CN115948417A - Barley HvFRF1 gene, protein, expression vector and application - Google Patents
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses a barley HvFRF1 gene, a protein, an expression vector and application. The nucleotide sequence of the barley HvFRF1 gene is shown in SEQ ID NO. 1. According to the protein coded by the barley HvFRF1 gene, the amino acid sequence of the protein is shown as SEQ ID NO. 2. The invention also provides application of the barley HvFRF1 gene in screening arabidopsis thaliana varieties and barley varieties by regulating and controlling the drought resistance of the arabidopsis thaliana and the barley. The invention verifies the function of the gene on arabidopsis thaliana and barley by cloning and analyzing the barley HvFRF1 gene and combining with arabidopsis thaliana and barley transgenic technology. The over-expression of the HvFRF1 gene is found to be capable of obviously improving the tolerance of arabidopsis thaliana and barley plants to drought stress. The invention provides theoretical basis and related genes for drought-resistant breeding and production of barley.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a barley HvFRF1 gene, a protein, an expression vector and application.
Background
Drought is the most prominent climate disaster affecting human production and crop growth, with high frequency of occurrence, long duration, and wide spread (Mosley et al, 2015 caine et al, 2019). In recent years, with global warming and extreme climate change abnormity, drought is increasingly aggravated, and becomes the most important limiting factor for worldwide food production, and the food safety is seriously threatened. According to the data of an emergency disaster database (EM-DAT, https:// www.emdat.be /), the frequency of drought occurrence accounts for one twentieth of the global natural disasters, and the direct loss caused by the drought accounts for more than one third of the direct loss caused by all the natural disasters. Therefore, the crop drought research is particularly concerned all over the world, and the cultivation of drought-resistant crop varieties to improve the drought resistance of crops has very important practical significance for fully utilizing land and natural resources and promoting agricultural sustainable development.
Barley (Hordeum vulgare l.) is one of the oldest diploid crops in the world, is suitable for being planted in high-altitude, cold, arid, saline-alkaline and barren lands (Carter et al, 2019 kebede et al, 2019), does not compete with staple grain crops for arable land resources, and has great significance in coping with global climate change and guaranteeing regional grain safety. In the last five years, the annual planting area of the barley in China is basically stabilized at 90-100 million hectares, the total yield is 450-500 million tons, the annual consumption of the barley is nearly 1500 million tons, and the annual maximum import quantity exceeds 1000 million tons (FAOSTAT, http:// FAOSTAT. Fao. Org /). The barley has wide application, and integrates various functions of eating, feeding, brewing, medicinal use and the like. Because the barley is rich in nutrition and has health care effect, the barley becomes an ideal raw material for processing popular, economical, convenient and dietetic food. Therefore, the method for mining the barley drought-resistant germplasm and the key drought-resistant gene to determine the drought-resistant mechanism has important significance for developing a new way for resisting drought stress and improving the drought-resistant capability and yield of crops.
FRS is a new class of transcription factors produced by "molecular acclimation" of one or more nonfunctional MULE transposons (Lin et al, 2007), whose family members regulate various cellular processes by activating or inhibiting the expression of different target genes through transcription, and exert important functions in plant growth and development and stress tolerance processes (Ouyang et al, 2011 li et al, 2016 ritter et al, 2017.
Through the above analysis, the problems and defects of the prior art are as follows:
FRF is a class of truncated FRS that competes with FRS for DNA binding sites to regulate expression of target genes. However, the research on such genes has mainly focused on arabidopsis thaliana, and the prior art on FRF genes of crops has been reported. Can not provide theoretical basis for drought-resistant breeding and production of barley; therefore, the identification of the barley FRF gene and the exploration of the function of regulating and controlling the drought stress of the barley FRF gene have important significance for improving the drought stress resistance of crops and the genetic improvement of the crops.
Disclosure of Invention
In order to overcome the problems in the related art, the disclosed embodiment of the invention provides a barley HvFRF1 gene, a protein, an expression vector and application. In particular to a barley HvFRF1 gene and application thereof.
The technical scheme is as follows: a barley HvFRF1 gene, the nucleotide sequence of the barley HvFRF1 gene is shown in SEQ ID NO. 1.
In one example, the barley HvFRF1 gene is a gene cloned from barley.
The invention also aims to provide a protein coded by the barley HvFRF1 gene, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
The invention also aims to provide an arabidopsis thaliana overexpression vector super1300 HvFRF1 constructed by homologous recombination according to the barley HvFRF1 gene. The nucleotide sequence is shown as SEQ ID NO. 26.
Another objective of the invention is to provide a barley overexpression vector pBract214 HvFRF1 constructed by the Gateway method based on the barley HvFRF1 gene. The nucleotide sequence is shown as SEQ ID NO. 29.
The invention also aims to provide application of the barley HvFRF1 gene in obtaining a drought-resistant arabidopsis strain.
The invention also aims to provide application of the barley HvFRF1 gene in obtaining a drought-resistant barley strain.
The invention also aims to provide application of the barley HvFRF1 gene in tissue expression in a barley root system under the induction of drought stress.
The invention also aims to provide the application of the barley HvFRF1 gene in screening arabidopsis thaliana varieties and barley varieties by regulating and controlling the drought resistance of the arabidopsis thaliana and the barley.
By combining all the technical schemes, the invention has the advantages and positive effects that:
first, aiming at the technical problems existing in the prior art and the difficulty in solving the problems, the technical problems to be solved by the technical scheme of the present invention are closely combined with results, data and the like in the research and development process, and some creative technical effects are brought after the problems are solved. The specific description is as follows:
with frequent and increasing drought caused by global warming, drought becomes the most important limiting factor for grain production worldwide, grain safety is seriously threatened, and crop drought resistance research is widely concerned worldwide. The method has the advantages that the drought-resistant related genes of crops are excavated, and drought-resistant crop varieties are cultivated to meet the requirements of global climate change and population growth on drought-resistant crops, and the method has important significance for agricultural sustainable development and guarantee of food safety. However, due to the genetic complexity of drought-resistant traits, the method has become an important bottleneck for analyzing the drought-resistant genetic basis of crops and cultivating breakthrough strong-resistance (drought-resistant) dry crop varieties. The barley is the fourth largest cereal crop in the world, is suitable for being planted in arid, saline-alkaline, barren and other lands, and is an important material for excavating excellent drought-resistant genes. The barley HvFRF1 gene is cloned and analyzed, and the gene is subjected to functional verification by combining an arabidopsis thaliana and barley transgenic overexpression technology, and the result shows that the drought resistance of the arabidopsis thaliana and barley can be remarkably improved by overexpressing the HvFRF1 gene. The invention can provide excellent gene resources for the drought-resistant genetic improvement of barley and other crops, innovates and enriches the drought-resistant genetic regulation theory of crops, and has important guiding significance for breeding new varieties of good drought-resistant crops.
Secondly, regarding the technical solution as a whole or from the perspective of products, the technical effects and advantages of the technical solution to be protected by the present invention are specifically described as follows:
the invention discloses a barley HvFRF1 gene and application thereof in regulating and controlling the tolerance of arabidopsis thaliana and barley to drought stress, wherein the CDS region nucleotide sequence of the barley HvFRF1 gene is shown as SEQ ID No. 1. The over-expression of the HvFRF1 gene is found to be capable of obviously improving the tolerance of arabidopsis thaliana and barley plants to drought stress. The invention provides theoretical basis and related genes for drought-resistant breeding and production of barley.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and together with the description, serve to explain the principles of the disclosure.
FIG. 1 is a flow chart of a super1300 HvFRF1 vector construction method provided by the invention embodiment;
FIG. 2A is a HvFRF1 domain and conserved sequence analysis predicted result of HvFRF1 domain according to the present invention, which contains a typical FAR1 domain map;
fig. 2B is a diagram showing the comparison result of barley HvFRF1 with arabidopsis thaliana AtFRF1, atFRF2, atFRF3, and AtFRF4 protein sequences in the analysis of the HvFRF1 functional domain and the conserved sequence provided in the embodiment of the present invention, where the box marks the position of the FAR1 functional domain, and the triangle marks conserved amino acids in the FAR1 functional domain;
fig. 3A is a phylogenetic tree analysis diagram of the HvFRF1 and homologous proteins in arabidopsis thaliana, rice and wheat in the evolutionary analysis of the HvFRF1 gene and the analysis of the expression in response to drought stress provided by the embodiments of the present invention, the gene has a close relationship with the FRF members in arabidopsis thaliana and rice;
FIG. 3B is a diagram showing the expression of the HvFRF1 gene after 7 days and 14 days of drought stress in the evolution analysis of the HvFRF1 gene and the expression analysis in response to drought stress provided in the embodiments of the present invention, wherein the greater the induced up-regulation expression multiple of the HvFRF1 gene is with the extension of the drought stress time;
FIG. 4A is a graph showing the expression of the HvHRF1 gene at the seedling stage and the tillering stage of barley by qRT-PCR analysis in the expression analysis of the HvHRF1 gene provided in the examples of the present invention, which shows a higher expression level in barley roots;
FIG. 4B is a diagram showing the localization of the HvFRF1 gene at the subcellular level in Arabidopsis protoplasts in the expression analysis of the HvHRF1 gene provided in the present invention, where the gene is mainly localized in the nucleus and red fluorescence is a nuclear localization marker; bar =10 μm;
FIG. 4C is a diagram showing the expression position of the HvHRF1 gene in barley roots and leaves, which is highly expressed mainly in the vascular bundles of epidermal cells of roots, roots and leaves, by in situ PCR analysis in the expression analysis of the HvHRF1 gene provided in the example of the present invention; e, epidermis; c, cortix, cortical parenchyma cells; en, endodermis, inner skin layer; x, xylem; xp, xylem parenchyma; p, phylem, phloem; bs, bundle shear; a vascular bundle sheath; xv, xylem vessel; pv, phloem vessel, phloem catheter; ms, mesophyll; bar =50 μm;
FIG. 5A is a schematic diagram of the construction of an Arabidopsis expression vector provided by the embodiment of the present invention and a schematic diagram of the construction of an Arabidopsis overexpression vector super1300: hvFRF1 in the seedling stage phenotypes of HvFRF1 transgenic Arabidopsis lines and WT under normal watering and drought treatment conditions; the super promoter drives HvFRF1 gene, the 35S promoter drives Hygromycin (Hygromycin) resistance gene, and NOS represents a terminator;
FIG. 5B is a schematic diagram of the construction of an Arabidopsis expression vector and the drought-resistant phenotype of over-expressing Arabidopsis lines OE1, OE2, OE3 and WT after drought treatment in the seedling-stage phenotypes of HvFRF1 transgenic Arabidopsis lines and WT under normal watering and drought treatment conditions, provided by an embodiment of the invention;
FIG. 5C is a schematic diagram of the construction of an Arabidopsis expression vector provided in the example of the present invention and a diagram of the MDA content of over-expressed Arabidopsis lines OE1, OE2, OE3 and WT after drought treatment in the seedling stage phenotypes of HvFRF1 transgenic Arabidopsis lines and WT under normal watering and drought treatment conditions;
FIG. 5D is a schematic diagram of an Arabidopsis expression vector construction provided by an embodiment of the present invention and a proline content map of over-expressed Arabidopsis strains OE1, OE2, OE3 and WT after drought treatment in the seedling stage phenotypes of HvFRF1 transgenic Arabidopsis strains and WT under normal watering and drought treatment conditions;
FIG. 6A is a schematic diagram showing the construction of a barley overexpression vector according to the embodiment of the present invention and the construction of a barley overexpression vector pBract214: hvFRF1 in a seedling stage phenotype of transgenic barley lines and WT under normal watering and drought treatment; the 35S promoter drives resistance gene Hygromycin (Hygromycin), the corn Ubi promoter (ZmUbi) drives HvFRF1 gene, attR1 and attR2 are LR reaction exchange positions in the Gateway method, and NOS represents a terminator;
FIG. 6B is a schematic diagram of the construction of a barley overexpression vector according to an embodiment of the present invention and the drought-resistant phenotype of the overexpressed barley lines OE1, OE2 and WT after drought treatment among the seedling-stage phenotypes of the transgenic barley lines and WT under normal watering and drought treatment;
FIG. 6C is a schematic diagram showing the construction of a barley overexpression vector according to an embodiment of the present invention and the expression amounts of the HvFRF1 genes in the overexpressed barley lines OE1, OE2 and WT in the seedling stage phenotypes of the transgenic barley lines and WT under normal watering and drought treatment;
FIG. 6D is a schematic diagram of construction of a barley overexpression vector according to an embodiment of the present invention and the dry weight of the aerial parts of overexpression barley lines OE1, OE2 and WT after drought treatment in seedling stage phenotypes of transgenic barley lines and WT under normal watering and drought treatment.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, embodiments accompanying figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
1. Illustrative examples are illustrated:
the embodiment of the invention provides a gene with drought resistance cloned from barley, and provides a related gene and a theoretical basis for cultivating a drought-resistant barley variety.
