CN115873891B - Dendrobium candidum DoObgC and application of alternative spliceosome thereof in promotion of hypocotyl elongation - Google Patents

Dendrobium candidum DoObgC and application of alternative spliceosome thereof in promotion of hypocotyl elongation Download PDF

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CN115873891B
CN115873891B CN202210946951.2A CN202210946951A CN115873891B CN 115873891 B CN115873891 B CN 115873891B CN 202210946951 A CN202210946951 A CN 202210946951A CN 115873891 B CN115873891 B CN 115873891B
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doobgc
dendrobium candidum
spliceosome
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hypocotyl
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CN115873891A (en
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陈稷
田孟良
罗新
万健
黄进
梁欢
刘帆
张浪
李林红
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Sichuan Agricultural University
Chengdu Univeristy of Technology
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Chengdu Univeristy of Technology
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Abstract

The invention discloses an application of dendrobium candidum DoObgC and an alternative spliceosome thereof in promoting hypocotyl elongation. The nucleotide sequence of dendrobium candidum DoObgC is KT359612.1, and the nucleotide sequence of the alternative spliceosome is any one sequence shown as SEQ ID NO. 1-5. The invention provides dendrobium candidum DoObgC and an alternative spliceosome thereof, the length of the arabidopsis hypocotyl of the overexpression dendrobium candidum DoObgC is higher than that of a wild plant under a dark condition, and the length of the arabidopsis hypocotyl of the overexpression dendrobium candidum DoObgC alternative spliceosome is also obviously higher than that of the wild plant.

Description

Dendrobium candidum DoObgC and application of alternative spliceosome thereof in promotion of hypocotyl elongation
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an application of dendrobium candidum DoObgC and an alternative spliceosome thereof in promotion of hypocotyl elongation.
Background
Dendrobium officinale is a traditional rare Chinese medicinal material in China, and in recent years, the demands of the domestic dendrobium officinale are increasing increasingly, but the natural fertility of the dendrobium officinale is low, and wild resources are endangered. In order to meet the huge market demand of dendrobium, under-forest and rock wall epiphytic wild-like cultivation techniques become a novel cultivation mode of dendrobium candidum. However, the growth of the dendrobium candidum and the accumulation of the effective components can be influenced by different conditions such as illumination, temperature and humidity, water and fertilizer under different cultivation modes.
The dendrobium candidum is used as an animate plant, the high illumination intensity can cause oxidative damage to the dendrobium candidum, the too weak illumination can slow down the growth and component accumulation of the dendrobium candidum, different illumination conditions can regulate and control the growth and development of the dendrobium candidum, polysaccharide accumulation, biosynthesis of secondary metabolites such as flavonoids and the like, so that the quality of the dendrobium candidum is influenced, and the visible illumination condition is one of key factors influencing the quality of the dendrobium candidum.
Dendrobium officinale is a facultative rhodiola acid metabolism (CAM) plant, and under normal conditions, photosynthesis proceeds by the C3 pathway, and under stress conditions, photosynthesis proceeds by the CAM pathway. Excessive illumination can trigger a light inhibition mechanism, and then the photosynthetic pathway of dendrobium candidum is converted into a CAM pathway. Conversely, the short light-dark period can induce the facultative CAM pathway to be converted into a separate C3 pathway, and can promote biomass accumulation and morphogenesis of dendrobium candidum.
Bacterial Obg is a Spo 0B-related GTP binding protein, a gtpase essential for bacterial survival, which is also well conserved in eukaryotes. Bacterial Obg studies have shown that a variety of obg mutants affect processes such as cell growth, morphological differentiation, sporulation, ribosome biosynthesis, chromosome segregation, regulation of DNA replication and repair processes, and stress response.
Plant ObgC is located in chloroplasts and orthologous to bacteria Obg, and AtObgC was found to be a necessary key gene in the early stages of embryogenesis in Arabidopsis. Also, obgC plays an important role in ribosome biosynthesis during chloroplast development. Plant ObgC protein consists of four domains: n-terminal chloroplast transit peptide, obg fold domain, gtpase domain, OCT (C-terminal domain of Obg). ObgC can be positioned in chloroplasts only when the N-terminal chloroplast transit peptide functional sequence is complete; the Obg folding domain is involved in ObgC binding to the ribosomal large subunit in the chloroplast; the GTPase domain can be combined with GTP/GDP protein, has GTP hydrolysis function, can influence the function of Obg folding domain, and can regulate and control the generation of chloroplast kernel glycomer; the C-terminal domain of ObgC plays an important role in the ppGpp signaling pathway of plant chloroplasts. Thus, different ObgC protein domains may have different effects on the normal development of chloroplasts. However, there is no report about the role of ObgC and its alternative spliceosome in hypocotyl elongation.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an application of dendrobium candidum DoObgC and an alternative spliceosome thereof in promoting the elongation of hypocotyls, and the provided dendrobium candidum DoObgC gene or the alternative spliceosome thereof can effectively promote the growth of hypocotyls.
