CN114107333B - Application of barley receptor kinase HvSERK1 in root hair growth - Google Patents

Application of barley receptor kinase HvSERK1 in root hair growth Download PDF

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CN114107333B
CN114107333B CN202111253390.XA CN202111253390A CN114107333B CN 114107333 B CN114107333 B CN 114107333B CN 202111253390 A CN202111253390 A CN 202111253390A CN 114107333 B CN114107333 B CN 114107333B
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黄琳丽
刘成洪
李颖波
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Shanghai Academy of Agricultural Sciences
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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 thereof in promoting plant root hair growth. According to the invention, the length of root hair of the arabidopsis thaliana can be remarkably improved by cloning the barley HvSERK1 gene and carrying out genetic transformation in the arabidopsis thaliana and overexpressing the exogenous barley HvSERK1 gene. Therefore, hvSERK1 is expected to be used for genetic engineering breeding, and is expected to improve the capability of barley for absorbing water and nutrients by introducing the HvSERK1 into short root hair barley varieties. The invention provides theoretical basis and related genes for drought-resistant breeding and production of barley.

Description

Application of barley receptor kinase HvSERK1 in root hair growth
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 cells of the plant root, so that the surface area of the root can be increased, and the capability of the plant for acquiring moisture and nutrient can be improved; the stress resistance of plants can be improved through self adaptive reaction and root secretions thereof. At present, in order to improve the yield of crops, a large amount of fertilizers and pesticides are applied to agricultural production, serious environmental pollution and energy waste are caused, and meanwhile, the cost input of farmers is increased, so that research on how to obtain new varieties of crops with more and longer root hairs, and improvement of the crop yield and efficient utilization of nutrient moisture are the basis and key for improving the crop yield, stress resistance and sustainable agricultural development. The research on genes related to root hair growth and development has important theoretical significance and practical application value for understanding the genetic regulation mechanism of plant growth and development.
Receptor kinases are a class of transmembrane proteins that are widely found in plants and they transduce extracellular signals to play an important role in regulating plant development, self-incompatibility and defense responses. SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) belong to the LRR-RLKs subfamily II, first to a class of receptor kinases found during carrot somatic embryogenesis. SERKs in Arabidopsis are involved in processes including somatic embryogenesis, seedling development, brassinolide (BRs) signal transduction, plant immunity, and Kong Fayo. The Arabidopsis 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 abortive due to the failure to form tapetum cells, which indicates that SERK1 and SERK2 are important for the formation of the tapetum of the anther of Arabidopsis thaliana. BRs can interact with auxins and other plant hormones to influence the development of arabidopsis roots, and the triple mutant serk1bak1bkk1 can not only block BR signal channels, but also influence the polar transport of auxins, cell cycle, endothelial layer development and down regulation of gene expression related to root meristem differentiation, thereby influencing the development of arabidopsis roots. SERK1, SERK2, SERK3/BAK1 and SERK4/BKK1 are all involved in regulating the development of Arabidopsis stomata and are independent of BR signals.
Wheat crops are important food crops in the world, and functional genome research of the wheat crops has extremely important significance for global food safety. Barley (Hordeum vulgare l.) belongs to the genus barley of the family gramineae, is a fourth major cereal crop in the world, is mainly used as a raw material for the beer industry, for animal husbandry, and for food processing, and is also an important model plant for genetic and physiological research. However, drought seriously affects the cultivation of barley, and becomes one of the most important abiotic factors restricting the yield increase of barley, so that the excavation of root hair related genes is urgently needed to cultivate multi-root hair and multi-root hair barley varieties.
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 the growth of Arabidopsis root hairs.
In a first aspect of the invention, there is provided a barley HvSERK1 gene having a CDS region nucleotide sequence shown in SEQ ID NO. 1.
In a second aspect of the present invention, there is provided a protein encoded by the barley HvSERK1 gene as described above, the amino acid sequence of which is shown in SEQ ID NO. 2.
Cloning and analysis of the HvSERK1 Gene: the invention identifies the gene HvSERK1 cloned from the cultivated barley flower 30, the CDS region of the gene has the total length of 1884bp and codes a 627aa protein sequence, the molecular weight of the protein is 69.0769kDa, and the isoelectric point pI=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 in SEQ ID NO.3 and SEQ ID NO.4, respectively.
In a fourth aspect of the present invention, there is provided a recombinant expression vector comprising 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, and the base sequence of the target gene is shown as SEQ ID NO. 1.
Further, the promoter that promotes the expression of the target gene is a 35S strong promoter.
Further, the original carrier is pH2GW7.
