CN112410370A - Application of corn 10kDa heat shock protein gene ZmHsp10 in changing stress resistance of plants - Google Patents

Application of corn 10kDa heat shock protein gene ZmHsp10 in changing stress resistance of plants Download PDF

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CN112410370A
CN112410370A CN202011112738.9A CN202011112738A CN112410370A CN 112410370 A CN112410370 A CN 112410370A CN 202011112738 A CN202011112738 A CN 202011112738A CN 112410370 A CN112410370 A CN 112410370A
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gene
zmhsp10
plant
corn
plants
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张举仁
李鹏
李朝霞
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Shandong University
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273Phenotypically 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
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance

Abstract

The invention discloses an application of a corn 10kDa heat shock protein gene ZmHsp10 in changing plant stress resistance, which is to clone a gene ZmHsp10 from corn, recombine the gene into a plant expression vector in a sense or antisense form, introduce a fusion gene into a plant by using a transgenic technology, and screen out a transgenic plant and progeny thereof with obviously improved or reduced resistance by carrying out drought resistance or heat resistance determination on the transgenic plant, thereby creating a new plant germplasm with application value. Wherein the cDNA sequence of the corn gene ZmHsp10 is shown in SEQ ID No.1, and the coded amino acid sequence is shown in SEQ ID No. 2; the plant is a crop or a turf grass. The application of the invention is of great significance to the improvement of crop yield under drought and heat stress.

Description

Application of corn 10kDa heat shock protein gene ZmHsp10 in changing stress resistance of plants
Technical Field
The invention belongs to the field of plant bioengineering breeding, and particularly relates to application of a corn 10kDa heat shock protein gene ZmHsp10 in changing drought resistance and heat resistance of plants.
Background
In recent years, drought and heat stress have increasingly more heavily influenced the yield of major crops with increasing global temperatures. The temperature in the south of Huaihe river and the Huang-Huai-Hai region of China is usually higher than the proper growth temperature of the corn within 7-9 months, and the high temperature can cause the serious yield reduction of the corn. In China, the area of about one half of the land is in arid and semiarid regions. Even in the main agricultural areas of non-arid regions, the plants are not affected by drought disasters, and the water resource shortage is one of the most important factors for limiting the development of agriculture and national economy in China. The corn plants are tall and large, the water consumption is high, the growth and development period is easy to be attacked by high-temperature drought, and the high-temperature drought is a main environmental factor influencing the corn yield in China. Severe high temperature or drought in the heading stage of maize results in substantial loss of production or loss of production of maize. The method has important strategic significance in cultivating the heat-resistant drought-resistant corn by adopting the gene operation technology, and is an important choice for coping with the continuous increase of the global temperature, improving the corn yield of China, and ensuring the grain safety and the sustainable development of agriculture.
Early in the 70-80 s of the last century, it has been discovered that cells, when subjected to heat stress, meet into a class of stress proteins with different molecular weights and with distinct differences in mechanism of action. Plant Heat Shock Proteins are classified into HSP60, HSP70, HSP90, HSP100 and small HSP families (small Heat Shock Proteins, sHSPs) according to their molecular weight. The molecular weight of the small-molecule heat shock protein is generally 12-34KD, and the small-molecule heat shock protein is extremely widely distributed and is generally present in all organisms. Small molecule heat shock proteins serve a variety of functions, including conferring thermotolerance to cells against high temperatures, acting as molecular chaperones to prevent protein aggregation, and opposing normal cell death, thereby regulating the balance of cell survival and death. The small-molecule heat shock protein is an important molecular chaperone, can be combined with a plurality of protein molecules and plays a plurality of physiological functions: help the amino acid chain fold into a three-dimensional correct structure, remove damaged amino acid chains that cannot be correctly folded, prevent protein aggregation, protect protein molecules from being interfered by other molecules and the like.
Chaperones are proteins that mediate the correct assembly of other proteins but do not themselves become the final functional structure, and they are composed of many unrelated proteins. Lasky first proposed the concept of molecular chaperones in 1987, which refer to nucleoxins (nucleolasmin) that bind to histones in the nucleus and mediate ordered assembly of nucleosomes. According to the definition of Ellis, the concept is extended to "a class of proteins with no sequence correlation but common functions, which help other polypeptide-containing structures to complete the correct assembly in the cell and separate from the polypeptide-containing structures after the assembly, and do not constitute the components of the protein structures for performing the functions". During protein synthesis, chaperones recognize and stabilize partially folded conformations of polypeptide chains, thereby participating in folding and assembly of nascent peptide chains. Heat shock proteins are a large class of molecular chaperones.
The number of heat shock proteins varies widely among plants, for example, 13 HSP20, 8 HSP70, 7 HSP90, 8 HSP100, and 21 heat shock protein transcription factors (Hsfs) in arabidopsis thaliana. In plants, small heat shock proteins (sHSPs) are the most abundant and widely distributed heat shock proteins, and the molecular weight of the heat shock proteins is generally 12-43 kD, and the C-terminal of most of the proteins contains 1 alpha-crystallin domain (ACD) with 80-100 amino acid sequences. The sHSPs are usually in low content under normal growth conditions of plants, but the expression of the sHSPs can be activated under adverse conditions, and the expression of small-molecule heat shock proteins can be induced by both biotic stress and abiotic stress under general conditions, wherein high-temperature stress is the most main inducing factor. And under high temperature stress, the content of the sHSPs is increased rapidly in a short time, and the types of the sHSPs are more than those of other heat shock proteins. The types and numbers of small heat shock proteins vary from plant to plant, and may be related to the type and life habit of the plant. Plant smHSP genes are all encoded by nuclear genes, have rich functions and genetic diversity, participate in a plurality of important processes of cell life activities, and are divided into different types according to subcellular localization and homology of amino acid sequences. Plant sHSPs are generally classified into 5 major subfamilies: (ii) CI, CII, CIII, CIV, CV, CVI and CVII subfamilies located in the cytoplasm or nucleus; MI and MII subfamilies localized to mitochondria; ③ localization to the P subfamily of plastids; (iv) ER subfamily localized to endoplasmic reticulum; and the Po subfamily of peroxisome. For example, there are 19 sHSPs genes in Arabidopsis thaliana, 13 sHSPs whose products are localized in cytoplasm/nucleus, 1 sHSP each localized in endoplasmic reticulum and peroxisome, and 2 sHSPs each localized in mitochondria and chloroplasts. 23 sHSPs genes are identified in rice, and the number of sHSPs distributed in different organelles is different.
