CN114107333A - Application of barley receptor kinase HvSERK1 in root hair growth - Google Patents
Application of barley receptor kinase HvSERK1 in root hair growth Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a barley HvSERK1 gene, a barley receptor kinase HvSERK1, a recombinant expression vector containing the gene and application of the recombinant expression vector in promoting the growth of plant root hair. The invention can obviously improve the root hair length of arabidopsis thaliana by cloning the barley HvSERK1 gene and carrying out genetic transformation in arabidopsis thaliana and over-expressing the exogenous barley HvSERK1 gene. Therefore, HvSERK1 is expected to be used for genetic engineering breeding, and the capacity of absorbing water and nutrients of barley is expected to be improved by introducing the HvSERK1 into a short-root-hair barley variety. The invention provides theoretical basis and related genes for barley drought-resistant breeding and production.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a barley HvSERK1 gene and an expression vector thereof, a barley receptor kinase HvSERK1 and application thereof.
Background
The root hair is a highly specialized cell in the epidermal cell of the plant root, and can increase the surface area of the root and improve the capability of the plant for acquiring water and nutrients; the stress resistance of the plant can be improved through self adaptive reaction and root secretion thereof. At present, in order to improve the yield of crops, a large amount of chemical fertilizers and pesticides are applied in agricultural production, so that serious environmental pollution and energy waste are caused, and the cost input of farmers is increased, therefore, the research on how to obtain new varieties of crops with more and longer roots and hairs, improve the yield of the crops and efficiently utilize nutrient and moisture is the basis and the key for improving the yield of the crops, improving the stress resistance and realizing sustainable agricultural development. The research on the genes related to the regulation of the growth and development of root hairs has important theoretical significance and practical application value for understanding the genetic regulation mechanism of the growth and development of plants.
Receptor kinases are transmembrane proteins widely present in plants, and they transduce extracellular signals to regulate plant development, self-incompatibility and defense response. The Somatie Embryo RECEPTOR KINASES (SERKs) belong to LRR-RLKs subfamily II, and are first a class of RECEPTOR KINASES found in SOMATIC EMBRYOGENESIS of carrot. SERKs are involved in Arabidopsis thaliana including somatic embryogenesis, seedling development, Brassinolide (BRs) signaling, plant immunity, and pore development. The Arabidopsis thaliana SERKs family includes 5 homologous genes, SERK1, SERK2, SERK3/BKA1, SERK4/BKK1, and SERK5, respectively. The amino acid sequences of SERK1 and SERK2 are quite similar, and the double mutant SERK1SERK2 is aborted due to the failure to form tapetum cells, indicating that SERK1 and SERK2 are important for the formation of the tapetum of Arabidopsis anther. BRs can interact with auxin and other plant hormones to influence the development of Arabidopsis roots, and the three-mutant serk1bak1bkk1 can block a BR signal channel and influence the down-regulation of gene expression related to auxin polarity transport, cell cycle, endothelial layer development and root meristem differentiation, thereby influencing the development of Arabidopsis roots. SERK1, SERK2, SERK3/BAK1, SERK4/BKK1 are all involved in regulating the development of Arabidopsis stomata independent of BR signals.
Wheat crops are important grain crops in the world, and the research on functional genomes of the wheat crops has extremely important significance on global grain safety. Barley (Hordeum vulgare L.) belongs to the genus Hordeum of the family gramineae, is the fourth largest cereal crop in the world, is mainly used as a raw material for beer industry, a feed for animal husbandry, and a raw material for food processing, and is also an important model plant for genetic and physiological research. However, the drought seriously affects the planting of barley and becomes one of the most important abiotic factors for restricting the yield increase of barley, so that the excavation of root hair related genes is urgently needed to cultivate barley varieties with multiple hair and long root hair.
Disclosure of Invention
The invention aims to provide a barley HvSERK1 gene, a barley receptor kinase HvSERK1, a recombinant expression vector containing the barley HvSERK1 gene and application of the gene in promoting growth of arabidopsis root hair.
In a first aspect of the invention, the barley HvSERK1 gene is provided, and the CDS region nucleotide sequence of the gene is shown in SEQ ID NO. 1.
In a second aspect of the invention, the barley HvSERK1 gene is coded by protein, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
Cloning and analysis of the HvSERK1 Gene: the invention identifies and clones a gene HvSERK1 from 'flower 30' of cultivated barley, the CDS region of the gene has the total length of 1884bp, and the gene encodes a 627aa protein sequence, the molecular weight of the protein is 69.0769KDa, and the isoelectric point pI is 5.70.
In a third aspect of the present invention, there is provided a primer pair for amplifying the barley HvSERK1 gene as described above, the nucleotide sequences of which are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.
In a fourth aspect of the invention, there is provided a recombinant expression vector containing the barley HvSERK1 gene as described above.
