CN116622766A - Application of poplar SPL16 and SPL23 genes in regulation and control of transformation in dormancy period of poplar - Google Patents

Abstract

The invention discloses application of poplar SPL16 and SPL23 genes in regulation and control of transformation of poplar terminal bud dormancy, wherein the poplar SPL16 gene codes protein of an amino acid sequence shown in SEQ ID No.1, the SPL23 gene codes protein of an amino acid sequence shown in SEQ ID No.2, and the SPL16 gene is knocked out or the SPL16/SPL23 gene is knocked out simultaneously through a CRISPR/Cas9 editing system, so that the time of terminal bud growth stopping and entering dormancy after sensing autumn photoperiod change (short sunlight condition) of a poplar is delayed. The growth period of the tree can be prolonged by delaying the dormancy time of the tree under the condition of short sunshine in autumn, so that the invention has a great application prospect in the aspects of improving the photosynthetic carbon fixation and biomass accumulation of the tree.

Description

Application of Poplar SPL16 and SPL23 Genes in Regulating Poplar Dormancy Transition

technical field

The invention belongs to the field of molecular biology, and in particular relates to the application of poplar SPL16 and SPL23 genes in regulating dormancy transition of poplar.

Background technique

The growth of perennial trees in the northern temperate zone has obvious seasonal characteristics. Under short-day conditions in autumn, the growth of terminal buds stops, dormancy is established, and then the leaves age and fall off, so as to increase the plant's ability to resist adversity and safely survive the winter environment that is unfavorable to tree survival. After a certain period of low temperature in winter, the dormancy is lifted, and the terminal buds undergo qualitative changes and begin to germinate in the next spring. The dormancy process of terminal buds can be divided into three stages: ecological dormancy (ecodormancy)-endodormancy (endodormancy)-ecological dormancy (Shim et al., 2014). In the ecological dormancy stage, the apical meristem cells maintain a certain ability to respond to growth factors, and the apical buds can resume growth if the environmental conditions are suitable. The stem cells in the internal dormancy stage have lost their ability to respond to the external environment, and only after a period of low temperature can they break the internal dormancy state and restore their ability to respond to the environment.

Photoperiod (the alternation of light and dark periods in the diurnal cycle) is a key environmental signal that determines the optimal time for a tree to stop seasonal growth. When the photoperiod is shorter than the critical threshold for growth (short day, SD), the growth of the shoot apical meristem stops, and the leaf primordia no longer form young leaves, but instead form bud scales that wrap the shoot apical meristem (Olsen, 2010) . After growth ceases, short days in turn cause the shoots to transition to internal dormancy, characterized by the inability of the shoot meristem to respond to growth-promoting signals, which is critical for plant overwintering (Tylewicz et al., 2008). Studies have shown that high temperature and long sunshine can delay the arrival of physiological dormancy (Begum et al., 2007, 2008; Tanino et al., 2010), low temperature can induce dormancy release, and the accumulation of low temperature is important for the release of internal dormancy and the growth of plants in the next spring. Budding is critical (Ding et al., 2014). In summary, the seasonal growth of trees is mainly regulated by photoperiod and temperature signals.

Light is an important environmental factor, which can not only participate in photosynthesis as energy, but also regulate plant growth and development as a signal. Studies have shown that there is a significant conservation between the genetic pathways involved in the regulation of growth arrest and dormancy in poplar and the photoperiod-mediated flowering regulation pathway in Arabidopsis. The light perception of higher plants depends on red light, far-red light receptor phytochrome and blue light receptor cryptochrome. There are three phytochrome genes in the poplar genome: PHYA, PHYB1 and PHYB2 (Howe et al., 1998). Recent studies have shown that poplar phyB1 and phyB2 are promoters of seasonal growth of terminal buds (Ding et al., 2021). Phytochrome-Interacting Factor 8 (PIF8), which belongs to the bHLH transcription factor family, is a seasonal growth inhibitor of poplar. Reducing the expression of PIF8 through RNAi interference reduced the sensitivity of terminal buds to short-day conditions. Transcript sequencing analysis showed that PIF8 controls the short-day response of terminal buds by indirectly regulating the expression of FLOWERING LOCUS T (FT) and BRANCHED 1 (BRC1) (Ding et al., 2021). However, the molecular elements acting downstream of the phyB-PIF8 module to mediate the regulation of FT2/BRC1 genes remain unclear.