The embodiment of the invention also provides application of the barley HvFRF1 gene in screening arabidopsis thaliana varieties and barley varieties in regulating and controlling the drought resistance of arabidopsis thaliana and barley, wherein the CDS region nucleotide sequence of the barley HvFRF1 gene is shown as SEQ ID No.1, and the amino acid sequence coded by the HvFRF1 gene is shown as SEQ ID No. 2.
Wherein the correlation sequences comprise:
CDS nucleotide sequence SEQ ID NO.1: ATGCCGGAGGTGATGTCCACGCCACAGCAACCCACAGCCATGATGAGATTTGACACTCTTGAGGATGCTGAGAAACACTACAAACTGTATGCGAGGCAGAAAGGTTTTGGAGTAAGGTATTGTTTCCGAAAAAGGTCAGAGGCTAGTGGTGAACTGATAAGAGCATCACTTGTCTGCCATAGAGCTGGGTTGAAGATCAAGAGGAAAGTAGACACCCAAAACCCGCAACCTATTGCCCCTGAGAGGAGTAGGAATACAACTGAAAGAACAAACTGCCCAGCCCGTATGTTTGTGAAGCGAAGAGATAATGCCTGGGTTGTAACAGAAATAAATGACAACCATAACCACCCTCTCATAAAGAAATGGTCGCTGACAGGATACCTACGATCACATAGACATATCCCTGAAGAAGAACAACAGTTTGTGAAGTTGCTTCATTCGTGCAATCTAGAACCTTCTAGACAGATGCAGTTGTTGACAGAGTTGCATGGCCAACGTGAAGCCATTGGTTACACTGACAAAGACTTGGCAAATTTGCTAGCAAAGTTCCGGGCTGAGCACAAATACACTGATATGCAGGACACGATTGAGTACTTCAAAAGCAGTCAACAGCTTGATAAAGACTTTTTTTACAAGTACAAGCTTGATGATGAAAATAAGGTCCAATGCATTTACTGGATTGATGGTTCAGCGAGAAGGGCTTACAAGTTTTTCAGTGATTGTGTCTCTTTTGACACAACATACTTGACCAATATATACAAGATGCCTTGTGCCCGTTTATTGGTATAA.
Amino acid sequence SEQ ID NO.2: MPEVMSTPQQPTAMMRFDTLEDAEKHYKLYARQKGFGVRYCFRKRSEASGELIRASLVCHRAGLKIKRKVDTQNPQPIAPERSRNTTERTNCPARMFVKRRDNAWVVTEINDNHNHPLIKKWSLTGYLRSHRHIPEEEQQFVKLLHSCNLEPSRQMQLLTELHGQREAIGYTDKDLANLLAKFRAEHKYTDMQDTIEYFKSSQQLDKDFFYKYKLDDENKVQCIYWIDGSARRAYKFFSDCVSFDTTYLTNIYKMPCARLLV.
The embodiment of the invention also provides an overexpression vector super1300 of HvFRF1 and pBract214 of HvFRF1.
Wherein, super1300 is an arabidopsis thaliana overexpression vector stored in the laboratory, and pBract214 is a barley overexpression vector provided by Peter Dominy, university of Glasgow, UK. The invention respectively joins the HvFRF1 gene into the two vectors through homologous recombination and Gateway methods, successfully constructs an arabidopsis thaliana overexpression vector super1300 of the HvFRF1 gene (SEQ ID NO. 26) and a barley overexpression vector pBract214 of the HvFRF1 gene (SEQ ID NO. 29), and can respectively verify the functions of the HvFRF1 gene on arabidopsis thaliana and barley.
As shown in fig. 1, an embodiment of the present invention provides a super1300: hvFRF1 vector construction method, including the following steps:
s101, designing a pair of primers HvFRF1-OE-F1 and HvFRF1-OE-R1 to amplify ORF fragments of the HvFRF1 gene by using a preserved pEASY-HvFRF 1 plasmid as a template; the primer sequence is as follows: SEQ ID NO.15, SEQ ID NO.16;
s102, adopt
The HD Cloning Kit homologous recombination Kit (Takara, japan) carries out homologous recombination reaction on the ORF fragment recovered and purified from the glue and the superl300 vector (stored in the laboratory); converting the reaction product into escherichia coli DH5 alpha, sending positive clones to a company for sequencing, and storing single-clone quality-improved particles with correct sequencing, wherein the obtained plasmids are superl300: hvFRF1;
s103, transforming the plasmid into agrobacterium-infected cells EHA105, coating a rifampicin + kanamycin-resistant YEB plate, culturing at 28 ℃ for about 40-48h, and verifying positive clones by PCR; adding the positive clone bacterial liquid into 30% of aseptic glycerol with the same volume, uniformly mixing, quickly freezing by liquid nitrogen, and storing at-80 ℃ for later use.
The embodiment of the invention also provides a pBract214: hvFRF1 vector construction method, which comprises the following steps:
an overexpression vector of the HvFRF1 gene, namely pBract214: hvFRF1, is constructed by adopting a Gateway method. A pair of primers HvFRF1-OE-F2 and HvFRF1-OE-R2 is designed to amplify the ORF fragment of the HvFRF1 gene by using the preserved pEASY: hvFRF1 plasmid as a template. The primer sequence is as follows: SEQ ID NO.21, SEQ ID NO.22;
detecting the PCR amplification product by using 1% agarose gel electrophoresis, carrying out gel recovery and purification on the target product, and measuring the concentration of the recovered product. Known concentrations of gene recovery products were then subjected to BP recombination with pDONR (Zeo) entry vector using Gateway BP clone II enzyme mix (Invitrogen, USA).
Reacting at 25 ℃ for 4h, adding 1 mu L of protease K, continuing to react at 37 ℃ for 10min, immediately converting a reaction product into escherichia coli DH5 alpha competent cells, coating a bleomycin resistant LB solid culture medium, culturing at 37 ℃ for about 16h, selecting monoclonals, shaking bacteria for carrying out bacteria liquid PCR, sending the bacteria liquid of positive clones to a company for sequencing, carrying out amplification on the monoclonals with correct sequencing, preserving the bacteria liquid glycerol and preserving quality-improved particles, wherein the plasmid is named pDONR (Zeo): hvFRF1.
The known concentrations of the above plasmids were subjected to LR recombination with the target vector pBract214 (supplied by Dr. Peter Dominy, university of Glassy, england) using Gateway LR clone II enzyme mix (Invitrogen, USA).
Reacting at 25 ℃ for 4h, adding 1 mul of protease K, continuing to react at 37 ℃ for 10min, immediately converting reaction products into escherichia coli DH5 alpha competent cells, coating a kanamycin-resistant LB solid culture medium, culturing at 37 ℃ for about 16h, selecting monoclonals, shaking bacteria, carrying out bacteria liquid PCR, sending the bacteria liquid with a target strip to a company for sequencing, carrying out amplification propagation on the monoclonals with correct sequencing, preserving the glycerol of the bacteria liquid and preserving quality-improved grains, wherein plasmids are named as pBract214 and HvFRF1 respectively;
the correctly sequenced pBract214 HvFRF1 plasmid and pSoup plasmid are jointly transformed into agrobacterium competent cells AGL1, coated with a rifampicin + kanamycin resistant YEB plate, cultured at 28 ℃ for about 40-48h, and positive clones are verified by PCR. Adding 100 μ L of the positive clone bacterial liquid into 10mL of MG culture solution (containing 25 μ g/mL rifampicin, 50 μ g/mL kanamycin, pH = 7.2) for culture, culturing at 28 ℃,180rpm/min, shaking to OD600=0.6-0.7, adding equal volume of 30% sterile glycerol, mixing uniformly, rapidly freezing with liquid nitrogen, and storing at-80 ℃ for later use.
Example 1
In the embodiment of the invention, the gene HvFRF1 with drought resistance is identified and cloned from a barley drought-resistant variety Hindmarsh, which is a drought-resistant high-yield cultivar from Australia.
Cloning and bioinformatics analysis of the HvFRF1 Gene: based on the barley FRS gene family analysis and qRT-PCR analysis, FRF members with high expression under drought treatment were identified, and the full-length CDS region sequence of the gene was cloned from Hindmarsh and named HvFRF1. The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO. 1.
The overall length of CDS region of HvFRF1 gene is 789bp, a protein sequence of 262aa is coded, the molecular weight of the protein is 31kDa, and the isoelectric point PI =9.2. The invention also provides the protein coded by the barley HvFRF1 gene, and the amino acid sequence of the protein is shown as SEQ ID NO. 2. The HvFRF1 protein sequence functional domain prediction analysis shows that the protein contains 1 FAR1 functional domain, therefore, the gene is classified as an FRF member in an FRS gene family. The result of multiple sequence alignment shows that the HvFRF1 protein contains conserved amino acids in the functional domain HMM logo map of FAR 1. The HvFRF1 protein sequence is subjected to evolutionary tree analysis, and the result shows that the protein and 3 FRF members in rice are positioned on the same clade.
Expression analysis of the HvFRF1 Gene: research results show that the HvFRF1 gene is expressed in roots, stems and leaves of the barley, but has the highest expression level in the roots; the gene is obviously up-regulated and expressed under the induction of drought stress. Subcellular localization results show that the HvFRF1 gene is localized in the nucleus; the tissue localization results show that the HvFRF1 gene is mainly expressed in epidermal cells and vascular bundles of root systems and in vascular bundles of leaves, and the gene is related to water absorption and transportation.
