ZmSht1 gene and application thereof in improving corn tolerance to high temperature and high light stress

Abstract

The application discloses application of a ZmSht1 gene in improving corn resistance to high-temperature and high-light double stress, and discloses a nucleotide sequence of the ZmSht1 gene, which is shown in a sequence table SEQ ID NO:1, and an amino acid sequence of an expression protein of the ZmSht1 gene, which is shown in a sequence table SEQ ID NO:2. The ZmSht1 gene provided by the application can improve the resistance of corn to high temperature and high light, and has a higher net photosynthetic rate under high-temperature and high-light conditions in summer, and is of great significance for corn stress resistance and high-quality breeding.

Description

Technical Field

[0001] This invention relates to the field of maize genetic breeding technology, and more specifically, to the ZmSht1 gene and its application in improving maize's tolerance to high temperature and strong light stress. Background Technology

[0002] Plants suffer from various abiotic stresses during their growth and development. Light and temperature are important environmental factors affecting plant growth and development. Strong light and high temperature stress typically have adverse effects on all stages of plant growth, such as hindering growth, reducing photosynthetic activity, deactivating photosystem II, causing stomatal closure, and inactivating rubisco enzymes, leading to a large accumulation of reactive oxygen species (ROS). ROS further damage proteins, DNA, and lipid molecules within the plant, affecting plant growth and development, and even causing plant death. Maize is one of the world's three major food crops. Maize production is of great significance to ensuring national food security. Field-grown maize is subjected to multiple abiotic stresses simultaneously, such as high temperature and light. During summer planting, especially during the flowering and grain-filling stages, maize often faces the dual stress of high temperature and strong light, affecting plant growth and yield. Therefore, elucidating the molecular mechanisms by which maize copes with the dual stresses of high temperature and strong light, and discovering key genes for maize's tolerance to combined high temperature and strong light stress, is of great significance for maize production and food security.

[0003] The domain of unknown function (DUF) protein family is a class of proteins encoding unknown functions, comprising thousands of families. Numerous studies have shown that various DUF protein families play important roles in life processes such as plant growth, development, and stress responses. However, in existing research, there are very few reports on the functions of gene family members encoding the DUF641 domain, and no reports have been found on their involvement in plant responses to high temperature and intense light stress. Therefore, studying the functions of DUF641 gene family members is of great significance for enriching the theoretical resources of DUF proteins. Summary of the Invention

[0004] This invention aims to improve the stress resistance of maize under high temperature and strong light stress; in order to achieve the above objective:

[0005] The first aspect of this invention discloses a ZmSht1 gene that can regulate maize's tolerance to high temperature and strong light stress, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] Zm00001d039444_T001 cdna chromosome:AGPv4:3:4497708:4499243:-1

[0007]

[0008] The second aspect of the present invention discloses the protein encoded by the ZmSht1 gene, characterized in that the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0009] Zm00001d039444_P001 transcript:Zm00001d039444_T001 gene_biotype:protein_coding transcript_biotype:protein_coding description:Zm00001d039444

