ZmbHLH175 gene related to corn kernel traits and application of ZmbHLH175 gene

Abstract

The invention discloses a ZmbHLH175 gene related to corn kernel traits and application thereof, and experiments prove that through overexpression of the ZmbHLH175 gene in corn, an overexpression strain has better kernel traits compared with a wild type, specifically, kernels of the overexpression strain have larger kernel width, kernel volume and hundred-kernel weight, and the corn kernel traits can be better improved. Compared with the prior art, the ZmbHLH175 gene has the advantages that the ZmbHLH175 gene has a positive influence on corn grain traits including grain width, grain volume, hundred-grain weight and starch content, so that corn plants can have larger grain width and grain volume through overexpression or high expression of the ZmbHLH175, the hundred-grain weight and the starch content in the grains are increased, and the corn yield is increased. The method effectively improves the yield and quality of the corn kernels, can provide a new direction for improving the quality and quantity of the corn, and has a reliable prospect when being applied to a biological breeding process for improving the characters of the corn kernels.

Description

Technical Field

[0001] The invention relates to the technical field of corn molecular breeding, and in particular to a ZmbHLH175 gene related to corn kernel traits and an application thereof. Background Art

[0002] The kernel-related traits of corn include protein content, kernel bulk density, kernel length, kernel width, kernel volume, and kernel weight per 100 kernels, which are important factors determining corn yield. Kernel traits have the advantages of high heritability, low environmental impact, and high accuracy of phenotypic evaluation, and can be used as important traits for analyzing genetic variation of yield traits. Tropical corn germplasm has the advantages of rich genetic diversity, high yield, high quality, and strong stress resistance, and can be used as raw materials for mining excellent genes. The bHLH gene family plays a key role in the growth and development of the entire plant and in adverse stress. Corn has 208 bHLH family members, and 157 and 17 bHLH genes were found to be highly expressed in endosperm and embryonic development during kernel formation, respectively. A key bHLH family gene for kernel development, ZmbHLH175, was mined through linkage populations of two commonly used tropical corn materials. The homologous gene of bHLH175 in Arabidopsis, rice and barley, also named ICE1a, was found to play a key role in plant tolerance to cold stress and can regulate seed germination and dormancy by regulating the abscisic acid signaling pathway, but there are currently no reports on the association of this gene with corn grain traits. Summary of the invention

[0003] Therefore, based on the above background, the present invention provides the ZmbHLH175 gene related to corn kernel traits and its application. In the present invention, ZmbHLH175 is used as a key gene related to kernel traits and is applied to biological breeding for improving corn kernel traits, which can provide a new direction for improving corn kernel shape and help accelerate the breeding process of high-yield and high-quality corn.

[0004] The first technical solution of the present invention is to provide a ZmbHLH175 gene related to corn grain traits, the nucleotide sequence of the ZmbHLH175 gene coding region is shown as SEQ ID NO.7, and the amino acid sequence of the protein encoded by the ZmbHLH175 gene is shown as SEQ ID NO.8.

[0005] Furthermore, the corn kernel traits include kernel width, kernel volume, 100-kernel weight and kernel starch content.

[0006] The second technical solution of the present invention is to provide a recombinant expression vector, which contains the ZmbHLH175 gene and a basic vector.

[0007] The third technical solution of the present invention is to provide a genetically engineered bacterium containing the ZmbHLH175 gene or the recombinant expression vector.

[0008] The fourth technical solution of the present invention is to provide the application of the ZmbHLH175 gene and the protein encoded by it, or the recombinant expression vector, or the genetically engineered bacteria, wherein the application includes at least one of the following:

[0009] 1) Application in improving corn kernel traits;

[0010] 2) Application in corn breeding or assisted breeding.

[0011] Furthermore, the improvement of corn grain traits is achieved by increasing the expression level of the ZmbHLH175 gene or its encoded protein in corn.

[0012] The fifth technical solution of the present invention provides the use of the ZmbHLH175 gene or its encoded protein in identifying or breeding or assisting in breeding corn plants, lines or varieties with excellent grain traits.

[0013] Furthermore, the expression level of ZmbHLH175 gene is detected by gene expression level, or the variation of ZmbHLH175 encoding protein is predicted by transcript, so as to screen out corn plants with excellent grain traits.