Functional identification of the HvFRF1 gene: through a homologous recombination method, the full-length CDS (with a stop codon) of the HvFRF1 gene is connected into an overexpression vector super1300, an arabidopsis thaliana overexpression vector super1300 of the HvFRF1 gene is constructed, hvFRF1 is used for transforming arabidopsis thaliana Columbia, and a stable transgenic strain is obtained. The drought resistance of the HvFRF1 transgenic arabidopsis strain under the drought treatment condition is obviously stronger than that of the wild type.
Under the drought treatment condition, the content of Malondialdehyde (MDA) in the HvFRF1 transgenic arabidopsis thaliana strain is obviously lower than that of a wild type, and the content of proline is obviously higher than that of the wild type, so that the HvFRF1 gene overexpression arabidopsis thaliana can enhance the drought resistance of the plant by reducing the membrane lipid peroxidation degree and accumulating the proline as an osmotic adjusting substance. The full-length CDS (with a stop codon) of the HvFRF1 gene is connected into an overexpression vector pBract214 by a Gateway method, a barley overexpression vector pBract214 of the HvFRF1 gene is constructed, hvFRF1 is used for transforming barley Golden protein, and a stable transgenic line is obtained.
The drought resistance of the HvFRF1 transgenic barley strain under the drought treatment condition is obviously stronger than that of a wild type, and the dry weight of the overground part of the HvFRF1 transgenic barley strain under the drought treatment condition is obviously higher than that of the wild type, so that higher biomass can be kept.
Example 2
The invention uses Australia drought-resistant and high-yield cultivar Hindmarsh as main material, clones and analyzes key genes for regulating and controlling the drought resistance of barley, and has important guiding significance for clarifying the molecular mechanism of barley responding to drought stress and the work of barley drought-resistant breeding and production.
In the present example, cloning and analysis of the CDS region of the HvFRF1 gene included:
(1) Cloning of CDS region sequence of HvFRF1 Gene
Based on the results of barley FRS gene family analysis and qRT-PCR expression analysis, an FRF member responding to drought stress was identified, and the full-length CDS region sequence of the gene (shown in SEQ ID No. 1) was cloned from Hindmarsh and named HvFRF1.
Total RNA from Hindmarsh leaves was extracted using SteadyPure plant RNA extraction Kit (Ai Kerui, china) and genomic DNA contamination of total RNA was removed with DNaseI, and the extracted total RNA was reverse transcribed into single stranded cDNA using Evo M-MLV Plus 1st Strand cDNA Synthesis Kit reverse transcription Kit (Ai Kerui, china). Designing a specific primer according to the sequence obtained by Blast, wherein the specific primer sequence is as follows:
SEQ ID NO.3：HvFRF1-CDS-F：
5'-GGTTTTGTATGTTATGCAGATCGTGAGAC-3'；
SEQ ID NO.4：
HvFRF1-CDS-R：5'-CCAAACTGAATTGACTGTCCATGGTTG-3'；
the CDS region of the gene was amplified using KOD OneTM PCR Master Mix-Blue (Toyo Boseki, japan). And (3) PCR system:
PCR procedure: first, pre-denaturation at 94 ℃ for 2min, then denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 5s, and extension at 68 for 5s, wherein the denaturation annealing and extension are circulated for 35 times, and then extension at 68 for 5min. Connecting the amplified product to pEASY (all-gold, china) vector, transforming Escherichia coli DH5 alpha, selecting positive clone, sending the positive clone to company for sequencing, extracting plasmid and preserving glycerol respectively when the sequencing is correct, and the obtained plasmid is named as pEASY: hvFRF1 plasmid. PCR primer synthesis and gene sequencing were performed by Biotech, inc., of Beijing Optimalaceae.
(2) Sequence analysis of HvFRF1 Gene
The HvFRF1 protein sequence was subjected to domain prediction and analysis by SMART (https:// SMART. Embl. De /) website, and the result showed that the protein contained a FAR1 domain (FIG. 2A). The HvFRF1 is FAR1-RELATED SEQUENCES-RELATED FACTOR (FRF). The HvFRF1 protein sequence and AtFRF1, atFRF2, atFRF3 and AtFRF4 protein sequences of Arabidopsis thaliana are subjected to conservative domain analysis, and the result shows that the protein sequences all contain the conserved amino acid of FAR1 (figure 2B), the same conserved amino acid can be displayed through an HMM logo picture, and the larger the letter is, the more the amino acid is conserved.
The FHY and FAR1 in Arabidopsis containing the HvFRF1 protein sequence, 12 FRSs, 4 FRFs, 1 FRS in rice, 3 FRFs and 1 FRS in wheat were constructed into phylogenetic trees by Clustal W and MEGA 7 software, and the result shows that the HvFRF1 indeed belongs to the FRF subfamily and is evolutionarily located in the same evolutionary branch as the FRFs of rice and Arabidopsis (FIG. 3A).
HvFRF1 Gene expression in response to drought stress
Selecting the barley Hindmarsh seeds with consistent plumpness, sowing the seeds in 5L plastic culture pots filled with equal amount of nutrient soil, sowing 6 seeds in each pot, watering equal amount of nutrient solution in each pot before sowing, fixing seedlings in each pot for 4 plants 7 days after germination, and watering equal amount in each pot in the period. Carrying out drought treatment in the two-leaf one-heart period, and setting two treatments: in contrast, each pot is watered normally in equal amount in the whole process, and the water content of the soil is kept to be 30-40%; drought treatment, stopping watering and subjecting the seedlings to drought stress. Functional leaves of control and drought-treated plants were taken 7 days and 14 days after the cessation of watering for HvFRF1 gene response drought stress expression analysis, respectively.
Total RNAs of each sample are respectively extracted by using a SteadyPure plant RNA extraction Kit (Ai Kerui, china), and are respectively reversely transcribed into single-stranded cDNAs by using an Evo M-MLV RT Kit with gDNA Clean for qPCR II reverse transcription Kit (Ai Kerui, china). The expression of HvFRF1 gene in the corresponding sample was subjected to qRT-PCR analysis using SYBR Green Fluoroscetase Complex (Ai Kerui, china) and CFX Connect Real-Time System PCR instrument (Bio-Red, USA), and the expression value was corrected using an internal reference Gene, actin. The PCR system is as follows:
the PCR procedure was: first pre-denatured at 94 ℃ for 30s, then denatured at 95 ℃ for 5s, and extended at 60 ℃ for 30s, with 40 cycles of denaturing extension. The dissolution curve program was: the reaction is carried out for 5s at 60-95 ℃ in each step, and the reaction is gradually increased according to 0.5 ℃. By use of 2 -ΔΔCq The gene expression value change was calculated by a relative quantification method. Each set of experiments was repeated three times. The qRT-PCR primer sequence is:
SEQ ID NO.5：HvFRF1-qRT-PCR-F：5'-CCCAAAACCCGCAACCTATTG-3'；
SEQ ID NO.6：HvFRF1-qRT-PCR-R：5'-GTAGGTATCCTGTCAGCGACC-3'；
SEQ ID NO.7：Actin-F：5'-TCGCTCCACCTGAGAGGAAG-3'；
SEQ ID NO.8：Actin-R：5'-GCTAGGATGGACCCTCCGAT-3'；
the qRT-PCR result shows that the HvFRF1 gene is up-regulated by drought induction, and the induction up-regulation expression multiple is higher along with the enhancement of the drought stress degree (figure 3B), which indicates that the HvFRF1 gene responds to the drought stress.
(3) Subcellular localization of HvFRF1 Gene
A pair of primers HvFRF1-super promoter-GFP-F and HvFRF1-super promoter-GFP-R is designed by using the preserved pEASY: hvFRF1 plasmid as a template to amplify the ORF fragment of the HvFRF1 gene (removing the termination codon TAA) for subcellular localization analysis. The primer sequences are (the underlined sections are enzyme sites):
SEQ ID NO.9：HvFRF1-super promoter-GFP-F：5'-CCAAATCGACTCTAGTCTAGAATGCCGGAGGTGATGTCCA-3'；
SEQ ID NO.10：HvFRF1-super promoter-GFP-R：5'-GCCCTTGCTCACCATGGTACCTACCAATAAACGGGCACAAGG-3'；
by using
The HD Cloning Kit homologous recombination Kit (Takara, japan) performed homologous recombination reaction of the ORF fragment purified by gel recovery with super1300-super promoter-GFP vector. And (3) transforming the reaction product into escherichia coli DH5 alpha, sending the positive clone to a company for sequencing, preserving the single clone with correct sequencing (ensuring no frame shift and being capable of translating into complete gene-GFP fusion expression protein) to obtain the quality-improved particle, and obtaining the plasmid which is super promoter-HvFRF1-GFP (SEQ ID NO. 27).
Leaves of 3-4 weeks old Arabidopsis thaliana were taken and placed in a 50ml flask, 5-10ml of an enzymatic hydrolysate (1.5% Cellulase R10,0.75% Macerozyme R10,0.6M Mannitol,10mM MES,40% 2-Hydroxy-1-ethano) was added to completely soak the leaves, the leaves were covered with a sealing film after evacuation, and subjected to weak light enzymatic hydrolysis at 23 ℃ for 3 hours. Filtering the enzymolysis product with 40 μm nylon gauze, collecting protoplast into 2ml centrifuge tube, centrifuging at 4 deg.C and 400rpm/min for 5min to collect precipitate, and adding pre-cooled W5 solution (154mM NaCl,125mM CaCl) 2 ，2mM KH 2 PO 4 2mM MES) was washed 2 times, resuspended and then allowed to stand on ice for 30min. The suspension after standing was centrifuged at 400rpm/min for 5min and MMG solution (0.4M Mannitol,15mM MgCl) 2 .6H 2 O,4mM MES), and adding super promoter-HvFRF1-GFP plasmid, nucleus localization marker plasmid and 40% PEG4000 solution as required, slowly reversing and mixing, and water bathing at 22.5 ℃ for 15-20min. Adding a volume of W5 solutionThe protoplasts were diluted, the reaction was stopped, centrifuged at 400rpm/min for 5min, washed 2 times with W5 solution and resuspended, and incubated overnight at 23 ℃ with low light. The overnight cultured protoplast suspension was centrifuged at 400rpm/min for 5min to collect protoplasts, and the fluorescence distribution was observed under a laser confocal microscope (LSM 780, carl Zeiss, germany).
The results showed that the HvFRF1 gene was localized in the nucleus, overlapping with the signal of the nuclear localization marker (FIG. 4B).
(4) Tissue localization of HvFRF1 Gene
Hindmarsh seed with 2% H 2 O 2 Sterilizing for 30min, washing with distilled water for 5-6 times, placing sterilized seeds in a germination box, culturing in dark (22 deg.C/18 deg.C) in a growth chamber until germination, and supplementing light (22 deg.C/18 deg.C, day/night) after germination. At 7d, seedlings with consistent growth vigor are selected and transferred to a 5L black plastic bucket containing barley basic culture solution (1/5 Hogland formula, PH = 5.8-6.0), covered by a 9-hole plastic cover, 1 seedling in each hole is fixed by using sponge, and cultured in a barley culture chamber, aeration is carried out by using an oxygenation pump in the whole culture process, and the culture solution is replaced every 5 days. And (3) taking the root, stem and leaf parts of the plant in the three-leaf stage and the tillering stage respectively to perform tissue expression analysis of the HvFRF1 gene. RNA extraction, reverse transcription, primer sequences, a qRT-PCR system and a program are the same as the expression condition of 3.HvFRF1 gene response drought stress.