microRNA156 (miR156) and its target gene SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) are important components of the age pathway. miR156 inhibits the expression of SPL and participates in the regulation of multiple growth and development processes such as the transition from juvenile to adult, floral transition, and plant type (branching). The expression level of miR156 is higher in embryos and seedlings, and the expression of miR156 decreases with age, while the expression of SPL is up-regulated, thereby promoting plant maturation and flowering (Xu et al., 2016). There are conserved MIR156 and SPL genes in poplar genome, but the research on poplar miR156-SPL module is still in its infancy, and its biological function is still rarely reported. Given that Arabidopsis SPL3, SPL4 and SPL5 transcription factors play an important role in regulating flowering and branching. Whether SPL16 and SPL23 transcription factors homologous to Arabidopsis SPL3/4/5 in poplar are involved in regulating the seasonal growth of poplar terminal buds deserves further exploration.

Contents of the invention

The technical problem to be solved by the present invention is: how to provide a method for weakening poplar's response to short-day sunshine conditions and delaying the stop of terminal bud growth and dormancy time.

The technical scheme of the present invention is: the application of poplar SPL16 or/and SPL23 gene in the dormancy period conversion in the process of regulating the seasonal growth of poplar, said poplar SPL16 gene encodes the amino acid sequence shown in SEQ ID No.1 Protein, the SPL23 gene encodes the protein with the amino acid sequence shown in SEQ ID No.2.

Further, the application is to knock out the SPL16 gene or simultaneously knock out the SPL16/SPL23 gene through the CRISPR/Cas9 system, thereby delaying the time for poplar to stop growing and establish dormancy after sensing autumn photoperiod changes (short-day conditions).

Further, the nucleotide sequence of the poplar SPL16 gene is shown in SEQ ID No.3, and the nucleotide sequence of the SPL23 gene is shown in SEQ ID No.4.

A method for constructing a poplar strain that postpones entering the autumn dormancy period, comprising the steps of:

(1) Using CRISPR/Cas9 editing technology to construct an editing vector targeting the poplar SPL16 gene or simultaneously targeting the SPL16 gene and the SPL23 gene;

(2) using the Agrobacterium-mediated leaf disc method to transfer the editing vector constructed in step (1) into poplar leaves;

(3) Through poplar tissue culture and screening of positive transgenic plants, the wild type and transgenic plants of Populus tomentosa cultivated under long-day conditions (16h light/8h dark) were transferred to short-day conditions (10h light/14h dark) for treatment, and observed And count the phenotype changes of terminal buds to obtain poplar lines with delayed terminal bud dormancy under short-day conditions.

Further, the sgRNA nucleotide sequence of the editing vector for the SPL16 gene is shown in SEQ ID No.5 and 6, and the sgRNA nucleotide sequence of the editing vector for the SPL23 gene is shown in SEQ ID No.7 and 8.

A sgRNA targeting poplar SPL16/SPL23 gene, the sgRNA nucleotide sequence is any one of SEQ ID No.5 to SEQ ID No.8.

A gene editing vector containing any sgRNA in SEQ ID No.5 to SEQ ID No.8.

The invention provides the application of SPL16 and SPL23 genes in regulating the conversion of poplar's seasonal growth period. The SPL16 and SPL23 genes in Populus tomentosa were edited using CRISPR/Cas9 gene editing technology, so that the target sites of SPL16 and SPL23 genes produced base deletions, insertions or large fragment deletions. The results showed that there was one base deletion in the T2 target site of the SPL16 gene in the spl16 L1 single mutation, and 31 base deletions in the T2 target site of the SPL16 gene in the spl16 L2 single mutation. In the spl16/23L1 double mutation, 6 bases were deleted at the T1 target site of the SPL16 gene, and 1 base was inserted at the T1 target site of the SPL23 gene. In the spl16/23L2 double mutation, there were 3 base deletions in the T1 target site of the SPL16 gene, and 5 base deletions in the T1 target site of the SPL23 gene. Under the long-day condition of normal culture, the number of side branches of the mutant plants was significantly increased compared with that of the wild type plants, and the number of side branches of the amphimorphic plants>the number of side branches of the monomorphic plants>the number of side branches of the wild type plants. The wild-type Populus tomentosa WT and spl16 single-horn and spl16/23 double-horn materials were cultured under long-day conditions (16h light/8h dark) for 2 months, and some materials were transferred to short-day conditions (10h light/14h dark) for treatment , the other part of the material (control group) continued to be cultured under long-day conditions. After about 15 days of short-day treatment, it was found that the spl16 single-horn and spl16/23 double-horn buds stopped growing and the dormancy time was delayed compared with WT poplar, indicating that the mutant plants’ terminal buds still survived short-day conditions. Maintain high growth activity.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention shows that knocking out SPL16 alone, or knocking out SPL16/23 genes simultaneously, can change the transition of dormancy period in the seasonal growth process of poplar. By delaying the dormancy time of forest trees under short-day conditions in autumn, the growth period of trees can be extended, so it has great application prospects in improving photosynthetic carbon sequestration and biomass accumulation of trees.

