Lily carotenoid metabolism regulation gene LnCCD4a, protein, VIGS system and application

Abstract

The invention discloses a lilium carotenoid metabolism regulation gene LnCCD4a, a protein, a VIGS system and application, and belongs to the technical field of gene engineering. VIGS experiments prove that the LnCCD4a gene has the function of regulating and controlling the flower color of the lily. A specific fragment is selected based on a lily endogenous LnCCD4a gene sequence, a silent expression vector is constructed, and a target gene is silent in a lily film by using a VIGS technology. Results show that after the LnCCD4a gene is silenced, the content of carotenoid in the flower tablets is increased, and the flower color is changed from white to yellow. The invention provides a gene resource and a technical basis for regulating the lily color through a molecular breeding technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and plant molecular breeding, and particularly relates to a lily carotenoid metabolism regulating gene LnCCD4a, a protein, a VIGS system and applications. Background Art

[0002] Flower color is an important feature that determines the commercial value of flower crops. It can meet people's demand for diversified and personalized flower varieties and improve the ornamental and commercial value of ornamental plants. Breeding new varieties with new flower colors is an important goal in the field of ornamental plants. Traditional hybridization and selective breeding have played an important role in improving flower color, but it is difficult to prevent reproductive isolation. In addition, for ornamental plants with complex genetic backgrounds, the separation range of hybrid offspring is wide, the workload is large, and it is difficult to breed new varieties.

[0003] Lily (Lilium spp.) is an important ornamental plant worldwide. Lily has rich flower colors and its flowering period is easy to control, making it an excellent material for studying flower color physiology and genetics. So far, research on the mechanism of genetic variation in lily flower color has mainly focused on analyzing red and pink lily varieties. The molecular mechanism of yellow and white lily flower formation is still unclear, and the key genes have not yet been identified, which limits the molecular breeding of lilies with different flower colors. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to solve the problem of long and unstable transformation period of existing lily white flowers to yellow tepals, and to provide gene resources for lily germplasm innovation and development and utilization.

[0005] The technical solution adopted by the present invention is: a lily LnCCD4a Gene, Its CDS sequence is shown in SEQ.ID.NO.1.

[0006] A lily LnCCD4a The amino acid sequence of the protein encoded by the gene is shown in SEQ.ID.NO.2.

[0007] The LnCCD4a protein is used for improving the metabolism of carotenoids in lily.

[0008] A kind for silent lily LnCCD4a A specific fragment of a gene, whose nucleotide sequence is shown in SEQ.ID.NO.3.

[0009] A recombinant vector comprising the lily according to claim 3 LnCCD4a Gene-specific fragments, which are used to silence lily LnCCD4a The specific fragment of the gene was cloned into the TRV2 vector EcoRI and BamH obtained between sites.

[0010] A silent lily LnCCD4a The VIGS system of the gene comprises an auxiliary vector and the recombinant vector according to claim 5, wherein the auxiliary vector is a TRV1 vector.

[0011] The Silent Lily LnCCD4a The VIGS system of the gene is to transform the recombinant vector and the TRV1 vector into Agrobacterium separately and mix them.

[0012] The Silent Lily LnCCD4a Application of VIGS system of genes in regulating flower color traits of lily, The application is to infect lily tepals with the Agrobacterium to silence nCCD4a Gene, which increases the content of carotenoid metabolites, resulting in the transformation of white lilies into yellow lilies. Beneficial effects of the present invention: The present invention provides a LnCCD4a protein related to lily carotenoid metabolism, the amino acid sequence of the LnCCD4a protein is shown in SEQ.ID.NO.2. It uses α-carotene and β-carotene as substrates to degrade carotenoids. The LnCCD4a protein of the present invention is encoded and synthesized by the carotenoid cleavage dioxygenase gene LnCCD4a, and is verified by VIGS experiments. LnCCD4a The gene has the function of regulating lily flower color. LnCCD4a The specific fragments of the gene sequence were selected, the silencing expression vector was constructed, and the target gene was silenced in the lily perianth using VIGS technology. The results showed that LnCCD4a After gene silencing, the carotenoid content in the tepals increases, and the flower color changes from white to yellow. The invention provides gene resources and technical basis for regulating the flower color of lilies through molecular breeding technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 The 'Nymph' lily and its mutants in Example 1 of the present invention; Figure 2 The content of carotenoid types accumulated in 'Nymph' and its mutants in Example 1 of the present invention; Figure 3 Lily perianth pieces in the embodiment of the present invention CCD4 Multiple sequence alignment analysis of genes; Figure 4 In the embodiment of the present invention CCD4 Gene phylogenetic analysis; Figure 5 Lily in the embodiment of the present invention LnCCDs Analysis of gene expression patterns; Figure 6 In the embodiment of the present invention, wild type lily, LnCCD4a Silent and LnCCD4a Mutant tepals. DETAILED DESCRIPTION