Further, construction of the recombinant expression vector: the barley HvSERK1 gene was ligated into the plant expression vector pH2GW7 using Gateway system (Invitrogen). The promoter for promoting the expression of the barley HvSERK1 gene 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 EHA105.
In a sixth aspect of the present invention, there is provided use of the above-mentioned barley HvSERK1 gene, a protein encoded by the above-mentioned barley HvSERK1 gene, the above-mentioned primer pair, the above-mentioned recombinant expression vector, or the above-mentioned transformant in plant breeding.
Further, the plant breeding is to regulate plant root hair growth.
Further, the plant is bred to promote plant root hair growth. In one embodiment of the invention, the plant is Arabidopsis thaliana.
The invention provides application of a barley HvSERK1 gene in regulating root hair growth, and the invention can obviously improve the root hair length of arabidopsis through cloning the barley HvSERK1 gene and carrying out genetic transformation in arabidopsis, and over-expressing an exogenous barley HvSERK1 gene. Therefore, hvSERK1 is expected to be used for genetic engineering breeding, and is expected to improve the capability of barley for absorbing water and nutrients by introducing the HvSERK1 into short root hair barley varieties. The invention provides theoretical basis and related genes for drought-resistant breeding and production of barley.
The invention has the advantages that:
according to the invention, through cloning and analyzing the barley HvSERK1 gene and combining with the phenotype analysis of the Arabidopsis, the barley HvSERK1 gene can obviously increase the root hair length of the Arabidopsis. The invention provides theoretical basis and related genes for breeding and production of barley long root hairs.
Drawings
FIG. 1 shows the expression level of HvSERK1 gene in an Arabidopsis transgenic material over-expressing HvSERK1 gene according to the invention.
FIG. 2 shows that the root hair length of the Arabidopsis transgenic material overexpressing the HvSERK1 gene is significantly increased.
Detailed Description
The following provides a detailed description of specific embodiments of the invention with reference to the examples and figures.
Example 1: construction of barley HvSERK1 gene overexpression vector and genetic transformation of arabidopsis thaliana
(1) RNA was extracted from barley "flower 30" young embryos, and Trizol method (Invitrogen) was used, and the procedure was performed according to instructions. 1. Mu.g of RNA was subjected to reverse transcription to obtain cDNA.
(2) Designing specific primers according to the sequence of the HvSERK1 gene, and adding restriction enzyme cutting sites at the 5' end of the primers respectively, wherein the sequence is 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 full-length cDNA as a template.
A KOD high-fidelity enzyme (TOYOBO) system is adopted:
Figure BDA0003323125890000041
sterilized distilled water was added to a total volume of 50. Mu.L.
The PCR cycle 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 extension at 68℃for 5min, the reaction was terminated.
(4) After the PCR product is recovered by running gel, the PCR product is connected to a Gateway cloning system entry vector NT007 through enzyme digestion, escherichia coli DH5 alpha is transformed, positive clones are 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, which was subjected to LR exchange reaction with the overexpression vector pH2GW7.
Using Gateway LR Clonase TM Enzyme Mix (Invitrogen corporation, cat. No. 11791-019), the system was as follows:
Figure BDA0003323125890000042
vortex twice briefly, slightly centrifuge, water bath at 25℃for 2h, then add 1. Mu. L Proteinase K solution (proteinase K solution) and mix, leave at 37℃for 10 min to finish the reaction.
And 5. Mu.L of the reaction product is transformed into escherichia coli DH5 alpha, PCR is selected to verify positive clones, the positive clones are sequenced and verified, and plasmids are extracted after the positive clones are sequenced correctly and named 35S: hvSERK1.
(6) LBA4404 competent cells were removed from-80℃refrigerator and Agrobacterium LBA4404 was transformed by heat shock.
Thawing competent cells on ice, adding 5 mu L of target plasmid into competent cells, mixing, and placing on ice for 30min; the tube was frozen in liquid nitrogen for 1min, then placed in a 37 ℃ water bath until melted. 1mL of LB liquid nutrient medium was added for shaking culture for 2-3 hours, and after centrifugation, the culture was spread on a resistant YEP plate containing 50mg/L of Rif (rifampicin) and 50mg/L of Spec (spectinomycin hydrochloride), and the culture was inverted at 28℃for 2 nights until single colonies were grown.
(7) The agrobacterium positive monoclonal after PCR verification is picked up and cultured for 48 hours at 28 ℃ in 5ml YEP liquid culture medium containing corresponding antibiotics. Adding the shaken bacteria into YEP liquid culture medium (containing corresponding antibiotics) according to the volume ratio of 1:50, and culturing at 28deg.C to OD 600 ＝1.0-1.2。
(8) Arabidopsis thaliana was transformed by the Arabidopsis thaliana flower dipping method.
The whole arabidopsis plant with the pod removed completely and only the inflorescence left is reversely buckled in the bacterial liquid of the transformed agrobacterium together with the plug tray, the seedling is immersed for 5min, and the bacterial liquid is continuously shaken during the period;
after infection, taking out plants, placing the plants in a tray, covering with dark black plastic cloth, placing the plants in an incubator, and uncovering the incubator after 24 hours;
culturing Arabidopsis plant under natural illumination, watering 1-2 times per week, and harvesting T after seed is mature 0 Seed generation.
Example 2: screening of barley HvSERK1 over-expression transgenic lines