Description of drawings

Figure 1. Creation of spl16 single mutant and spl26/23 double mutant materials in Populus tomentosa. (a) Gene structure of poplar SPL16 and SPL23 and design of target sites (T1, T2) in the first exon. (b) Genotype sequencing results of spl16 single mutant (two lines, L1 and L2) of Populus tomentosa.

Figure 2. Creation of spl26/23 double mutant material of Populus tomentosa.

Fig. 3. Observation of phenotypes of poplar spl16 single mutant and spl26/23 double mutant materials. (a) The collateral phenotypes of wild-type P. tomentosa, spl16 single-horn and spl26/23 double-horn plants under long-day conditions. (b) Terminal bud phenotypes of wild-type P. tomentosa, spl16 monomorph and spl26/23 bimorph materials under long-day (LD) and short-day (SD) conditions. (c) Statistical analysis of the number of side branches. Different letters represent significant differences (p<0.05, ANOVA analysis). (d) Comparative analysis of the terminal bud activity of poplar materials with different genetic backgrounds under short-day conditions, showing that the growth arrest and dormancy time of the mutant lines are delayed compared with the wild type.

Detailed ways

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from commercial sources unless otherwise specified.

(1) Construction of CRISPR/Cas9 knockout vectors for SPL16 and SPL23 genes

First, download poplar SPL16 gene (gene sequence such as SEQ ID No.3, encoded protein such as SEQ ID No.1) and SPL23 gene (gene sequence such as SEQ ID No.4, encoded protein such as SEQ ID No.2) from the Phytozome database ), using the SnapGene Viewer software to design SPL16 and SPL23 gene-specific target sequences (design sgRNA). To ensure gene editing efficiency, two optimal sgRNA target sequences were designed for each gene.

Table 1 The designed sgRNA sequence

target gene T1 T2 SPL16 ATGGAAACAAGCAAAAGCAGAAGG ATGGCGTCTATGGTATCGCTTGG SPL23 ATGGGAACAAGCAAAGCTGAAGG ATGTCGTTTATGGTATCGCTTGG

These target sequences were then ligated into the pYLCRISPR_Cas9P35S-H vector by homologous recombination, and transformed into Agrobacterium after verifying that the vector was correct by PCR sequencing.

(2) Genetic transformation and genotype identification of poplar

Using poplar (Populus tomentosa Carrière) as the recipient material, the successfully constructed CRISPR/Cas9 vector was introduced into poplar leaves by Agrobacterium-mediated leaf disc method, and positive transgenes with corresponding resistance were screened by poplar tissue culture plants.

First, the successfully constructed CRISPR/Cas9 vector was transformed into Agrobacterium, cultured in a 28°C incubator for 2 days, and then single clones were cultured overnight in a YEP medium supplemented with kanamycin at 28°C. The Agrobacterium cultured overnight was added to the YEP medium supplemented with kanamycin at a ratio of 1:100, and the OD was grown to 0.4-0.6 in a shaker at 28°C. Then centrifuge in a low-temperature centrifuge at 4°C, 4000rpm, 10min, collect the bacteria, resuspend the bacteria with the resuspension liquid, add the resuspension liquid to 30mL, shake and cultivate at 28°C for 45min. Then the poplar leaves were cut into leaf disks of 4-6mm, infected in the Agrobacterium solution for 10 minutes, and oscillated every 3-5 minutes. After 10 minutes, blot the bacterial solution on the surface of the leaf disk with sterile paper, then place it on the co-cultivation medium, culture in dark for 2 days, and then transfer to the medium with kanamycin for cultivation.

After the whole plant is obtained, the leaves of the plant are taken, and the plant DNA is extracted by the CTAB method. In order to identify its genotype, specific primers for the SPL16 and SPL23 genes were used to amplify the gene fragments containing the target sequences, and accurate gene editing information was obtained by PCR sequencing. The specific results were as follows: one base was deleted in the T2 target of the SPL16 gene in the spl16 L1 single mutation, and 31 bases were deleted in the T2 target of the SPL16 gene in the spl16L2 single mutation. In the spl16/23L1 double mutation, 6 bases were deleted at the T1 target site of the SPL16 gene, and 1 base was inserted at the T1 target site of the SPL23 gene. In the spl16/23L2 double mutation, there are 3 base deletions in the T1 target of the SPL16 gene, and 5 base deletions in the T1 target of the SPL23 gene, as shown in Figure 1 and Figure 2.