[0015] To make the above-mentioned objects, features and advantages of the present invention more obvious, the following is a brief description of the present invention in conjunction with the attached Figure 1-Figure 6 The present invention is further described in detail with specific implementation modes.

[0016] Example 1: Identification of the LnCCD4a gene.

[0017] The LnCCD4a gene comes from the OT lily hybrid line variety 'Nymph'. It is considered one of the most successful OT hybrid lily varieties in recent years, with large flowers and strong resistance to pathogens. The petals of 'Nymph' lily (WF) are milky white to light yellow-green, and the inner bottom of the petals are dark purple-red. During the cultivation process of this example, a natural (YF) mutant of 'Nymph' lily was found. The petals of the mutant plant are golden yellow, which is obviously different from the milky white petals of the wild-type plant, while no change is observed in the purple-red part of the base of the petals ( Figure 1 ).

[0018] In order to clarify the mechanism of lily flower color variation, this example used 'Nymph' lily and its yellow flower mutant as materials for metabolome analysis. The results of flavonoid metabolome analysis showed that there were 25 differentially accumulated flavonoids between WF and YF, of which only three colorless flavonoids, epicatechin, pinotene-7-O-neohesperidin and vitexin-2'-O-galactoside, were upregulated in YF. This shows that flower color variation does not come from differentially accumulated flavonoids. The results of carotenoid metabolome analysis showed that there were significant differences in the types and contents of carotenoids in the petals of YF and WF. Among the 57 carotenoids, 47 were differentially accumulated between WF and YFs, of which 40 were upregulated and 7 were downregulated. The upregulated carotenoid metabolites included α-carotene, β-carotene and 38 luteins. The down-regulated carotenoids included three carotenes (γ-carotene, (E / Z)-phytoene, and lycopene) and four xanthophylls (zeaxanthin palmitate stearate, lutein stearate, lutein dioleate, and zeaxanthin dipalmitate). Among them, 19 lutein types were specifically accumulated in YF, while 1 carotene (lycopene) and 4 Lutein Accumulated specifically in WF ( Figure 2 ). KEGG pathway analysis showed that the nine differentially accumulated carotenoids were related to the carotenoid biosynthesis pathway. The types and contents of carotenoids in YF were greater than those in WF. Lutein, in particular, had the highest content in both lily varieties, and the carotenoid content in YF increased significantly relative to that in WF. To clarify the cause of the difference in carotenoid content, this example used the petals of 'Nymph' lily and its yellow flower mutant as materials for comparative transcriptome analysis. In the carotenoid metabolic pathway (ko00906), a gene differentially expressed in WF and YF was obtained. LnCCD4a gene (cluster-39081.1).

[0019] Example 2: Cloning of LnCCD4a gene.

[0020] (1) The total RNA from the perianth segments of Lilium 'Fairy' was extracted by CTAB method, and the quality of total RNA from lily embryos was detected by 1.5% agarose gel electrophoresis and microplate reader. RNA with an A260 / A280 ratio of about 2.0 and clear electrophoresis bands was selected for reverse transcription.

[0021] The reverse transcription reaction system is 50 μl (see Table 1). Place the PCR tube on an ice box and add 1.0 μg of total RNA, 4.0 μL of dNTP (2.5 mM), and 4.0 μL of Oligo (dT) in order, mix well, put it into the PCR instrument, denature at 70℃ for 5 min, take out the PCR tube and ice bath, add other chemicals after sufficient cooling, and react at 42℃ for 60 min. After the reaction is completed, it can be used directly for subsequent reactions or stored at -20℃ for later use.

