CN116622766A - Application of poplar SPL16 and SPL23 genes in regulation and control of transformation in dormancy period of poplar - Google Patents
Application of poplar SPL16 and SPL23 genes in regulation and control of transformation in dormancy period of poplar Download PDFInfo
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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
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to application of poplar SPL16 and SPL23 genes in regulation and control of transformation in a dormancy period of poplar.
Background
Perennial forest growth in the North temperate zone has obvious seasonal characteristics. The growth of the terminal buds is stopped under the condition of short sunshine in autumn, dormancy is established, and leaves are aged and fall off, so that the capability of plants for resisting stress is improved, and the winter environment which is unfavorable for tree survival is safely spent. After a certain period of low temperature in winter, dormancy is released, and the terminal bud becomes to sprout in spring in the next year. The dormancy process of the terminal buds can be divided into 3 phases: ecological dormancy (ecodormanny) -internal dormancy (endodormanny) -ecological dormancy (Shim et al, 2014). The apical meristematic cells maintain a certain ability to respond to growth factors during the ecological dormancy stage, and can resume growth if environmental conditions are appropriate for the terminal buds. And the stem cells in the internal dormancy stage lose the reaction capacity to the external environment, and can break the internal dormancy state only after a period of low temperature, and resume the reaction capacity to the environment.
Photoperiod (alternating of light and dark periods in the circadian cycle) is a critical environmental signal that determines the optimal time for the tree to stop seasonally. When the photoperiod is shorter than the critical threshold for growth (short sun, SD), shoot apical meristem growth ceases and leaf primordia no longer form young leaves, but form shoot scales surrounding shoot apical meristems (Olsen, 2010). After growth ceases, short sun exposure in turn causes the buds to transition to internal dormancy, which is characterized by the inability of the bud meristem to respond to growth promoting signals, which is critical to plant overwintering (Tylewicz et al, 2008). Studies have shown that high temperature and long sunlight can delay the arrival of physiological dormancy (Begum et al 2007,2008;Tanino et al, 2010), low temperature can induce dormancy release, and low temperature accumulation is critical to release internal dormancy and germination of plants in the next spring (Ding et al 2014). In summary, seasonal growth of trees is primarily regulated by photoperiod and temperature signals.
Light is an important environmental factor, and can be used as energy to participate in photosynthesis, and can also be used as a signal to regulate plant growth and development. Studies have shown that there is significant conservation between the genetic pathways involved in regulating the cessation of poplar growth and dormancy and the photoperiod-mediated flowering regulatory pathways in Arabidopsis. The perception of light by higher plants depends on red, far-red receptor photopigments and blue receptor cryptomelane. The genome of poplar has 3 photopigment (phytochrome) genes: PHYA, PHYB1 and PHYB2 (Howe et al, 1998). Recent studies have shown that poplar phyB1 and phyB2 are promoters of terminal bud seasonal growth (Ding et al, 2021). The photopigment interaction factor (Phytochrome-Interacting Factor, PIF 8) belonging to the bHLH transcription factor family is an inhibitor of seasonal growth of poplar. Decreasing PIF8 expression by way of RNAi interference results in a decrease in the sensitivity of the terminal buds to short-day conditions. Transcriptional sequencing analysis showed that PIF8 controls the induction of short-day sunlight by terminal buds through indirect regulation of FLOWERING LOCUS T (FT), and expression of BRANCHED 1 (BRC 1) (Ding et al, 2021). However, the molecular elements that act downstream of the phyB-PIF8 module that mediate the regulation of the FT2/BRC1 gene are still unclear.
microRNA156 (miR 156) and target gene SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) thereof are important components of the age pathway. miR156 inhibits SPL expression, and participates in regulating and controlling a plurality of growth and development processes such as plant juvenile period to adult period transition, flower formation transition, plant type (branching) and the like. miR156 expression levels are higher in seed embryos and seedlings, miR156 expression decreases with age, and SPL expression is upregulated, promoting plant maturation and flowering (Xu et al, 2016). The poplar genome has conserved MIR156 and SPL genes, but research on the poplar miR156-SPL module is still in a starting stage, and biological functions of the poplar miR156-SPL module are still rarely reported. Whereas 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 seasonal growth of poplar terminal buds is worth exploring deeply.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: how to provide a method for weakening poplar response to short sunlight conditions and delaying the growth stop and dormancy time of terminal buds.