In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
The application of dendrobium candidum DoObgC in promoting plant hypocotyl elongation is provided, wherein the sequence number of the dendrobium candidum DoObgC nucleotide is KT359612.1.
An alternative spliceosome of dendrobium candidum DoObgC, the nucleotide sequence of which is any one sequence shown in SEQ ID NO. 1-5.
The protein sequence coded by the alternative spliceosome is shown as SEQ ID NO. 6-10.
The nucleotide sequence of the alternative spliceosome and the specific sequence of the protein encoded by the same are as follows:
Alternative spliceosome D2E:
ATGGCGTGTGCATCCGTAGCTCTCTCCTGCTACATTCTCAGTGCTGGCAGCAGCCATAAGTCCAGAAAAAGTAGAAAATTTTATCGCGAAAAACCTAAACCCTCTGATTCTCTTCACTTTCCTCCGGCCATTCAGTCCGCCGGCGGGGAGGCTACTTCTTACACCCGGCTTCCCTTAAAGGACGACTTCCAACTCGATCTTTCTTCGGGTCTCTCTACTCCATTTTATGACGTCATCAAGCTCCGCGAAGCGGCAGTCCTGGCCGTGGATTCTCGGTCGAAAAAGCCTTCTAAACCCACAGGTTTTTTGGAAGAGATCGGTGAAGGATACGACGTAGAAGAAGAGTTTCTTGGATTTGACTCGGATGATGATGATGAGGTCATCTTTGGAATTGAGTCGGACGACGTTGGCGTGGAAGGGCTCGATGAGGACGAGGAGTTGAAGGAGGATGATGTCAAGGAGAAAGGTGTGCCTGCGGTTATGCGGTGCTTCGATCGAGCAAAGATTTACGTGAAGGCTGGGGATGGAGGTAACGGAGTCGTGGCGTTCCGGAGAGAGAAATATGTGCCCTATGGCGGCCCTTCTGGCGGGGACGGTGGTAAAGGAGGCGATGTTTATGTGGAGGTTGATGGGTCGATGAATTCCCTTCTTCCTTTTAGGAAAAATGTCCACTTTAGGGCTGGGAGAGGCGGGCATGGCCTGGGGAAGAAGCAGGCAGGGGGGAAGGGAGAGGATGTAGTGGTGAAGGTGGCTCCAGGGACTGTCATCAGGGAATCGGGGAAGGGACCAGCGGAAGGGGTGTTGTTACTGGAGCTGTTGAATCCTGGACAAAGAGCATTGCTTTTGCCCGGTGGAAGGGGCGGGAGGGGTAATGCTTCATTCAAGACAGGGACAAGGAAGGCCCCAAAGATTGCAGAGAACGGGGAAGCTGGCGCTGAAATGTGCATGTTGTTGATGGTTCCAGTCAACGGCCAGATTATGAGTTTGATGCAGTTCGTTTGGAACTGGAACTATTTAGCCCGGGGTTGTCAGAAAAGCCATACATTGTTGCATTTAATAAAATGGATCTTCCAGAAGCATATGAAAAATGGCATTCATTTAAAGAATATCTTAAAAACCGTGGAATTGAGCCTTTATGCATGA(SEQ ID NO.1)
Protein encoded by alternative spliceosome D2E ：MACASVALSCYILSAGSSHKSRKSRKFYREKPKPSDSLHFPPAIQSAGGEATSYTRLPLKDDFQLDLSSGLSTPFYDVIKLREAAVLAVDSRSKKPSKPTGFLEEIGEGYDVEEEFLGFDSDDDDEVIFGIESDDVGVEGLDEDEELKEDDVKEKGVPAVMRCFDRAKIYVKAGDGGNGVVAFRREKYVPYGGPSGGDGGKGGDVYVEVDGSMNSLLPFRKNVHFRAGRGGHGLGKKQAGGKGEDVVVKVAPGTVIRESGKGPAEGVLLLELLNPGQRALLLPGGRGGRGNASFKTGTRKAPKIAENGEAGAEMCMLLMVPVNGQIMSLMQFVWNWNYLARGCQKSHTLLHLIKWIFQKHMKNGIHLKNILKTVELSLYA*(SEQ ID NO.6)
Alternative spliceosome D3E:
ATGGCGTGTGCATCCGTAGCTCTCTCCTGCTACATTCTCAGTGCTGGCAGCAGCCATAAGTCCAGAAAAAGTAGAAAATTTTATCGCGAAAAACCTAAACCCTCTGATTCTCTTCACTTTCCTCCGGCCATTCAGTCCGCCGGCGGGGAGGCTACTTCTTACACCCGGCTTCCCTTAAAGGACGACTTCCAACTCGATCTTTCTTCGGGTCTCTCTACTCCATTTTATGACGTCATCAAGCTCCGCGAAGCGGCAGTCCTGGCCGTGGATTCTCGGTCGAAAAAGCCTTCTAAACCCACAGGTTTTTTGGAAGAGATCGGTGAAGGATACGACGTAGAAGAAGAGTTTCTTGGATTTGACTCGGATGATGATGATGAGGTCATCTTTGGAATTGAGTCGGACGACGTTGGCGTGGAAGGGCTCGATGAGGACGAGGAGTTGAAGGAGGATGATGTCAAGGAGAAAGGTGTGCCTGCGGTTATGCGGTGCTTCGATCGAGCAAAGATTTACGTGAAGGCTGGGGATGGAGGTAACGGAGTCGTGGCGTTCCGGAGAGAGAAATATGTGCCCTATGGCGGCCCTTCTGGCGGGGACGGTGGTAAAGGAGGCGATGTTTATGTGGAGGTTGATGGGTCGATGAATTCCCTTCTTCCTTTTAGGAAAAATGTCCACTTTAGGGCTGGGAGAGGCGGGCATGGCCTGGGGAAGAAGCAGGCAGGGGGGAAGGGAGAGGATGTAGTGGTGAAGGTGGCTCCAGGGACTGTCATCAGGGAATCGGGGAAGGGACCAGCGGAAGGGGTGTTGTTACTGGAGCTGTTGAATCCTGGACAAAGAGCATTGCTTTTGCCCGGTGGAAGGGGCGGGAGGGGTAATGCTTCATTCAAGACAGGGACAAGGAAGGCCCCAAAGATTGCAGAGAACGGGGAAGCTGGCGCTGAAATGTGGTTGGACCTGGAGTTAAAGTTGGTTGCTGATGTTGGTATAGTAGGTGCTCCAAATGCTGGGAAAAGCACACTTTTGAGTGCTATAACTGCTGCTCAGCCATCAATAGCTGATTATCCCTTCACCACACTACTTCCAAATTTAGGTGTGGTGTCAATGGATTATGATGAAACCATGGTTGTTGCAGATTTGCCAGGTTTGCTTGAAGGTGCACATAAAGGTTTTGGTTTAGGGCATGAATTTCTACGGCATACTGAAAGATGTGCTGTGCTGAAGAATGGGCGCAGTCAGAAAATCTAA(SEQ ID NO.2)
Alternatively, the spliceosome D3E encodes a protein:
MACASVALSCYILSAGSSHKSRKSRKFYREKPKPSDSLHFPPAIQSAGGEATSYTRLPLKDDFQLDLSSGLSTPFYDVIKLREAAVLAVDSRSKKPSKPTGFLEEIGEGYDVEEEFLGFDSDDDDEVIFGIESDDVGVEGLDEDEELKEDDVKEKGVPAVMRCFDRAKIYVKAGDGGNGVVAFRREKYVPYGGPSGGDGGKGGDVYVEVDGSMNSLLPFRKNVHFRAGRGGHGLGKKQAGGKGEDVVVKVAPGTVIRESGKGPAEGVLLLELLNPGQRALLLPGGRGGRGNASFKTGTRKAPKIAENGEAGAEMWLDLELKLVADVGIVGAPNAGKSTLLSAITAAQPSIADYPFTTLLPNLGVVSMDYDETMVVADLPGLLEGAHKGFGLGHEFLRHTERCAVLKNGRSQKI*(SEQ ID NO.7)
alternative spliceosome D2ED3E:
ATGGCGTGTGCATCCGTAGCTCTCTCCTGCTACATTCTCAGTGCTGGCAGCAGCCATAAGTCCAGAAAAAGTAGAAAATTTTATCGCGAAAAACCTAAACCCTCTGATTCTCTTCACTTTCCTCCGGCCATTCAGTCCGCCGGCGGGGAGGCTACTTCTTACACCCGGCTTCCCTTAAAGGACGACTTCCAACTCGATCTTTCTTCGGGTCTCTCTACTCCATTTTATGACGTCATCAAGCTCCGCGAAGCGGCAGTCCTGGCCGTGGATTCTCGGTCGAAAAAGCCTTCTAAACCCACAGGTTTTTTGGAAGAGATCGGTGAAGGATACGACGTAGAAGAAGAGTTTCTTGGATTTGACTCGGATGATGATGATGAGGTCATCTTTGGAATTGAGTCGGACGACGTTGGCGTGGAAGGGCTCGATGAGGACGAGGAGTTGAAGGAGGATGATGTCAAGGAGAAAGGTGTGCCTGCGGTTATGCGGTGCTTCGATCGAGCAAAGATTTACGTGAAGGCTGGGGATGGAGGTAACGGAGTCGTGGCGTTCCGGAGAGAGAAATATGTGCCCTATGGCGGCCCTTCTGGCGGGGACGGTGGTAAAGGAGGCGATGTTTATGTGGAGGTTGATGGGTCGATGAATTCCCTTCTTCCTTTTAGGAAAAATGTCCACTTTAGGGCTGGGAGAGGCGGGCATGGCCTGGGGAAGAAGCAGGCAGGGGGGAAGGGAGAGGATGTAGTGGTGAAGGTGGCTCCAGGGACTGTCATCAGGGAATCGGGGAAGGGACCAGCGGAAGGGGTGTTGTTACTGGAGCTGTTGAATCCTGGACAAAGAGCATTGCTTTTGCCCGGTGGAAGGGGCGGGAGGGGTAATGCTTCATTCAAGACAGGGACAAGGAAGGCCCCAAAGATTGCAGAGAACGGGGAAGCTGGCGCTGAAATAAGAATGGGCGCAGTCAGAAAATCTAAATCATGTTGCTGA(SEQ ID NO.3)
Alternatively, the spliceosome D2ED3E encodes a protein:
MACASVALSCYILSAGSSHKSRKSRKFYREKPKPSDSLHFPPAIQSAGGEATSYTRLPLKDDFQLDLSSGLSTPFYDVIKLREAAVLAVDSRSKKPSKPTGFLEEIGEGYDVEEEFLGFDSDDDDEVIFGIESDDVGVEGLDEDEELKEDDVKEKGVPAVMRCFDRAKIYVKAGDGGNGVVAFRREKYVPYGGPSGGDGGKGGDVYVEVDGSMNSLLPFRKNVHFRAGRGGHGLGKKQAGGKGEDVVVKVAPGTVIRESGKGPAEGVLLLELLNPGQRALLLPGGRGGRGNASFKTGTRKAPKIAENGEAGAEIRMGAVRKSKSCC*(SEQ ID NO.8)
alternative spliceosome D2Ep3E:
ATGGCGTGTGCATCCGTAGCTCTCTCCTGCTACATTCTCAGTGCTGGCAGCAGCCATAAGTCCAGAAAAAGTAGAAAATTTTATCGCGAAAAACCTAAACCCTCTGATTCTCTTCACTTTCCTCCGGCCATTCAGTCCGCCGGCGGGGAGGCTACTTCTTACACCCGGCTTCCCTTAAAGGACGACTTCCAACTCGATCTTTCTTCGGGTCTCTCTACTCCATTTTATGACGTCATCAAGCTCCGCGAAGCGGCAGTCCTGGCCGTGGATTCTCGGTCGAAAAAGCCTTCTAAACCCACAGGTTTTTTGGAAGAGATCGGTGAAGGATACGACGTAGAAGAAGAGTTTCTTGGATTTGACTCGGATGATGATGATGAGGTCATCTTTGGAATTGAGTCGGACGACGTTGGCGTGGAAGGGCTCGATGAGGACGAGGAGTTGAAGGAGGATGATGTCAAGGAGAAAGGTGTGCCTGCGGTTATGCGGTGCTTCGATCGAGCAAAGATTTACGTGAAGGCTGGGGATGGAGGTAACGGAGTCGTGGCGTTCCGGAGAGAGAAATATGTGCCCTATGGCGGCCCTTCTGGCGGGGACGGTGGTAAAGGAGGCGATGTTTATGTGGAGGTTGATGGGTCGATGAATTCCCTTCTTCCTTTTAGGAAAAATGTCCACTTTAGGGCTGGGAGAGGCGGGCATGGCCTGGGGAAGAAGCAGGCAGGGGGGAAGGGAGAGGATGTAGTGGTGAAGGTGGCTCCAGGGACTGTCATCAGGGAATCGGGGAAGGGACCAGCGGAAGGGGTGTTGTTACTGGAGCTGTTGAATCCTGGACAAAGAGCATTGCTTTTGCCCGGTGGAAGGGGCGGGAGGGGTAATGCTTCATTCAAGACAGGGACAAGGAAGGCCCCAAAGATTGCAGAGAACGGGGAAGCTGGCGCTGAAATGTGGTTGGACCTGGAGTTAAAGTTGGTTGCTGATGTTGGTATAGTAGGTGCTCCAAATGCTGGGAAAAGCACACTTTTGAGTGCTATAACTGCTGCTCAGCCATCAATAGCTGATTATCCCTTCACCACACTACTTCCAAATTTAGGTGTGGTGTCAATGGATTATGATGAAACCATGGTTGAAGAATGGGCGCAGTCAGAAAATCTAAATCATGTTGCTGATTCCATAGAAAAGCAAAGAAGTGCCTCTATGAATGATTTTGAAATATTTCATGAAAGTAGTTCAAACACATGGCGTGTAGTTGGAGCTGGAATTGAACGATTTGTTCAGATGACAAATTGGCGGTATAATGAATCTCTTAGAAGGTTCCAACATGCTTTGGAGGCATGTGGTGTGAACAAAGCTTTGCGTAAAGGGGGTGTAAGAGAAGGAGATACAGTAATTGTGGGTGAGATGGAGATGGTTTGGAACGATGGGAATGAACAGCCAGGGCCTTCGGGCACaaaaaaaGGAGTATCTGGATCTGTTAGATGGCCTCAATTTGGTTAA(SEQ ID NO.4)
alternative spliceosome D2Ep3E encodes a protein:
MACASVALSCYILSAGSSHKSRKSRKFYREKPKPSDSLHFPPAIQSAGGEATSYTRLPLKDDFQLDLSSGLSTPFYDVIKLREAAVLAVDSRSKKPSKPTGFLEEIGEGYDVEEEFLGFDSDDDDEVIFGIESDDVGVEGLDEDEELKEDDVKEKGVPAVMRCFDRAKIYVKAGDGGNGVVAFRREKYVPYGGPSGGDGGKGGDVYVEVDGSMNSLLPFRKNVHFRAGRGGHGLGKKQAGGKGEDVVVKVAPGTVIRESGKGPAEGVLLLELLNPGQRALLLPGGRGGRGNASFKTGTRKAPKIAENGEAGAEMWLDLELKLVADVGIVGAPNAGKSTLLSAITAAQPSIADYPFTTLLPNLGVVSMDYDETMVEEWAQSENLNHVADSIEKQRSASMNDFEIFHESSSNTWRVVGAGIERFVQMTNWRYNESLRRFQHALEACGVNKALRKGGVREGDTVIVGEMEMVWNDGNEQPGPSGTKKGVSGSVRWPQFG*(SEQ ID NO.9)
alternative splice body 2Ein:
ATGGCGTGTGCATCCGTAGCTCTCTCCTGCTACATTCTCAGTGCTGGCAGCAGCCATAAGTCCAGAAAAAGTAGAAAATTTTATCGCGAAAAACCTAAACCCTCTGATTCTCTTCACTTTCCTCCGGCCATTCATTCCGCCGGCGGGGAGGCTACTTCTTACACCCGGCTTCCCTTAAAGGACGACTTCCAACTCGATCTTTCTTCGGGTCTCTCTACTCCATTTTATGACGTCATCAAGCTCCGCGAAGCGGCAGTCCTGGCCGTGGATTCTCGGTCGAAAAAGCCTTCTAAACCCACAGGTTTTTTGGAAGAGATCGGTGAAGGATACGACGTAGAAGAAGAGTTTCTTGGATTTGACTCGGATGATGATGATGAGGTCATCTTTGGAATTGAGTCGGACGACGTTGGCGTGGAAGGGCTCGATGAGGACGAGGAGTTGAAGGAGGATGATGTCAAGGAGAAAGGTGTGCCTGCGGTTATGCGCTGCTTCGATCGAGCAAAGATTTACGTGAAGGCTGGGGATGGAGGTAACGGAGTCGTGGCGTTCCGGAGAGAGAAATATGTGCCCTATGGCGGCCCTTCTGGCGGGGACGGTGGTAAAGGAGGCGATGTTTATGTGGAGGTTGATGGGTCGATGAATTCCCTTCTTCCTTTTAGGAAAAATGTCCACTTTAGGGCTGGGAGAGGCGGGCATGGCCTGGGGAAGAAGCAGGCAGGGGGGAAGGGAGAGGATGTAGTGGTGAAGGTGGCTCCAGGGACTGTCATCAGGGAATCGGGGAAGGGACCAGCGGAAGGGGTGTTGTTACTGGAGCTGTTGAATCCTGGACAAAGAGCATTGCTTTTGCCCGGTGGAAGGGGCGGGAGGGGTAATGCTTCATTCAAGACAGGGACAAGGAAGGCCCCAAAGATTGCAGAGAACGGGGAAGCTGGCGCTGAAATGTGGTTGGACCTGGAGTTAAAGTTGGTTGCTGATGTTGGTATAGTAGGTGCTCCAAATGCTGGGAAAAGCACACTTTTGAGTGCTATAACTGCTGCTCAGCCATCAATAGCTGATTATCCCTTCACCACACTACTTCCAAATTTAGGTGTGGTGTCAATGGATTATGATGAAACCATGGTTGTTGCAGATTTGCCAGGTTTGCTTGAAGGTGCACATAAAGGTTTTGGTTTAGGGCATGAATTTCTACGGCATACTGAAAGATGTGCTGTGCTGGTATGCTATATTTCTTGCAAGTATCTTCCCTGTGTTGCACTGGAACTCAATCATTTCTAGAGGTTCTCAACTTTATATATAATGCAGAAAGAATCTAATTTAGCT(SEQ ID NO.5)
alternatively, splice 2Ein encodes a protein:
MACASVALSCYILSAGSSHKSRKSRKFYREKPKPSDSLHFPPAIHSAGGEATSYTRLPLKDDFQLDLSSGLSTPFYDVIKLREAAVLAVDSRSKKPSKPTGFLEEIGEGYDVEEEFLGFDSDDDDEVIFGIESDDVGVEGLDEDEELKEDDVKEKGVPAVMRCFDRAKIYVKAGDGGNGVVAFRREKYVPYGGPSGGDGGKGGDVYVEVDGSMNSLLPFRKNVHFRAGRGGHGLGKKQAGGKGEDVVVKVAPGTVIRESGKGPAEGVLLLELLNPGQRALLLPGGRGGRGNASFKTGTRKAPKIAENGEAGAEMWLDLELKLVADVGIVGAPNAGKSTLLSAITAAQPSIADYPFTTLLPNLGVVSMDYDETMVVADLPGLLEGAHKGFGLGHEFLRHTERCAVLVCYISCKYLPCVALELNHF*RFSTLYIMQKESNLA(SEQ ID NO.10)
Use of the above-described alternative spliceosome to promote elongation of plant hypocotyls.
An engineering bacterium comprises the dendrobium candidum DoObgC or the alternative spliceosome.
A plasmid comprising dendrobium candidum DoObgC or an alternative spliceosome as described above.
A recombinant expression vector comprising dendrobium candidum DoObgC or a variant spliceosome as described above.
A gene chip comprises the dendrobium candidum DoObgC or the alternative spliceosome.
A preparation for promoting plant hypocotyl elongation, comprising the protein encoded by dendrobium candidum DoObgC or the protein encoded by a variable spliceosome, or an agent for promoting expression of dendrobium candidum DoObgC or dendrobium candidum DoObgC variable spliceosome.