The sHSPs can maintain the stability of natural proteins and play an important role in protecting membrane structures, etc., in coping with biotic or abiotic stress in organisms. The function of any protein is determined by its conformation and the way in which it folds in three-dimensional structure, and sHSPs regulate the folding, accumulation and localization and degradation of proteins. Aggregates formed by the sHSPs are capable of binding to damaged proteins generated after stress in vivo to prevent their aggregation; can interact with HSP100 or HSP70 in the presence of ATP to help the damaged protein refold and restore its biological function. The sHSPs play an important role when plants face stress, and have diverse stress resistance. Research shows that after heat induction, the micromolecule heat shock protein sHSP17.4 is expressed in large quantity, the drought resistance of arabidopsis is improved, the expression quantity of sHSP17.4 of a drought-sensitive mutant strain is very low, and the sHSP17.4 can maintain the growth and development of plants by protecting cell components under drought stress. The overexpression of the CII AtHSP17.6A in Arabidopsis can enhance the salt tolerance and drought resistance of plants, and the expression of the AtHSP17.6A is induced by heat, osmotic stress and a seed development process. Heat shock treatment is carried out on peanuts (the temperature of a growth chamber is higher than the ambient temperature by more than 10 ℃), and heat shock proteins are rapidly induced (0.5 hour), particularly small heat shock proteins (HSP 17 and HSP 40) are rapidly induced in a heat-resistant genotype, and the long duration time (2 hours) is probably a physiological adaptation mechanism of plants to high-temperature stress. After the AtHSP22.0 gene is over-expressed, the capability of the arabidopsis seeds for tolerating the tunicamycin stress is obviously improved, and the seed germination rate is obviously higher than that of the wild type. Cytoplasmic CI-type shps are expressed in elevated amounts during embryogenesis as well as during maturation of seeds. Research also shows that the micromolecule heat shock protein gene LimHSP16.45 of the lily which is ectopically overexpressed in arabidopsis thaliana can improve the resistance of a transgenic plant to adversity stress through ways of preventing irreversible protein aggregation and removing active oxygen and the like in cells. The overexpression of the small-molecule heat shock protein gene sHSP17.7 in rice can enhance the heat resistance and the resistance to B-type ultraviolet rays of transgenic plants, and the gene also plays an important role in drought resistance of rice. The rice OsHSP18.6 can be caused by various stresses such as drought, salt and cold, especially at high temperature. The gene is expressed in roots, stems, leaves, internodes and spikelets, and the heat resistance and the capability of resisting other abiotic stresses of transgenic plants over expressing the OsHSP18.6 are obviously improved. Under the conditions of high temperature and drought, the over-expressed plant Malondialdehyde (MDA) level is low, the activities of Catalase (CAT) and superoxide dismutase (SOD) are high, the sterility rate is reduced, but other agronomic characters are not obviously changed. RNAi interferes with OsHSP18.0 of rice to reduce the resistance of the plant to bacteria, and the heat resistance and the salt tolerance. Alfalfa MsHSP17.7 can be induced and expressed in high-temperature, high-salt, oxidative stress and drought environments, and the transgenic plant for overexpressing MsHSP17.7 obviously improves the resistance of the plant to high-salt stress, which indicates that the gene plays an important role in resisting salinization stress of the plant.
Under high temperature stress, the photosynthetic efficiency of the plant decreases and growth slows down or stops. Chloroplast small-molecule heat shock proteins (CP-shps), which are the most abundant heat shock proteins in plastids, protect photosynthetic organelles when plants are subjected to heat stress. CP-sHSP protects the chloroplast membrane, membrane-bound proteins, and stabilizes the membrane structure of the cell. CP-sHSP production can alleviate photoinhibition of photosystem II (PS II), and the intensity of thermotolerance is correlated with the expression level of CP-sHSP in different genotypes of the same plant. In addition, transgenic experiments prove that CP-sHSPs play an important protective role in plants against heat stress. Overexpression of CP-sHSPs in tobacco increases photosystem II stability under high temperature stress. In tomato, CP-sHSP only protects PSII under high temperature stress (47 ℃), but not at normal temperature (25 ℃).
The overexpression of the mitochondrial heat shock protein gene can obviously inhibit the programmed cell death of plants, and a complex formed by the small molecular heat shock protein has an important function in the plant. The small-molecule heat shock proteins interact with each other to form a complex to participate in heat-resistant reaction of plants, such as rice HSP17.7 which can interact with HSP16.9A. The mitochondrial sHSPs play an important role in the heat resistance of plants, can relieve the damage of protein complexes in mitochondria, ensure the normal electron transfer and ATP synthesis of cells, and promote the plants to maintain normal growth under the condition of high-temperature stress. Molecular cloning and stress expression analysis are carried out on a small heat shock protein gene CaHSP24 of the hot pepper, and the expression quantity of the gene under abiotic stress is found to be obviously increased, which indicates that the gene participates in stress response of plants. If the CaHSP26 gene of the pepper is ectopically overexpressed in arabidopsis thaliana, the heat resistance of a transgenic plant is enhanced. In order to elucidate the role of mitochondrial small heat shock protein (MT-sHSP) in heat shock response, the tomato MT-sHSP22 gene under the control of the 35S promoter was introduced into tobacco, and the heat resistance of the transformed plants was examined. Regardless of the sense or antisense of the gene, the transgenic plants exhibit normal morphology and growth rate during vegetative growth. The 4-week-old seedlings show heat resistance by over-expressing MT-sHSP22 plants under sudden heat stress, and the accumulation of sHSP22 protein can improve the oxidation stress resistance and adaptability of cells; and the antisense plant with the suppressed expression shows sensitivity.
The expression level of the endoplasmic reticulum small-molecule heat shock protein ER-sHSP can be obviously increased at high temperature, for example, LeHSP21.3 of tomato and AtHSP22.0 of Arabidopsis have certain resistance to high temperature. The induced expression of the peanut small heat shock protein gene (sHSP) can enhance the physiological adaptability of plants at high temperature. Researchers combine transcriptome sequencing of peony under high temperature stress to determine the resistance of the peony to high temperature by the small heat shock protein. In addition, the endoplasmic reticulum small heat shock protein gene is over-expressed, and the tunicamycin resistance of the transgenic plant is obviously enhanced compared with that of a control, so that the stress pressure of the outside on the endoplasmic reticulum can be relieved to a certain extent by the gene.
Different abiotic stress factors seriously affect the growth and development of plants, and the breeding of excellent stress-resistant varieties is a main way for improving the stress resistance of crops. Plant response to high temperature, drought, and other adversity stresses is a complex biological process controlled by multiple genes. Although the small heat shock protein can be activated and expressed when a plant is stressed by adversity such as biotic stress, heat stress and the like, and participates in improvement of stress resistance of the plant, the main work is concentrated on cytoplasmic-positioned sHSPs, the work of influence on the stress resistance of the plant by the change of the expression quantity of CP-sHSP, MT-sHSP and ER-sHSP genes is concentrated on the change of the heat resistance, the work of influence on the drought resistance of the plant by the change of the expression quantity of MT-sHSP is not reported, the work of change of the stress resistance of corn by the sHSPs is not reported, and the functional research of the plant heat shock 10kD protein (sHsp10) in crops such as corn is also not reported.
