Application of heat shock transcription factor gene ZmHsf05 in improving single ear yield of corn inbred lines

Abstract

The application discloses a corn heat shock transcription factor ZmHsf05 gene in improving corn inbred pollen viability, ear grain number and single ear grain weight. ZmHsf05 Firstly, a plant overexpression vector containing the gene is constructed to obtain a corn material overexpressing the gene. ZmHsf05 Through continuous backcrossing and selfing, an improved corn inbred overexpressing the gene is obtained. ZmHsf05 The pollen viability, ear grain number and single ear grain weight of the improved inbred are significantly improved. ZmHsf05 Through pollen transcriptome sequencing, it is found that ZmHsf05 improves pollen viability by regulating target genes in endoplasmic reticulum protein processing, cysteine and methionine metabolism and flavonoid biosynthesis pathways. Therefore, the improved corn inbred overexpressing the gene has good breeding potential in production.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and crop genetic breeding technology, specifically to maize heat shock transcription factors. ZmHsf05 Novel applications of genes in improving yield-related traits such as pollen viability, number of kernels per ear, and kernel weight per ear in maize. Background Technology

[0002] corn( Zea mays Maize (L.) is a globally important crop for food, feed, and industrial raw materials. However, against the backdrop of intensifying climate change, mainstream maize varieties generally suffer from problems such as low pollen viability, asynchronous development of male and female ears, and kernel abortion, becoming a significant bottleneck restricting yield improvement. Studies have shown that the number of kernels per ear and the weight of kernels per ear depend on successful ovule pollination, and pollen viability is one of the key factors determining pollination success rate. Decreased pollen viability and reduced pollination efficiency directly lead to a reduction in the number of kernels per ear and a decrease in kernel weight, ultimately resulting in significant yield loss. Therefore, identifying and utilizing key genes that regulate pollen viability and yield-related traits, and using molecular breeding methods to improve the stress resistance and yield potential of maize varieties, has become an important direction for current maize genetic improvement.

[0003] Heat shock transcription factors (Hsf) are a highly conserved class of transcriptional regulatory proteins in eukaryotes. They participate in plant responses to various abiotic stresses such as high temperature, drought, and salinity by activating the expression of downstream target genes such as heat shock proteins (HSPs). However, research on Hsf family members has largely focused on their functions in stress tolerance, with very little attention paid to their direct regulation of reproductive organ development and yield components. Currently, the application of transgenic technology in improving maize yield traits remains relatively scarce. Most reported transgenic maize research focuses on insect resistance, herbicide resistance, or stress resistance traits, while there are few cases of genetic engineering aimed at directly improving pollen viability, ear grain number, and single ear grain weight.

[0004] Based on the above background, the present invention will ZmHsf05 The application of genes has expanded to the field of maize yield traits. This is achieved through the construction of… ZmHsf05 By overexpressing maize material and repeatedly backcrossing and self-pollinating, improved maize inbred lines were obtained. The pollen viability, number of grains per ear, and grain weight per ear were systematically evaluated. This invention aims to provide a new method for improving the yield potential of maize inbred lines using this gene, which has important theoretical and applied value. Summary of the Invention

[0005] The purpose of this invention is to provide a maize heat shock transcription factor gene. ZmHsf05The new applications of this gene in improving maize yield traits specifically involve its use in enhancing maize pollen viability, increasing the number of kernels per ear, and increasing the weight of kernels per ear, thus expanding its application value in high-yield breeding and providing excellent genetic resources for breeding new high-yield and stable-yield maize varieties.

[0006] This invention employs the following technical solution: maize heat shock transcription factor gene ZmHsf05 Its application in improving corn pollen viability, ear kernel number, and single ear kernel weight. ZmHsf05 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0007] Furthermore, the application is to... ZmHsf05 Genes are introduced into maize and overexpressed, thereby increasing maize pollen viability, number of kernels per ear, and kernel weight per ear.

[0008] Furthermore, the maize in question is a maize inbred line.

[0009] Furthermore, the aforementioned ZmHsf05 Gene overexpression was achieved by constructing a plant expression vector containing the nucleotide sequence shown in SEQ ID No. 1 and transforming it into maize.

[0010] This invention also provides a method for improving corn pollen viability, ear kernel number, and single ear kernel weight, characterized by comprising the following steps: (1) Construct a structure containing the SEQ ID No. 1 shown ZmHsf05 Plant expression vectors for gene nucleotide sequences; (2) The plant expression vector was introduced into maize cells; (3) Culture the maize cells to obtain overexpression ZmHsf05 A corn plant with a specific gene; (4) Screen maize plants with improved pollen viability, number of kernels per ear, and weight of kernels per ear.

[0011] Further, in step (2), the plant expression vector is introduced into maize cells by Agrobacterium-mediated transformation or gene gun bombardment.

