; and by constructing a transgenic peanut strain, the function of the AhGPAT9A gene is investigated, and the AhGPAT9A gene is found to have a remarkable influence on the oil content of peanut seeds. Therefore, the method has important theoretical guidance significance and practical application value for analyzing the mechanism of the peanut high-oil molecules, improving the breeding technical level of the high-oil molecules and cultivating new high-oil species.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided an AhGPAT9A gene derived from the genome of arachis hypogaea, wherein the nucleotide sequence of the AhGPAT9A gene is as shown in any one of the following (1) to (3):

(1) a nucleotide sequence shown as SEQ ID NO. 1;

(2) a nucleotide sequence shown as SEQ ID NO. 2;

(3) a nucleotide sequence other than (2) encoding the amino acid composition shown in SEQ ID NO. 3.

In a second aspect of the invention, a recombinant expression vector, a genetically engineered bacterium or a transgenic strain carrying the AhGPAT9A gene is provided.

In a third aspect of the present invention, there is provided a use of the AhGPAT9A gene as shown in any one of (1) to (3) below for regulating oil content in peanut seeds;

(1) the AhGPAT9A gene has a DNA sequence shown in SEQ ID NO. 1;

(2) the cDNA sequence of the AhGPAT9A gene is shown in SEQ ID NO. 2;

(3) the AhGPAT9A gene, which has a nucleotide sequence coding for the amino acid composition shown in SEQ ID NO.3 except for (2).

In a fourth aspect of the invention, the invention provides an application of a recombinant expression vector, a genetically engineered bacterium or a transgenic strain carrying the AhGPAT9A gene in regulating the oil content of peanut seeds.

In a fifth aspect of the present invention, there is provided a use of a protein as described in any one of (1) to (3) below for regulating oil content of peanut seeds;

1) the amino acid sequence is a protein shown as SEQ ID NO. 3;

2) the protein which has the same function with the protein shown in SEQ ID NO.3 is obtained by replacing, deleting or inserting one, a plurality of or dozens of amino acids in the amino acid sequence shown in SEQ ID NO. 3;

3) and (3) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in SEQ ID NO. 3.

In a sixth aspect of the present invention, there is provided a method for improving oil content of peanut seeds by using AhGPAT9A gene, comprising: a step of overexpressing the AhGPAT9A gene shown in any one of the following (1) to (3) in a peanut plant;

(1) the AhGPAT9A gene has a DNA sequence shown in SEQ ID NO. 1;

(2) the cDNA sequence of the AhGPAT9A gene is shown in SEQ ID NO. 2;

(3) the AhGPAT9A gene, which has a nucleotide sequence coding for the amino acid composition shown in SEQ ID NO.3 except for (2).

In a seventh aspect of the invention, there is provided a method of breeding high oil transgenic peanut lines comprising the steps of:

(1) constructing a overexpression vector pGB-GPAT9A-OE of the peanut AhGPAT9A gene, transferring the overexpression vector pGB-GPAT9A-OE into agrobacterium LBA4404 competent cells by a freeze-thaw method, and screening to obtain a recombinant agrobacterium strain carrying a plasmid pGB-GPAT 9A-OE;

(2) selecting a single colony of the recombinant agrobacterium strain obtained in the step (1), performing activated culture in a YEB liquid culture medium, centrifuging, collecting thalli, and suspending the thalli by using an MS liquid culture medium until OD600 is 0.6-0.7 to obtain a bacterial suspension; taking young leaves of seeds of a peanut cultivar Fenghua No.2 as transgenic infection explants, inoculating the transgenic infection explants in a bud cluster induction culture medium under an aseptic condition for pre-culture, placing the pre-cultured explants in a bacterial suspension for oscillation infection for 10-20min, sucking a bacterial solution on the surfaces of the explants by using filter paper, transferring the bacterial solution to the bud cluster induction culture medium, performing dark culture for 3-5d, transferring the bacterial solution to a bud cluster induction culture medium added with cephalo, performing culture for 28-32d, transferring the bacterial solution to a differentiation culture medium, and continuing subculture until seedlings grow out to obtain regenerated seedlings; transferring the regenerated seedlings into a screening culture medium containing a herbicide, and screening to obtain resistant seedlings; transferring the resistant seedlings to an MS culture medium for rapid propagation;

(3) and carrying out Bar gene detection on the fast propagated resistant seedlings, transplanting the T0 transgenic plants with positive gene detection into a field, continuously planting the harvested seeds in the second year, continuously carrying out Bar gene detection on DNA of the T1 transgenic plants for three or more generations, and obtaining the transgenic peanut lines with stable high-oiliness.