[0014] Furthermore, the nucleotide sequences of the primers for detecting gene expression are shown in SEQ ID NO.3 and SEQ ID NO.4:

[0015] Left primer F: 5′-GTGCCGAAGATCAGCAAGAT-3′;

[0016] Right primer R: 5′-TCGGTGTTGATGGGTTGAAG-3′;

[0017] The nucleotide sequences of the primers used for transcript prediction are shown in SEQ ID NO.5 and SEQ ID NO.6:

[0018] Left primer F: 5′-GAGTTGCCAGTGAACCAGGAT-3′;

[0019] Right primer R: 5′-CTACATTGCGTTGTGGAGGC-3′.

[0020] The sixth technical solution of the present invention is to provide a corn breeding method for improving corn kernel traits, wherein the corn gene ZmbHLH175 is transformed into corn immature embryos, and then a corn transgenic strain overexpressing the ZmbHLH175 gene is obtained after cultivation.

[0021] Furthermore, the obtained transgenic strains overexpressing the maize gene ZmbHLH175 are subjected to continuous self-pollination or hybridization to obtain homozygous offspring that stably overexpress the ZmbHLH175 gene.

[0022] The beneficial effects achieved by the present invention are as follows:

[0023] The present invention has been verified through experiments that by overexpressing the ZmbHLH175 gene in corn, the overexpression strain has better grain traits than the wild type. Specifically, the grains of the overexpression strain have larger grain width and grain volume and 100-grain weight, and the starch content in the grains is higher. The ZmbHLH175 gene has a positive effect on the grain traits of corn including grain width, grain volume, 100-grain weight, and starch content. Therefore, overexpression or high expression of ZmbHLH175 can enable corn plants to have better grain traits, effectively improve the yield and quality of corn grains, provide a new direction for improving the quality and quantity of corn, and apply it to the biological breeding process for improving corn grain traits, with reliable prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Attached Figure 1 The transcript amplification bands (upper figure) and gene structure diagram (lower figure) of the ZmbHLH175 gene in the present invention in the maize inbred lines B73 and B104 are shown.

[0025] Attached Figure 2 The figure is a diagram showing the genotype of the ZmbHLH175 gene overexpressing transgenic strain (upper figure) and the expression level of ZmbHLH175 in the plant (lower figure) in the examples of the present invention, with the wild type B104 (WT) as the control, among which OE1, OE2 and OE3 are homozygous ZmbHLH175 overexpressing strains.

[0026] Attached Figure 3 Statistical analysis of grain phenotypic variation of overexpressing transgenic lines.

[0027] Attached Figure 4 Analysis of starch content in grains of overexpressing transgenic lines. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0031] Example 1: Construction and identification of ZmbHLH175 overexpressing transgenic strains

[0032] The ZmbHLH175 gene sequence was cloned from the test material:

[0033] The extraction of total RNA from corn leaves and the synthesis of cDNA were performed according to the following steps:

[0034] The leaves of B73 plants at the 2-week seedling stage were cut off and the leaf tissues were quickly ground into powder in liquid nitrogen using a mortar.

[0035] Total RNA was extracted using QIAGEN RNA preparation kit (QIAGEN).

[0036] Using PrimeScript TM The cDNA sequence was synthesized using 1st Strand cDNA Synthesis Kit (Takara).

[0037] The coding region sequence of the ZmbHLH175 gene was cloned and sequenced using primers for transcript detection, wherein the primers include the following nucleotide sequence:

[0038] SEQ ID NO.1: GAGTTGCCAGTGAACCAGGAT;

[0039] SEQ ID NO. 2: CTACATTGCGTTGTGGAGGC.

[0040] The ZmbHLH175 gene was cloned from the leaves of wild-type B104. The full length of its coding region was 1129 bp (shown in SEQ ID NO.7). This gene encodes a basic helix-loop-helix (bHLH) protein, belongs to the bHLH transcription factor family, and is homologous to rice OsbHLH175, so it was named ZmbHLH175.

[0041] The protein encoded by the ZmbHLH175 gene contains 346 amino acids (SEQ ID NO.8).

