Application of knocking out phagl17 gene in improving the traits of petunia hybrida

Abstract

This invention relates to the field of plant genetic engineering technology, specifically to a knockout method. PhAGL17 Application of genes in improving petunia traits. This invention is the first to discover that knocking out wild-type petunias... PhAGL17 Genes can significantly reduce plant height and increase calyx size, which helps control the overall compact shape of the plant and beautify the landscape.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a knockout method. PhAGL17 New applications of genes in improving petunia traits. Background Technology

[0002] Petunia ( Petunia hybrida Petunia, a perennial herbaceous plant belonging to the Solanaceae family, is an important ornamental horticultural flower. Plant height is a core horticultural trait of petunias, directly affecting their potted plant shape, landscape configuration, and commercial value. Currently, the regulation of petunia plant height mainly focuses on gibberellin synthesis and signaling pathways, TFL1, and miR156-SPL pathways, but these pathways are often accompanied by defects such as delayed flowering and smaller floral organs. In addition, the functional research on the AGL17-like subfamily gene in the MADS-box family is still insufficient. Currently, there are no reports on the regulation of petunia plant height and calyx development by this gene, and its biological function and breeding application potential need to be further explored and elucidated. Summary of the Invention

[0003] This invention provides knockout PhAGL17 Application of genes in improving petunia traits.

[0004] The present invention adopts the following technical solution: This invention provides knockout PhAGL17 Application of genes in improving petunia traits. Compared to wild-type petunias, the changes in said traits include at least one or more of the following: (1) PhAGL17 Gene knockout petunia plants have reduced plant height; (2) PhAGL17 Gene knockout petunia plants have enlarged calyxes; (3) PhAGL17 Gene knockout petunia plants have a compact layered distribution of branches and leaves.

[0005] In some embodiments, the wild-type petunia is selected from W115, whose PhAGL17 Gene knockout plants have a height reduction of more than 50%.

[0006] In some embodiments, CRISPR / Cas9 gene editing technology is preferably used to knock out [the gene]. PhAGL17 Gene, specific methods and steps include: constructing targeted knockout PhAGL17 CRISPR / Cas9 gene editing vector; transformation of wild-type petunias; screening and breeding of phenotypically optimized petunia mutant plants.

[0007] In some embodiments, targeted knockout is constructed. PhAGL17The target sequence of the CRISPR / Cas9 gene editing vector is: 5'-ATACTCTGCGATGCTGAGGT-3'.

[0008] In some embodiments, the primer sequences used in CRISPR / Cas9 gene editing technology are as follows: sg-F: 5'-TGATTGTACTCTGCGATGCTGAGGT-3'; sg-R: 5'-AAACACCTCAGCATCGCAGAGTACA-3'.

[0009] In some embodiments, the primer pair sequences for identifying gene-edited positive plants are: F: 5'-GACAAGAAGTACAGCATCGGCCTGG-3'; R: 5'-TCAGGTCCTGGTGGTGCTCGT-3'.

[0010] In some embodiments, the primer sequences for identifying mutant mutation sites are: F: 5'-TAGTGTGTGTGAGATCCGTG-3'; R: 5'-TCTCGTTAGTCTACATACAC-3'.

[0011] In some embodiments, the step of screening and cultivating petunia mutant plants with improved traits includes: cultivating and screening heterozygous mutants with improved traits, and then self-pollinating and screening the heterozygous mutants to obtain homozygous mutants with stable improved traits.

[0012] Compared with the prior art, the core technical advantages and beneficial effects of the present invention are as follows: In the model plant Arabidopsis thaliana AGL17 The primary function of the gene is to promote flowering. This invention is the first to discover in petunias that, compared to wild-type petunias, knocking out… PhAGL17 Genes can achieve novel functions in improving traits such as significantly reduced petunia plant height, larger calyx development, and more compact branch and leaf distribution.

[0013] In particular, this invention is the first to confirm the knockout of wild-type petunia W115. PhAGL17 The gene can significantly reduce plant height. The height of the homozygous mutant is more than 50% lower than that of the wild type, and the plant is compact, which is conducive to controlling the overall shape of the plant. At the same time, the calyx is significantly enlarged and the number of sepals increases.

[0014] The petunia mutant plants obtained by gene editing in this invention have stable traits and do not affect the flowering period or other ornamental value compared with the wild type. They can be directly used for the cultivation of new petunia varieties with dwarf and compact plant types and large calyxes, and have both landscape configuration and commercial value. Attached Figure Description

[0015] Figure 1 for PhAGL17 A schematic diagram of the gene sequence composition.