Preferably, in the step (1), the construction method of the overexpression vector pGB-GPAT9A-OE comprises the following steps:

specifically amplifying peanut AhGPAT9A gene by using primers shown in SEQ ID NO.4 and SEQ ID NO.5, connecting the amplified product with a cloning vector pEASY-T1 to obtain a recombinant plasmid pEASY-AhGPAT 9A;

and carrying out double digestion on the recombinant plasmids pEASY-AhGPAT9A and pGBVE expression vectors by using restriction enzymes Bam H I and Not I respectively, recovering corresponding target fragments, and connecting the recovered target gene AhGPAT9A to the rear of a 35S promoter in the expression vector pGBVE by using T4DNA ligase to obtain a super-expression vector pGB-GPAT 9A-OE.

Preferably, in step (2), the composition of the bud plexus induction medium is: MS culture medium +5.0mg/L6-BA +0.75mg/L NAA.

Preferably, in step (2), the composition of the differentiation medium is: MS culture medium +6 mg/L6-BA.

Preferably, in step (2), the composition of the screening medium is: MS culture medium +1.2mg/L PPT.

Preferably, in step (3), the method for detecting Bar gene is as follows: the primers shown in SEQ ID NO.8 and SEQ ID NO.9 are used for PCR amplification, and the positive transgenic plant can generate a 442bp target band.

The invention has the beneficial effects that:

the AhGPAT9A gene is cloned from a cultivated peanut A chromosome group for the first time, an overexpression vector pGB-GPAT9A-OE and an inhibition expression vector pGB-GPAT9A-AE of a peanut AhGPAT9A gene are constructed, a peanut receptor is transformed by an optimized agrobacterium-mediated gene transformation method, and a peanut transgenic strain with stable character expression is obtained, wherein the oil content of seeds of an AhGPAT9A gene overexpression plant is remarkably improved, and the oil content of seeds of an AhGPAT9A gene inhibition expression plant is remarkably reduced. Through the oil content detection of the seeds of 3 generations, a transgenic line with obviously improved oil content and a transgenic line with obviously reduced oil content are screened out. The invention provides reference for deeply researching the GPAT9 gene function of the peanut, and has important theoretical and practical significance for improving the oil content of the peanut and cultivating new varieties of high-oil peanuts by utilizing genetic engineering.

Drawings

FIG. 1: the total RNA of the young peanut leaves is subjected to a 1% agarose gel electrophoresis picture.

FIG. 2: the RNA reverse transcription product was electrophoresed through 1% agarose gel to give a band in a diffuse shape.

FIG. 3: the AhGPAT9A gene PCR amplification product is detected by 1% agarose gel electrophoresis, and the fragment size is about 1400 bp. M is DL5000 marker.

FIG. 4: a is the restriction enzyme BamH I and Not I which are used for double digestion of pGBVE expression vector and recombinant plasmid pEASY-T1, B is the electrophoresis pattern of recombinant plasmid pGB-GPAT9A-OE identified by BamH I and Not I digestion.

FIG. 5: the plant expression vector of the AhGPAT9A gene comprises a construction schematic diagram of an overexpression vector pGB-GPAT9A-OE and an inhibition expression vector pGB-GPAT 9A-AE.

FIG. 6: the PCR detection result of the marker gene Bar gene is carried out on the resistant seedlings, and the positive T0 generation transgenic plants can generate a 442bp target strip. The G and R numbers represent resistant seedlings obtained by overexpression and suppression of expression, respectively.

FIG. 7: and carrying out PCR detection on marker gene Bar gene of T1 transgenic plants. The G and R numbers represent overexpression and repression expression transgenic plants respectively. G represents bacteria liquid control, and FH2 is wild type Fenghua No. 2.

FIG. 8: and carrying out PCR detection on marker gene Bar gene of T2 transgenic plants. + represents the positive control.

FIG. 9: and carrying out PCR detection on marker gene Bar gene of T3 transgenic plants. + and-positive bacteria control and negative wild type Fenghua No.2 control, respectively. Through the screening of two generations of T1 and T2, the positive detection rate in the T3 generation reaches more than 90%.

FIG. 10: and carrying out PCR detection on marker gene Bar gene of T4 transgenic plants. The positive detection rate in the T4 generation reaches more than 95 percent. Indicating that the transgenic fragment has been essentially stably inherited.

FIG. 11: and analyzing the expression quantity of the AhGPAT9A gene of seeds of T2 transgenic plants. The G and R numbers represent transgenic plants of the overexpression and suppression expression T2 generations respectively. FH2 was wild type control floret No. 2.

FIG. 12: the comparative analysis of the oil content of T1 transgenic plants and the wild control Fenghua No.2 shows that G and R represent overexpression and suppression transgenic plants respectively. FH2 was Fenghua No. 2.