Further, the recombinant expression vector comprises an original vector and a target gene inserted into the original vector, wherein the base sequence of the target gene is shown as SEQ ID NO. 1.
Further, the promoter for promoting the expression of the target gene is a 35S strong promoter.
Further, the original carrier is pH2GW 7.
Further, the construction of the recombinant expression vector: the barley HvSERK1 gene was ligated into a plant expression vector pH2GW7 using the Gateway system (Invitrogen corporation). The promoter for promoting the barley HvSERK1 gene expression is a strong promoter. The strong promoter can promote the overexpression of the barley HvSERK1 gene. Preferably, the strong promoter is 35S.
In a fifth aspect of the present invention, there is provided a transformant containing the recombinant expression vector as described above.
Further, the host bacteria of the transformant are agrobacterium GV3101, LBA4404, AGL1 or EHA 105.
In a sixth aspect, the present invention provides the use of the barley HvSERK1 gene, the protein encoded by the barley HvSERK1 gene, the primer set, the recombinant expression vector, or the transformant for plant breeding.
Further, the plant breeding is to regulate and control the growth of plant root hair.
Further, the plant is bred to promote the growth of plant root hair. In one embodiment of the invention, the plant is Arabidopsis thaliana.
The invention provides application of a barley HvSERK1 gene in regulation and control of root hair growth, and the barley HvSERK1 gene is cloned and genetically transformed in arabidopsis thaliana to over-express an exogenous barley HvSERK1 gene, so that the root hair length of arabidopsis thaliana can be remarkably increased. Therefore, HvSERK1 is expected to be used for genetic engineering breeding, and the capacity of absorbing water and nutrients of barley is expected to be improved by introducing the HvSERK1 into a short-root-hair barley variety. The invention provides theoretical basis and related genes for barley drought-resistant breeding and production.
The invention has the advantages that:
the barley HvSERK1 gene is proved to be capable of obviously increasing the root hair length of arabidopsis thaliana by cloning and analyzing the barley HvSERK1 gene and combining with the phenotype analysis of arabidopsis thaliana. The invention provides theoretical basis and related genes for barley long root hair breeding and production.
Drawings
FIG. 1 shows the HvSERK1 gene expression level in Arabidopsis transgenic material overexpressing the HvSERK1 gene.
FIG. 2 shows that the root hair length of the Arabidopsis transgenic material overexpressing HvSERK1 gene is obviously increased.
Detailed Description
The following detailed description of the present invention will be made with reference to the accompanying drawings.
Example 1: construction of overexpression vector of barley HvSERK1 Gene and genetic transformation of Arabidopsis thaliana
(1) Barley "flower 30" embryos were harvested and RNA extracted using Trizol (Invitrogen) protocol, with the procedure described. Mu.g of RNA was taken for reverse transcription to obtain cDNA.
(2) Specific primers are designed according to the HvSERK1 gene sequence, and restriction enzyme sites are respectively added at the 5' ends of the primers, and the sequences are as follows:
HvSERK1-CDS-F:5’-CCATCGATCTGCGGCTTTGAGACGG-3’(SEQ ID NO.3)；
HvSERK1-CDS-R:5’-CCCCCGGGAGAAATTCTGCAACGAGGGG-3’(SEQ ID NO.4)；
(3) PCR amplification was performed using the full-length cDNA as a template.
Using KOD high fidelity enzyme (TOYOBO) system:
sterile distilled water was added to a total volume of 50. mu.L.
The PCR cycling conditions were: pre-denaturation at 94 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 68 ℃ for 90s, amplification for 35 cycles, and finally extension at 68 ℃ for 5min, thus finishing the reaction.
(4) After PCR products are recovered by running gel, the products are connected to an entry vector NT007 of a Gateway cloning system through enzyme digestion, escherichia coli DH5 alpha is transformed, a positive clone is sequenced and analyzed, and the HvSERK1 gene sequence is shown as SEQ ID NO. 1.
(5) The correctly sequenced NT007 plasmid containing the full length CDS of HvSERK1 was designated HvSERK1-NT007, and this plasmid was subjected to LR exchange reaction with the over-expression vector pH2GW 7.
The Gateway LR clone TM Enzyme Mix (Invitrogen, Cat. No.11791-019) was used as follows:
vortex twice and mix, centrifugate slightly, in 25 deg.C water bath for 2h, then add 1. mu.L protease K solution and mix, stand for 10 minutes at 37 deg.C and finish the reaction.
Transforming 5 mu L of reaction product into escherichia coli DH5 alpha, selecting PCR to verify positive clone, verifying the positive clone by sequencing, extracting plasmid after the sequencing is correct, and naming the plasmid as 35S: HvSERK 1.