[0012] This invention also provides a method for breeding high-yield maize varieties, characterized by comprising the following steps: (1) Obtain overexpression using the aforementioned method ZmHsf05 A corn plant with a specific gene; (2) The overexpression ZmHsf05 The maize plant with the gene was crossed with the target maize variety; (3) Through continuous backcrossing and self-crossing, ZmHsf05 Genes were introduced into the target maize variety; (4) Screen maize varieties with improved pollen viability, number of kernels per ear, and weight of kernels per ear.

[0013] Furthermore, the target maize variety is a maize inbred line.

[0014] Furthermore, the pollen viability of the maize varieties obtained by the method is increased by more than 10% under normal growth conditions.

[0015] Furthermore, the maize varieties obtained by the method have an increase of more than 10% in the number of kernels per ear under normal growth conditions.

[0016] Furthermore, the corn varieties obtained by the method have a single ear grain weight increase of more than 5% under normal growth conditions.

[0017] The present invention also provides a maize variety, characterized in that it is obtained by the aforementioned breeding method.

[0018] The present invention also provides a corn seed, characterized in that it is derived from the aforementioned corn variety.

[0019] The present invention also provides a plant expression vector, characterized in that it comprises the one shown in SEQ ID No. 1. ZmHsf05 Gene nucleotide sequence for overexpression in maize ZmHsf05 Genes are used to improve pollen viability, number of kernels per ear, and weight of kernels per ear in maize.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) First revealed ZmHsf05 The function of genes in improving maize yield traits has been expanded to include multiple yield-related traits such as pollen viability, number of kernels per ear, and kernel weight per ear. (2) The yield-increasing effect of this gene in improved maize inbred lines was clarified, providing new gene resources for high-yield maize breeding; (3) System validation confirmed the overexpression ZmHsf05 The gene can significantly enhance corn pollen viability, increase the number of kernels per ear, and increase the weight of kernels per ear without affecting normal growth and development; (4) The provided technical solutions are characterized by strong operational feasibility and wide application range, and can be directly used for the improvement of maize backbone inbred lines and the breeding of high-yield hybrids.

[0021] Using the PCR-identified positive maize Hi-II transgenic line as the male parent and the maize backbone inbred line Zheng58 as the female parent, positive lines in the F1 generation were hybridized and screened. Using the F1 generation positive lines as the female parent and the maize backbone inbred line Zheng58 as the male parent, backcrossing was performed for 3 consecutive generations. The BC3 generation positive lines were then self-crossed for 5 consecutive generations to obtain the BC3F5 generation homozygous and stable improved maize inbred line Zheng58.

[0022] Example 1: Gene Cloning and Vector Construction Specific primers were designed using Primer 3.0 software. The upstream primer incorporated a BamHI restriction site, and the downstream primer incorporated a SacI restriction site. Using maize inbred line H21 cDNA as a template, PCR amplification was performed using PrimeSTAR HS DNA Polymerase with GC Buffer. The reaction mixture consisted of: 25 μL of 2×PrimeSTAR GC Buffer, 4 μL of dNTP™ extract (2.5 mol / L), 1 μL of upstream primer (10 mmol / L), 1 μL of downstream primer (10 mmol / L), 0.5 μL of template cDNA, 0.5 μL of PrimeSTAR HS DNA Polymerase (2.5 U / μL), and sterile water to a final volume of 50 μL. The amplification program was: 94℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 10 s, 72℃ extension for 60 s, for a total of 30 cycles; and a final extension at 72℃ for 5 min. After amplification and recovery by gel electrophoresis, the amplified products were mixed with the pCUB10800-Ubiquitin1-Flag vector, which had been digested with BamHI / SacI, and homologous recombination ligation was performed using the ClonExpress II One Step Cloning Kit. The ligation products were transformed into E. coli DH5α, and single clones were selected for colony PCR verification. Positive clones were sent to a sequencing company for sequencing identification. After confirmation, they were used for subsequent maize genetic transformation.

[0023] Example 2: Maize genetic transformation and identification of positive lines The recombinant expression vector was constructed and genetically transformed into maize hybrid Hi-II by Jiangsu Weimi Biotechnology Co., Ltd. T0 generation regenerated plants were transplanted to a greenhouse for cultivation. Leaf samples were collected at the three-leaf-one-heart stage, and genomic DNA was extracted using the CTAB method. Specific primers for the ZmHsf05-Flag fusion gene (Ubi-F: CCCTGTTGTTTGGTGTTAC; NOS-R: TAATCATCGCAAGACCGGC) were designed for PCR amplification. The amplification system consisted of 12.5 μL of 2×TaqPCR MasterMix, 1 μL each of forward and reverse primers, 1 μL of template DNA, and ddH2O to a final volume of 25 μL. Amplification conditions were as follows: 94℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 60 s, for a total of 30 cycles; and a final extension at 72℃ for 5 min. The amplified products were detected by 1.0% agarose gel electrophoresis; plants showing the expected band were considered positive. The expression level of the ZmHsf05-flag fusion protein in the total protein of positive lines was further identified by Western blot. Under optimal temperature conditions, there was no significant difference in the growth of seedlings between the ZmHsf05 improved maize inbred lines and wild-type maize. Figure 1 The protein expression levels of ZmHsf05 differed among the three improved maize inbred lines. Figure 1 ).