Heat shock 10kD protein (Hsp10) also known as chaperonin 10(Cpn10, chaperonin 10) and the homologue in E.coli is GroES. Cpn10 is a member of the heat shock protein family and has been isolated and purified from bacteria, plastids or mitochondria. In E.coli, GroEL is sometimes referred to as a chaperone, while GroES is referred to as a Co-chaperone (Co-chaperonin), since GroEL plays an important role in directing the folding process, while GroES assists in its active function. The molecular weight of the whole GroEL/GroES is 106 daltons, and is similar to the small subunit of ribosome. The Cpn family are oligomeric proteins with unique double-layered 7-9 membered cyclic structures that promote protein folding in vivo in an ATP-dependent manner. Cpns can be divided into two groups, the Hsp60(GroEL) family and the TriC family. Cpns of the Hsp60 type are present in eubacteria, mitochondria and chloroplasts and consist of double-layered circular loops with 7 isotypic subunits per layer, each subunit having a molecular weight of about 60 Kd. They bind in vivo to a cofactor, the 7-mer made up of Hsp10 subunits, e.g. to GroES in e. These proteins are stress-induced, except for the analogs in chloroplasts. It has been reported that Cpn60 and Cpn10 form stable binary complexes in the presence of ATP. This phenomenon was used as a basis for the identification of a Cpn10 homologue in potato mitochondria. When GroES binds to GroEL, it induces a conformational change in the proximal GroEL, increasing the volume of the central cavity, changing the environment in which the substrate is located. ATP plays an important role in the functioning of GroEL. Each subunit of GroEL contains one ATP molecule. ATP on the subunit on the proximal loop is essential for folding of the substrate, and release of GroES and substrate requires ATP hydrolysis of the distal loop. Little work has been done on Hsp10 function in plants, and it is only known that cpn10 is found to be upregulated in cold-treated roots and in hypoxic plant leaves, and appears to be a calmodulin-binding protein (Yang and poovaah, 2000), possibly involved in cold signaling. No reports of the effect of Hsp10 or cpn10 on plant stress resistance were found.
The maize ZmHsp10 gene (GRMZM2G035063) encodes a 10Kd heat shock protein (GRMZM2G035063_ P01(GenBank accession: DAA 41590); Unit accession(s): GRMZM2G035063-B6SLX1) of 2254bp in length, located on chromosome 214323983..214329237 in maize 2, containing 2 introns and 3 exons. Registered in GenBank as ZEAMMB73_172782 or ZEAMMB73_100400, and 103647892 in NCBI Gene (Entrez Gene). The classical transcript is GRMZM2G035063_ T01, the cDNA length is 779bp, and 98 amino acids of the encoded protein. According to the sequence similarity, the homologous gene of ZmHsp10 in sorghum is Sb02g040870, the homologous gene in rice is LOC _ Os07g44740, and the homologous gene in Arabidopsis is AT1G23100.1, which encodes a GroES-like family protein. ZmHsp10 is a gene with high expression abundance, the highest transcript level appears in maize endosperm, and the second is the meristematic region and the elongation region of embryo and root, the expression levels of the primary root, the young leaf and the immature tassel of the plant in seedling stage are also high, and the expression levels in the mature leaf and internode are low. ZmHsp10 has a homologous Gene GRMZM2G013652(GenBank: ZEAMMB73_881784, ZEAMMB73_ 298840; NCBI Gene (Entrez Gene):100193174) in maize, the latter being present on chromosome 7 (Chr7:169306985..169311988), classical length 2003 bp. The homologous gene in sorghum was Sb02g040870, the homologous gene in rice was LOC _ Os07g44740, presumably encoding a chaperonin protein, while the homologous gene in arabidopsis was still AT1G23100.1. GRMZM2G013652 encodes a GroES-like family protein with 3 different transcripts, the classical transcript is GRMZM2G013652_ T01, the cDNA is 866bp in length and comprises 3 exons and 2 introns, and the classical protein is GRMZM2G013652_ P01(GenBank access: DAA 63865; UniProcess(s): GRMZM2G013652-B4FE30) with 98 amino acids. The 2 nd Transcript GRMZM2G013652_ T02(Transcript ID: DAA63867) is 1217bp long, comprising 2 exons and 2 introns, and encodes a polypeptide 83 amino acids in length. The 3 rd Transcript is GRMZM2G013652_ T03(Transcript ID: DAA63866), is 712bp in length, comprises 3 exons and 2 introns, encodes a polypeptide of 98 amino acids, and is completely identical to the sequence of GRMZM2G013652_ P01. GRMZM2G013652 is a ubiquitous gene with high expression level, abundant expression of transcripts in maize endosperm, young embryo, root meristematic region and elongation region, V7-V9 internode, leaf base, stem tip and meristem, immature tassel and ear, high protein intensity in embryo and endosperm, ear primordium, germinating kernel and meristem in vegetative growth period, namely, large amount of synthesis in rapidly dividing and growing organs. In Arabidopsis, a gene similar to ZmHsp10 also has AT1G14980.1, which may encode a mitochondrially localized chaperone 10, which complements the groES mutation in E.coli, whose mRNA abundance is up-regulated by heat stress and is expressed in different organs.
No report is found about the functions and the functions of the ZmHsp10 gene and the 10Kd heat shock protein coded by the gene in plant stress resistance through searching.
Disclosure of Invention
Aiming at the current research situation, the invention aims to provide application of a corn 10kDa heat shock protein gene ZmHsp10 in changing drought resistance and heat resistance of plants.
The application of the corn 10kDa heat shock protein gene ZmHsp10 in changing the stress resistance of plants; wherein: the cDNA nucleotide sequence of the corn 10kDa heat shock protein gene ZmHsp10 is shown in SEQ ID No. 1; the coded amino acid sequence is shown as SEQ ID No. 2; the stress resistance refers to drought resistance and heat resistance.
In the above application: the plant is a crop or a turf grass.
Wherein: the crop is preferably corn or sorghum and the turf grass is preferably bluegrass or ryegrass.
The applicant discovers that ZmHsp10 gene transcripts are obviously up-regulated and expressed after a corn plant suffers drought stress by screening cDNA deduction libraries of different genotypes of corn before and after drought treatment, and the up-regulation of expression abundance is large in a drought-resistant inbred line. Sequencing and identifying that the whole length of cDNA is 779bp, coding 98 amino acids, and sequence searching to obtain that the gene transcript codes a heat shock protein (ZmHsp10) with 10 Kd. The corn 10kDa heat shock protein gene ZmHsp10 has similarity with 10kD heat shock proteins of other plants on the amino acid level, and has the highest identity with XP _006658032.1 of wild rice (Oryza brachyantha) on the amino acid level, wherein the identity is 91 percent; the identity with rice XP _015647372.1 was 90%. The cDNA is recombined into a plant expression vector pCUA, transcription is started by a maize Ubquitin1 promoter or an inducible promoter RD29A/B, and the transcription is stopped by a nos terminator, so that an expression cassette and a plant transformation vector are constructed.