[0042] The coding region nucleotide sequence of ZmbHLH175 gene is SEQ ID NO.7:

[0043]

[0044] The amino acid sequence of the protein encoded by the ZmbHLH175 gene is SEQ ID NO.8:

[0045] MGSGSYPAFPFSRDVLSTPTSASLLLSMDPAALFDFNGTFPPSSAAAATAGSSLSAFHDFSCINPFDDAGHFLGAPPPVPAAAAPQQQGQKGGFFAPLPASDFNDAGMSWDDEDEIDQSVDASSMAISASMENAAGAVAGASGAGGGSGRGKKKGMPAKNLMAERRRRKKLND RLYMLRSVVPKISKMDRASILGDAIEYLKELLQRISDLHNELESAPSSSLVGPTSASFNPSTPTLQTFPGQVKEELCPGSFPSPTGQQATVEVRMREGHAVNIHMFCARRPGILLSTMTALDSLGLDIEQAVISCFNGFAMDVFRAECADGPGMVPEEIKAVLMHTAGLHNAM

[0046] Generation and identification of ZmbHLH175 overexpressing transgenic lines

[0047] The coding region sequence of ZmbHLH175 gene was used to construct an overexpression vector through the Gateway system. The specific steps are as follows:

[0048] (1) Through BP reaction kit Gateway TM BP Clonase TM II Enzyme mix (Invitrogen, California, USA) was used to link the purified PCR amplification product to the pDONOR-Zeo vector. A 10 μL reaction system was used: 1 to 7 μL of PCR purified product (150 ng / μL), 1 μL of pDONR-Zeo vector (150 ng / μL) and 2 μL of BP reaction enzyme mixture, which was supplemented to 8 μL with 1×TE buffer, mixed and reacted in a 25°C water bath for 1 hour, then 1 μL of Proteinase K solution was added, mixed and reacted in a PCR at 37°C for 10 minutes.

[0049] (2) The BP ligation reaction was transformed through E. coli competent cells. Take out the DH5α competent cells prepared in advance and stored at -80℃ and thaw on ice for 5 minutes. Add 50μl competent cells to 5μL BP ligation reaction, mix gently and incubate on ice for 20 minutes. Take out and put it in a 42℃ water bath for 1 minute, take it out quickly, and put it in ice for 2 minutes. Then add 250μL LB liquid culture medium to the EP tube of the reaction and incubate at 37℃ and 200xg for 45 minutes. Finally, take out 150μL of bacterial solution and inoculate it on the LB plate containing Zeocin antibiotics and culture it overnight for 14 hours.

[0050] (3) Use Taq enzyme to perform colony PCR positive screening, inoculate the positive clones into 5 mL of liquid culture medium containing antibiotics and culture overnight, and use a small-scale plasmid extraction kit ( Plasmid was extracted according to the instructions of PlasmidMini Kit I (Omega Bio-Tek Inc., Georgia, USA) and stored or sent for sequencing.

[0051] (4) Insert the target fragment into the overexpression vector pMDC83 through LR recombination reaction. Use a 10 μL reaction system: 1 to 7 μL of the entry vector pDONOR-Zeo-ZmbHLH175 (150 ng / μL) containing the ZmbHLH175 coding region and 1 μL of the expression vector pMDC83, then add 1×TE buffer to make up to 8 μL system, and then add 2 μL of LR reaction enzyme mixture Gateway TM LRClonase TM II Enzyme Mix (Invitrogen, California, USA), mix well and react in a 25°C water bath for 1 hour, finally add 1 μL Proteinase K solution and mix well and react in a PCR instrument at 37°C for 10 minutes. As in step (2), take 1 μL LR reaction solution for transformation, positive screening, plasmid extraction and sequencing. The positive plasmid of the overexpression vector pMDC83-ZmbHLH175 containing the ZmbHLH175 coding region was obtained and stored for later use.

[0052] (5) Cooperated with Weimi Company to genetically transform the pMDC83-ZmbHLH175 vector through corn callus tissue.