[0022] Table 1 Reverse transcription reaction system Reagents Volume (µL) Lilium tepals total RNA 1.0 dNTP Mixture (2.5 mM) 4.0 Oligo (dT) 4.0 Reverse Transcriptase M-MLV 2.0 M-MLV 5×Reaction Buffer 10.0 Recombinant RNase Inhibitor 2.0 <![CDATA[RNase-free ddH2O]]> 27.0 (2) Gene cloning Designed with Primer Premier 5.0 software LnCCD4a Gene amplification primers, the primer sequences are as follows: F: 5'-CTCCGTCTAGCTCGCCCATA-3' R: 5'-TCCGTCACGATCTCCAACTCC-3' Using the cDNA prepared in step (1) as a template, PCR amplification was performed using the above primers. The PCR reaction system is: Table 2 LnCCD4a Gene PCR amplification reaction system Reagents Volume (µL) TaKaRa LA Taq 0.5 <![CDATA[10×LA PCR Buffer II(Mg 2+ )]]> 3.0 dNTP Mixture 0.8 F 0.8 R 0.8 cDNA 1.0 <![CDATA[ddH2O]]> 18.1 The PCR reaction program was as follows: 95℃ for 3 min; 95℃ for 30 s, 65℃ for 30 s, 72℃ for 45 s, 10 cycles (+0.3℃); 95℃ for 30 s, 65℃ for 30 s, 72℃ for 45 s, 30 cycles (-0.5℃); 72℃ for 5 min; stored at 4℃. After the reaction, 1.5% agarose gel electrophoresis was used for detection. PCR products with clear, bright, and single target bands were selected and purified using TaKaRa MiniBEST DNA Fragment Purification Kit Ver. 4.0. After the PCR product was purified, 1.5% agarose gel electrophoresis was performed again, and the product concentration was determined using an enzyme reader. According to the concentration of the purified product, the product was connected to the intermediate cloning vector using the pMD™18-T Vector Cloning Kit and transformed into Escherichia coli. After the bacterial solution PCR detection, sequencing verification was performed.

[0023] (3) Bioinformatics analysis of gene sequences The NCBI database was used to search for the conserved protein domain of LnCCD4a and predict the gene family to which it belongs. DNAman software was used to predict the cloned LnCCD4a Multiple sequence alignment analysis of the coding amino acid sequences was performed, and the phylogenetic tree was constructed using MEGA6.0.6 software (Neighbor Joining method). Figure 3 ). LnCCD4a The CDS length of the gene is 1767 bp, encoding 589 amino acids. CCD4 The sequence alignment of the genes showed significant differences in length. Lilium brownii var. viridulum, Lilium formosanum × Lilium longiflorum and 'Nymph' CCD4 The genes encode 501, 604 and 589 amino acid residues respectively. The C-terminal region sequences of different lily varieties have poor similarity ( Figure 4 ).

[0024] (4) Analysis of CCD gene expression patterns in lily In the lily perianth transcriptome, a total of four CCD family genes were retrieved. Using qRT-PCR, their expression patterns were identified ( Figure 5 ).in, LnCCD4a (cluster-39081.1) and LnCCD4b (cluster-39081.0) is mainly expressed in petals and leaves, with the highest expression level in leaves. LnCCD1-I) is mainly expressed in petals and leaves, with the highest expression level in petals. LnCCD1-I , with the highest expression levels in leaves and similar levels in stems and flowers. As the color of lily petals increases, LnCCD4a The expression level gradually increased.

[0025] Embodiment 3: LnCCD4a Construction of gene silencing expression vector.

[0026] (1) Selection of specific fragments choose LnCCD4a The specific sequence of the 3' end fragment of the gene was detected by BioEdit to determine the specificity of the sequence fragment in the homologous gene in the transcriptome. There was no completely identical sequence greater than or equal to 20 bp in length. The sequence of the specific fragment was SEQ.ID.NO.2.