[0024] Example 3: Detection of pollen viability, number of grains per ear, and grain weight per ear in improved inbred lines. Seeds of the wild-type Zheng 58 inbred line and the improved BC3F5 generation maize inbred line were sown in experimental fields at the Hebei Academy of Agricultural and Forestry Sciences Comprehensive Experimental Park. The experimental fields were under closed management, and real-time temperature and humidity were monitored. At 60-70 days of age, during the pollen shedding stage, all maize lines were bagged and strictly self-pollinated. Pollen samples were collected for TTC staining, and pollen viability was assessed under a microscope. Specifically, an appropriate amount of pollen was added to a 0.5% TTC solution and incubated at 37℃ in the dark for 15 min. Red (viable) and colorless (non-viable) pollen were observed and counted under a microscope, and the viability percentage was calculated. Each sample was measured three times, and the average value was taken. At maturity, 18 uniformly growing plants were selected from each plot, strictly bagged, and self-pollinated. The number of grains per ear and the weight of a single ear were measured after maturity. All data were analyzed using SPSS 26.0 software using one-way ANOVA, with P < 0.05 considered statistically significant. The pollen viability of the BC3F5 generation improved maize inbred lines was significantly higher than that of the wild type on days 5 and 8 after flowering, with an increase of 11%-25% compared to the wild type. Figure 2After the female ears matured, the number of kernels per ear and the weight of kernels per ear in improved maize inbred lines and wild type were measured. The results showed that the number of kernels per ear and the weight of kernels per ear in improved maize inbred lines were significantly higher than those in wild type. Among them, the average number of kernels per ear in improved lines increased by about 50% and the average weight of kernels per ear increased by about 35% compared with wild type. Figure 3 and 4 ).

[0025] Example 4 ZmHsf05 Pollen transcriptome analysis of overexpression lines To elucidate the mechanism by which ZmHsf05 enhances pollen viability at the molecular level, [the study focused on...]. ZmHsf05 Transcriptome analysis was performed on pollen from the overexpression line and the wild-type Zheng 58. The results showed that... ZmHsf05 Overexpression led to significant changes in a series of differentially expressed genes. This was analyzed using a volcano plot. Figure 5 A total of 393 significantly upregulated genes and 85 significantly downregulated genes were identified (screening criteria: |log2FC|>1, p<0.05). The distribution of these differentially expressed genes indicates... ZmHsf05 Overexpression had a wide range of effects on the pollen transcriptome. KEGG pathway enrichment analysis ( Figure 6 Further analysis showed that differentially expressed genes were significantly enriched in multiple metabolic pathways closely related to pollen viability, including: (1) Endoplasmic reticulum protein processing pathway, involving protein folding, modification and quality control processes; (2) Cysteine ​​and methionine metabolic pathways are related to the synthesis of sulfur-containing amino acids and antioxidant defense; (3) Flavonoid biosynthesis pathway, involved in pollen wall formation and stress resistance regulation; (4) Sulfur metabolism pathway, affecting redox balance and cell signal transduction; (5) Plant hormone signal transduction pathways, including hormone responses such as auxin and brassinolide.

[0026] KEGG gene-pathway network analysis ( Figure 7 This study revealed the interactions between these pathways, suggesting that ZmHsf05 may enhance pollen viability by coordinating multiple metabolic pathways. This molecular-level evidence provides a mechanistic explanation for ZmHsf05's ability to improve maize pollen viability. Attached Figure Description

[0027] Figure 1 As described in the embodiments of the present invention ZmHsf05 Overexpression of the growth phenotype of maize inbred lines.

[0028] Figure 2 As described in the embodiments of the present invention ZmHsf05A graph showing the comparison of pollen viability between overexpression maize inbred lines and wild-type maize.

[0029] Figure 3 As described in the embodiments of the present invention ZmHsf05 A graph showing the comparison of kernel number per ear between overexpression maize inbred lines and wild-type maize.

[0030] Figure 4 As described in the embodiments of the present invention ZmHsf05 A comparative data graph of ear and grain weight between overexpression maize inbred lines and wild type.

[0031] Figure 5 for ZmHsf05 Volcano plot of pollen transcriptome comparison analysis between overexpression lines and wild-type Zheng 58, showing the distribution of differentially expressed genes.

[0032] Figure 6 for ZmHsf05 KEGG pathway enrichment analysis of differentially expressed genes in pollen of overexpression lines shows significantly enriched metabolic pathways.

[0033] Figure 7 This is a KEGG gene-pathway network analysis diagram, revealing the association between differentially expressed genes and enriched pathways.

Claims

1. A maize heat shock transcription factor gene ZmHsf05 Applications in improving maize pollen viability, increasing ear kernel number, and increasing single ear kernel weight include: Construct containing ZmHsf05 Plant overexpression vectors for genes, to obtain ZmHsf05 Maize materials with overexpressed genes were obtained through successive backcrosses and self-pollination. ZmHsf05 Improved inbred lines with overexpressed genes, the aforementioned ZmHsf05 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

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