(1) T to be harvested 0 After the seeds are sterilized and disinfected, the seeds are planted in a solid culture medium of MS+35mg/L hygromycin, after vernalization for 3d in the dark at 4 ℃, a culture dish is placed in an artificial climate box for culture, and the growth condition of arabidopsis plants is observed. Transgenic seeds that are hygromycin resistant will grow in the screening medium, rather than the transgenic seeds will not grow after germination.
(2) Transplanting the selected transgenic plants into nutrient soil, cutting about 100mg of Arabidopsis leaves when the plants are bolting, placing the Arabidopsis leaves into a 1.5ml centrifuge tube, extracting DNA, carrying out PCR verification, and detecting whether the selected plants are positive.
(3) Harvesting T of single plant of plant positive to detection 1 After seed generation, sterilizing and disinfecting, and planting in MS+35mg/L hygromycinIn a solid culture medium, after vernalization for 3d in darkness at 4 ℃, the culture dish is placed in a climatic chamber for culture.
(4) Transgenic plants with hygromycin resistance are transplanted into nutrient soil. When the plants are bolting, about 100mg of Arabidopsis leaves are cut out from each plant line to extract RNA, and cDNA is obtained by reverse transcription. And performing fluorescence quantitative PCR analysis on the expression of HvSERK1 genes in different Arabidopsis transgenic lines by using SYBR green and a PCR instrument, and correcting the expression values by using an internal reference gene TUB 2.
The fluorescent quantitative PCR system is as follows:
Figure BDA0003323125890000061
the PCR procedure was: 95℃1min,95℃5s,60℃20s,40 cycles. Each set of experiments 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 amounts of HvSERK1 expressed in the different Arabidopsis transgenic lines are shown in FIG. 1. Selecting 3 strains 35S:HvSERK1-4, 35S:HvSERK1-7 and 35S:HvSERK1-9 with highest expression level, and harvesting T 2 Seed generation.
Example 3: phenotype observation of Arabidopsis thaliana root
(1) Wild type Arabidopsis thaliana and T 2 The transgenic seeds of generation HvSERK1 (35S: hvSERK1-4, 35S: hvSERK1-7, 35S: hvSERK 1-9) are inoculated on MS solid culture medium after sterilization and disinfection, and after vernalization for 3d in dark at 4 ℃, the culture dish is vertically cultivated and placed in a climatic chamber for cultivation (temperature: 20+/-2 ℃ C.; light intensity: 90-120 mu molm) -2 sec -1 The method comprises the steps of carrying out a first treatment on the surface of the Photoperiod: 16h light/8 h dark).
(2) After 7d cultivation in a climatic chamber, the root hair phenotype with the root tip up to about 4mm was photographed with a split microscope.
(3) Statistical analysis was performed on root hair length using Image pro plus software, with the longest 20 roots measured per root and at least 20 roots per strain. As shown in fig. 2. As a result, it was found that overexpression of HvSERK1 was able to promote root hair growth.
While the preferred embodiments of the present invention have been illustrated and described, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by one skilled in the art without departing from the spirit of the present invention, and these equivalent modifications and substitutions are intended to be included in the scope of the present invention as defined in the appended claims.
SEQUENCE LISTING
<110> Shanghai national academy of sciences of agriculture
<120> application of barley receptor kinase HvSERK1 in root hair growth
<130> /
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 1884
<212> DNA
<213> barley (Hordeum vulgare)
<400> 1
atggctgcgt cgccggagat gctgcggagg tgctggtggg cggcggcggc ggtcctgtca 60
ctggtgctgg ccgtcagtcg ggtggccgcc aacacagagg gtgatgctct gtacagtctg 120
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
attccacagt ccttgaccaa cattagcacc ctccaagttc tggatctatc aaacaacaat 540
ctctctggag aggttccatc aactggctcg ttttcactct ttacccctat aagttttggt 600
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
aacctgcttc gactccgtgg attctgtatg acacctacag aacggctact agtctatcca 1140
tacatggcta atggtagcgt tgcatcacgt ttgcgagagc gagggccaaa tgagccagct 1200
cttgagtggg aaaagagaac tcggatcgca ctgggatctg ccagaggact gtcttacttg 1260
catgatcact gtgatcccaa gatcattcat cgtgatgtca aagctgcaaa cattctcttg 1320
gatgaagact ttgaggcggt tgtgggcgac tttggactgg ccaagcttat ggactacaag 1380
gacactcatg taaccacagc tgtccgtgga acgatcgggc acattgctcc cgagtaccta 1440
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
<210> 2
<211> 627
<212> PRT
<213> barley (Hordeum vulgare)
<400> 2
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Gly Ala Ile Ala Gly Gly Val Ala Ala Gly Ala Ala Leu Ile Phe Ala
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Leu Ala Asn Asp Asp Asp Val Met Leu Leu Asp Trp Val Lys Gly Leu
515 520 525
Leu Lys Glu Lys Lys Val Glu Met Leu Val Asp Pro Asp Leu Gln Ser
530 535 540
Val Tyr Val Glu His Glu Val Glu Ala Leu Ile Gln Val Ala Leu Leu
545 550 555 560
Cys Thr Gln Gly Ser Pro Met Asp Arg Pro Lys Met Ser Glu Val Val
565 570 575
Arg Met Leu Glu Gly Asp Gly Leu Ala Glu Arg Trp Glu Glu Trp Gln
580 585 590
Lys Val Glu Val Val Arg Gln Glu Ala Glu Leu Ala Pro Arg His Asn
595 600 605
Asp Trp Ile Val Asp Ser Thr Phe Asn Leu Arg Ala Val Glu Leu Ser
610 615 620
Gly Pro Arg
625
<210> 3
<211> 25
<212> DNA
<213> Artificial sequence (Artifical)
<400> 3
ccatcgatct gcggctttga gacgg 25
<210> 4
<211> 28
<212> DNA
<213> Artificial sequence (Artifical)
<400> 4
cccccgggag aaattctgca acgagggg 28
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artifical)
<400> 5
cttggcagac ggaactcttg 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (Artifical)
<400> 6
tctcggtcgg tgtcatacag 20
<210> 7
<211> 21
<212> DNA
<213> Artificial sequence (Artifical)
<400> 7
gttctcgatg ttgttcgtaa g 21
<210> 8
<211> 21
<212> DNA
<213> Artificial sequence (Artifical)
<400> 8
tgtaaggctc aaccacagta t 21