Further, the agent is siRNA, miRNA, shRNA, an antibody polypeptide, or a fusion protein.
The dendrobium candidum DoObgC or the dendrobium candidum DoObgC alternative spliceosome is applied to improving the quality or germplasm resources of the dendrobium candidum.
The invention has the beneficial effects that:
the invention provides dendrobium candidum DoObgC and an alternative spliceosome thereof, the length of the arabidopsis hypocotyl of the overexpression dendrobium candidum DoObgC is higher than that of a wild plant under a dark condition, and the length of the arabidopsis hypocotyl of the overexpression dendrobium candidum DoObgC alternative spliceosome is also obviously higher than that of the wild plant.
Drawings
FIG. 1 shows the DoObgC.AS gene structure;
FIG. 2 is a phenotypic observation after dark treatment of DoObgC.AS Arabidopsis over-expressed;
FIG. 3 is a comparison of the length of the hypocotyl of an Arabidopsis thaliana overexpressing DoObgC.AS;
FIG. 4 is a quantitative analysis of DoObgC.AS and AtObgC;
FIG. 5 shows the length of the hypocotyl after shading treatment of the DoObgC.AS Arabidopsis.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.
Example 1
1. RNA extraction and cDNA Synthesis
Extracting total RNA of dendrobium candidum leaves according to the operation instruction of an RNA extraction kit of Edley company, and synthesizing cDNA by taking RNA as a template according to the operation instruction of a reverse transcription kit of Noruzan company after product concentration measurement and PCR detection. After the reaction was completed, 180. Mu.L of ddH 2 O was added thereto for dilution and stored at-20℃for later use.
2. Primer design
CDS base sequences of arabidopsis AtObgC and NCBI are obtained through TAIR websites respectively, CDS base sequences of dendrobium candidum DoObgC are obtained through NCBI, and homologous recombination primers are designed for primer3Plus by using a primer design tool. The length of the fluorescent quantitative PCR primer is generally 20-24bp, the annealing temperature is 58-60 ℃, and the size of the product is 101-200bp. The primers used in the experiments are shown in Table 1:
TABLE 1 primer list
3. Amplification of target Gene
The Takara high-fidelity KOD DNA polymerase was used to design primer sequences DoObgC-F, doObgC-R (see Table 1), and the target gene fragments were amplified by PCR, the reaction system was shown in Table 2, to obtain different spliceosomes and DoObgC full-length sequences.