The results showed that the HvFRF1 gene was expressed in the roots, stems, and leaves of the plants, but was expressed in higher amounts in the roots (FIG. 4A).
Three-leaf stage hydroponic Hindmarsh roots and leaves were harvested, tissue fragments of about 1 cm were cut, immediately placed in fresh formaldehyde fixing solution (63% Ethanol,5% acetic acid,2% formaldehyde) and evacuated. Wash Buffer 1 (63% Ethanol,5% glacial acetic acid) was washed three times and leaves and roots were embedded with 5% agarose and 5% low melting agarose, respectively. The embedded tissues were sectioned (50-80 μm) using a shaking microtome, and each of the sectioned tissue samples was placed in a PCR tube containing an RNase inhibitor (Takara, japan) and digested with RNase free DNase I (Takara, japan) overnight at 37 ℃. Using PrimeScript TM 1st Strand cDNA Synthesis Kit (Takara, japan) and In situ HvFRF1-R primers were reverse transcribed and used for In situ PCR studies, with the 18S internal reference gene as a positive control and no primer added during reverse transcription as a negative control. The In situ PCR primer sequence is:
SEQ ID NO.11：In situ HvFRF1-F：
5'-AACACTACAAACTGTATGCG-3'；
SEQ ID NO.12：In situ HvFRF1-R：
5'-GTTCTAGATTGCACGAATGA-3'；
SEQ ID NO.13：In situ Hv18S-F：
5'-GGTAATTCCAGCTCCAAT-3'；
SEQ ID NO.14：In situ Hv18S-R：
5'-GTTTATGGTTGAGACTAG-3'；
using each tissue section as a template, the gene was amplified using dUTP (Roche, germany) containing digoxin marker, PCR system:
PCR procedure: first, pre-denaturation at 95 ℃ for 2min, then denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 5s, and extension at 68 for 5s, wherein the denaturation annealing and extension are circulated for 30 times, and then extension at 68 for 5min. Tissue sections after PCR were washed with 1 XPBS buffer (10 mM Na) 2 HPO 4 130mM NaCl, pH = 7.5.) was washed twice, and 1 XBock solution (10 mg BSA dissolved in 1mL 10xPBS) was added and ice-cooled for 30min. Adding Anti-DIG-AP antibody (Roche, germany), incubating at 37 ℃ for 1h, adding BM-pure (Roche, germany) for color reaction, washing, fixing with 40% (v/v) glycerol, spreading on a glass slide, and observing tissue staining condition of HvFRF1 gene under a body type microscope.
The results show that blue color appears mainly in the vascular bundles of epidermal cells of roots, roots and leaves, indicating that the HvFRF1 gene is mainly expressed in these tissue parts, and the epidermal cells and vascular bundles are closely related to water absorption and transportation of plants, indicating that the expression of the HvFRF1 gene is beneficial to the absorption and utilization of water by barley.
Example 3
In the embodiment of the invention, the functional verification of the HvFRF1 gene comprises the following steps:
(1) super1300 HvFRF1 vector construction
A pair of primers HvFRF1-OE-F1 and HvFRF1-OE-R1 are designed to amplify the ORF fragment of the HvFRF1 gene by using the preserved pEASY: hvFRF1 plasmid as a template. The primer sequence is as follows:
SEQ ID NO.15：HvFRF1-OE-F1：5'-CGACTCTAGTCTAGAAAGCTTATGCCGGAGGTGATGTCC-3'；
SEQ ID NO.16：HvFRF1-OE-R1：5'-CGATCGGGGAAATTCGAGCTCTTATACCAATAAACGGGC-3'；
by using
The HD Cloning Kit homologous recombination Kit (Takara, japan) performed homologous recombination reaction of the ORF fragment purified by gel recovery with superl300 vector (stored in this laboratory). The reaction product is transformed into Escherichia coli DH5 alpha, the positive clone is sent to the company for sequencing, the single clone with correct sequencing is upgraded grains for storage, and the obtained plasmid is superl300: hvFRF1 (figure 5A).
Transforming the plasmid into agrobacterium competent cells EHA105, coating a rifampicin + kanamycin resistant YEB plate, culturing at 28 ℃ for about 40-48h, and verifying positive clones by PCR. Adding the positive clone bacterial liquid into 30% of aseptic glycerol with the same volume, uniformly mixing, quickly freezing by liquid nitrogen, and storing at-80 ℃ for later use.
(2) Arabidopsis genetic transformation and positive plant validation:
agrobacterium containing the super1300: hvFRF1 plasmid was inoculated into 5ml of liquid LB medium containing rifampicin + kanamycin antibiotic, and shaken overnight at 220rpm/min under dark culture conditions at 28 ℃ (about 18 h). Taking 1ml of overnight bacteria according to the proportion of 1. The collected cells were resuspended with the transformant permeate (1/2MS, 5% sucrose, 20% silwet L-77,0.05% MES,1% AS,1%6 BA) to an OD600 of 0.8-1.0. Soaking the bud part of wild Arabidopsis thaliana (Columbia type) in soil culture flowering period in the above penetrating fluid for 1-2h. After infection, the plants were placed upside down in wet trays, placed in dark for 48 hours in the dark, and then cultured upright (23 ℃,16/8h, day/night). And finally, collecting seeds as many as possible, wherein the collected seeds are T1 generation. After the T1 generation seeds are disinfected, a large number of plates are paved on a flat plate containing 25mg/L hygromycin, and seedlings with good and robust root systems are selected and transferred to a nutrition pot (nutrient soil: vermiculite: pearl salt = 6. Extracting leaf DNA before bolting to carry out positive plant verification, and taking superl300: hvFRF1 plasmid as a positive control and wild type arabidopsis thaliana as a negative control. And (3) extracting leaf RNA from the positive plant, carrying out reverse transcription, carrying out expression quantity analysis, and taking Actin of arabidopsis thaliana as an internal reference gene. The positive plant verification primer and the expression analysis primer have the sequences as follows:
SEQ ID NO.17：super1300-F：
5'-GCTCGCGGTGACGCCATTTCGCCTTTTC-3'；
SEQ ID NO.18：HvFRF1-R1：
5'-GTGTAACCAATGGCTTCACGTTGGCCATG-3'；
SEQ ID NO.19：AtActin-F：
5'-AGGAAGGATCTGTACGGTAAC-3'；
SEQ ID NO.20：AtActin-R：
5'-TTCTGTGAACGATTCCTGGAC-3'；
and selecting a positive plant with high HvFRF1 gene expression quantity to harvest seeds to obtain T2 seeds. And (3) spreading the T2 seeds on a plate containing 25mg/L hygromycin, recording resistance expression according to the seedling rate, and reserving strains of which positive seedlings and negative seedlings accord with the segregation ratio of 3:1 for use. Selecting healthy and strong seedlings from a culture dish of a strain for the T2 generation, transferring the healthy and strong seedlings to a nutrition pot for continuous culture, and harvesting T3 seeds from a single plant after maturation. And (3) taking 100 seeds of the T3 generation strain respectively, paving the seeds on a plate containing 25mg/L hygromycin, recording resistance expression according to the seedling rate, and reserving the strain according with 95.
(3) Drought resistance phenotype identification of HvFRF1 gene overexpression Arabidopsis strains:
seeds of wild arabidopsis thaliana and over-expression strains OE1, OE2 and OE3 are taken and disinfected with 75% alcohol for 2min, then 10% sodium hypochlorite is added for disinfection for 5min, and the seeds are washed with sterilized water for 4-5 times, and each time lasts for 2min. The sterilized seeds were spread evenly on a 1/2MS solid medium (containing 1.5% sucrose, 0.4% plant gel, pH 5.8) and excess water on the surface of the medium was aspirated. After vernalization for 3 days at 4 ℃, the 1/2MS culture dish with the sowed seeds is placed in an arabidopsis culture room for culture (23 ℃,16/8h, day/dark), and is transplanted in a nutrition pot in a four-leaf period. Carrying out drought treatment 15 days after transplanting, and setting two treatments: in contrast, in the whole process, each pot is watered normally and equivalently, and the water content of the soil is kept at 30-40%; drought treatment, stopping watering and subjecting the seedlings to drought stress. Drought-resistant phenotype observation is carried out on the plants on the 12 th day after watering is stopped, and the functional leaves of the control plants and the drought-treated plants are taken for physiological index measurement.
The results showed that under the control conditions, there was no significant difference in the growth vigor of wild type and overexpressed arabidopsis thaliana, but the growth vigor of the overexpressed plants was significantly better than that of the wild type after drought stress, showing a smaller degree of wilting (fig. 5B). After drought treatment, the MDA content of wild-type arabidopsis thaliana was significantly increased, while the MDA content in the over-expressed lines was significantly decreased (fig. 5C). In addition, the accumulation of proline in the over-expressed strain was significantly higher than that of the wild type (fig. 5D).
The results show that the expression of the HvFRF1 gene can reduce the peroxidation degree of membrane lipid and accumulate proline as an osmoregulation substance to enhance the drought resistance of plants.
Example 4
The embodiment of the invention provides a pBract214: hvFRF1 vector construction method, which comprises the following steps:
an overexpression vector of the HvFRF1 gene, namely pBract214: hvFRF1, is constructed by adopting a Gateway method. A pair of primers HvFRF1-OE-F2 and HvFRF1-OE-R2 is designed to amplify the ORF fragment of the HvFRF1 gene by using the preserved pEASY: hvFRF1 plasmid as a template. The primer sequence is as follows:
SEQ ID NO.21：HvFRF1-OE-F2：
5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGCCGGAGGTGATGTCCACGCC-3'；
SEQ ID NO.22：HvFRF1-OE-R2：
5'-GGGGACCACTTTGTACAAGAAAGCTGGGTGTTATACCAATAAACGGGCACAAG-3'；
detecting the PCR amplification product by using 1% agarose gel electrophoresis, carrying out gel recovery and purification on the target product, and measuring the concentration of the recovered product. The known concentrations of gene recovery products were then subjected to BP recombination reaction with pDONR (Zeo) entry vector using Gateway BP clone II enzyme mix (Invitrogen, USA). BP recombination reaction system:
reacting at 25 ℃ for 4h, adding 1 mu L of protease K, continuing to react at 37 ℃ for 10min, immediately converting reaction products into escherichia coli DH5 alpha competent cells, coating a bleomycin resistant LB solid culture medium, culturing at 37 ℃ for about 16h, selecting monoclonals, performing bacterium liquid PCR (polymerase chain reaction) by bacterium liquid shaking, sending the bacterium liquid of positive clones to a company for sequencing, performing amplification and propagation on the monoclonals with correct sequencing, storing the bacterium liquid glycerol and preserving quality-improved grains, wherein the plasmid is named as pDONR (Zeo): hvFRF1.