The corn ZmHsp10 gene can be recombined into a plant transformation vector in a genome gene form besides a cDNA form; its coding sequence can be used in sense form or antisense form or gene editing structure form to construct fusion gene, which can be used for plant transgene.
The invention provides application of a corn drought-tolerant new strain which is bred by a corn 10kDa heat shock protein gene ZmHsp10 through a transgenic overexpression technology and has a yield remarkably higher than that of a control group after suffering from drought stress.
The invention provides application of a new heat-resistant Poa annua strain which is cultured by using a corn 10kDa heat shock protein gene ZmHsp10 through a transgenic overexpression technology and has obviously better plant survival rate and leaf color than wild type plants after being subjected to heat stress.
The basic method comprises the following steps: the ZmHsp10 gene is cloned from corn and used for constructing an expression vector for plant transformation, and then a transgenic technology is utilized to produce a plant with transgenic over-expression or reduced expression, or a gene editing technology is utilized to increase or knock out the expression of the ZmHsp10 gene. By carrying out drought resistance measurement on the transgenic plant, the transgenic plant with obviously improved or reduced drought resistance and the descendant thereof are screened out, and a new plant germplasm with application value is created. The method comprises the following steps:
cloning and analysis of maize ZmHsp10 Gene
First, the ZmHsp10 gene was cloned from maize genomic DNA or cDNA, which when expressed results in a small protein of 98 amino acids. If a complete cDNA sequence is cloned from a cDNA library of the zearals 319 by adopting a PCR method, a nucleotide sequence identified by sequencing is shown as SEQ ID No.1, namely the cDNA form of the ZmHsp10 gene is determined. And (3) designing a primer by using the obtained cDNA sequence, and amplifying a genome sequence of the ZmHsp10 gene from the genome of the maize inbred line 319 by using a PCR method. BLAST the 5' end sequence of ZmHsp10 gene cDNA in NCBI to obtain a series of sequences of different lengths for analyzing the transcriptional regulatory elements of the gene, and then replacing the transgene overexpression operation by inserting an enhancer at a fixed point to promote gene expression.
Construction of plant expression vector of corn ZmHsp10 gene
The high expression of ZmHsp10 in plants requires that ZmHsp10 is recombined into a plant expression vector and applied to plant transgenes to generate engineering plants. Firstly, the corn ZmHsp10 gene is inserted into an insert box of a target gene (transgene) of a plant transformation vector by a conventional gene recombination technology to form a fusion gene, then the generated plasmid is transferred into a receptor cell, and an engineering strain is obtained through selection and identification. The fusion gene is a gene expression structure formed by connecting a ZmHsp10 gene coding frame with a promoter and a transcription termination region, wherein the ZmHsp10 coding frame can be positioned at the 3 'end of the promoter and the 5' end of the transcription termination region in the positive direction or the reverse direction. The promoter can have different promoter characteristics, such as a stress inducible promoter RD29A/B promoter and a constitutive promoter Ubiquitin1 promoter. The acceptor cells of the recombinant plasmid are Escherichia coli and Agrobacterium tumefaciens cells.
Corn ZmHsp10 gene transformed plant
In the present invention, plants are transformed with a plant expression vector containing the maize ZmHsp10 gene. Different transgenic methods are selected according to the characteristics of transgenic receptor materials to obtain transgenic plants in batches. The following briefly describes the process of obtaining and screening transgenic plants using maize as an example.
Common methods for maize transformation are: 1) direct transformation, i.e., extracting recombinant plasmid DNA, and introducing the plasmid DNA into cells by gene gun bombardment, electrofusion, etc. 2) Agrobacterium-mediated genetic method. In addition, there is a transformation procedure in which different kinds of methods are used in combination, such as a gene gun bombardment method and Agrobacterium are used in combination to improve transformation efficiency. The following description will be given by taking embryogenic callus of maize inbred line as material and using gene gun bombardment method to perform genetic transformation.
9-15 days after selfing and pollination, picking up ears, removing bracts, soaking in 70% alcohol for 5min, picking up young embryos with the size of about 1.0-1.5mm under aseptic condition, inoculating on an induction culture medium, culturing for 4-6 weeks to obtain crisp and light yellow II-type callus, and then carrying out subculture. Subcultured every 10-15 days. The obtained II type callus is used as receptor material for genetic transformation.
In the present invention, genetic transformation may be carried out by gene gun bombardment. High quality plasmid DNA was first extracted from recipient cells containing the maize ZmHsp10 recombinant plasmid, e.coli, which is typically about 4-5 kb. The engineering bacteria are cultured and the plasmid is extracted and purified by a conventional method. Then preparing a micro-bullet, namely weighing gold powder with the size of 1.0 mu m, washing by 70% ethanol, standing for 15 minutes, and centrifuging to remove supernatant; washed with sterile water 3 times and then stored in 50% sterile glycerol (final concentration 60mg/ml microprojectiles) for future use. In use, the mixture was vortexed for 5 minutes to break the gold powder aggregates, and 5. mu.l of plasmid DNA (1. mu.g/. mu.l), 50. mu.l of 12.5M CaCl2, and 20. mu.l of 0.1M spermidine were added in this order while vortexing the mixture. And then, continuing swirling for 2-3 minutes, and standing for 1 minute. Centrifuging to remove supernatant, adding 70% ethanol, standing, centrifuging to remove supernatant, re-suspending with anhydrous ethanol, and sampling and adding on a micro-elastic carrier. The dosage of the micro-pill is 0.5mg per pill.
In the invention, a sterile culture medium with the thickness of 0.3cm is poured into a culture dish with the diameter of 9cm, and then the callus is put into the culture dish in high density and bombarded once per dish. And (3) obtaining bombardment parameters: the distance between the breakable wafer and the carrier is 2.5cm, the distance between the carrier and the blocking net is 0.8cm, and the micro-bullet flying distance is 6-9 cm. Other parameters are valued according to the instruction. And (3) after bombardment, restoring and culturing the material in the dark for 3 days, and then transferring the material into a new culture medium with unchanged components for culturing for 3 weeks to ensure that the transferred target gene is fully expressed. The material is then transferred to a medium supplemented with a selection agent (e.g. the 0.09% herbicide glufosinate) for screening. Continuously screening for 2 generations, each generation being 15 days, transferring the resistant callus to a culture medium without a screening agent, recovering and culturing for 1 generation under illumination for 16 hours/day, and transferring to a differentiation culture medium to differentiate seedlings. And (3) rooting the plantlets generated by the callus in a rooting culture medium, transplanting the strong plantlets into a flowerpot, planting the strong plantlets in a field or a greenhouse when the strong plantlets grow to about 10cm high, and selfing and fructifying. The transgenic plant generates a pure line through several generations of molecular detection and selfing fructification, and then obtains a transgenic excellent material through resistance identification and field selection.
