CN107988234B - Application of GRMZM2G701063 gene in adversity stress - Google Patents

Application of GRMZM2G701063 gene in adversity stress Download PDF

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CN107988234B
CN107988234B CN201711145351.1A CN201711145351A CN107988234B CN 107988234 B CN107988234 B CN 107988234B CN 201711145351 A CN201711145351 A CN 201711145351A CN 107988234 B CN107988234 B CN 107988234B
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corn
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CN107988234A (en
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宋纯鹏
李保珠
彭雷
樊若楠
郭竞选
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Henan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/13—Abiotic stress
    • Y02A40/132—Plants tolerant to drought

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a patent application of a corn GRMZM2G701063 gene in regulation of plant adversity stress reaction. The maize GRMZM2G701063 gene has CDS length of 822bp, and codes R2R3 MYB transcription factor with 273 amino acids. Research shows that the gene is related to growth and development of corn leaf sheaths and tassels, and compared with wild type B73, the gene is abnormally expressed, the corn phenotype shows the phenotypic characteristics of green leaf sheaths (the wild type B73 specifically shows that the leaf sheaths gradually become purple under illumination) and colorless tassels (the wild type B73 shows that anthers make purple), and meanwhile, the drought tolerance is obviously enhanced. Based on the characteristics, a certain theory and application foundation can be laid for the cultivation of new plant varieties with specific phenotypes and the cultivation of new plant varieties with better stress resistance.