Among them, the spliceosomes include D2E (271 bp deletion of the second exon), D3E (289 bp deletion of the third exon), D2E3E (simultaneous deletion of the second and third exons), D2E3E (92 bp partial deletion of the 3' -end of the second exon and complete deletion of the third exon) and 2Ein (107 bp retention of the rear part of the second exon) (see FIG. 1).
TABLE 2 Gene cloning reaction System
After the PCR is finished, the reaction product is mixed with 6X LoadingDye, whether the size of the target band meets the expectations or not is detected by 1% agarose gel electrophoresis, and the target fragment with the correct size is recovered and stored according to the operation instructions of the Novain recovery kit.
EXAMPLE 2 construction of pMDC-DoObgC.AS expression vector
1. Construction of intermediate vector pENTR-DoObgC.AS
The gel recovery product was prepared according to pENTR TM direct from Invitrogen corporationThe Cloning kit was ligated to pENTR/D-TOPO vector, and the ligation reaction system is shown in Table 3. After connection, converting the product into escherichia coli DH5 alpha, performing bacterial inspection, extracting plasmids, performing enzyme digestion verification, detecting whether the size of a target band accords with the expected or not by 1% agarose gel electrophoresis, sequencing and comparing the plasmids after verification, and performing sequencing and comparison on the plasmids after verification, wherein the successfully constructed pENTR-DoobgC.AS plasmids and 30% glycerol are subjected to 600 mu L bacterial liquid; after mixing 400 mu L of glycerin, the mixture was frozen in liquid nitrogen and stored at-80℃for later use.
TABLE 3 ligation reaction System
2. Construction of pMDC-DoObgC.AS expression vector
The plasmid was extracted from the intermediate vector pENTR-Doobj C.AS which was constructed successfully, and then subjected to single digestion with PvuII, and after digestion at 37℃overnight, 1% agarose gel electrophoresis was performed to recover the target band of about 2.5 Kb. pMDC83 empty and gum recovery products were mixed for gateway reaction: after 2h at 25℃in a linker, proteinase K. Mu.L of the mixture was added, mixed and centrifuged, and after 10min at 37℃the product was placed on ice and transformed into E.coli DH 5. Alpha. After the transformation, the culture was performed on a plate with LB medium containing 50. Mu.g/mLKan in an ultra clean bench. And performing bacterial detection verification, propagating positive strains after the verification is successful, extracting plasmids, performing enzyme digestion electrophoresis detection, sequencing, and converting to agrobacterium after the verification is successful.
Table 4 enzyme digestion System
TABLE 5 gateway reaction system
3. Obtaining of overexpressing transgenic plants
PRC primers ObgC-P1, obgC-P2 and ObgC-P3 (see Table 1) are designed to detect the genotype of the Arabidopsis obgc-T mutant, the obtained agrobacterium transformed with the expression vector is further transformed into the Arabidopsis obgc-T heterozygous mutant, PCR detection is carried out on the obtained transgenic T0 generation plant to obtain a transgenic plant, the transgenic plant is propagated to the T3 generation, and the seed collection is used for the phenotype statistics of the light and dark treatment of the offspring.
Example 3
1. Light treatment of overexpressing doobgc.as arabidopsis mutants
(1) After transgenic over-expression arabidopsis is screened to the generation T3, wild type and over-expression DoobgC.AS heterozygous mutant arabidopsis seeds are sterilized in an ultra-clean workbench and sown on a 1/2MS solid culture medium for vernalization at 4 ℃ for 24 hours. And (5) carrying out dark culture for 6d after the seeds germinate, and measuring physiological indexes of wild type and over-expressed DoObgC.AS Arabidopsis after the culture is finished. The treated Arabidopsis thaliana was sampled to extract RNA, reverse transcribed, and then an internal reference gene primer AtUBQ was designed, and simultaneously primers AtUBQ-qRT-F/R, atObgC-qRT-F3/R3, doObgC-qRT-F1/R1, doD2E-qRT-F/R, and Do2Ein-qRT-F/R (see Table 1) were designed, and then fluorescent quantitative PCR was performed to detect the expression level of the relevant gene, and the experiment was repeated 3 times, with the result shown in FIG. 4.
Fluorescent quantitative PCR (10. Mu.L) reaction system: the cDNA is diluted 20 times and then detected; cDNA 2.5. Mu.L, qPCR Mastermix 5. Mu.L, primerF/primerR each 0.4. Mu.L, ddH 2 O1.7. Mu.L. Reaction conditions: pre-denaturation at 95℃for 2min; denaturation at 95℃for 15sec, annealing at 55℃for 15sec, elongation at 72℃for 20sec,39 cycles, denaturation at 95℃for 10sec. Repeated 3 times.