The identification of the transgenic plant in the invention adopts PCR detection method screening and Southern blotting verification. Namely, DNA of the leaves of the herbicide resistant plantlets is extracted for PCR detection. And designing primers according to the cDNA sequences of the screening marker gene and the target gene ZmHsp10, and respectively carrying out PCR detection. The transgenic target band is amplified from the transgenic plant, while the non-transgenic control plant does not have the target band with the same size, and the PCR positive plants are preliminarily deduced to be transgenic plants. And extracting DNA from the positive plant leaves for Southern blotting verification, and selecting transgenic plants for selfing propagation.
Selection and utilization of corn ZmHsp10 transgenic plant
In the invention, in order to know the relationship between transgenosis and plant stress resistance, a T2 generation transgenic plant and a gene editing plant which are positive in PCR and stable in heredity are selected, non-transgenic control is taken as a material, the RNA of leaves is extracted, and the expression strength of a target gene is detected by Real time RT-PCR. In the detection of corn transgenic material, the corn Ubiquitin1 gene is used as an internal standard gene. In the maize ZmHsp10 gene overexpression strain, the mRNA abundance of ZmHsp10 is 2.4-6.83 times that of the control strain; in the ZmHsp10 antisense structure-transferred strain, the mRNA abundance of the ZmHsp10 gene is 0.41-0.75 times that of the control strain. ZmHsp10 transcripts were not detected in gene-edited plants, indicating efficient expression of the transgene in these transgenic lines. In the invention, the selection of the transgenic plant progeny and the determination of drought resistance are carried out in stages.
Under normal growth conditions, the 3-leaf transgenic, gene-edited and recipient inbred (non-transgenic control) plants were not significantly phenotypically different. The biomass of the 3-leaf stage plants under normal growth conditions was measured, and it was found that the ZmHsp10 over-expressed plants grew faster. When the plants grow to 11-14 leaf stage, the difference in phenotype between the transgenic plants and the recipient inbred plants is not obvious. Drought death experiments were performed on transgenic maize of the T2 generation at the 3-leaf stage and on shoots from the recipient inbred line. After most of maize seedlings are subjected to drought stress treatment for 7 days, leaves are nearly dry, stem bases become soft, seedlings which are frequently died due to the drought stress are picked out and watered again, and the recovery conditions of the seedlings of different strains are observed. The differences of the drought stress resistance of different strains are obvious, watering is resumed for 2 days after the different strains are subjected to the same degree of drought stress, most plants of the transgenic overexpression strain resume normal growth, most plants of the control and antisense strains die after being dried, and all plants edited by the ZmHsp10 gene die.
Controlling the soil water content when the corn plants grow to 10-leaf stage, maintaining the relative water content between 50% and 55%, carrying out stress treatment for 8 days, then recovering normal watering, and carrying out emasculation after the plants grow for one week. At the same time, a control area under normal watering conditions was set. And observing the phenotype changes of the transgenic corn plant and the receptor inbred line plant in the drought stress process, and respectively taking materials and measuring various physiological indexes at 0, 2,4, 6 and 8 days of stress treatment and one week after the recovery of watering. The morphological characteristics and growth and development rate of the ZmHsp10 gene strain under normal growth conditions were not much different from WT. After the drought stress of 5 days, the leaves of the receptor inbred line plant, the plant with the ZmHsp10 antisense structure and the gene editing plant are seriously curled and yellowed, while the leaf wilting degree of the plant with the ZmHsp10 gene overexpression is lighter and the leaf color is emerald. The cell membrane ion leakage rate and the malonaldehyde content of the maize leaf under different conditions and sampling time are respectively measured, and the malonaldehyde content and the cell membrane ion leakage of transgenic plants and receptor inbred line plants and gene editing plants under normal growth conditions are obtained without significant difference. Along with the prolonging of the drought stress time, the content of malondialdehyde and the cell membrane ion leakage rate are obviously increased, wherein the increasing amplitude of the ZmHsp10 gene overexpression strain is smaller. After 8 days of stress, the malondialdehyde content and the cell membrane ion leakage rate of the receptor inbred line are 21.18nmol/g FW and 39.6%, the malondialdehyde content and the ion leakage rate of the ZmHsp10 gene overexpression strain are obviously lower than those of the receptor inbred line, and the malondialdehyde content and the ion leakage rate of the ZmHsp10 antisense structure strain and the gene editing strain are obviously higher than those of the receptor inbred line. Namely, in the drought stress process, the damage of the cell membrane of an overexpression strain is light, the oxidation degree of membrane lipid is low, and the drought resistance is strong. To understand the extent of leaf water loss under drought stress conditions, the relative water content in leaves of different maize lines was determined. Before drought stress, the relative water content RWC has no obvious difference among the strains, after the drought stress, the relative water content of all the strains of leaves is reduced, but after the drought stress for 3 days, the RWC reduction amplitude is different in different strains of leaves. Compared with the ZmHsp10 gene overexpression strain, the leaf water loss of the receptor inbred line, the antisense structure-transferred strain and the gene editing strain is more. On day 8 of drought stress, leaf RWC of the ZmHsp10 gene over-expressed strain was significantly higher than that of the receptor inbred, and those of the ZmHsp10 antisense construct-transferred strain and the gene editing strain were significantly lower than that of the receptor inbred. After normal watering was resumed, RWC of the ZmHsp10 gene over-expressed line was still higher than that of the recipient inbred line, but leaf water content of the antisense and gene editing lines also returned faster. Prior to drought stress treatment, the soluble total sugar content of maize leaves did not differ significantly between the transgenic lines and the recipient inbred lines. Drought stress treatment caused an increase in soluble sugar content in all leaves of the lines, but at different amplitudes. Under the drought stress condition, the ZmHsp10 gene overexpression strain leaves accumulate more soluble sugar content than the receptor inbred line; compared with the recipient inbred line, the ZmHsp10 antisense construct and gene editing line had less soluble sugars. These results indicate that ZmHsp10 gene overexpression strain leaves have better water retention capacity under drought stress conditions, and the normal cell turgor is well maintained, which is beneficial to the growth of plants.