Description

Application of GRMZM2G701063 gene in adversity stress
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to corn GRMZM2G7010633The application of the gene in regulating plant stress response.
Background
Corn is the first crop in the world, and its yield and quality directly affect the survival and development of human society. Drought is the most important limiting factor for crop yield, and is currently one of the central topics of plant adversity stress.
In long-term plant drought resistance studies, many components that can play important roles in plant drought stress responses have been identified, but the identification of these components and the studies on the ways in which they function have been mainly achieved by the model arabidopsis thaliana and the like. For important crops such as corn, rice, wheat and the like, the research on the drought stress function and the regulation and control component in the genome is relatively less, so that the identification of the important component responding to the drought stress and the drought reaction path in the genome has very important theoretical and practical significance.
For the maize GRMZM2G701063 gene (see maize bioinformatics database: https:// www.maizegdb.org/gene _ center/gene/GRMZM2G 701063), studies have shown that: the CDS length of the GRMZM2G701063 gene is 822bp, and the CDS encodes a 273-amino-acid R2R3 MYB transcription factor; the function of the homologous gene is predicted to be related to the generation of secondary metabolites of plants, but the function is not reported to be related to stress response.
Disclosure of Invention
The application aims to preliminarily understand the functions of the GRMZM2G701063 gene and the plant phenotype correlation and the stress response correlation through preliminary research on the GRMZM2G701063 gene, and further lay a certain theoretical and application foundation for new crop variety cultivation.
The technical solution adopted in the present application is detailed as follows.
The CDS length of the corn GRMZM2G701063 gene is 822bp, an R2R3 MYB transcription factor with 273 amino acids is coded, the gene is related to the growth and development of corn leaf sheaths and tassels, and after the gene is abnormally expressed due to fragment deletion, insertion mutation and the like, the corn phenotype shows the phenotypic characteristics of the green leaf sheaths (the wild type corn B73 variety specifically shows that the corn leaf sheaths gradually purple under the illumination condition) and the colorless tassels (the wild type corn B73 variety specifically shows that anthers purple).
A method for cultivating a new corn variety with specific phenotypic characteristics, namely leaf sheath greening and tassel colorless phenotypic characteristics, comprises the step of inactivating a corn GRMZM2G701063 gene by a gene insertion or gene silencing technology to enable the gene to be abnormally expressed.
The GRMZM2G701063 gene is applied to adversity stress, the gene is related to plant drought stress reaction, the gene is abnormal due to deletion mutation, insertion mutation and other reasons, the plant drought tolerance is obviously enhanced, and the plant is specifically corn.
A method for improving the drought tolerance of plant features that the GRMZM2G701063 gene or the homologous gene to GRMZM2G701063 gene is abnormal by gene insertion or gene silencing technique to change its activity.
In the prior art, functional prediction of a gene homologous with a corn GRMZM2G701063 suggests that the gene may play a certain role in the generation of plant secondary metabolite flavonoids (based on homologous gene functional research), but the actual function of the gene is lack of experimental verification, particularly the function of the corn GRMZM2G701063 gene in adversity.
In this application, based on the intensive research of specific mutant materials, the inventors obtained the mutant materials with deletion of GRMZM2G701063 gene fragment from the corn mutant library and further made preliminary analysis and research on the function of the gene. Research shows that the gene is related to growth and development of corn leaf sheaths and tassels, compared with wild type B73, after the gene is abnormal due to segment deletion and the like, the corn phenotype shows the phenotypic characteristics of green leaf sheaths (the wild type B73 specifically shows that the leaf sheaths gradually become purple under illumination) and colorless tassels (the wild type B73 shows that anthers become purple), and meanwhile, the drought tolerance is obviously enhanced. Based on the characteristics, a certain theory and application foundation can be laid for the cultivation of new plant varieties with specific phenotypes and the cultivation of new plant varieties with better stress resistance.
Drawings
FIG. 1 is a drawing ofdos57Screening the mutant; wherein A is a pairdos57Leaf temperature detection of mutants: (dos57The mutant is an infrared leaf temperature abnormal mutant), B isdos57Quantitative analysis of leaf temperature difference between the mutant and the corn B73;
FIG. 2 isdos57Comparing the phenotype difference of the mutant and the maize B73; wherein A is maize B73 anddos57comparing the water loss of the mutant in vitro leaves, wherein B represents that the mutant grows on the culture soil for 10 days and is dried for 5 daysComparing the phenotypes of (1); c is the contrast of the mature spike type;