[0053] The vector plasmid was transferred into Agrobacterium EHA105 by electroporation and identified by PCR. Freshly peeled maize embryos of about 1 mm from the B104 inbred line were used as materials. The peeled maize embryos were placed in a 2 mL plastic centrifuge tube containing 1.8 mL of suspension for half an hour; the suspension was aspirated, 1.0 mL of Agrobacterium suspension was added, and it was placed for 5 minutes; the co-culture medium was poured, and the excess bacterial liquid on the surface was aspirated with a pipette, and the embryos were cultured in the dark at 23°C for 3 days; the embryos were transferred to the resting culture medium and cultured in the dark at 28°C for 6 days; the embryos were transferred to the screening culture medium containing bialaphos and continued to be cultured in the dark at 28°C for two weeks; then the embryos were transferred to the differentiation culture medium and cultured at 25°C light (5000lx) for 3 weeks; the differentiated seedlings were transferred to the rooting culture medium and cultured at 25°C light (5000lx) until roots were formed; the seedlings were transferred to the soil matrix for growth, and the offspring seeds were harvested after 3-4 months. The positive overexpression plants of the T0 generation were screened by PCR detection of the vector and fluorescence quantitative PCR detection of the expression level of ZmbHLH175. Figure 2 .

[0054] The nucleotide sequences of the primers for the PCR detection of positive overexpression strains are as follows:

[0055] SEQ ID NO:3: GATTTGTGTACGCCCGACAGT;

[0056] SEQ ID NO: 4: GTGGATATGTCCTGCGGGTAA.

[0057] The nucleotide sequence of the primers used to detect the expression of ZmbHLH175 is as follows:

[0058] SEQ ID NO:5:GTGCCGAAGATCAGCAAGAT;

[0059] SEQ ID NO: 6: TCGGTGTTGATGGGTTGAAG.

[0060] Attached Figure 3 and attached Figure 4 The grains of the OE group were obtained from the T4 generation homozygous overexpression lines of the above overexpression plants, with the B104 inbred line as the control, i.e. the wild-type material (WT). The WT and three overexpression lines were planted in the experimental field of Guizhou Academy of Agricultural Sciences in Guiyang, Guizhou Province, and the conventional management strategies for corn fields were used.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. ZmbHLH175 gene associated with maize kernel traits, characterized in that: The nucleotide sequence of the coding region of the ZmbHLH175 gene is shown as SEQ ID NO.7, and the amino acid sequence of the protein encoded by the ZmbHLH175 gene is shown as SEQ ID NO.

8.

2. A recombinant expression vector, characterized in that: It contains the ZmbHLH175 gene according to claim 1 and a basic vector.

3. A genetically engineered bacterium, characterized in that: Contains the ZmbHLH175 gene according to claim 1 or the recombinant expression vector according to claim 2.

4. The use of the ZmbHLH175 gene and the protein encoded by it according to claim 1, or the recombinant expression vector according to claim 2, or the genetically engineered bacteria according to claim 3, characterized in that: The application includes at least one of the following: 1) Application in improving corn kernel traits; 2) Application in corn breeding or assisted breeding.

5. The use according to claim 4, characterized in that: The invention improves the corn grain traits by increasing the expression level of the ZmbHLH175 gene or its encoding protein in the corn.

6. Use of the ZmbHLH175 gene or its encoded protein according to claim 1 in identifying or breeding or assisting in breeding of corn plants, lines or varieties with excellent grain traits.

7. The use according to claim 6, characterized in that: By detecting the expression level of ZmbHLH175 gene through gene expression level, or predicting the variation of ZmbHLH175 encoding protein through transcripts, corn plants with excellent grain traits can be screened out.

8. The use according to claim 7, characterized in that: The nucleotide sequences of the primers for detecting gene expression are shown in SEQ ID NO.3 and SEQ ID NO.4: Left primer F: 5′-GTGCCGAAGATCAGCAAGAT-3′; Right primer R: 5′-TCGGTGTTGATGGGTTGAAG-3′; The nucleotide sequences of the primers used for transcript prediction are shown in SEQ ID NO.5 and SEQ ID NO.6: Left primer F: 5′-GAGTTGCCAGTGAACCAGGAT-3′; Right primer R: 5′-CTACATTGCGTTGTGGAGGC-3′.

9. A corn breeding method for improving corn kernel traits, characterized in that: After the maize gene ZmbHLH175 was transformed into maize immature embryos, a maize transgenic strain overexpressing the ZmbHLH175 gene was obtained after cultivation.

10. The method for cultivating corn for improving corn kernel traits according to claim 9, characterized in that: The obtained transgenic strains overexpressing the maize gene ZmbHLH175 are subjected to continuous self-pollination or hybridization to obtain homozygous offspring that stably overexpress the ZmbHLH175 gene.

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