The technical scheme of the invention is as follows: use of a poplar SPL16 or/and SPL23 gene encoding a protein having an amino acid sequence shown in SEQ ID No.1 for modulating the conversion of dormancy stages during seasonal growth of poplar, said SPL16 gene encoding a protein having an 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 postponing the time for poplar growth to stop and establish dormancy after sensing autumn photoperiod changes (short sun conditions).
Further, the nucleotide sequence of the poplar SPL16 gene is shown as SEQ ID No.3, and the nucleotide sequence of the SPL23 gene is shown as SEQ ID No. 4.
A method of constructing a Yang Shuzhu line that delays entry into an autumn sleep stage comprising the steps of:
(1) Constructing an editing vector aiming at the poplar SPL16 gene or aiming at the SPL16 gene and the SPL23 gene simultaneously by using a CRISPR/Cas9 editing technology;
(2) Transferring the editing vector constructed in the step (1) into poplar leaves by adopting an agrobacterium-mediated leaf disc method;
(3) The method comprises the steps of culturing poplar tissues, screening positive transgenic plants, transferring wild-type aspen and transgenic plants cultured under long-day conditions (16 h light/8 h dark) to short-day conditions (10 h light/14 h dark) for treatment, observing and counting the variation of the terminal bud phenotype, and obtaining a Yang Shuzhu line with delayed terminal bud dormancy period under the short-day conditions.
Further, the nucleotide sequence of the sgRNA of the editing vector for the SPL16 gene is shown in SEQ ID No.5 and 6, and the nucleotide sequence of the sgRNA of the editing vector for the SPL23 gene is shown in SEQ ID No.7 and 8.
An sgRNA of a targeted poplar SPL16/SPL23 gene, wherein the nucleotide sequence of the sgRNA is shown in any one of SEQ ID No. 5-SEQ ID No. 8.
A gene editing vector comprising any sgRNA of SEQ ID No.5 to SEQ ID No. 8.
The invention provides application of SPL16 and SPL23 genes in regulating and controlling transformation of seasonal growth period of poplar. The CRISPR/Cas9 gene editing technology is utilized to carry out gene editing on two genes of SPL16 and SPL23 in populus tomentosa, so that base deletion, insertion or large fragment deletion are generated on target points of the SPL16 and SPL23 genes. The results show that the SPL16 gene T2 target in the SPL16L 1 single process has one base deletion, and the SPL16 gene T2 target in the SPL16L2 single process has 31 base deletion. The SPL16 gene T1 target point in the SPL16/23L1 double-process has 6 base deletion, and the SPL23 gene T1 target point has 1 base insertion. The SPL16 gene T1 target point in the SPL16/23L2 double-process has 3 base deletion, and the SPL23 gene T1 target point has 5 base deletion. Under the condition of normal culture and long sunlight, the number of the lateral branches of the mutant plant is obviously increased compared with that of a wild plant, and the number of the lateral branches of the double-mutant plant is larger than that of the lateral branches of the single-mutant plant. After wild type WT of populus tomentosa and spl16 single-process and spl16/23 double-process materials were cultured under long-day conditions (16 h light/8 h dark) for 2 months, one part of materials was transferred to short-day conditions (10 h light/14 h dark) for treatment, and the other part of materials (control group) was continued to be cultured under long-day conditions. After about 15 days of short-day treatment, spl16 single-process, spl16/23 double-process materials were found to have a delayed growth stop and dormancy time compared to the WT poplar terminal buds, indicating that the terminal buds of the mutant plants still maintained higher growth activity under short-day conditions.
Compared with the prior art, the invention has the following beneficial effects:
the invention shows that after the SPL16 is singly knocked out or the SPL16/23 genes are knocked out simultaneously, the dormancy period conversion in the seasonal growth process of poplar can be changed. 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 method has a great application prospect in the aspects of improving photosynthetic carbon fixation and biomass accumulation of the tree.