[0027] (2) PCR amplification LnCCD4a Gene-specific fragment TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0 was used to extract the plasmid DNA of the cloning vector as a cloning template. The amplification primer sequences were designed as follows: F: 5'-gtgagtaaggttaccgaattcGGGTATGTTGGGAGGCGG-3' R:5'-cgtgagctcggtaccggatccGATTTCCGTCACGATCTCCAAC-3' The PCR reaction system is: Table 3 PCR amplification reaction system Reagents Volume (µL) TaKaRa Premix Taq 25.0 Forward Primer 1.6 Reverse Prime 1.6 ddH2O 20.8 The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, 55°C for 30 s, extension at 72°C for 2 min, for a total of 30 cycles; extension at 72°C for 15 min; and storage at 4°C.

[0028] The PCR amplification products were detected by 1.0% agarose gel electrophoresis and then purified according to the instructions of the DNA purification kit of Tiangen Company.

[0029] (3) Linearization of expression vector The multiple cloning sites on the expression vector TRV2 sequence were analyzed using Snap Gene software, and restriction sites without repetitive sequences and with a GC content of 40% to 60% in the 20 bp region upstream and downstream of the vector cloning site were selected for cloning. EcoR I and BamHThe vector was linearized by enzyme digestion. The double enzyme digestion reaction system is as follows: Table 4 Double enzyme digestion system Reagents Volume (µL) EcoR I 2 BamH 2 10×NE Buffer 5 Vector plasmid 2000 / concentration ddH2O To 50 μl The reaction conditions were metal bath at 37 °C for 1 h. The digestion product was detected by 1.0% agarose gel electrophoresis. Two long and short bands were observed on the gel imager, indicating that the circular vector plasmid was successfully linearized by double digestion. After gel electrophoresis detection, the large fragment was purified and recovered and stored at -20 °C for later use.

[0030] (4) Connection After the concentration of the specific fragment amplification and purification product and the linearized vector was determined, they were connected according to the method provided by the In Fusion HD Rapid Seamless Cloning Kit.

[0031] Table 5 Cloning and ligation reaction system Reagents Volume (µL) 5X In-Fusion HD Enzyme Premix 2 Linearized vector fragment 10-200 ng Specific fragment 50-200 ng ddH2O up to 10 µL The mixed reaction system was incubated at 50°C for 15 min to connect and obtain the recombinant plasmid. 10 µL of the connection product was added to 100 µL of DH5α competent cells (thawed on ice), pipetted and mixed, and then ice-bathed for 30 min; heat-shocked at 42°C for 90 s and then ice-bathed for 5 min; 500 μL of LB liquid culture medium preheated at 37°C was added to the centrifuge tube, and shaken and cultured at 180 rpm and 37°C for 1 h; centrifuged at 5,000 rpm for 30 s, discarded 400 μL of the supernatant, and the remaining part was pipetted and mixed; the mixed bacterial solution was evenly spread on a plate containing 50 mg·L -1 The cells were plated on LB plates and incubated upside down at 37 °C for 24 h.

[0032] Positive monoclonal bacteria were selected, and the cultured bacterial solution was used as a template. After verification by PCR of the bacterial solution, sequencing was performed for verification. Plasmids were extracted from those with correct sequencing and named TRV2-LnCCD4a.

[0033] Example 4: Lily was infected with the silencing expression vector.

[0034] Add the TRV2-LnCCD4a recombinant plasmid to the competent cells of Agrobacterium GV3101 in a volume of 1:10 on an ice bath and mix well by pipetting; sequentially place in an ice bath, quick-freeze in liquid nitrogen, water bath at 28°C, and ice bath for 5 min each, add an appropriate amount of YEB liquid culture medium without antibiotics, and culture at 28°C with shaking for 2-3 h; spread on YEB solid culture medium containing kanamycin and rifampicin resistance, and culture inverted at 28°C for about 50 h; pick monoclonal colonies for PCR amplification, and detect positive bacterial liquid by 1.0% agarose gel electrophoresis.