[0016] Figure 2 The map shows the pBI121-PhAGL17 overexpression recombinant vector.

[0017] Figure 3 The analysis of different transformed plants after transformation of W115 wild-type petunia with the 35S:PhAGL17 overexpression vector was performed. PhAGL17 Statistical graph of relative expression levels.

[0018] Figure 4 Map of the pcambia1300-PhAGL17 knockout vector plasmid.

[0019] Figure 5 Comparison of plant height and calyx of wild-type petunia W115 and mutant lines #27 and #18.

[0020] Figure 6 This is a sequence diagram of the gene-edited mutant.

[0021] Figure 7 A comparison of traits such as plant height between wild-type petunia W115 and homozygous mutant #18 (-1bp).

[0022] Figure 8 A comparison of traits such as plant height between wild-type petunia W115 and homozygous mutant #27 (-1bp). Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. The following embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention. In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art. Example 1

[0024] The inventors' team previously conducted transcriptome sequencing on leaves and terminal buds of non-flowering petunias and wild-type petunias, identifying differences in gene expression levels. The analysis and statistics are shown in the table below: Table 1. Summary of differentially expressed genes in the leaf and apical bud transcriptomes of non-flowering petunias

[0025] As can be seen from the table above: The expression level of the petunia AGL17-like gene was significantly upregulated in the leaves and terminal buds of non-flowering lines, with a fold difference of log2FC>7.

[0026] This result does not match the flowering-promoting function of AGL17 in Arabidopsis thaliana, so the inventors' team analyzed and verified the gene as a candidate gene.

[0027] This embodiment is further based on the petunia genome. P. axillaris N and P. inflata Using the S6 database (https: / / solgenomics.net / ) (Bombarely, et al. 2016) and transcriptome databases, we analyzed petunias. PhAGL17 The gene sequence (gene ID: PEAXI162SCF01138), and a schematic diagram of the candidate gene composition are shown in [reference needed]. Figure 1 : PhAGL17 The gene consists of eight exons and seven introns, with the first intron being the longest at 17483 bp. The 5'UTR and 3'UTR are 343 bp and 275 bp, respectively, and the full-length CDS is 717 bp, encoding 238 amino acids. Example 2

[0028] This embodiment constructs the 35S:PhAGL17 overexpression vector and transforms it into wild-type petunia W115. The specific steps include: S1, constructing a system containing PhAGL17 Recombination vector of full-length coding sequence.

[0029] Using Primer Premier 6 software, a screening program was designed targeting... PhAGL17 Gene-specific amplification primer pairs, cloned PhAGL17 The full-length cDNA sequence. Specifically, it includes: The primer sequences synthesized by the commissioned team are as follows: F: 5'-ATGGGAAGAGGAAAGATAGTGATAAG-3' (SEQ ID NO: 1); R: 5'-CTAATGCAGTTGTAGCCCCAATTCAG-3' (SEQ ID NO: 2).

[0030] PCR amplification system: 10×KOD Buffer: 5μL, dNTP: 15μL, upstream primer F: 1μL, downstream primer R: 1μL, KOD DNA Polymerase: 0.5μL, template DNA: 0.21μL, ddH2O: 41.5μL.

[0031] The PCR amplification program was as follows: pre-denaturation at 95℃ for 3 min, 36 cycles of 95℃ for 20 s, 56℃ for 30 s, 72℃ for 1 min, total extension at 72℃ for 10 min, and incubation at 4℃ for 10 min.

[0032] Using leaf nucleic acid extracted from wild-type petunia W115 as a template, amplification was performed using the aforementioned commercial kit's protocol to obtain... PhAGL17 The full-length cDNA sequence was ligated into the pMD18-T vector to obtain a vector containing... PhAGL17 The pMD18-T recombinant vector with a full-length coding sequence.

[0033] S2, construct the 35S:PhAGL17 overexpression vector.

[0034] The pMD18-T recombinant vector carrying the full-length coding sequence of PhAGL17 was digested with XbaI and SmaI restriction endonucleases. The target fragment was ligated into the pBI121 vector (containing the CaMV 35S promoter and GFP fluorescent tag) to construct the pBI121-PhAGL17 overexpression recombinant vector. See [link to documentation]. Figure 2 .