FIG. 3 identification of mutantsdos57The mutation-associated phenotype of (a) is under monogenic recessive control; wherein A is backcross F1Comparing leaf sheath and drought tolerance phenotype of generations, wherein B is a comparison graph of the color of the stem and the drought tolerance in the hybrid population;
FIG. 4 is a graph showing the results of locating a mutant gene from a clonal population;
FIG. 5 shows the results of related gene sequencing and gene identification; wherein A is the structure of GRMZM2G701063 gene; b is the gene sequencing result (the first row is B73 sequence determination, the next two rows are mutant sequence determination); c is B73 and the peak diagram of the difference sequence measured by the mutant.
Detailed Description
The present invention will be further explained with reference to the following examples, which are intended to briefly describe the background of some of the biomaterials involved in the following examples before describing the specific examples.
Biological material:
corn mutant pool: a mutant library which is created by EMS mutagenesis and takes a corn natural inbred line B73 as background material can be used for screening corn target phenotype material;
maize B73, a published, most commonly used inbred line of maize for research;
maize PH4CV, a public and commonly used inbred line of maize for research, is the male parent of pioneer maize 335 bred by pioneer america, has characteristics such as drought tolerance similar to B73, and its genomic sequence is not yet published;
the related sequencing in this application was done by Biotechnology engineering (Shanghai) Inc.
Examples
Due to the results related to the present application with maize mutants: (dos57Mutant) was obtained in direct correlation, so this example was first for maizedos57The process of obtaining mutants by screening and cloning the maize GRMZM2G701063 gene is briefly described as follows.
Corn, corndos57Screening for mutantsTo obtain
The corn mutant bank is cultivated in a greenhouse by corn seeds under the following cultivation conditions: light/dark cycle 14/10h, temperature day/night: 30/24 ℃, the relative humidity is about 70%, after the seeds germinate and grow for 15 days, the phenotype of the corn seedlings in the mutant library is observed, and meanwhile, a far infrared imager is used for detecting the leaf temperature.
The results show that: mutantsdos57The comparison of the abnormal leaf temperature (shown in figure 1A) with the leaf temperature of the background material (wild type B73) shows that the difference of the leaf temperature exceeds 1 degree (shown in figure 1B), and the method has certain research value.
Second, corndos57Phenotype of the mutant
Further selfing for multiple generations to obtain puredos57And (4) mutant seeds. Will be provided withdos57When the mutant and the wild type B73 are planted separately (the cultivation conditions refer to the above), and the phenotype is compared, it can be seen that:
（1）dos57the mutant exhibits an infrared leaf temperature abnormality phenotype of temperature;
b73 anddos57simultaneously sowing the seeds in the same culture pot (10 cm multiplied by 10 cm) (nutrient soil: vermiculite =1: 1) to ensure that the water content of the soil is between 50 and 70 percent; normally culturing the mutant for 15 days (3-4 leaf stage)dos57And B73 seedlings were subjected to the in vitro leaf water loss test, the results are shown in FIG. 2A, and it can be seen that, dos57the water loss rate of the mutant is obviously lower;
in the drought treatment experiment, the watering of the corn seedlings which are normally cultured for about 10 days (to 3-leaf stage) is stopped, and phenotype observation and related infrared detection and the like are carried out; about 5 days after water cut, B73 anddos57the mutants showed significant differences in drought stress phenotype (wilting), where soil moisture was below 30% and the mutants showed stable drought tolerance phenotype (as shown in FIG. 2B)
（2）dos57The mutant seedlings were clearly distinguished from their background material B73 in leaf sheath color and adult tassel (as shown in fig. 2B and 2C); furthermore, the color of the leaf sheath, the development phenotype such as the tassel of the adult plant and the drought tolerance phenotype are stably inherited (over 7 generations of selfing), which shows that the color of the mutant gene and the leaf sheath andthe development phenotype and drought tolerance phenotype of adult tassels are related.
III, mutantdos57Is recessively controlled by a single gene
To identify mutantsdos57Whether the mutation-associated phenotype is regulated by a single gene or by multiple gene interactions, the inventors willdos57The mutant was backcrossed with wild type B73 to obtain backcross F1Whereas, backcrossed F1 seedlings were identical to B73 in leaf sheath color and drought-resistant phenotype (as shown in FIG. 3A), indicating that the mutant phenotype is controlled by a recessive gene.
Then F is mixed1F is produced by strict selfing2. The identification result shows that F2Phenotypic segregation, F2The seedling generation part is compared with the mutant on the stem color and the drought tolerance phenotypedos57Coincidences (as shown in fig. 3B). Meanwhile, the statistical data shows that the corn seedlings with the stem color and the drought tolerance phenotype account for 1/4 of the seedlings of the population, and accord with monogenic recessive inheritance, namely: mutantsdos57Is recessively controlled by a single gene.