Drought stress is continuously carried out on the corn plants before flowering for 6 weeks in a rainproof greenhouse, and then normal watering is resumed. During this period, phenotypic changes of transgenic and recipient inbred plants under drought stress were observed: the leaves of the ZmHsp10 gene-transferred over-expression strain plants are relatively extended, and the drought resistance is obviously better than that of receptor inbred lines. The ZmHsp10 antisense structural strain and the gene editing strain under drought stress show similar symptoms as WT, and the leaf curl is severe and the leaf color is light. The average ear length of the ZmHsp10 gene overexpression strain after drought stress is obviously larger than that of the receptor inbred line, and the average ear length of the antisense strain and the gene editing strain is obviously smaller than that of the receptor inbred line. From the single-ear grain weight, the single-ear grain weight of the ZmHsp10 gene overexpression sense line is obviously higher than that of the receptor inbred line, while the single-ear grain weight of the antisense line and the gene editing line is obviously lower than that of the receptor inbred line, and the difference reaches a significant degree or a very significant degree (P <0.05 or P <0.01(n ═ 10)). From the hundred grain weight, although differences exist between each transgenic line and the receptor inbred line, the differences do not reach a significant degree. These results indicate that ZmHsp10 transgenic over-expressed plants have significantly improved resistance to drought stress encountered before flowering, and the reproductive organ development is less affected, resulting in significantly higher grain yield than that of receptor inbred lines.
Based on the above results, the differences in physiological indexes and yields of different lines of plants of maize under the conditions of drought stress were synthesized to obtain: the ZmHsp10 gene-transferred overexpression strain has larger increase amplitude of cell soluble substances when meeting drought stress, reduces the loss of cell water, can better keep the water content of cells, has small damage to cell membranes, stronger photosynthetic capacity and less influence on the growth and development of plants, so that the grain yield is high, the drought resistance is obviously improved, and the ZmHsp10 gene-transferred overexpression strain can better adapt to the drought stress environment compared with a receptor inbred line; on the contrary, when the ZmHsp10 antisense structure-transferred strain or the gene editing strain encounters drought stress, the increase of the cytosol is small, the water loss of cells is large, the damage of cell membranes is large, the photosynthetic capacity is weakened, the yield of the plant is reduced, and the drought resistance of the plant is poor. The fact shows that the drought resistance of the corn can be obviously changed by over-expressing the ZmHsp10 gene in the corn, provides an example for plant stress-resistant breeding, and has important significance for improving the yield of crops in an environment easy to dry.
Experiments also confirm that transgenic plants show significantly improved heat and cold tolerance.
Transgenic homozygous maize plants grown at 28 ℃ (light, 13h/d)/22 ℃ (dark, 11h/d) were transferred to 36 ℃ (light) for 2h of growth, followed by 4 consecutive heat treatments at 39 ℃ (light 13h/d, dark 11h/d) and then restored to growth at 28 ℃. Almost all the plants of the control inbred line die, while the heat resistance of most of the transgenic overexpression lines is obviously better than that of the control, and partial transgenic plants are not obviously damaged.
Sowing corn in a greenhouse, performing continuous 5-day high-temperature treatment when the corn grows to 9-11 leaf stage under normal growth conditions, then restoring to the normal growth conditions to grow and develop until the fruit cluster is mature, and performing open pollination and seed setting on plants in the same line. During the high-temperature treatment, the temperature is 20-24 ℃ between 22 hours and 6 hours of the next day, the temperature is increased from 20-24 ℃ to about 37 ℃ between 6 and 8 hours, the temperature is maintained at 38-39 ℃ between 9 and 17 hours, the temperature is maintained at 36-37 ℃ between 17 and 19 hours, and the temperature is reduced to 22-24 ℃ between 19 and 22 hours. Natural illumination, the illumination time is 14 h/d. Transgenic over-expression lines and non-transgenic control partitions were grown. During the high temperature treatment, the damage of the transgenic over-expression plants is not obvious, the leaves without the transgenic control are slightly wilted at the time of 11-17 days, the leaves begin to yellow after 3 days of treatment, and the new leaves grow slowly. The plant height of the non-transgenic control plant after high-temperature treatment is obviously lower than that of the transgenic over-expression plant, the tassel development is poor, and about half of pollen is sterile. Compared with the yield of single plant grains, the yield of the seeds is reduced by 31 to 19 percent compared with that of 3 transgenic over-expression strains without transgenes. Namely, the transgenic overexpression strain shows obviously improved heat resistance.
These results indicate that increasing the expression level of the maize ZmHsp10(10kDa heat shock protein) gene significantly increases the heat resistance of maize plants, making maize more tolerant in high temperature environments.
In conclusion, the invention discloses application of a corn 10kDa heat shock protein gene ZmHsp10 in changing plant stress resistance, namely cloning the gene ZmHsp10 from corn, recombining the gene into a plant expression vector in a sense or antisense form, introducing a fusion gene into a plant by using a transgenic technology, and screening a transgenic plant with obviously improved or reduced resistance and progeny thereof by carrying out drought resistance or heat resistance determination on the transgenic plant to create a new plant germplasm with application value. The application of the invention is of great significance to the improvement of crop yield under drought and heat stress.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples are merely illustrative of the preferred embodiments of the present invention and are not intended to limit the present invention in any way, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention fall within the scope of the technical solution of the present invention.
Example 1: over-expression of corn ZmHsp10 gene to create drought-tolerant inbred line of corn
1) The receptor system is established by using backbone inbred lines used in agricultural production in China as materials, such as Zheng 58, Chang 7-2, 6WC and the like. Soaking the seeds in 70% ethanol for 8 min, soaking in 0.1% mercuric chloride for 8-12 min, and washing with sterile water for 3-5 times. The sterilized seeds germinate in a sterile triangular flask under dark conditions (25-28 ℃) for 1-2 days, and after the seeds germinate, the seeds are placed on an improved MS culture medium to germinate under dark conditions. When the coleoptile is extended to 3-4 cm, the coleoptile and 2-3 young leaves are timely peeled off to expose the stem tip growth cone.