The above-mentioned plasmids of known concentration were subjected to LR recombination reaction with a target vector pBract214 (SEQ ID NO. 28) (supplied by Dr. Peter Dominy, university of Glasgow, england) using Gateway LR clone II enzyme mix (Invitrogen, USA). LR recombination reaction system:
reacting for 4h at 25 ℃, adding 1 mul of protease K, continuing to react for 10min at 37 ℃, immediately converting a reaction product into escherichia coli DH5 alpha competent cells, coating a kanamycin-resistant LB solid culture medium, culturing for about 16h at 37 ℃, selecting a monoclonal antibody, shaking bacteria to perform bacteria liquid PCR, sending the bacteria liquid of a target strip to a company for sequencing, performing amplification propagation on the monoclonal antibody with correct sequencing, storing bacteria liquid glycerol and improving plasmid, and respectively naming plasmids as pBract214: hvFRF1 (figure 6A).
The correctly sequenced pBract214, hvFRF1 plasmid and pSoup plasmid are jointly transformed into Agrobacterium tumefaciens competent cell AGL1, coated with a YEB plate resistant to rifampicin and kanamycin, cultured at 28 ℃ for about 40-48h, and the positive clone is verified by PCR. Adding 100 μ L of the positive clone bacterial liquid into 10mL of MG culture solution (containing 25 μ g/mL rifampicin, 50 μ g/mL kanamycin, pH = 7.2) for culture, culturing at 28 ℃,180rpm/min, shaking to OD600=0.6-0.7, adding equal volume of 30% sterile glycerol, mixing uniformly, rapidly freezing with liquid nitrogen, and storing at-80 ℃ for later use.
Example 5
The embodiment of the invention provides a genetic transformation method of barley immature embryos, which comprises the following steps:
selecting young embryo of Golden hope (Golden Promise) of barley cultivated 2-3 weeks after flowering as genetic transformation material. Peeling off the seeds meeting the standard from the ears, removing the awns, sterilizing the surfaces of the seeds with 70% alcohol for 30s, and washing with sterilized water for several times. Soaking in 10% sodium hypochlorite for 4min, and washing with sterilized water several times. Separating immature embryo on sterile filter paper, removing embryonic axis, placing on callus induction medium with shield plate facing upwards, and culturing in dark at 23-24 deg.C for 2-3d. Adding 400 mu L of the preserved agrobacterium liquid into 10mL of MG liquid culture medium without antibiotics, shaking the bacteria to OD600=1.3-1.4 at 28 ℃ and 180rpm/min, and using the mixture for infection of immature embryos. And dripping the prepared agrobacterium tumefaciens staining solution on each young embryo, and airing. Sealing the plate with sealing film, and co-culturing at 23-24 deg.C in dark for 2 days. Transfer of immature embryos to fresh callus induction medium plates for selection (containing 50mg/L hygromycin, 160mg/L Terminacticin). After culturing at 23-24 ℃ in the dark for 56 days (medium plates were changed every 14 days), the calli isolated from the young embryos were transferred to a transfer medium and cultured at 24 ℃ for 21 days under low light, at which time green spots were produced. The green spot was transferred to subculture medium for further culture, and when the leaves at the upper part of the ground reached 2-3cm, the roots began to build up. The plantlets were transferred to rooting medium without any growth regulator and without antibiotic change. Then taking out the plantlets with the established root systems, washing the culture medium, transferring the plantlets into a plastic culture pot containing vermiculite and nutrient media, and growing in an artificial climate chamber (22 ℃/18 ℃, day/night) until the seeds are harvested.
In the present example, the verification of transgenic plants and the phenotypic identification of HvFRF1 overexpressing barley lines included:
and (3) extracting leaf DNA of a transgenic plant in a seedling stage by adopting a CTAB method, and verifying whether a vector carrying a target fragment is transferred into a barley genome or not by taking wild Golden Promise as a negative control and pBract214-HvFRF1 plasmid as a positive control. Selecting plants with positive DNA verification, extracting leaf RNA, carrying out reverse transcription, verifying the expression level of the HvFRF1 gene by fluorescent quantitative PCR (polymerase chain reaction) by taking the expression level of the HvFRF1 gene in wild Golden Promise as a reference, and selecting two strains OE1 and OE2 with higher expression levels (figure 6C). The qRT-PCR primers for verifying the expression quantity of the positive plants are the HvFRF1-qRT-PCR-F, hvFRF-qRT-PCR-R, actin-F and Actin-R primers in the part of the HvFRF1 gene response drought stress expression condition. The DNA amplification primer sequence for verifying positive plants is as follows:
SEQ ID NO.23：pBract214-HvFRF1-F：5'-ACAAGTTTGTACAAAAAAGCAGGCTTC-3'；
SEQ ID NO.24：pBract214-HvFRF1-R：
5'-TTATACCAATAAACGGGCACAAG-3'；
soil culture drought stress tests are carried out on a wild type Golden Promise and a stable HvFRF1 overexpression strain of barley in a three-leaf period, and the results show that under the control condition, the growth vigor of the wild type and the overexpression strain is not obviously different, but the growth vigor of the overexpression strain after drought stress is obviously better than that of the wild type, shows a smaller wilting degree (figure 6B) and a larger biomass accumulation (figure 6D), and indicate that the HvFRF1 gene is positively used for regulating the drought resistance of the barley.
In conclusion, the HvFRF1 gene of barley is separated, cloned and analyzed, and the gene is functionally verified on barley GP by combining a heterologous overexpression technology and a homologous overexpression technology, and the result shows that the HvFRF1 overexpression obviously enhances the drought resistance of plants. The invention provides theoretical basis and related genes for drought-resistant breeding and production of barley.
The super1300 vector sequence is SEQ ID NO.25, wherein the sequence of the multiple cloning site comprises: TCTAGAAAGCTTCTGCAGGGGCCCGGGGTCGACATTTAAATACTAGTGGATCCGGTACCGAGCTC;
the specific sequence is as follows:
GGATCCCTGAAAGCGACGTTGGATGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTACGTAAGCGCTTACGTTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTTCACCTACGATGGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGGATCCCTGAAAGCGACGTTGTGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTACGTAAGCGCTTACGTTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTACGATGGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGGATCCCTGAAAGCGACGTTGGATGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTACGTAAGCGCTTACGTTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTTCACCTACGATGGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGGATCCGCGAGCTGCTCAATCCCATTGCTTTTGAAGCAGCTCAACATTGATCTCTTTCTCGATCGAGGGAGATTTTTCAAATCAGTGCGCAAGACGTGACGTAAGTATCCGAGTCAGTTTTTATTTTTCTACTAATTTGGTCGTTTATTTCGGCGTGTAGGACATGGCAACCGGGCCTGAATTTCGCGGGTATTCTGTTTCTATTCCAACTTTTTCTTGATCCGCAGCCATTAACGACTTTTGAATAGATACGCTGACACGCCAAGCCTCGCTAGTCAAAAGTGTACCAAACAACGCTTTACAGCAAGAACGGAATGCGCGTGACGCTCGCGGTGACGCCATTTCGCCTTTTCAGAAATGGATAAATAGCCTTGCTTCCTATTATATCTTCCCCCAAATTACCAATACATTACACTAGCATCTGAATTTCATAACCAATCTCGATACACCAAATCGACTCTAGTCTAGAAAGCTTCTGCAGGGGCCCGGGGTCGACATTTAAATACTAGTGGATCCGGTACCGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGCTTTCGCAGATCCGGGGGGGCAATGAGATATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTTAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTACAACCGGTCGCGGAGGCTATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCACGACTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGAAATAGAGTAGATGCCGACCGGATCTGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATCCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTCAATTTGTTTACACCACAATATATCCTGCCACCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCACTCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAAGGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCTGCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGATGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATACTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTTTTCTGGTATTTAAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCTTCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAATGGCTAAAATGAGAATATCACCGGAATTGAAAAAACTGATCGAAAAATACCGCTGCGTAAAAGATACGGAAGGAATGTCTCCTGCTAAGGTATATAAGCTGGTGGGAGAAAATGAAAACCTATATTTAAAAATGACGGACAGCCGGTATAAAGGGACCACCTATGATGTGGAACGGGAAAAGGACATGATGCTATGGCTGGAAGGAAAGCTGCCTGTTCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGCAATCTGCTCATGAGTGAGGCCGATGGCGTCCTTTGCTCGGAAGAGTATGAAGATGAACAAAGCCCTGAAAAGATTATCGAGCTGTATGCGGAGTGCATCAGGCTCTTTCACTCCATCGACATATCGGATTGTCCCTATACGAATAGCTTAGACAGCCGCTTAGCCGAATTGGATTACTTACTGAATAACGATCTGGCCGATGTGGATTGCGAAAACTGGGAAGAAGACACTCCATTTAAAGATCCGCGCGAGCTGTATGATTTTTTAAAGACGGAAAAGCCCGAAGAGGAACTTGTCTTTTCCCACGGCGACCTGGGAGACAGCAACATCTTTGTGAAAGATGGCAAAGTAAGTGGCTTTATTGATCTTGGGAGAAGCGGCAGGGCGGACAAGTGGTATGACATTGCCTTCTGCGTCCGGTCGATCAGGGAGGATATCGGGGAAGAACAGTATGTCGAGCTATTTTTTGACTTACTGGGGATCAAGCCTGATTGGGAGAAAATAAAATATTATATTTTACTGGATGAATTGTTTTAGTACCTAGAATGCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCGCCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTCGGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTAATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATCAGTCACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGGTTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAAAGAGACCTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCCGTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATGACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGTCATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTGCGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAAAAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGCGGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCTCTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGGCGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACCGAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCAGAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCCCTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGTACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCTACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCTTCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCACGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTTCCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGATCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTTGGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGGCCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGTTCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTCATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTGCAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTTGCTCATTTTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGCCAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCAAGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGCGTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGACCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTAAGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACACAGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCCGATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAGCGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGAATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGTTGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTCATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAGCGACCGCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCTCGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACAAACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGAACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGGTTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTCGGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGGGTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCTGGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGGGGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTCGATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCATGCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCCCGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGGGCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTCAAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAAAACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCTGGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGTTCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAAACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAACTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAAGCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGGGGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCACGCCCTTTTAAATATCCGTTATTCTAATAAACGCTCTTTTCTCTTAGGTTTACCCGCCAATATATCCTGTCAAACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAGCTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCC。
super1300, hvFRF1 vector sequence (red is enzyme cutting site, blue is HvFRF1 gene sequence) SEQ ID NO.26, wherein the enzyme cutting site comprises: AAGCTT, GAGCTC; the HvFRF1 gene sequence is as follows: atgccggaggtgatgtccacgccacagcaacccacagccatgatgagatttgacactcttgaggatgctgagaaacactacaaactgtatgcgaggcagaaaggttttggagtaaggtattgtttccgaaaaaggtcagaggctagtggtgaactgataagagcatcacttgtctgccatagagctgggttgaagatcaagaggaaagtagacacccaaaacccgcaacctattgcccctgagaggagtaggaatacaactgaaagaacaaactgcccagcccgtatgtttgtgaagcgaagagataatgcctgggttgtaacagaaataaatgacaaccataaccaccctctcataaagaaatggtcgctgacaggatacctacgatcacatagacatatccctgaagaagaacaacagtttgtgaagttgcttcattcgtgcaatctagaaccttctagacagatgcagttgttgacagagttgcatggccaacgtgaagccattggttacactgacaaagacttggcaaatttgctagcaaagttccgggctgagcacaaatacactgatatgcaggacacgattgagtacttcaaaagcagtcaacagcttgataaagactttttttacaagtacaagcttgatgatgaaaataaggtccaatgcatttactggattgatggttcagcgagaagggcttacaagtttttcagtgattgtgtctcttttgacacaacatacttgaccaatatatacaagatgccttgtgcccgtttattggtataa;
the specific sequence of SEQ ID NO.26 is:
GGATCCCTGAAAGCGACGTTGGATGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTACGTAAGCGCTTACGTTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTTCACCTACGATGGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGGATCCCTGAAAGCGACGTTGTGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTACGTAAGCGCTTACGTTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTACGATGGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGGATCCCTGAAAGCGACGTTGGATGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTACGTAAGCGCTTACGTTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTTCACCTACGATGGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGGATCCGCGAGCTGCTCAATCCCATTGCTTTTGAAGCAGCTCAACATTGATCTCTTTCTCGATCGAGGGAGATTTTTCAAATCAGTGCGCAAGACGTGACGTAAGTATCCGAGTCAGTTTTTATTTTTCTACTAATTTGGTCGTTTATTTCGGCGTGTAGGACATGGCAACCGGGCCTGAATTTCGCGGGTATTCTGTTTCTATTCCAACTTTTTCTTGATCCGCAGCCATTAACGACTTTTGAATAGATACGCTGACACGCCAAGCCTCGCTAGTCAAAAGTGTACCAAACAACGCTTTACAGCAAGAACGGAATGCGCGTGACGCTCGCGGTGACGCCATTTCGCCTTTTCAGAAATGGATAAATAGCCTTGCTTCCTATTATATCTTCCCCCAAATTACCAATACATTACACTAGCATCTGAATTTCATAACCAATCTCGATACACCAAATCGACTCTAGTCTAGAAAGCTTatgccggaggtgatgtccacgccacagcaacccacagccatgatgagatttgacactcttgaggatgctgagaaacactacaaactgtatgcgaggcagaaaggttttggagtaaggtattgtttccgaaaaaggtcagaggctagtggtgaactgataagagcatcacttgtctgccatagagctgggttgaagatcaagaggaaagtagacacccaaaacccgcaacctattgcccctgagaggagtaggaatacaactgaaagaacaaactgcccagcccgtatgtttgtgaagcgaagagataatgcctgggttgtaacagaaataaatgacaaccataaccaccctctcataaagaaatggtcgctgacaggatacctacgatcacatagacatatccctgaagaagaacaacagtttgtgaagttgcttcattcgtgcaatctagaaccttctagacagatgcagttgttgacagagttgcatggccaacgtgaagccattggttacactgacaaagacttggcaaatttgctagcaaagttccgggctgagcacaaatacactgatatgcaggacacgattgagtacttcaaaagcagtcaacagcttgataaagactttttttacaagtacaagcttgatgatgaaaataaggtccaatgcatttactggattgatggttcagcgagaagggcttacaagtttttcagtgattgtgtctcttttgacacaacatacttgaccaatatatacaagatgccttgtgcccgtttattggtataaGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGCTTTCGCAGATCCGGGGGGGCAATGAGATATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTTAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTACAACCGGTCGCGGAGGCTATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCACGACTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGAAATAGAGTAGATGCCGACCGGATCTGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATCCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTCAATTTGTTTACACCACAATATATCCTGCCACCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCACTCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAAGGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCTGCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGATGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATACTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTTTTCTGGTATTTAAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCTTCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAATGGCTAAAATGAGAATATCACCGGAATTGAAAAAACTGATCGAAAAATACCGCTGCGTAAAAGATACGGAAGGAATGTCTCCTGCTAAGGTATATAAGCTGGTGGGAGAAAATGAAAACCTATATTTAAAAATGACGGACAGCCGGTATAAAGGGACCACCTATGATGTGGAACGGGAAAAGGACATGATGCTATGGCTGGAAGGAAAGCTGCCTGTTCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGCAATCTGCTCATGAGTGAGGCCGATGGCGTCCTTTGCTCGGAAGAGTATGAAGATGAACAAAGCCCTGAAAAGATTATCGAGCTGTATGCGGAGTGCATCAGGCTCTTTCACTCCATCGACATATCGGATTGTCCCTATACGAATAGCTTAGACAGCCGCTTAGCCGAATTGGATTACTTACTGAATAACGATCTGGCCGATGTGGATTGCGAAAACTGGGAAGAAGACACTCCATTTAAAGATCCGCGCGAGCTGTATGATTTTTTAAAGACGGAAAAGCCCGAAGAGGAACTTGTCTTTTCCCACGGCGACCTGGGAGACAGCAACATCTTTGTGAAAGATGGCAAAGTAAGTGGCTTTATTGATCTTGGGAGAAGCGGCAGGGCGGACAAGTGGTATGACATTGCCTTCTGCGTCCGGTCGATCAGGGAGGATATCGGGGAAGAACAGTATGTCGAGCTATTTTTTGACTTACTGGGGATCAAGCCTGATTGGGAGAAAATAAAATATTATATTTTACTGGATGAATTGTTTTAGTACCTAGAATGCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCGCCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTCGGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTAATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATCAGTCACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGGTTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAAAGAGACCTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCCGTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATGACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGTCATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTGCGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAAAAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGCGGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCTCTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGGCGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACCGAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCAGAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCCCTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGTACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCTACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCTTCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCACGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTTCCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGATCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTTGGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGGCCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGTTCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTCATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTGCAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTTGCTCATTTTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGCCAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCAAGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGCGTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGACCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTAAGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACACAGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCCGATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAGCGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGAATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGTTGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTCATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAGCGACCGCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCTCGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACAAACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGAACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGGTTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTCGGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGGGTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCTGGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGGGGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTCGATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCATGCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCCCGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGGGCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTCAAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAAAACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCTGGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGTTCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAAACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAACTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAAGCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGGGGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCACGCCCTTTTAAATATCCGTTATTCTAATAAACGCTCTTTTCTCTTAGGTTTACCCGCCAATATATCCTGTCAAACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAGCTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCC。
super promoter-HvFRF1-GFP SEQ ID NO.27, wherein the sequence of the restriction enzyme cutting site comprises: TCTAGA, GGTACC and HvFRF1 gene sequences are as follows:
atgccggaggtgatgtccacgccacagcaacccacagccatgatgagatttgacactcttgaggatgctgagaaacactacaaactgtatgcgaggcagaaaggttttggagtaaggtattgtttccgaaaaaggtcagaggctagtggtgaactgataagagcatcacttgtctgccatagagctgggttgaagatcaagaggaaagtagacacccaaaacccgcaacctattgcccctgagaggagtaggaatacaactgaaagaacaaactgcccagcccgtatgtttgtgaagcgaagagataatgcctgggttgtaacagaaataaatgacaaccataaccaccctctcataaagaaatggtcgctgacaggatacctacgatcacatagacatatccctgaagaagaacaacagtttgtgaagttgcttcattcgtgcaatctagaaccttctagacagatgcagttgttgacagagttgcatggccaacgtgaagccattggttacactgacaaagacttggcaaatttgctagcaaagttccgggctgagcacaaatacactgatatgcaggacacgattgagtacttcaaaagcagtcaacagcttgataaagactttttttacaagtacaagcttgatgatgaaaataaggtccaatgcatttactggattgatggttcagcgagaagggcttacaagtttttcagtgattgtgtctcttttgacacaacatacttgaccaatatatacaagatgccttgtgcccgtttattggta；