[0035] S3, the pBI121-PhAGL17 recombinant plasmid was electroporated into Agrobacterium tumefaciens AGL0, and then transformed into wild-type petunia W115 by leaf disc method.

[0036] The pBI121-PhAGL17 overexpression recombinant vector was electroporated into Agrobacterium AGL0 to obtain Agrobacterium culture containing the target gene.

[0037] Wild-type petunia W115 explant leaves were obtained, disinfected, cut into pieces, and incubated with Agrobacterium tumefaciens containing the target gene for 10-15 minutes. After conventional petunia genetic transformation tissue culture technology, callus formation, germination, rooting, seedling strengthening, hardening, and transplanting were induced to obtain T0 generation transgenic lines, and positive seedlings were detected and identified.

[0038] The primer sequences used for identification PCR are as follows: F: 5'-CGAGGAGCATCGTGGAAAAAGAAGA-3' (SEQ ID NO: 3); R: 5'-TTGCCCGGTGGTGCAGATGAACT-3' (SEQ ID NO: 4).

[0039] Using the above PCR amplification system and procedure, a total of 16 positive lines of 35S:PhAGL17 transformed petunia were obtained after identification.

[0040] See the results of transgenic line expression analysis. Figure 3 .

[0041] Observations and statistics showed that no phenotypic differences were observed in the 16 T0 generation transgenic lines compared with wild-type petunias.

[0042] Therefore, strains 19 and 23, which had high expression levels, and strains 18 and 30, which also had high expression levels, were selected for subsequent phenotypic observation and related experiments. Cultivation methods and phenotypic observation: The 35S:PhAGL17 T2 generation transgenic positive line and wild-type petunia W115 were simultaneously planted in the LD greenhouse, with 25 plants in each line. Phenotypic characteristics such as flowering time, plant height, internode spacing, size and number of floral organs, number of branches, and root length were recorded.

[0043] The results show that: Compared to the wild-type petunia W115 plant, PhAGL17 Petunia plants transformed with gene overexpression vectors showed no significant changes in traits including plant height, number of branches, flowering time, and floral organs, and root development was not affected. Example 3

[0044] This embodiment describes the construction of a CRISPR / Cas9 gene editing vector and its transformation into W115 wild-type petunias. The specific steps include: S1, constructing gene editing vectors.

[0045] Referring to the above examples, cDNA was prepared by reverse transcription using W115 wild-type petunia RNA as a template.

[0046] according to PhAGL17 Genomic and CDS sequences were used to screen for specific sgRNA targets, and amplification primers were designed using PrimerPremier 6 software.

[0047] The target sequence was determined to be: 5'-ATACTCTGCGATGCTGAGGT-3' (SEQ ID NO:5).

[0048] The amplification primers were determined to be: sg-F: 5'-TGATTGTACTCTGCGATGCTGAGGT-3' (SEQ ID NO: 6); sg-R: 5'-AAACACCTCAGCATCGCAGAGTACA-3' (SEQ ID NO: 7).

[0049] The target sequence was ligated into the CRISPR / Cas9 vector pcambia1300 to obtain the pcambia1300-PhAGL17 knockout vector plasmid. (See [link to documentation]) Figure 4 .

[0050] S2, the pcambia1300-PhAGL17 knockout vector plasmid was electroporated into LBA4404 Agrobacterium and then transformed into wild-type petunia W115 using the leaf disc method.

[0051] Referring to the transformation and culture methods described in the above embodiments, T0 generation transgenic mutant lines were obtained, and positive seedlings were detected and identified.

[0052] The primers for identifying positive plants are: F: 5'-GACAAGAAGTACAGCATCGGCCTGG-3' (SEQ ID NO: 8); R: 5'-TCAGGTCCTGGTGGTGCTCGT-3' (SEQ ID NO: 9).

[0053] PCR amplification of the target region, using the following primers: F: 5'-TAGTGTGTGTGAGATCCGTG-3' (SEQ ID NO: 10); R: 5'-TCTCCGTTAGTCTACATACAC-3' (SEQ ID NO: 11).

[0054] Sequencing was used to detect base mutations, and mutant plants with effective editing were screened. A total of 14 positive plants were obtained, of which 8 were effectively edited and all were heterozygous mutants.

[0055] The phenotypes of heterozygous mutant lines #27 and #18 are shown in [reference needed]. Figure 5 .