Claims (4)

1. The application of a recombinant expression vector containing a barley HvSERK1 gene in promoting the growth of Arabidopsis root hair is characterized in that 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; the promoter for promoting the expression of the target gene is a 35S strong promoter.
2. The use according to claim 1, wherein the original carrier is pH2GW7.
3. Use of a transformant containing a recombinant expression vector of barley HvSERK1 gene for promoting growth of root hairs of arabidopsis; 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; the promoter for promoting the expression of the target gene is a 35S strong promoter.
4. The use according to claim 3, wherein the host bacterium of the transformant is Agrobacterium GV3101, LBA4404, AGL1 or EHA105.
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CN108085334B (en) * 2016-11-17 2021-04-30 上海市农业科学院 Improved method for transforming barley microspore by agrobacterium
CN109280671B (en) * 2018-09-07 2021-06-01 南京农业大学 Wheat cell wall related receptor protein kinase gene and expression vector and application thereof
CN109652424B (en) * 2018-12-27 2019-11-08 山东农业大学 A kind of methods and applications that somatic embryos directly occur
CN110128516B (en) * 2019-04-16 2022-04-22 扬州大学 Barley moisture-resistant regulatory gene HvERF2.11, protein and application thereof in breeding
CN110129291B (en) * 2019-04-16 2021-06-08 扬州大学 Barley moisture-resistant regulatory gene HvACO1, protein and application thereof in breeding
WO2021015616A1 (en) * 2019-07-22 2021-01-28 Wageningen Universiteit Lrr-rlkii receptor kinase interaction domains
CN110564737A (en) * 2019-09-10 2019-12-13 上海市农业科学院 Transcription factor HvTTG1 derived from barley genotype SLB and application thereof
CN110982835A (en) * 2019-11-14 2020-04-10 上海市农业科学院 Method for reducing callus pollution of barley and highland barley microspores in agrobacterium transformation process

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