Drawings
FIG. 1. Creation of Spl16 single mutant and spl26/23 double mutant materials from Populus tomentosa. (a) Design of target sites (T1, T2) in the first exon, the genetic structure of poplar SPL16 and SPL 23. (b) Genotyping results of aspen spl16 single mutant (two lines, L1 and L2).
FIG. 2. Creation of aspen spl26/23 double mutant material.
FIG. 3 phenotypic observations of poplar spl16 single mutant and spl26/23 double mutant material. (a) Aspen wild type, spl16 single process and spl26/23 double process materials have side branch phenotype under long sunshine condition. (b) Populus tomentosa wild type, spl16 monosynaptic and spl26/23 double-synaptic material terminal bud phenotype under long-day (LD) and short-day (SD) conditions. (c) statistical analysis of the number of side shoots. The different letters represent significant differences (p <0.05, anova analysis). (d) Comparative analysis of the activity of terminal buds of different genetic background poplar materials under short sunlight conditions showed that mutant lines had a delay in growth arrest and dormancy compared to wild type.
Detailed Description
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from commercial sources.
(1) Construction of CRISPR/Cas9 knockout vectors of SPL16 and SPL23 genes
First, genomic sequences of the poplar SPL16 gene (gene sequence shown as SEQ ID No.3, encoded protein shown as SEQ ID No. 1) and SPL23 gene (gene sequence shown as SEQ ID No.4, encoded protein shown as SEQ ID No. 2) were downloaded from the Phytozome database, and specific target sequences of SPL16 and SPL23 genes were designed (sgrnas were designed) using snap gene Viewer software. To ensure gene editing efficiency, 2 optimal sgRNA target sequences were designed for each gene.
Table 1 shows the sgRNA sequences
| Targeting genes | T1 | T2 |
| SPL16 | ATGGAAACAAGCAAAGCAGAAGG | ATGGCGTCTATGGTATCGCTTGG |
| SPL23 | ATGGGAACAAGCAAAGCTGAAGG | ATGTCGTTTATGGTATCGCTTGG |
And then the target sequences are connected into a pYLCRISPR_Cas9P35S-H vector in a homologous recombination mode, and the agrobacterium is transformed after the PCR sequencing verifies that the vector is error-free.
(2) Genetic transformation and genotyping of poplar
The successfully constructed CRISPR/Cas9 vector is introduced into poplar leaves by an agrobacterium-mediated leaf disc method by taking aspen (Populus tomentosa Carri re) as a receptor material, and positive transgenic plants with corresponding resistance are selected by poplar tissue culture.
First, the successfully constructed CRISPR/Cas9 vector was transferred into agrobacterium, cultured in a 28 ℃ incubator for 2 days, and then selected for monoclonal culture in YEP medium supplemented with kanamycin, shaking overnight at 28 ℃. The overnight Agrobacterium was added to the kanamycin-supplemented YEP medium at a ratio of 1:100 and the OD was shake-cultured at 28℃to 0.4-0.6. Subsequently, centrifugation was performed in a low-temperature centrifuge at 4℃at 4000rpm for 10min, the cells were collected, the cells were resuspended in the suspension, and then added to 30mL of the suspension, followed by shaking culture at 28℃for 45min. The poplar leaves were then cut into 4-6mm leaf discs and infested in agrobacterium liquid for 10min with shaking every 3-5 min. After 10min, the leaf disk surface bacteria liquid was blotted with sterile paper, then placed on a co-culture medium, dark-cultured for 2 days, and then transferred to a medium with kanamycin for culturing.
After obtaining the whole plant, taking plant leaves, and extracting plant DNA by using a CTAB method. In order to identify the genotypes, specific primers of SPL16 and SPL23 genes are utilized to amplify the gene fragments containing target sequences, and accurate gene editing information is obtained through PCR sequencing. The specific results are as follows: the SPL16 gene T2 target in the SPL16L 1 single process has one base deletion, and the SPL16 gene T2 target in the SPL16L2 single process has 31 base deletion. The SPL16 gene T1 target point in the SPL16/23L1 double-process has 6 base deletion, and the SPL23 gene T1 target point has 1 base insertion. The SPL16 gene T1 target point in the SPL16/23L2 double-process has a 3-base deletion, and the SPL23 gene T1 target point has a 5-base deletion, as shown in figures 1 and 2.