[0056] turn out: Compared with the wild-type W115 petunia line, the heterozygous mutants #27 and #18 petunia lines showed a significantly dwarfed phenotype (≥ 50%), with a compact plant type and increased calyx size and sepals. Example 4

[0057] In this embodiment, homozygous mutant plants were obtained by self-pollination of heterozygous mutants #18 and #27 petunia lines. The specific steps included are as follows: S1, the heterozygous mutants #18 and #27 petunia lines were self-pollinated, and the pods were harvested after they matured and dried in a cool, ventilated place.

[0058] S2, the self-crossed progeny of heterozygous mutants #18 and #27 were sown simultaneously with wild-type petunia W115 in an LD culture room at a temperature of 26℃.

[0059] S3. DNA was extracted from the T1 line, the progeny of the heterozygous mutants #18 and #27, respectively. The target region was amplified by PCR, and the base mutation was detected by sequencing to screen for homozygous mutants.

[0060] See sequencing screening results Figure 6 Mutants #18 and #27 both yielded homozygous mutant plants with -1bp and -6bp respectively.

[0061] Phenotypic statistical results show that: The average plant height of wild-type petunia W115 was 54.1 ± 2.85 cm. The homozygous mutant #18 (-1bp) had an average plant height of 18.32 ± 2.45 cm, which was approximately 66% lower than the wild type. See [link to plant height and other traits] for details. Figure 7 The plant has a compact shape.

[0062] The homozygous mutant #27 (-1bp) had an average plant height of 18.45 ± 2.77 cm, which was about 65% lower than that of the wild type. For plant height and other traits, please refer to [link to relevant documentation]. Figure 8 The plant has a compact shape.

[0063] The above experiments confirmed that: wild-type petunia W115 knockout PhAGL17 Genes can significantly reduce plant height; homozygous mutants are more than 50% shorter than wild-type, resulting in a more compact plant shape, which is beneficial for controlling the overall plant morphology. Simultaneously, the calyx is significantly enlarged and the number of sepals increases. This gene-editing technology can be used to selectively knock out petunias. PhAGL17 The mutant plants obtained through gene breeding have stable traits and do not affect the flowering period or other ornamental value compared with the wild type. They can be directly used for the breeding of new petunia varieties with dwarf and compact plant types and large calyxes, and have both landscape configuration and commercial value.

[0064] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Knockout PhAGL17 The application of genes in improving petunia traits is characterized by, Compared to wild-type petunias, the changes in the aforementioned traits include at least one or more of the following: (1) PhAGL17 Gene knockout petunia plants have reduced plant height; (2) PhAGL17 Gene knockout petunia plants have enlarged calyxes; (3) PhAGL17 Gene knockout petunia plants have a compact layered distribution of branches and leaves.

2. The application according to claim 1, characterized in that, The wild-type petunia was selected from W115. PhAGL17 Gene knockout plants have a height reduction of more than 50%.

3. The application according to claim 1 or 2, characterized in that, Knockout using CRISPR / Cas9 gene editing technology PhAGL17 Gene.

4. The application according to claim 3, characterized in that, Knockout using CRISPR / Cas9 gene editing technology PhAGL17 The steps involved in gene-based methods include: Build targeted knockout PhAGL17 CRISPR / Cas9 gene editing vectors; Transforming wild-type petunias; Screening and breeding of petunia mutant plants with improved traits.

5. The application according to claim 4, characterized in that, Build targeted knockout PhAGL17 The target sequence in the CRISPR / Cas9 gene editing vector is: 5'-ATACTCTGCGATGCTGAGGT-3'.

6. The application according to claim 5, characterized in that, The primer sequences used in CRISPR / Cas9 gene editing technology are as follows: sg-F: 5'-TGATTGTACTCTGCGATGCTGAGGT-3'; sg-R: 5'-AAACACCTCAGCATCGCAGAGTACA-3'.

7. The application according to claim 5, characterized in that, The primer pair sequences for identifying gene-edited positive plants are: F: 5'-GACAAGAAGTACAGCATCGGCCTGG-3'; R: 5'-TCAGGTCCTGGTGGTGCTCGT-3'.

8. The application according to claim 5, characterized in that, The primer sequences for identifying mutation sites are: F: 5'-TAGTGTGTGTGAGATCCGTG-3'; R: 5'-TCTCGTTAGTCTACATACAC-3'.

9. The application according to claim 5, characterized in that, The steps of screening and cultivating petunia mutant plants with improved traits include: screening and cultivating heterozygous mutants with improved traits, and then self-pollinating the heterozygous mutants to obtain homozygous mutants with improved traits and genetic stability.

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