2) Corn stem tip transformation and plant regeneration construct a fusion gene (in a sense form) carrying a corn ZmHsp10 cDNA sequence, and the transgenic ZmHsp10 is started by a stress inducible promoter RD29A/B or a corn Ubiquitin1 promoter. And recombining the fusion gene into a Mini-Ti plasmid of the agrobacterium tumefaciens with a T-DNA zone having a plant herbicide resistance gene bar to obtain a genetic transformation vector. Agrobacterium tumefaciens (e.g., LBA4404, etc.) harboring a binary vector (Mini-Ti plasmid carrying a selection agent resistance gene and a ZmHsp10 gene) was shake-cultured in LB medium (containing, per liter of medium, tryptone 10g, yeast extract 5g, NaCl 10g, pH 7.0, autoclaved) supplemented with antibiotics at 28 ℃ at a shaking rate of 110rpm to obtain a bacterial concentration OD6000.45 to 0.6. Then, the mixture was centrifuged at 3000rpm for 10 minutes, and the supernatant was discarded. The mycelia were washed with 1/2 modified MS liquid medium and collected by centrifugation. Then suspending the thallus in 1/2 modified MS liquid culture medium added with 100 mu mol/l of acetosyringone (As), and diluting 5-20 times for transformation. Pouring the bacteria liquid into culture dish, inclining culture dish to make the aseptic seedling with stem tip growth cone exposed soaked in the bacteria liquid at 0.5X 10%5Processing for 8-12 minutes under the pressure of Pa atmosphere. Then the impregnated bud tip is usedThe bacteria filter paper is sucked dry, and the germinated seeds are placed on an improved MS solid culture medium to be cultured for 2-3 days in the dark, wherein the culture temperature is 22-24 ℃. Then the aseptic seedlings are put under scattered light to be cultured for 2-3 days, and then the aseptic seedlings are transplanted into a flowerpot paved with upper vermiculite and lower loam, and the top of the plant is covered with vermiculite. The transplanted plants are grown under natural illumination, the daily temperature is 22-28 ℃, the night temperature is 15-21 ℃, and 1/2 inorganic salt in the improved MS culture medium is poured every other day. After the transformed plant grows 3 leaves, 0.125% of herbicide glufosinate-ammonium aqueous solution is sprayed, and preferably, the plant drops. Untransformed control plants stopped growing after 4 days post-spray and started dying after 9 days. After the transformed plants are sprayed, some individuals have similar changes to the control plants, and other individuals continue to grow and have insignificant changes. When the survival plants grow to 5 leaves, the plants are planted in the field, and the plants are bagged and selfed to form seeds. Taking the leaves of the transplanted survived plants to carry out PCR detection and screening of transgenic plants.
3) Resistance testing and selection of transgenic plants a candidate transgenic plant (T0) was bagged for selfing. T1 seeds from different T0 plants were sown in a greenhouse or in a field with protective facilities and sprayed with 0.18% aqueous herbicide glufosinate solution to observe the plant resistance. And (4) continuously bagging and selfing herbicide-resistant plants screened from the T1 generation, and continuously performing molecular biological identification and resistance detection on filial generations of the herbicide-resistant plants. Through several generations of selfing homozygosis and resistance detection and selection, the transgenic corn homozygosis line is finally obtained. In the drought resistance identification and selection, the transgenic plant lines are respectively planted in a flowerpot, a greenhouse and a field, drought stress treatment is respectively carried out in a 3-leaf stage, an elongation stage and a flowering early stage, a emasculation and powder scattering stage and a grain filling stage, the change of physiological parameters and grain yield are measured, and the plant lines with strong drought resistance, grain yield remarkably increased compared with that of a non-transgenic control material and slightly increased grain yield with a receptor inbred line under the appropriate cultivation condition are screened. The strain can be used for preparing corn drought-resistant herbicide-resistant hybrid.
Example 2: ZmHsp10 gene transfer to create new heat-resistant poa annua strain
Poa pratensis L is one of the most important turf grass species in temperate regions. Because of its advantages of beautiful color, strong cold resistance, shade resistance and pruning resistance, it is widely used as lawn-building grass seed in northern area and southern cool area of China. The grassland bluegrass also has the defects of poor high temperature resistance, weak drought resistance, easy damage by diseases and insects, and the like, and the grassland bluegrass stops growing in the midsummer and even partially dies. These disadvantages greatly restrict the widespread use of Poa pratensis in hot summer areas. Since the meadow bluegrass has the parthenogenesis characteristic, the difficulty of improving the unfavorable characters by adopting the conventional breeding technology is high, and the success rate is low, so the work of cultivating the new meadow bluegrass variety by adopting a genetic engineering means is emphasized. The invention creates the drought-tolerant germplasm of the Poa pratensis by transferring the ZmHsp10 gene.
1) Construction of transformation vectors: construction of plasmids used for genetic transformation As shown in example 1, the selection marker gene may be herbicide resistance genes als (herbicide resistance chlorsulfuron), bar (herbicide resistance glufosinate) and the like.
2) The grass-bluegrass transformation receptor is established by sterilizing grass-bluegrass seeds with 70% alcohol for 2 minutes and 0.2% mercuric chloride for 14 minutes, washing with sterile water for 3-5 times, and germinating on sterile wet filter paper in the dark. Transferring the seedlings to an induction culture medium (MS culture medium +3 mg/L6-BA +0.5 mg/L2, 4-D +200mg/L hydrolyzed casein +30g/L sucrose +6.5g/L agar) after 10-15D to induce the base parts of the seedlings to expand, and transferring the seedlings to a cluster bud generation culture medium (MS culture medium +2 mg/L6-BA +200mg/L hydrolyzed casein +30g/L sucrose +6.5g/L agar) to induce the seedlings to form cluster buds after culturing for 18D. And transferring the cluster buds to a subculture medium for subculture at the temperature of 24 +/-2 ℃ under the light intensity of 500-1000 Lx for 14 h/d. In order to ensure that the clumped buds can be subcultured for a long time, MS culture medium +3 mg/L6-BA +0.07 mg/L2, 4-D +200mg/L hydrolyzed casein +30g/L sucrose +6.5g/L agar is taken as a suitable subculture medium for most varieties. When the concentration of 2,4-D is higher, callus is generated at the base of the cluster bud block, and the capability of differentiating buds is low. When the concentration of 2,4-D is lower, the seedlings are easy to age and are not easy to generate new buds. Even on a suitable subculture medium, clumped bud mass plantlets after 5 subcultures are aged and the proliferative capacity is reduced. To keep the cluster buds of the Poa pratensis in tender state, the cluster buds can be divided into single buds or small blocks of 2-3 mm after subculture for 4-5 times, inoculated on an induction culture medium for induction and expansion, and then transferred to a cluster bud generation culture medium to generate cluster bud blocks. And taking the clumpy buds cultured on a subculture medium for about 5 days in a dark environment as a transformation receptor.