the GFP gene sequence is:
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA；
the specific sequence of SEQ ID NO.27 is:
GGATCCCTGAAAGCGACGTTGGATGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTACGTAAGCGCTTACGTTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTTCACCTACGATGGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGGATCCCTGAAAGCGACGTTGTGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTACGTAAGCGCTTACGTTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTACGATGGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGGATCCCTGAAAGCGACGTTGGATGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTACGTAAGCGCTTACGTTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTTCACCTACGATGGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGGATCCGCGAGCTGCTCAATCCCATTGCTTTTGAAGCAGCTCAACATTGATCTCTTTCTCGATCGAGGGAGATTTTTCAAATCAGTGCGCAAGACGTGACGTAAGTATCCGAGTCAGTTTTTATTTTTCTACTAATTTGGTCGTTTATTTCGGCGTGTAGGACATGGCAACCGGGCCTGAATTTCGCGGGTATTCTGTTTCTATTCCAACTTTTTCTTGATCCGCAGCCATTAACGACTTTTGAATAGATACGCTGACACGCCAAGCCTCGCTAGTCAAAAGTGTACCAAACAACGCTTTACAGCAAGAACGGAATGCGCGTGACGCTCGCGGTGACGCCATTTCGCCTTTTCAGAAATGGATAAATAGCCTTGCTTCCTATTATATCTTCCCCCAAATTACCAATACATTACACTAGCATCTGAATTTCATAACCAATCTCGATACACCAAATCGACTCTAGTCTAGAatgccggaggtgatgtccacgccacagcaacccacagccatgatgagatttgacactcttgaggatgctgagaaacactacaaactgtatgcgaggcagaaaggttttggagtaaggtattgtttccgaaaaaggtcagaggctagtggtgaactgataagagcatcacttgtctgccatagagctgggttgaagatcaagaggaaagtagacacccaaaacccgcaacctattgcccctgagaggagtaggaatacaactgaaagaacaaactgcccagcccgtatgtttgtgaagcgaagagataatgcctgggttgtaacagaaataaatgacaaccataaccaccctctcataaagaaatggtcgctgacaggatacctacgatcacatagacatatccctgaagaagaacaacagtttgtgaagttgcttcattcgtgcaatctagaaccttctagacagatgcagttgttgacagagttgcatggccaacgtgaagccattggttacactgacaaagacttggcaaatttgctagcaaagttccgggctgagcacaaatacactgatatgcaggacacgattgagtacttcaaaagcagtcaacagcttgataaagactttttttacaagtacaagcttgatgatgaaaataaggtccaatgcatttactggattgatggttcagcgagaagggcttacaagtttttcagtgattgtgtctcttttgacacaacatacttgaccaatatatacaagatgccttgtgcccgtttattggtaGGTACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGCTTTCGCAGATCCGGGGGGGCAATGAGATATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTTAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTACAACCGGTCGCGGAGGCTATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCACGACTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGAAATAGAGTAGATGCCGACCGGATCTGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATCCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTCAATTTGTTTACACCACAATATATCCTGCCACCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCACTCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAAGGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCTGCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGATGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATACTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTTTTCTGGTATTTAAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCTTCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAATGGCTAAAATGAGAATATCACCGGAATTGAAAAAACTGATCGAAAAATACCGCTGCGTAAAAGATACGGAAGGAATGTCTCCTGCTAAGGTATATAAGCTGGTGGGAGAAAATGAAAACCTATATTTAAAAATGACGGACAGCCGGTATAAAGGGACCACCTATGATGTGGAACGGGAAAAGGACATGATGCTATGGCTGGAAGGAAAGCTGCCTGTTCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGCAATCTGCTCATGAGTGAGGCCGATGGCGTCCTTTGCTCGGAAGAGTATGAAGATGAACAAAGCCCTGAAAAGATTATCGAGCTGTATGCGGAGTGCATCAGGCTCTTTCACTCCATCGACATATCGGATTGTCCCTATACGAATAGCTTAGACAGCCGCTTAGCCGAATTGGATTACTTACTGAATAACGATCTGGCCGATGTGGATTGCGAAAACTGGGAAGAAGACACTCCATTTAAAGATCCGCGCGAGCTGTATGATTTTTTAAAGACGGAAAAGCCCGAAGAGGAACTTGTCTTTTCCCACGGCGACCTGGGAGACAGCAACATCTTTGTGAAAGATGGCAAAGTAAGTGGCTTTATTGATCTTGGGAGAAGCGGCAGGGCGGACAAGTGGTATGACATTGCCTTCTGCGTCCGGTCGATCAGGGAGGATATCGGGGAAGAACAGTATGTCGAGCTATTTTTTGACTTACTGGGGATCAAGCCTGATTGGGAGAAAATAAAATATTATATTTTACTGGATGAATTGTTTTAGTACCTAGAATGCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCGCCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTCGGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTAATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATCAGTCACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGGTTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAAAGAGACCTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCCGTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATGACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGTCATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTGCGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAAAAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGCGGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCTCTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGGCGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACCGAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCAGAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCCCTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGTACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCTACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCTTCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCACGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTTCCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGATCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTTGGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGGCCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGTTCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTCATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTGCAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTTGCTCATTTTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGCCAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCAAGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGCGTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGACCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTAAGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACACAGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCCGATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAGCGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGAATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGTTGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTCATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAGCGACCGCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCTCGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACAAACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGAACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGGTTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTCGGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGGGTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCTGGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGGGGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTCGATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCATGCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCCCGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGGGCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTCAAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAAAACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCTGGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGTTCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAAACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAACTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAAGCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGGGGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCACGCCCTTTTAAATATCCGTTATTCTAATAAACGCTCTTTTCTCTTAGGTTTACCCGCCAATATATCCTGTCAAACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAGCTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCC。
pBract214 vector SEQ ID NO.28, the sequence of attR arm includes: ACAAGTTTGTACAAAAAAGCTGAAC, GTTCAGCTTTCTTGTACAAAGTGG;
the specific sequence of SEQ ID NO.28 is:
TTTTTATCCCCGGAAGCCTGTGGATAGAGGGTAGTTATCCACGTGAAACCGCTAATGCCCCGCAAAGCCTTGATTCACGGGGCTTTCCGGCCCGCTCCAAAAACTATCCACGTGAAATCGCTAATCAGGGTACGTGAAATCGCTAATCGGAGTACGTGAAATCGCTAATAAGGTCACGTGAAATCGCTAATCAAAAAGGCACGTGAGAACGCTAATAGCCCTTTCAGATCAACAGCTTGCAAACACCCCTCGCTCCGGCAAGTAGTTACAGCAAGTAGTATGTTCAATTAGCTTTTCAATTATGAATATATATATCAATTATTGGTCGCCCTTGGCTTGTGGACAATGCGCTACGCGCACCGGCTCCGCCCGTGGACAACCGCAAGCGGTTGCCCACCGTCGAGCGCCAGCGCCTTTGCCCACAACCCGGCGGCCGGCCGCAACAGATCGTTTTATAAATTTTTTTTTTTGAAAAAGAAAAAGCCCGAAAGGCGGCAACCTCTCGGGCTTCTGGATTTCCGATCCCCGGAATTAGATCTTGGCAGGATATATTGTGGTGTAACGTATCACAAGTTTGTACAAAAAAGCAGGCTCCGCGGCCGCCCCCTTCACCTAGACTCGACGCGTCCTAGAGATCCGTCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACGACCCCGATATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGGTAAATTTCTAGTTTTTCTCCTTCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGATCTTTCTTTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAACTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGATGATAACTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGAGTAGATGCCGACCGGGATCCGGAGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCATCGATGATATCAGATCAAGGGTGGGCGCGCCGAACCAGCTTTCTTGTACAAAGTGGTGATCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCGTGCAGCGTGACCCGGTCGTGCCCCTCTCTAGAGATAATGAGCATTGCATGTCTAAGTTATAAAAAATTACCACATATTTTTTTTGTCACACTTGTTTGAAGTGCAGTTTATCTATCTTTATACATATATTTAAACTTTACTCTACGAATAATATAATCTATAGTACTACAATAATATCAGTGTTTTAGAGAATCATATAAATGAACAGTTAGACATGGTCTAAAGGACAATTGAGTATTTTGACAACAGGACTCTACAGTTTTATCTTTTTAGTGTGCATGTGTTCTCCTTTTTTTTTGCAAATAGCTTCACCTATATAATACTTCATCCATTTTATTAGTACATCCATTTAGGGTTTAGGGTTAATGGTTTTTATAGACTAATTTTTTTAGTACATCTATTTTATTCTATTTTAGCCTCTAAATTAAGAAAACTAAAACTCTATTTTAGTTTTTTTATTTAATAATTTAGATATAAAATAGAATAAAATAAAGTGACTAAAAATTAAACAAATACCCTTTAAGAAATTAAAAAAACTAAGGAAACATTTTTCTTGTTTCGAGTAGATAATGCCAGCCTGTTAAACGCCGTCGACGAGTCTAACGGACACCAACCAGCGAACCAGCAGCGTCGCGTCGGGCCAAGCGAAGCAGACGGCACGGCATCTCTGTCGCTGCCTCTGGACCCCTCTCGAGAGTTCCGCTCCACCGTTGGACTTGCTCCGCTGTCGGCATCCAGAAATTGCGTGTCGGACGGCAGACGTGAGCCGGCACGGCAGGCGGCCTCCTCCTCCTCTCACGGCACCGGCAGCTACGGGGGATTCCTTTCCCACCGCTCCTTCGCTTTCCCTTCCTCGCCCGCCGTAATAAATAGACACCCCCTCCACACCCTCTTTCCCCAACCTCGTGTTGTTCGGAGCGCACACACACACAACCAGATCTCCCCCAAATCCACCCGTCGGCACCTCCGCTTCAAGGTACGCCGCTCGTCCTCCCCCCCCCCCCCTCTCTACCTTCTCTAGATCGGCGTTCCGGTCCATGGTTAGGGCCCGGTAGTTCTACTTCTGTTCATGTTTGTGTTAGATCCGTGTTTGTGTTAGATCCGTGCTGCTAGCGTTCGTACACGGATGCGACCTGTACGTCAGACACGTTCTGATTGCTAACTTGCCAGTGTTTCTCTTTGGGGAATCCTGGGATGGCTCTAGCCGTTCCGCAGACGGGATCGATTTCATGATTTTTTTTTGTTTCGTTGCATAGGGTTTGGTTTGCCCTTTTCCTTTATTTCAATATATGCCGTGCACTTGTTTGTCGGGTCATCTTTTCATGCTTTTTTTTGTCTTGGTTGTGATGATGTGGTCTGGTTGGGCGGTCGTTCTAGATCGGAGTAGAATTAATTCTGTTTCAAACTACCTGGTGGATTTATTAATTTTGGATCTGTATGTGTGTGCCATACATATTCATAGTTACGAATTGAAGATGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGCGGGTTTTACTGATGCATATACAGAGATGCTTTTTGTTCGCTTGGTTGTGATGATGTGGTGTGGTTGGGCGGTCGTTCATTCGTTCTAGATCGGAGTAGAATACTGTTTCAAACTACCTGGTGTATTTATTAATTTTGGAACTGTATGTGTGTGTCATACATCTTCATAGTTACGAGTTTAAGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGTGGGTTTTACTGATGCATATACATGATGGCATATGCAGCATCTATTCATATGCTCTAACCTTGAGTACCTATCTATTATAATAAACAAGTATGTTTTATAATTATTTTGATCTTGATATACTTGGATGATGGCATATGCAGCAGCTATATGTGGATTTTTTTAGCCCTGCCTTCATACGCTATTTATTTGCTTGGTACTGTTTCTTTTGTCGATGCTCACCCTGTTGTTTGGTGTTACTTCGCCCATCACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGGCGGCCGCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAACGCGTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGGCTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTTCTTGTACAAAGTGGTGATGGGGGATCCACTAGTTCTAGAATTCGATTGAGTCAAGCAGGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGACCGGCATGCAAGCTGATATCAATCACTAGTGAATTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGAAGGCCTTGACAGGATATATTGGCGGGTAAACTAAGTCGCTGTATGTGTTTGTTTGAGATCTCATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAGAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGTGTAACATTGGTCTAGTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCTGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACAACATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGGTAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTTGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCGATCCCCATCCAACAGCCCGCCGTCGAGCGGGCT。