[0010] Met Glu Thr Gln Val Ala Ala Pro Pro His Gln Gln Gln GlnLys Ala Ala Asn Leu Ala Arg Thr Phe Thr Lys Leu Leu Arg Arg Lys Arg Ala GluAla Ala Ala Gln Gly Gly Ala Pro Glu Ala Pro Ala Ser Val Ala Pro Gly Asp AlaLeu Glu Glu Gln Arg Thr Glu Pro Pro Pro Ala Ile Pro Ser Leu Ser Lys Leu LysLeu Ser Gly Asn Leu Ala Ala Tyr Ser Phe Asp Ala Phe Phe Arg Asn Ala AlaGlu Lys Lys Ala Ala Ala Gly Gly Gly Ala Val Arg Gln Pro Ala Gly Ala GlyGlu Val Thr Leu Glu Ala Ala Asp Ala Leu Leu Ala Asn Leu Phe Ala Gly ValSer Ala Val Lys Ala Ala Tyr Ala Gln Leu Gln Leu Ala Gln Phe Pro Tyr Asp AlaGlu Ala Ile Gln Ser Ala Asp Ala Val Val Ala Glu Leu Thr Arg Leu Ser AspThr Lys Arg Arg Tyr Leu Arg Asp Pro Ala Ala Ala Ala Arg Gly Ala Ala Ala AlaGly His Thr Ala Leu Ala Ala His Ala Glu Glu Gln Arg His Leu Leu Lys Thr TyrGln Ile Thr Ala Arg Lys Leu Glu Ser Asp Leu Arg Ala Arg Asp Ala Glu Ala GluArg Ala Arg Ser Ser Leu Thr Gly Glu Leu Arg Ala Glu Arg Ala Leu Glu Ala ArgLeu His Pro Gly Arg Thr Leu AlaSer Leu Asp Glu Leu His Leu Ser Gly Leu AsnPro Thr His Phe Leu Thr Ala Leu Arg His Thr Val Lys Ser Ile Arg Ser Phe SerArg Ser Met Leu Ser Ser Met Gln Ser Ala Gly Trp Asp Leu Gly Ala Ala Ala AlaAla Val His Pro Gly Val Pro Leu Arg Arg Ala Gly Asp Ala Lys Phe Val Phe GluSer Tyr Val Ala Met Lys Met Phe Ala Asn Phe His Arg Arg Asp Leu Phe Asn Phe SerPhe Gly Glu Arg Glu Phe Glu Arg Arg Arg Phe Phe Glu Glu Phe Thr GluLeu Lys Ala Glu Pro Ala Ser Ala Phe Leu Asp Ala Arg Ser Pro Arg Trp Gly GlyLeu Gly Lys Phe Leu Arg Ala Lys Tyr Leu Ser Leu Val His Ala Arg Met Glu ThrAla Phe Phe Gly Arg Leu Glu Gln Arg Gly Ile Val Ser Ala Gly Pro Gly Phe ProGlu Ser Ser Trp Phe Ala Asp Phe Ala Glu Met Ala Arg Arg Val Trp Leu Leu HisCys Leu Phe Phe Ala Phe Asp Gly Gly Ala Glu Glu Asp Gly Ala Ser Ile Phe GlnVal Arg Thr Gly Ala Arg Phe Ser Glu Val Tyr Met Glu Ser Ala Ser Asp Gly SerAla Gly Asp Asp Ala Glu Asp Arg Val Gly Phe Thr Val Leu Pro Gly Phe ArgVal Gly Arg Thr Leu Ile Gln Cys Arg Val Tyr Ser LysPro Gly Arg Arg Pro(SEQ ID NO.2)

[0011] The third aspect of this invention is the application of the aforementioned ZmSht1 gene and its encoded protein in improving maize's tolerance to high temperature and strong light stress; specifically, its application in cultivating maize plants resistant to high temperature and strong light stress.

[0012] The fourth aspect of this invention discloses a recombinant expression vector containing the ZmSht1 gene described above.

[0013] The fifth aspect of this invention discloses a genetically engineered bacterium containing the above-described recombinant expression vector.

[0014] The sixth aspect of this invention discloses a method for improving the tolerance of maize to high temperature and strong light stress by overexpressing the ZmSht1 gene in maize. This can be achieved by using the aforementioned recombinant vector and genetically engineered bacteria to transform the maize into plants, and then cultivating and screening transgenic homozygous plant lines.

[0015] The present invention achieves the following beneficial technical effects:

[0016] The ZmSht1 gene provided by this invention can participate in regulating the tolerance of maize to the dual stress of high temperature and high light intensity, and transgenic plants overexpressing ZmSht1 have a higher net photosynthetic rate under the conditions of high temperature and high light intensity in summer. Attached Figure Description

[0017] Figure 1 Analysis of the expression pattern of the maize ZmSht1 gene induced by high temperature and strong light.

[0018] Figure 2 The phenotypic and physiological indicators of H2O2 content of maize ZmSht1 knockout mutant after high temperature and strong light treatment.

[0019] Figure 3 The phenotypic and physiological indicators of H2O2 content in transgenic maize plants overexpressing ZmSht1 after being treated with high temperature and strong light.

[0020] Figure 4 Determination of kernel phenotype and net photosynthetic rate of ZmSht1 transgenic maize planted in Hefei in summer. Detailed Implementation

[0021] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0022] ZmSht1 is a DUF-like protein containing the unknown functional domain DUF641. Studies have reported that DUF-like proteins are widely involved in various physiological processes in plants, especially in plant growth and development and responses to abiotic stresses. However, no functional reports have been found for DUF641-type proteins in maize. Whether ZmSht1 can respond to the dual stresses of high temperature and intense light has not yet been studied. Therefore, this invention conducts an in-depth study on the biological function of ZmSht1 in improving the tolerance of maize to high temperature and intense light, providing important genetic resources for breeding high-quality, stress-resistant maize varieties. The following are examples of the main research process and verification experiments regarding ZmSht1's ability to improve the tolerance of maize to the dual stresses of high temperature and intense light.