3) Genetic transformation of grass early maturing clump buds
A monoclonal culture of Agrobacterium (carrying a plasmid for transformation) was picked and inoculated into liquid LB liquid medium containing 25mg/L rifampicin and 50mg/L kanamycin, and cultured at 28 ℃ with shaking (180r/min) to logarithmic phase. Centrifuging the bacterial liquid at 4000r/min, pouring out the supernatant, re-suspending the thallus with liquid culture medium (modified MS culture medium +2 mg/L6-BA +200mg/L hydrolyzed casein +30g/L sucrose), and diluting to OD600And (4) keeping the standard value at 0.6 for standby. 0.2% (final concentration) Acetosyringone (AS) was added to the bacterial suspension before the dip dyeing. Cutting off leaves of the cluster buds of the Poa pratensis, peeling off leaf sheaths, cutting into small bud blocks of about 1mm, exposing meristems of bud tips as much as possible, putting the small bud blocks into a culture dish, pouring agrobacterium liquid, and dip-dyeing for 4-6min under the negative pressure treatment of 0.05 MP. After the bacterial liquid was poured out, the residual bacterial liquid was blotted dry with sterile filter paper, and the small bud pieces were transferred to a cluster bud generation medium and cultured in the dark at 24. + -. 2 ℃ for 3 days. Transferring the co-cultured bud blocks to a cluster bud generation culture medium added with 100mg/L of cefamycin to inhibit the growth of agrobacterium, dividing the cluster bud blocks into single buds after 10 days, placing the single buds on a cluster bud generation culture medium containing 0.1-0.2% of herbicide glufosinate (the concentration is greatly different due to different genotypes) to screen resistant buds, and continuously screening for 3 generations, wherein each generation is screened for 15 days. And transferring the resistant plantlets obtained after 3 generations of screening to a cluster bud generation culture medium for recovery and multiplication culture. Then transferring the obtained product into a rooting culture medium, and growing roots after about 8-15 days. And when the root grows to be 2-5 cm long, culturing for 1-2 days under natural light, removing the sealing film, hardening the seedling for 1-2 days, and transplanting into a flowerpot. The lower part of the flowerpot is soil, the upper part of the flowerpot is vermiculite with the thickness of 6-8 cm, 1/2MS nutrient solution is poured once every 5 days, and the water is poured once every other day. The transplanting survival rate is 95-99%. Taking leaves for PCR detection after the leaves survive for 2 months.
4) Molecular detection of transformed plants
DNA of the leaf blade of the Poa pratensis regenerated plant is extracted by a CTAB method and used as a template to carry out PCR reaction to detect transgenosis. The transgenic plant is subjected to plant division propagation or sister cross-breeding. The asexual clone seedling or the filial generation seedling is continuously sprayed with herbicide glufosinate-glufosinate (0.15 percent concentration) for screening, the resistant plant adopts PCR to detect transgene and RT-PCR to measure the transgene expression level, and the plant with high transgene expression level is selected for plant division propagation. The latter was used for resistance detection experiments.
5) Detection and utilization of heat resistance of transgenic strain
Cloning the stable transgenic plant and the plant of the receptor variety, selecting small seedlings with similar sizes, transplanting the small seedlings into small flowerpots with the same sizes respectively, and growing under proper conditions. When plants produced 3-4 tillers, they were divided into 2 groups of about 8 pots per line, with plantlets of the recipient variety as the control material. Transgenic homozygous plants grown at 18 ℃ (light, 13h/d)/12 ℃ (dark, 11h/d) were transferred to 30 ℃ (light) for 2h growth, followed by a continuous heat treatment at 35 ℃ for 3 days (light 13h/d, dark 11h/d), and then restored to growth at 20 ℃ (light, 13h/d)/15 ℃ (dark, 11 h/d). After heat treatment, almost all of the non-transgenic control (wt) dies, only individual strains in the transgenic strains have small difference with the non-transgenic control, the heat resistance of part of the strains is obviously superior to that of the control, part of the leaves are still green, the growth is gradually recovered under the normal growth condition, and the damage symptoms are not obvious after 5 days, namely the initially selected transgenic heat-resistant strains. Breeding seeds of the selected transgenic plant, sowing the seeds in the field for character observation, and breeding the new strain of the excellent heat-resistant bluegrass. The latter plants have significantly better survival rates and leaf color after hot summer and also show significantly improved cold tolerance than the wild-type control.
Sequence listing
<110> Shandong university
<120> corn 10kDa heat shock protein gene ZmHsp10 and application thereof in changing plant stress resistance
<141>2020-10-15
<160>2
<210>1
<211>779
<212>cDNA
<213> corn (Zea mays L.)
<221> nucleotide sequence of maize 10kDa heat shock protein gene ZmHsp10
<222>(1)…(779)
<400> 1
gcacgctggc acgttgcggc ggcgcagagc acatccccaa ttccccagga acaggctcgt 60
aatttttcca ctgccatcgc accctttcat aaaaccctaa acccaggacc caggtttatc 120
ggtgaggcga gccgcgagtg gaactgtgga agcgtcgagc ttttttcttc tttgtcttgc 180
tgcgcaagaa gaagggaaac gagaggaggg cggcgatggc gaagaggctg ctcccgtcgc 240
tgaaccgggt gctggtggag aagctggtgc agcccaagaa gaccgccggc ggcatcctcc 300
tcccggaaac atccaagcag ctgaatgctg ctaaagtggt ggctgttggc cctggtgagc 360
gcgacaaggc aggcaatctg atcccagttg ctctgaagga aggcgacact gttcttctgc 420
ccgagtatgg tggatctgaa gtcaagcttg cggctgataa agagtacctc ctcttcagag 480
aggatgacat tttgggcaca cttgtggact gattcagatc gcgggcaagc gggtggtggt 540
gatatgcgta ggatttgaag ctgggaacaa actccagaaa gaactgtgcc ctgtcgtgat 600
gtcataaatt tgtggtgaaa catcatatca tgaggataaa acttgttgct tttttgtgat 660
gcaatttaga aaactttcga atttgaactg gttcccaata ttttgttctc ctccctgtcc 720
cctgttgaag tgctctctgt tttaagttag caattaacgg gatcggattt acttgtgaa 779
<210> 2
<211> 98
<212> PRT
<213> Artificial sequence
<221> amino acid sequence encoded by maize 10kDa heat shock protein gene ZmHsp10
<222>(1)…(98)
<400> 2
MAKRLLPSLN RVLVEKLVQP KKTAGGILLP ETSKQLNAAK VVAVGPGERD KAGNLIPVAL 60
KEGDTVLLPE YGGSEVKLAA DKEYLLFRED DILGTLVD 98

Claims (5)

1. The application of a corn 10kDa heat shock protein gene ZmHsp10 in changing the stress resistance of plants; wherein: the cDNA nucleotide sequence of the corn 10kDa heat shock protein gene ZmHsp10 is shown in SEQ ID No. 1; the coded amino acid sequence is shown as SEQ ID No. 2; the stress resistance refers to drought resistance and heat resistance.
2. The use of claim 1, wherein: the plant is a crop or a turf grass.
3. Use according to claim 2, characterized in that: the crop is corn or sorghum, and the turf grass is bluegrass or ryegrass.
4. The application of the maize 10kDa heat shock protein gene ZmHsp10 in breeding a new maize drought-tolerant strain with the yield remarkably higher than that of a control group after drought stress by using a transgenic overexpression technology.
5. The application of the corn 10kDa heat shock protein gene ZmHsp10 in cultivating the new heat-resistant Poa annua strain with the survival rate and the leaf color obviously superior to those of the wild type after suffering from heat stress by using a transgenic overexpression technology.
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