(6) LBA4404 competent cells were removed from the-80 ℃ freezer and Agrobacterium LBA4404 was transformed using heat shock.
Thawing the competent cells on ice, adding 5 μ L of the objective plasmid into the competent cells, mixing, and placing on ice for 30 min; the tube was frozen in liquid nitrogen for 1min and then placed in a 37 ℃ water bath until thawed. 1mL of LB liquid medium was added and shake-cultured for 2-3 hours, centrifuged, and spread on resistant YEP plates containing Rif (rifampicin) 50mg/L and Spec (spectinomycin hydrochloride) 50mg/L, and inverted-cultured at 28 ℃ for 2 nights until single colonies were grown.
(7) The Agrobacterium positive monoclonal after PCR validation was picked up and put into 5ml YEP liquid medium containing the corresponding antibiotic and shake-cultured at 28 ℃ for 48 h. Adding the shaken bacteria into YEP liquid culture medium (containing corresponding antibiotics) at a volume ratio of 1:50, culturing at 28 deg.C to OD600＝1.0-1.2。
(8) Arabidopsis thaliana was transformed by floral dip method.
Removing fruit pods, and reversely buckling the whole arabidopsis thaliana plant only with inflorescences and a plug disc into the bacterial liquid of the transformed agrobacterium, soaking seedlings for 5min, and continuously shaking the bacterial liquid in the period;
after infection, taking out the plant, laterally placing the plant in a tray, covering a dark black plastic cloth, placing the plant in an incubator, and uncovering the plant after 24 hours;
culturing Arabidopsis plant under natural illumination, watering 1-2 times per week, and harvesting T after seed maturation0And (5) seed generation.
Example 2: screening of barley HvSERK1 overexpression transgenic line
(1) T to be harvested0And (3) sterilizing and disinfecting the seeds, planting the seeds in a solid culture medium of MS +35mg/L hygromycin, performing vernalization in the dark at 4 ℃ for 3 days, placing a culture dish in an artificial climate box for culture, and observing the growth condition of the arabidopsis thaliana plants. Transgenic seeds with hygromycin resistance will grow in the selection medium, while non-transgenic seeds will not grow after germination.
(2) Transplanting the screened transgenic plants into nutrient soil, when the plants are bolting, shearing about 100mg of arabidopsis leaves, placing the arabidopsis leaves into a 1.5ml centrifugal tube, extracting DNA, carrying out PCR verification, and detecting whether the screened plants are positive.
(3) Harvesting T for single plants with positive detection1After seed generation, sterilization and disinfection, planting in a solid culture medium of MS +35mg/L hygromycin, performing vernalization in the dark at 4 ℃ for 3 days, and placing the culture dish in an artificial climate box for culture.
(4) The transgenic plants with hygromycin resistance are transplanted into nutrient soil. When the plants are bolting, each plant line is cut about 100mg of arabidopsis leaves to extract RNA, and cDNA is obtained by reverse transcription. The expression of HvSERK1 gene in different Arabidopsis transgenic lines was subjected to fluorescent quantitative PCR analysis using SYBR green and PCR instrument, and the expression value was corrected using internal reference gene TUB 2.
The fluorescent quantitative PCR system is as follows:
the PCR procedure was: 1min at 95 ℃, 5s at 95 ℃, 20s at 60 ℃ and 40 cycles. Each experiment was repeated three times, and the fluorescent quantitative PCR primer sequences were as follows:
HvSERK1-RT-PCR-F：5’-CTTGGCAGACGGAACTCTTG-3’(SEQ ID NO.5)；
HvSERK1-RT-PCR-R：5’-TCTCGGTCGGTGTCATACAG-3’(SEQ ID NO.6)；
AtTUB2-F：5’-GTTCTCGATGTTGTTCGTAAG-3’(SEQ ID NO.7)；
AtTUB2-R：5’-TGTAAGGCTCAACCACAGTAT-3’(SEQ ID NO.8)。
(5) the relative expression level of HvSERK1 in different transgenic lines of Arabidopsis thaliana is shown in FIG. 1. Selecting 3 strains with the highest expression quantity, namely 35S HvSERK1-4, 35S HvSERK1-7 and 35S HvSERK1-9, and harvesting T2And (5) seed generation.
Example 3: root hair phenotype Observation of Arabidopsis
(1) Combining Arabidopsis thaliana wild type with T2Generating HvSERK1 transgenic seeds (35S: HvSERK1-4, 35S: HvSERK1-7, 35S: HvSERK1-9), sterilizing, dibbling on MS solid culture medium, vernalizing in dark at 4 deg.C for 3d, vertically culturing in culture dish, culturing in artificial climate box (temperature: 20 + -2 deg.C, light intensity 90-120 μmolm)-2sec-1(ii) a The photoperiod: 16h light/8 h dark).