pBract214 HvFRF1 vector SEQ ID NO.29, sequence of attB arm: ACAAGTTTGTACAAAAAAGCAGGCTTCACC, CACCCAGCTTTCTTGTACAAAGTGGT;
the sequence of the HvFRF1 gene sequence is:
atgccggaggtgatgtccacgccacagcaacccacagccatgatgagatttgacactcttgaggatgctgagaaacactacaaactgtatgcgaggcagaaaggttttggagtaaggtattgtttccgaaaaaggtcagaggctagtggtgaactgataagagcatcacttgtctgccatagagctgggttgaagatcaagaggaaagtagacacccaaaacccgcaacctattgcccctgagaggagtaggaatacaactgaaagaacaaactgcccagcccgtatgtttgtgaagcgaagagataatgcctgggttgtaacagaaataaatgacaaccataaccaccctctcataaagaaatggtcgctgacaggatacctacgatcacatagacatatccctgaagaagaacaacagtttgtgaagttgcttcattcgtgcaatctagaaccttctagacagatgcagttgttgacagagttgcatggccaacgtgaagccattggttacactgacaaagacttggcaaatttgctagcaaagttccgggctgagcacaaatacactgatatgcaggacacgattgagtacttcaaaagcagtcaacagcttgataaagactttttttacaagtacaagcttgatgatgaaaataaggtccaatgcatttactggattgatggttcagcgagaagggcttacaagtttttcagtgattgtgtctcttttgacacaacatacttgaccaatatatacaagatgccttgtgcccgtttattggtataa
the specific sequence of SEQ ID NO.29 is:
TTTTTATCCCCGGAAGCCTGTGGATAGAGGGTAGTTATCCACGTGAAACCGCTAATGCCCCGCAAAGCCTTGATTCACGGGGCTTTCCGGCCCGCTCCAAAAACTATCCACGTGAAATCGCTAATCAGGGTACGTGAAATCGCTAATCGGAGTACGTGAAATCGCTAATAAGGTCACGTGAAATCGCTAATCAAAAAGGCACGTGAGAACGCTAATAGCCCTTTCAGATCAACAGCTTGCAAACACCCCTCGCTCCGGCAAGTAGTTACAGCAAGTAGTATGTTCAATTAGCTTTTCAATTATGAATATATATATCAATTATTGGTCGCCCTTGGCTTGTGGACAATGCGCTACGCGCACCGGCTCCGCCCGTGGACAACCGCAAGCGGTTGCCCACCGTCGAGCGCCAGCGCCTTTGCCCACAACCCGGCGGCCGGCCGCAACAGATCGTTTTATAAATTTTTTTTTTTGAAAAAGAAAAAGCCCGAAAGGCGGCAACCTCTCGGGCTTCTGGATTTCCGATCCCCGGAATTAGATCTTGGCAGGATATATTGTGGTGTAACGTATCACAAGTTTGTACAAAAAAGCAGGCTCCGCGGCCGCCCCCTTCACCTAGACTCGACGCGTCCTAGAGATCCGTCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACGACCCCGATATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGGTAAATTTCTAGTTTTTCTCCTTCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGATCTTTCTTTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAACTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGATGATAACTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGAGTAGATGCCGACCGGGATCCGGAGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCATCGATGATATCAGATCAAGGGTGGGCGCGCCGAACCAGCTTTCTTGTACAAAGTGGTGATCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCGTGCAGCGTGACCCGGTCGTGCCCCTCTCTAGAGATAATGAGCATTGCATGTCTAAGTTATAAAAAATTACCACATATTTTTTTTGTCACACTTGTTTGAAGTGCAGTTTATCTATCTTTATACATATATTTAAACTTTACTCTACGAATAATATAATCTATAGTACTACAATAATATCAGTGTTTTAGAGAATCATATAAATGAACAGTTAGACATGGTCTAAAGGACAATTGAGTATTTTGACAACAGGACTCTACAGTTTTATCTTTTTAGTGTGCATGTGTTCTCCTTTTTTTTTGCAAATAGCTTCACCTATATAATACTTCATCCATTTTATTAGTACATCCATTTAGGGTTTAGGGTTAATGGTTTTTATAGACTAATTTTTTTAGTACATCTATTTTATTCTATTTTAGCCTCTAAATTAAGAAAACTAAAACTCTATTTTAGTTTTTTTATTTAATAATTTAGATATAAAATAGAATAAAATAAAGTGACTAAAAATTAAACAAATACCCTTTAAGAAATTAAAAAAACTAAGGAAACATTTTTCTTGTTTCGAGTAGATAATGCCAGCCTGTTAAACGCCGTCGACGAGTCTAACGGACACCAACCAGCGAACCAGCAGCGTCGCGTCGGGCCAAGCGAAGCAGACGGCACGGCATCTCTGTCGCTGCCTCTGGACCCCTCTCGAGAGTTCCGCTCCACCGTTGGACTTGCTCCGCTGTCGGCATCCAGAAATTGCGTGTCGGACGGCAGACGTGAGCCGGCACGGCAGGCGGCCTCCTCCTCCTCTCACGGCACCGGCAGCTACGGGGGATTCCTTTCCCACCGCTCCTTCGCTTTCCCTTCCTCGCCCGCCGTAATAAATAGACACCCCCTCCACACCCTCTTTCCCCAACCTCGTGTTGTTCGGAGCGCACACACACACAACCAGATCTCCCCCAAATCCACCCGTCGGCACCTCCGCTTCAAGGTACGCCGCTCGTCCTCCCCCCCCCCCCCTCTCTACCTTCTCTAGATCGGCGTTCCGGTCCATGGTTAGGGCCCGGTAGTTCTACTTCTGTTCATGTTTGTGTTAGATCCGTGTTTGTGTTAGATCCGTGCTGCTAGCGTTCGTACACGGATGCGACCTGTACGTCAGACACGTTCTGATTGCTAACTTGCCAGTGTTTCTCTTTGGGGAATCCTGGGATGGCTCTAGCCGTTCCGCAGACGGGATCGATTTCATGATTTTTTTTTGTTTCGTTGCATAGGGTTTGGTTTGCCCTTTTCCTTTATTTCAATATATGCCGTGCACTTGTTTGTCGGGTCATCTTTTCATGCTTTTTTTTGTCTTGGTTGTGATGATGTGGTCTGGTTGGGCGGTCGTTCTAGATCGGAGTAGAATTAATTCTGTTTCAAACTACCTGGTGGATTTATTAATTTTGGATCTGTATGTGTGTGCCATACATATTCATAGTTACGAATTGAAGATGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGCGGGTTTTACTGATGCATATACAGAGATGCTTTTTGTTCGCTTGGTTGTGATGATGTGGTGTGGTTGGGCGGTCGTTCATTCGTTCTAGATCGGAGTAGAATACTGTTTCAAACTACCTGGTGTATTTATTAATTTTGGAACTGTATGTGTGTGTCATACATCTTCATAGTTACGAGTTTAAGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGTGGGTTTTACTGATGCATATACATGATGGCATATGCAGCATCTATTCATATGCTCTAACCTTGAGTACCTATCTATTATAATAAACAAGTATGTTTTATAATTATTTTGATCTTGATATACTTGGATGATGGCATATGCAGCAGCTATATGTGGATTTTTTTAGCCCTGCCTTCATACGCTATTTATTTGCTTGGTACTGTTTCTTTTGTCGATGCTCACCCTGTTGTTTGGTGTTACTTCGCCCATCACAAGTTTGTACAAAAAAGCAGGCTTCACCatgccggaggtgatgtccacgccacagcaacccacagccatgatgagatttgacactcttgaggatgctgagaaacactacaaactgtatgcgaggcagaaaggttttggagtaaggtattgtttccgaaaaaggtcagaggctagtggtgaactgataagagcatcacttgtctgccatagagctgggttgaagatcaagaggaaagtagacacccaaaacccgcaacctattgcccctgagaggagtaggaatacaactgaaagaacaaactgcccagcccgtatgtttgtgaagcgaagagataatgcctgggttgtaacagaaataaatgacaaccataaccaccctctcataaagaaatggtcgctgacaggatacctacgatcacatagacatatccctgaagaagaacaacagtttgtgaagttgcttcattcgtgcaatctagaaccttctagacagatgcagttgttgacagagttgcatggccaacgtgaagccattggttacactgacaaagacttggcaaatttgctagcaaagttccgggctgagcacaaatacactgatatgcaggacacgattgagtacttcaaaagcagtcaacagcttgataaagactttttttacaagtacaagcttgatgatgaaaataaggtccaatgcatttactggattgatggttcagcgagaagggcttacaagtttttcagtgattgtgtctcttttgacacaacatacttgaccaatatatacaagatgccttgtgcccgtttattggtataaCACCCAGCTTTCTTGTACAAAGTGGTGATGGGGGATCCACTAGTTCTAGAATTCGATTGAGTCAAGCAGGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGACCGGCATGCAAGCTGATATCAATCACTAGTGAATTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGAAGGCCTTGACAGGATATATTGGCGGGTAAACTAAGTCGCTGTATGTGTTTGTTTGAGATCTCATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAGAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGTGTAACATTGGTCTAGTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCTGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACAACATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGGTAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTTGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCGATCCCCATCCAACAGCCCGCCGTCGAGCGGGCT。
in the above embodiments, the descriptions of the respective embodiments have respective emphasis, and reference may be made to the related descriptions of other embodiments for parts that are not described or illustrated in a certain embodiment.
2. The application example is as follows:
application example
The application example of the invention provides a barley HvFRF1 gene, and the nucleotide sequence of the barley HvFRF1 gene is shown in SEQ ID NO. 1. The barley HvFRF1 gene is a gene cloned from barley.
The application example of the invention also provides a protein coded by the barley HvFRF1 gene, and the amino acid sequence of the protein is shown in SEQ ID NO. 2.
The application example of the invention also provides an arabidopsis thaliana overexpression vector super1300 HvFRF1 constructed by homologous recombination according to the barley HvFRF1 gene.
The application example of the invention also provides a barley overexpression vector pBract214: hvFRF1 constructed by a Gateway method according to the barley HvFRF1 gene.
The application example of the invention also provides application of the barley HvFRF1 gene in obtaining a drought-resistant arabidopsis strain.
The application example of the invention also provides application of the barley HvFRF1 gene in obtaining a drought-resistant barley strain.
The application example of the invention also provides an application of the barley HvFRF1 gene in tissue expression in a barley root system under the induction of drought stress.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention, and the scope of the present invention should not be limited thereto, and any modifications, equivalents and improvements made by those skilled in the art within the technical scope of the present invention as disclosed in the present invention should be covered thereby.
Claims (10)
1. A barley HvFRF1 gene is characterized in that the nucleotide sequence of the barley HvFRF1 gene is shown in SEQ ID NO. 1.
2. The barley HvFRF1 gene according to claim 1, characterized in that the barley HvFRF1 gene is a gene cloned from barley.
3. The barley HvFRF1 gene according to any one of claims 1 to 2, which encodes a protein having the amino acid sequence shown in SEQ ID No. 2.
4. An Arabidopsis thaliana overexpression vector super1300: hvFRF1 constructed by homologous recombination according to the barley HvFRF1 gene of claim 1, the nucleotide sequence of which is shown as SEQ ID No. 26.
5. A barley overexpression vector pBract214 HvFRF1 constructed by the Gateway method according to the barley HvFRF1 gene of claim 1, the nucleotide sequence of which is shown as SEQ ID NO. 29.
6. An application of barley HvFRF1 gene in obtaining drought-resistant arabidopsis thaliana strains.
7. An application of a barley HvFRF1 gene in obtaining a barley strain with drought resistance.
8. An application of barley HvFRF1 gene in tissue expression in barley root system under the induction of drought stress.
9. An application of barley HvFRF1 gene in controlling arabidopsis thaliana to screening arabidopsis thaliana varieties.
10. An application of a barley HvFRF1 gene in screening barley varieties by regulating and controlling the drought resistance of barley.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112795545A (en) * | 2021-01-29 | 2021-05-14 | 浙江大学 | Barley HvHMT3 gene and application thereof |
| CN113151299A (en) * | 2021-04-22 | 2021-07-23 | 沈阳农业大学 | Gene for improving low temperature resistance of tomato plant and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112795545A (en) * | 2021-01-29 | 2021-05-14 | 浙江大学 | Barley HvHMT3 gene and application thereof |
| CN113151299A (en) * | 2021-04-22 | 2021-07-23 | 沈阳农业大学 | Gene for improving low temperature resistance of tomato plant and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| MASCHER等: "Long-read sequence assembly: a technical evaluation in barley", THE PLANT CELL, vol. 33, no. 6, 12 March 2021 (2021-03-12), pages 1888 * |
| MA等: "FAR1-RELATED SEQUENCE (FRS) and FRS-RELATED FACTOR (FRF) Family Proteins in Arabidopsis Growth and Development", FRONTIERS IN PLANT SCIENCE, vol. 9, 7 June 2018 (2018-06-07), pages 1 - 10 * |
| 强治全;杨文博;张帅;于正阳;史学英;王鑫;朱维宁;张林生;: "异源表达WZY2-1基因提高拟南芥植株抗旱性", 草业学报, no. 06, 30 June 2018 (2018-06-30), pages 92 - 99 * |
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