[0023] Example 1: Analysis of the expression pattern of the ZmSht1 gene under high temperature and strong light induction

[0024] To determine whether the ZmSht1 gene is affected by high temperature and strong light, maize inbred line KN5585 seedlings at the three-leaf stage were subjected to high temperature and strong light stress (45℃, 3000 μmol / (m²·s)) for 0 h, 0.5 h, 1 h, 3 h, and 6 h, respectively. Total RNA was then extracted from the third leaf, and its expression was analyzed by qRT-PCR. The results are as follows: Figure 1 As shown, the ZmSht1 gene was significantly induced by the dual stresses of high temperature and strong light, and its expression level reached its highest point after 1 h of further treatment. These results indicate that ZmSht1 can respond to high temperature and strong light stress in plants.

[0025] Example 2: Identification of the high temperature and strong light tolerance of transgenic plants based on ZmSht1

[0026] To further investigate the function of ZmSht1 under the dual stress of high temperature and strong light, homozygous knockout mutants and homozygous overexpression lines of ZmSht1 were obtained using maize genetic transformation technology.

[0027] 1. Phenotypic identification of ZmSht1 transgenic plants under high temperature and strong light stress

[0028] To further investigate the role of ZmSht1 in maize's tolerance to high temperature and strong light, seedling experiments were conducted to observe and statistically analyze the phenotype and leaf damage rate of transgenic plants after high temperature and strong light treatment.

[0029] Wild-type, knockout mutant, and overexpression were divided into two groups and kept at 45℃ and 3000 μmol / (m²). 2 The product was treated under high temperature and high light intensity for 11 h, and the results are as follows: Figure 2 As shown in Figure 3. Figure 2 As can be seen, the leaves of the treated knockout mutants exhibited significant curling, withering, and scorching compared to the wild type. A statistical analysis of the leaf damage rates after treatment in both wild type and knockout mutants revealed that the degree of leaf damage in the knockout mutants was far greater than that in the wild type. Figure 3 The results were the opposite. Compared to the wild type, ZmSht1 overexpressing plants showed slight leaf wilting after high temperature and strong light treatment, but still grew well. Statistical results on leaf damage rate also indicated that the damage to overexpressing maize was less than that to the wild type. These results suggest that the ZmSht1 gene can improve maize's heat and high light intensity tolerance, and that ZmSht1 overexpressing plants exhibit stronger tolerance.

[0030] 2. Under high temperature and strong light stress, the H2O2 content in ZmSht1 transgenic plants was measured.

[0031] To further clarify the protective effect of ZmSht1 against high temperature and strong light stress, the H2O2 content of its transgenic plants before and after high temperature and strong light treatment was measured. The results are as follows: Figure 2 As shown in Figure 3, the H2O2 content of the knockout mutant after high temperature and strong light treatment was significantly increased and higher than that of the wild type, while the overexpression plant significantly inhibited the increase of H2O2 content induced by high temperature and strong light, thus effectively resisting the damage caused by high temperature and strong light stress.

[0032] Example 3: Grain size statistics and net photosynthetic rate determination of ZmSht1 transgenic plants

[0033] To investigate whether ZmSht1 overexpression affects maize kernel size, the kernel size of ZmSht1 transgenic maize was observed and statistically analyzed. The results are as follows: Figure 4 As shown in A, B, and C, the overexpressing plants exhibited larger kernel size and 100-kernel weight compared to the wild type. This indicates that ZmSht1 not only responds to the dual stresses of high temperature and strong light in maize but also influences kernel size.

[0034] On a sunny summer day at midday, the photosynthetic rate of the ZmSht1 transgenic plants was measured using a photosynthesis meter. The results are as follows: Figure 4 As shown in Figure D, under high temperature and strong light conditions in the field, overexpression of ZmSht1 can increase the net photosynthetic rate of maize.

[0035] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Overexpression ZmSht1 The application of genes in improving the tolerance of maize to high temperature and strong light stress is characterized by, The ZmSht1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The ZmSht1 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

3. A method for improving the tolerance of maize to high temperature and strong light stress, characterized in that, The as described in claim 1 ZmSht1 The gene is overexpressed in maize.

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