(2) After culturing in an artificial climate box for 7 days, the root hair phenotype of about 4mm of the root tip is photographed by a body type microscope.
(3) And (3) carrying out statistical analysis on the length of the root hairs by adopting Image pro plus software, wherein each root measures the longest 20 root hairs, and each strain measures at least 20 root hairs. As shown in fig. 2. As a result, it was found that overexpression of HvSERK1 promoted root hair growth.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
SEQUENCE LISTING
<110> Shanghai city academy of agricultural sciences
<120> application of barley receptor kinase HvSERK1 in root hair growth
<130> /
<160> 8
<170> PatentIn version 3.3
<210> 1
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cgccaaagcc ttaaagatgc taacaatgtg ctgcagagtt gggatcccac tctggttaat 180
ccatgcacat ggttccatgt tacttgtaac accgacaaca gtgtaatcag agttgatctt 240
ggaaacgcac aactgtcagg tgcattggtg tcccaacttg gacagttgaa aaatctacaa 300
tatttggagc tgtacagcaa caatataagt gggataatac ctttggaact tgggaacttg 360
acaaacctgg tcagtttgga tctgtacctg aacaaattca ctggcggtat tccagacaca 420
ttggggcaac tcttaaagtt gcgattcctc cgtcttaaca acaacagtct ttctggtcaa 480
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aataatccaa atctttgtgg accgggtact acgaaaccat gtcctggggc acctcctttt 660
tctccgcctc ccccattcaa tcctccaaca ccagtcacga accaaggtga ctctaaaacc 720
ggggcaattg ctggaggtgt tgctgcgggt gctgcattga tatttgctgt tcctgcaatt 780
ggatttgcac tgtggcggcg acgtaaacct gaagagcatt tctttgatgt ccctgccgag 840
gaggatccag aagtgcacct gggccagcta aagaggttct cactaaggga gcttcaagtt 900
gctagcgata acttcagcaa taagaacatt ctaggaagag gtggctttgg aaaggtctac 960
aaggggagac tgacggatgg tacattggta gcagttaaaa gattaaaaga agaacgtact 1020
cctggtggcg aactccaatt ccaaacagaa gttgaaatga ttagtatggc agtgcatagg 1080
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catgatcact gtgatcccaa gatcattcat cgtgatgtca aagctgcaaa cattctcttg 1320
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tccaccggga agtcctctga gaagacggat gttttcggtt acggcatcat gcttctggag 1500
cttatcactg gacagagggc gtttgacctc gcacgtcttg cgaatgacga cgatgtcatg 1560
ctgcttgact gggtgaaagg gctgctgaaa gagaagaagg tggagatgct ggtggacccg 1620
gacctgcaga gcgtgtacgt ggagcacgag gtggaggcgc tgatccaggt ggcgctgctg 1680
tgcacgcagg ggtcaccgat ggaccggccc aagatgtcgg aggtggtgag gatgctggag 1740
ggcgacgggc tggcggagcg ctgggaggag tggcagaagg tggaggtggt ccggcaggag 1800
gcggagctgg ctccgcgaca caacgactgg atcgtcgact ccactttcaa cctccgggcg 1860
gtggagctgt ccggcccgag gtaa 1884
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<213> Artificial sequence (Artificial)
<400> 7
gttctcgatg ttgttcgtaa g 21
<210> 8
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 8
tgtaaggctc aaccacagta t 21
Claims (10)
1. The nucleotide sequence of the CDS region of the barley HvSERK1 gene is shown as SEQ ID NO. 1.
2. The barley protein HvSERK1 gene according to claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID No. 2.
3. The primer pair for amplifying the barley HvSERK1 gene as claimed in claim 1, wherein the nucleotide sequences are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively.
4. A recombinant expression vector comprising the barley HvSERK1 gene of claim 1.
5. The recombinant expression vector of claim 4, wherein the recombinant expression vector comprises an original vector and a target gene inserted into the original vector, and the base sequence of the target gene is shown as SEQ ID NO. 1.
6. The recombinant expression vector according to claim 5, wherein the promoter for promoting the expression of the target gene is a 35S strong promoter; the original carrier is pH2GW 7.
7. A transformant containing the recombinant expression vector according to any one of claims 4 to 6.
8. The transformant according to claim 7, wherein the host bacterium of the transformant is Agrobacterium GV3101, LBA4404, AGL1 or EHA 105.
9. Use of the barley HvSERK1 gene according to claim 1, the protein encoded by the barley HvSERK1 gene according to claim 2, the primer pair according to claim 3, the recombinant expression vector according to any one of claims 4 to 6, or the transformant according to claim 7 or 8 in plant breeding.
10. The use of claim 9, wherein the plant is bred to promote root hair growth in plants.
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