[0035] Two days before infection, the Agrobacterium containing the LnCCD4a-TRV2 recombinant plasmid and the empty TRV1 were propagated and shaken, and the bacteria were collected by centrifugation using a high-speed centrifuge. The supernatant was discarded, and the resuspension solution (0.01M MES+0.01M MgCl2+0.001M AS) was added for resuspending, and the OD600 value was adjusted to about 1.0. It was left to stand in the dark at 28℃ for 3 h. A 1 ml sterile syringe was used to inject the base of the vascular bundle of the 'Nymph bud close to the third stage (S3, which is about 4~5cm long). Three biological replicates were performed for each treatment, with 10 buds per replicate. The treated plants were placed in a growth chamber (22℃, 60% humidity, 16h light / 8h dark) for phenotypic identification. Digital photos were taken 1 week after the pressure injection ( Figure 6 The total carotenoid content was detected using a kit. The results showed that the average carotenoid content in the perianth segments of the wild-type and silent plants was 48.45 μg / g and 97.06 μg / g, respectively, and the silent plants were significantly higher than the wild type.

[0036] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

[0037] SEQ.ID.NO .1 SEQ.ID.NO.2 MGPHLLSTTNSFSSASSSFVATYQSSFFPTLLSQSSPASLQSISAVQTEEKPETQANPKKTTLTGPKLYKKNALKTSRSLPPASLPVTICNALDDLINNFIDPPILRPSVDPRHILSNNFSPVTELPPTPCPVVRGSIPPCLRGGAYIRNGPNPQYLPRGPHHLFDGDGMLHSLLLPTTGSAPATLCSRHVHTYKYLLERDAGAPVLPNVFSGFHGTAGIARGAITAIRVLTGQLNPLEGVGLANTSLSFFGGRLFALGESDLPYSVHLSTNNGDISTLGRHDFDKKVVMGMTA HPKKDPVTGELFAFRYGPVPPYVTLFRFDKEGNKVGPDVPIFSMRDPSFLHDFAITEHYTVLPDIQMVMKPMDMVLGGGSPVGSDRAKVPRLGVLPRYATSEADIRWFEVPGFNIMHALNAWEEDGGSTIVLVAPNVLSIEHALERLELVHSCVEMVRIDLRSGAVSRTPLSADNLDFGVIHPGYVGRRNRYGYLGVGDPMPKICGVVKLDFEQAGHGDCVVARREFGPGCFGGEPFFVPEEDSEGEEDKGYVVSYVHDEGRGESKFVVMDAQSPELEIVTEISRGSPGRAQSV SEQ.ID.NO.3 GGGTATGTTGGGAGGCGGAACAGGTATGGTTATCTTGGGGTGGGGGATCCATGCCGAAGATTTGTGGTGTGGTGGAAGCTGGATTTTGAGCAGGCTGGGCACGGGGATTGTGTGGTGGCGCGGCGGGAGTTCGGGCCTGGTTTCGGTGGAGAGCCGTTCTTTGTCGGAGGAGGACTCGGAGGGGGAGGAGGACAAAGGGTATGTGGTGTCATATTGCACGACGAAGGGAGAGGGGAATCTAAGTTTGTGGTGATGGATGCACAGTCGCCGGAGTTGGAGATCGTGACGGAAATC。

Claims

1. A kind of lily LnCCD4a Gene, whose CDS sequence is shown in SEQ.ID.NO.

1.

2. A lily according to claim 1 LnCCD4a The amino acid sequence of the protein encoded by the gene is shown in SEQ.ID.NO.

2.

3. Use of the LnCCD4a protein according to claim 2 in regulating carotenoid metabolism in lily.

4. A method for silencing lilies LnCCD4a A specific fragment of a gene, whose nucleotide sequence is shown in SEQ.ID.NO.

3.

5. A recombinant vector, characterized in that: The lily according to claim 4 LnCCD4a Gene-specific fragments, which are used to silence lily LnCCD4a The specific fragment of the gene was cloned into the TRV2 vector EcoRI and BamH obtained between sites.

6. A Silent Lily LnCCD4a A VIGS system for genes, characterized in that It comprises an auxiliary vector and the recombinant vector according to claim 5, wherein the auxiliary vector is a TRV1 vector.

7. The silent lily according to claim 6 LnCCD4a A VIGS system for genes, characterized in that The recombinant vector and the TRV1 vector are transformed into Agrobacterium respectively and mixed.

8. The Silent Lily according to claim 7 LnCCD4a Application of the VIGS system for regulating flower color traits in lily.

9. The use according to claim 8, wherein the Agrobacterium is used to infect lily tepals to silence nCCD4a Gene, which increases the content of carotenoid metabolites, resulting in the transformation of white lilies into yellow lilies.

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