Fourthly, constructing clone population and pairing mutantsdos57Localization of a single-gene recessive control gene (mutant gene)
The mutantdos57With another natural wild type maize PH4CV (withdos57The maize inbred line with relatively similar indexes such as drought tolerance and the like can be used for map-based cloning of a target gene). Further, F is1Strict selfing was performed to form a population of F2 clones.
From F2Selection of mutant phenotypes in progeny populations (and mutants)dos57Phenotypically identical). SSR markers released by a MaizeGDB website (https:// www.maizegdb.org /) are utilized to pick out codominant SSR markers which are uniformly distributed in 10 chromosomes of corn, and the codominant SSR markers are subjected to single plant amplification and exchange rate statistics on 300 target character single plants screened from clone groups. According to the linkage interchange genetic rule, the SSR markers with lower interchange rate and the target gene present closer linkage relation, namely the farther from the target gene the SSR markers areAnd (4) approaching.
According to the above rules, the target gene is initially located in the range of the sixth chromosome lower arm 100-115M in maize. More SSR markers are searched in the range, and a larger number of single plants (about 4800) are utilized to perform the crossover law statistics and the single plant tendency experiment.
Finally, the mutants are combineddos57The single-gene recessive control gene (mutant gene) is positioned between 108.2 and 108.6M of the sixth chromosome of the maize (as shown in figure 4).
Fifthly, confirmation of corn GRMZM2G701063 gene mutation
Research shows that 9 genes are shared among 108.2-108.6M of the sixth chromosome of maize, and further sequencing work is carried out on the 9 genes in order to determine the phenotypic change caused by mutation of which gene.
Sequencing results show that only the third exon of the maize GRMZM2G701063 gene is lost, and sequencing signal peaks at the deleted fragment are clear and single (as shown in FIG. 5).
To further confirm the reliability of the results, 10 individuals randomly selected from the mutants were subjected to repeated sequencing and showed the same results. This result indicates that the mutant is compared to wild type B73dos57The third exon of the middle GRMZM2G701063 gene has base loss and fragment deletion, and the part codes the important functional region of the gene.
The existing research shows that: the GRMZM2G701063 gene encodes a transcription factor type R2R3 in maize. The study of the GRMZM2G701063 gene in Arabidopsis thaliana homologous to the GRMZM2G701063 gene of maize showed that there were abnormalities in Arabidopsis thaliana in terms of the anthocyanins following mutation of the gene. However, there is no specific validation and study on the specific role of the gene in maize and in relation to stress resistance.
In summary, based on the present application, it can be concluded that:
in corn, the corn GRMZM2G701063 gene is related to the growth and development of corn leaf sheaths and tassels, and after the gene is abnormal due to deletion mutation, insertion mutation and the like, the corn phenotype shows the phenotypic characteristics of green leaf sheaths (the wild corn B73 variety specifically shows that the corn leaf sheaths gradually become purple under the illumination condition) and colorless tassels (the wild corn B73 variety specifically shows that the anthers make purple); based on the characteristic, the GRMZM2G701063 gene of the corn is changed by utilizing related genetic engineering technical means, and a new corn new variety with specific phenotype (green sheath and colorless tassel phenotype) characteristics can be cultivated.
The GRMZM2G701063 gene has relevance to adversity stress, and by utilizing relevant gene engineering technical means, after the GRMZM2G701063 gene or a gene homologous to the GRMZM2G701063 gene is abnormally expressed, the drought tolerance of the plant can be obviously improved, and the gene can be further used for cultivating a new plant variety with better drought tolerance.

Claims (2)

  1. The application of the GRMZM2G701063 gene in adversity stress is characterized in that the gene is related to plant drought stress reaction, and after the gene is functionally deleted, the plant drought tolerance is obviously enhanced; the plant is corn.
  2. 2. A method for improving drought tolerance of a plant, which is characterized in that the GRMZM2G701063 gene is abnormally expressed in a plant genome by a gene silencing technology, wherein the plant is maize.
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CN109666679A (en) * 2019-03-01 2019-04-23 中国农业大学 Chinese rose transcription factor RhPTM and its application
CN112680472B (en) * 2019-10-17 2023-08-01 华南农业大学 Application of ZmSPL gene in regulation and control of maize crown root or aerial root development
CN112795552B (en) * 2021-03-10 2022-10-04 河南大学 Application of Zm00001d024568 gene and encoding protein thereof in drought stress resistance of corn
CN115700282B (en) * 2022-12-21 2023-11-03 河南大学三亚研究院 Maize drought-resistant gene Zm00001d030678 and application thereof

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CN106755076A (en) * 2016-12-29 2017-05-31 新乡医学院 Applications of the arabidopsis gene COLD1 in terms of Genes For Plant Tolerance drought stress

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