(3) Phenotype observation of transgenic plants

The wild-type Populus tomentosa WT and spl16 single-horn and spl16/23 double-horn materials were cultured under long-day conditions (16h light/8h dark) for 2 months, and some materials were transferred to short-day conditions (10h light/14h dark) for treatment , the other part of the material (control group) continued to be cultured under long-day conditions. According to the scoring criteria for the growth state of terminal buds in previous studies (Rohde et al., 2011; Johansson et al., 2022), the changes in the growth state of the terminal buds of poplars with different genetic backgrounds after short-day treatment were observed, recorded and evaluated.

The results are shown in Figure 3. Under the long-day conditions of normal culture, the number of side branches of the mutant plants was significantly increased compared with the wild-type plants, and the double-horn plants>single-horn plants>wild-type plants. The wild-type Populus tomentosa WT and spl16 single-horn and spl16/23 double-horn materials were cultured under long-day conditions (16h light/8h dark) for 2 months, and some materials were transferred to short-day conditions (10h light/14h dark) for treatment , the other part of the material (control group) continued to be cultured under long-day conditions. After about 15 days of short-day treatment, it was found that the spl16 single-horn and spl16/23 double-horn buds stopped growing and the dormancy time was delayed compared with WT poplar, indicating that the mutant plants’ terminal buds still survived short-day conditions. Maintain high growth activity. The present invention shows that knocking out SPL16 alone, or knocking out SPL16/23 genes simultaneously, can change the transition of dormancy period in the seasonal growth process of poplar. Under short-day conditions in autumn, delaying the growth stop and dormancy time of tree top buds can promote the growth period of trees, so it has a great application prospect in improving photosynthetic carbon sequestration and biomass accumulation of trees.

Claims (7)

1. the application of poplar SPL16 or/and SPL23 gene in the dormant phase conversion of terminal buds in the process of regulating poplar seasonal growth, the protein of the amino acid sequence shown in the SPL16 gene encoding SEQ ID No.1, the SPL23 The gene encodes a protein with the amino acid sequence shown in SEQID No.2. 2. The application according to claim 1, characterized in that the application is to knock out the SPL16 gene or simultaneously knock out the SPL16/SPL23 gene through the CRISPR/Cas9 editing system, thereby delaying the poplar's perception of the autumn photoperiod change after the terminal bud The time of growth cessation and entry into dormancy. 3. The application according to claim 1 or 2, wherein the nucleotide sequence of the SPL16 gene is as shown in SEQ ID No.3, and the nucleotide sequence of the SPL23 gene is as shown in SEQ ID No.4 Show. 4. a kind of construction weakens poplar response short-day sunshine condition, delays the method for the poplar strain of terminal bud growth stop and dormancy time, is characterized in that, comprises the steps: (1) Utilize the CRISPR/Cas9 gene editing technology to construct an editing vector aimed at the poplar SPL16 gene or at the same time for the SPL16 and SPL23 genes; the nucleotide sequence of the SPL16 gene is shown in SEQ ID No.3, and the SPL23 gene The nucleotide sequence is shown in SEQ ID No.4; (2) using the Agrobacterium-mediated leaf disc method to transfer the editing vector constructed in step (1) into poplar leaves; (3) Through poplar tissue culture and screening of positive transgenic plants, the wild-type and transgenic plants of Populus tomentosa cultivated under long-day conditions were transferred to short-day conditions for treatment, and the changes in terminal bud phenotype were observed and counted, and obtained under short-day conditions. For poplar strains with delayed terminal bud dormancy, the long-day conditions refer to 16h light/8h darkness, and the short-day conditions refer to 10h light/14h darkness. 5. The method according to claim 4, characterized in that, the sgRNA nucleotide sequence of the editing carrier for the SPL16 gene is as shown in SEQ ID No.5 and 6, and the sgRNA nucleotide sequence of the editing carrier for the SPL23 gene As shown in SEQ ID No.7 and 8. 6. A sgRNA targeting poplar SPL16/SPL23 gene, characterized in that, the sgRNA nucleotide sequence is as shown in any one of SEQ ID No.5 to SEQ ID No.8. 7. A gene editing vector, characterized in that it contains any sgRNA in SEQ ID No.5 to SEQ ID No.8.