(3) Phenotypic observation of transgenic plants
After wild type WT of populus tomentosa and spl16 single-process and spl16/23 double-process materials were cultured under long-day conditions (16 h light/8 h dark) for 2 months, one part of materials was transferred to short-day conditions (10 h light/14 h dark) for treatment, and the other part of materials (control group) was continued to be cultured under long-day conditions. The change in the terminal bud growth status of poplars of different genetic backgrounds after short-day treatment was observed, recorded and evaluated according to the scoring criteria for terminal bud growth status of previous studies (Rohde et al 2011;Johansson et al, 2022).
The results are shown in fig. 3, in which the mutant plants have significantly increased numbers of side shoots compared to wild type plants under long sunlight conditions of normal culture, and the double plants > single plants > wild type plants. After wild type WT of populus tomentosa and spl16 single-process and spl16/23 double-process materials were cultured under long-day conditions (16 h light/8 h dark) for 2 months, one part of materials was transferred to short-day conditions (10 h light/14 h dark) for treatment, and the other part of materials (control group) was continued to be cultured under long-day conditions. After about 15 days of short-day treatment, spl16 single-process, spl16/23 double-process materials were found to have a delayed growth stop and dormancy time compared to the WT poplar terminal buds, indicating that the terminal buds of the mutant plants still maintained higher growth activity under short-day conditions. The invention shows that after the SPL16 is singly knocked out or the SPL16/23 genes are knocked out simultaneously, the dormancy period conversion in the seasonal growth process of poplar can be changed. Under the condition of short sunshine in autumn, the growth stop and dormancy time of the terminal buds of the trees can be delayed, so that the method has a great application prospect in the aspects of improving photosynthetic carbon fixation and biomass accumulation of the trees.
Claims (7)
1. Use of the SPL16 or/and SPL23 genes of poplar in regulating the dormant period transformation of terminal buds during seasonal growth of poplar, wherein the SPL16 genes encode a protein of the amino acid sequence shown in SEQ ID No.1, and the SPL23 genes encode a protein of the amino acid sequence shown in SEQ ID No. 2.
2. The use according to claim 1, wherein the use is to delay the time for terminal bud growth to stop and enter sleep phase after poplar perception of autumn photoperiod changes by knocking out SPL16 gene or simultaneously knocking out SPL16/SPL23 gene by CRISPR/Cas9 editing system.
3. The use according to claim 1 or 2, wherein the nucleotide sequence of the SPL16 gene is shown in SEQ ID No.3 and the nucleotide sequence of the SPL23 gene is shown in SEQ ID No. 4.
4. A method of constructing a poplar line that attenuates the response of the poplar to short sunlight conditions, delays the cessation of terminal bud growth and dormancy, comprising the steps of:
(1) Constructing an editing vector for the poplar SPL16 gene or for the SPL16 and SPL23 genes simultaneously by using a CRISPR/Cas9 gene editing technology; the nucleotide sequence of the SPL16 gene is shown as SEQ ID No.3, and the nucleotide sequence of the SPL23 gene is shown as SEQ ID No. 4;
(2) Transferring the editing vector constructed in the step (1) into poplar leaves by adopting an agrobacterium-mediated leaf disc method;
(3) Through poplar tissue culture and screening of positive transgenic plants, the wild type and transgenic plants of populus tomentosa cultivated under long sunlight conditions are transferred to short sunlight conditions for treatment, and the variation of terminal bud phenotype is observed and counted to obtain the poplar strain with delayed terminal bud dormancy under the short sunlight conditions, wherein the long sunlight conditions are 16h illumination/8 h darkness, and the short sunlight conditions are 10h illumination/14 h darkness.
5. The method according to claim 4, wherein the nucleotide sequence of the sgRNA of the editing vector for the SPL16 gene is shown in SEQ ID No.5 and 6, and the nucleotide sequence of the sgRNA of the editing vector for the SPL23 gene is shown in SEQ ID No.7 and 8.
6. The sgRNA targeting the poplar SPL16/SPL23 gene is characterized in that the nucleotide sequence of the sgRNA is shown in any one of SEQ ID No. 5-SEQ ID No. 8.
7. A gene editing vector comprising any one of SEQ ID No.5 to SEQ ID No. 8.
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