FIG. 13: the comparative analysis of the oil content of T2 generation transgenic plants and the wild type control Fenghua No.2, wherein A is the result analysis of the oil content of the seeds of the overexpression transgenic plants; b, result analysis of oil content of the seeds of the transgenic plants with the suppressed expression. FH2 was Fenghua No. 2.

FIG. 14: and comparing the oil content of the screened T3 generation transgenic line seeds with that of the wild type control floret No. 2. The G and R numbers represent overexpression and repression expression transgenic plants respectively. FH2 was Fenghua No. 2.

FIG. 15: comparative analysis of partial agronomic traits of T3 generation transgenic line with wild type control Toyobo No. 2. The G and R numbers represent overexpression and repression expression transgenic plants respectively. FH2 was Fenghua No. 2.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As described in the background section, current studies on the cultivated peanut GPAT9 gene do not define the chromosome group to which the gene belongs. The homologous genes between the A chromosome set and the B chromosome set of the cultivated peanuts have various complex relationships in function. Therefore, only by respectively carrying out specific cloning and function analysis on the genes of the two genomes of the cultivated peanuts, the functions of the genes of different genomes and the action relationship between homologous genes can be determined. However, because some homologous gene sequences between group A and group B have very high similarity, it is difficult to clone the gene sequences of group A and group B separately.

In addition, peanuts are one of crops which are difficult to be genetically transformed, and in the process of establishing a peanut genetic transformation system, the problems of low regeneration and transformation rate, limitation of variety genotypes, difficult stable existence of transformed genes in transgenic plants and the like exist, so that the function of AhGPAT9A genes derived from a peanut A genome is very difficult to clone and verify.

The inventor separates the full-length cDNA sequence of AhGPAT9A gene from the variety Fenghua No.2 of the cultivated species, constructs an over-expression vector and an inhibition expression vector, transforms Fenghua No.2 by using an agrobacterium-mediated method, researches the expression condition of the AhGPAT9A gene in a transgenic strain and the influence on the oil content of seeds, screens out 2 transgenic peanut strains of which the AhGPAT9A gene is over-expressed and the oil content of the seeds is remarkably improved, and screens out 2 transgenic peanut strains of which the AhGPAT9A gene is inhibited and the oil content of the seeds is remarkably reduced.

AhGPAT9A gene DNA sequence 5426bp (SEQ ID NO. 1). The cDNA sequence has total length of 1395bp (SEQ ID NO.2), including 127bp 5 '-UTR and 137bp 3' -UTR, and coding region 1131 bp. Contains 13 exons and 12 introns. The encoded protein comprises 376 amino acids (SEQ ID NO. 3).

Aiming at the difficulties existing in the process of establishing a peanut genetic transformation system, the peanut genetic transformation system is established by adopting an agrobacterium-mediated method, the concentration of a bacterial liquid, the infection time, the culture conditions and the like are optimized, and the transferred gene fragment can be stably expressed in transgenic crops through continuous multi-generation planting.

Based on the AhGPAT9A gene found above, the scope of the present invention also includes DNA fragments homologous to the AhGPAT9A gene as long as they encode a protein functionally equivalent to the protein shown in SEQ ID NO. 3. The phrase "functionally equivalent to the protein shown in SEQ ID NO. 3" as used herein means that the protein encoded by the target DNA fragment is identical or similar to the protein shown in SEQ ID NO.3 in terms of biological functions, physiological and biochemical characteristics, etc. The typical biological function of the protein shown in SEQ ID NO.3 is to promote the increase of the oil content in peanut seeds.

The DNA fragments homologous with the AhGPAT9A gene comprise alleles, homologous genes, mutant genes and derivative genes corresponding to the nucleotide sequences (SEQ ID NO.1 and SEQ ID NO.2) of the invention; the encoded proteins are similar to the protein shown in SEQ ID NO.3 of the invention, or have substitution, deletion or insertion phenomena of one, a plurality of or dozens of amino acids, and belong to the content of the invention.

The nucleotide sequence of the non-critical site of the AhGPAT9A gene of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the AhGPAT9A gene of the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as the encoded protein is functionally equivalent to the protein represented by SEQ ID NO. 3.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.3 of the present invention. The identity of amino acid or nucleotide sequences can be determined using the BLAST algorithm (Altschul et al 1990.journal of Molecular Biology 215: 403. sup. 410; Karlin and Daltschul.1993.proceedings of the National Academy of Sciences 90: 5873. sup. 5877).

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples and comparative examples of the present invention are conventional in the art and are commercially available. The experimental procedures, for which no detailed conditions are indicated, were carried out according to the usual experimental procedures or according to the instructions recommended by the supplier. Wherein: the kit and the reagent adopted in the invention are purchased from Beijing Tiangen (TIABGEN) company, Huayuyo company and Dalibao (TaKaRa) company; the peanut cultivar Fenghua No.2 used in the experiment was provided by peanut research institute of Shandong agricultural university.