2. Light-dark induced DoObgC transcript gene structure
Inducing tissue culture Miao Yizhou of the dendrobium candidum at the age of three months at different illumination intensities (0μmol·m-2·s-1、20μmol·m-2·s-1、70μmol·m-2·s-1、120μmol·m-2·s-1), and taking dendrobium candidum leaves after illumination for 8 hours for transcriptome sequencing. It was found by analysis that a total of 4 alternative splice types were detected, including: D2E (273 bp deletion of the second exon), D3E (289 bp deletion of the third exon), D2E3E (simultaneous deletion of the second and third exons), D2Ep3E (92 bp partial deletion of the 3' end of the second exon and complete deletion of the third exon) and 2Ein (107 bp retention of the rear part of the second exon). As a result of cleavage, both the GTPase domain (GTPase), OCT deletion occurred in the protein structure of D2E, D3E, D2E3E, 2Ein, whereas D2Ep3E only deleted part of the GTPase (see fig. 1).
3. Effect of dark treatment on the growth of over-expressed DoObgC.AS Arabidopsis mutant
Under normal light conditions, there was no significant difference in hypocotyl length between wild type and overexpressed doobgc.as transgenic arabidopsis. In the dark condition, the length of the hypocotyl of the over-expressed Arabidopsis thaliana is higher than that of a wild type plant, and the lengths of the hypocotyls of the D3E, D E3E and 2Ein plants are obviously higher than that of the wild type plant, and the results are shown in figures 2 and 3, wherein the WT in figure 3 represents the wild type plant; do represents DoObgC full-length over-expression lines; D2E, D3E, D2E3E, D Ep3E, 2Ein represent the doobgc.as over-expression strain. Measurement was done by ImageJ software. Light: normal light growth 6d; dark: and the dark growth is carried out for 6d.
To verify the relationship between the difference in elongation of the hypocotyl of Arabidopsis after dark treatment and the expression level of Doobj C.AS, the expression levels of Arabidopsis Doobj C.AS and AtObgC were analyzed by fluorescent quantitative PCR (see FIG. 4).
As shown in FIG. 4, doObgC.AS has higher expression levels, while Arabidopsis itself AtObgC was significantly inhibited, indicating the hypocotyl elongation phenotype by DoObgC.AS, regardless of light or dark conditions.
4. Effect of shading on the growth of overexpressed doobgc.as arabidopsis mutants
The arabidopsis seeds of the same strain are subjected to low temperature treatment at 4 ℃ for 24 hours, are grown for 6d under shading conditions (R/fr=0.75), the hypocotyl length is observed, and the sampling and the preservation are carried out, and the result is shown in fig. 5, wherein WT in fig. 5 represents a wild type plant; do represents DoObgC full-length over-expression lines; D2E, D3E, D2E3E, D Ep3E, 2Ein represent the doobgc.as over-expression strain.
Shading is a common light environment in nature where the ratio of red to far-red light (R/FR) is reduced. When young plants are subjected to shading stress, the plants will receive more light through the elongation of the hypocotyl in order to adapt to the change of the external environment. As shown in fig. 5, the germinated wild type and doobgc.as overexpressing plants were grown under shading conditions (R/fr=0.75) for 6d, their phenotypes were observed and recorded. Under shading conditions, the arabidopsis has the phenomena of cotyledon petiole elongation and cotyledon enlargement, and the hypocotyl length of the DoobgC.AS over-expressed plant is obviously higher than that of a wild plant, which indicates DoObgC and an alternative splice body thereof can regulate and control the elongation of the hypocotyl of the over-expressed plant.

Claims (10)

1. The application of dendrobium candidum DoObgC in promoting elongation of embryonal axis of dendrobium candidum is characterized in that the sequence number of the dendrobium candidum DoObgC nucleotide is KT359612.1.
2. An alternative spliceosome of dendrobium candidum DoObgC is characterized in that the nucleotide sequence of the alternative spliceosome is any one sequence shown in SEQ ID NO. 1-5.
3. The protein encoded by the alternative spliceosome of claim 2, wherein the amino acid sequence of the protein is shown in SEQ ID nos. 6-10.
4. Use of the alternative spliceosome of claim 2 for promoting elongation of the embryonal axis of dendrobium candidum.
5. An engineered bacterium comprising the alternative spliceosome of claim 2.
6. A plasmid comprising the alternative spliceosome of claim 2.
7. A recombinant expression vector comprising the alternative spliceosome of claim 2.
8. A gene chip comprising the alternative spliceosome of claim 2.
9. A formulation for promoting elongation of the embryonal axis of dendrobium candidum, comprising a protein encoded by dendrobium candidum DoObgC of claim 1 or an alternatively spliced body of claim 2.
10. Use of the dendrobium candidum DoObgC of claim 1 or the dendrobium candidum DoObgC alternative spliceosome of claim to promote quality or germplasm resource improvement related to elongation of dendrobium candidum hypocotyl.
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