Application and method of knockout poplar LUX1 gene in maintaining bud growth and wood development under short day

Abstract

The invention discloses an application and a method for knocking out a poplar LUX1 gene in delaying growth stop or dormancy time under short day, and the poplar LUX1 gene is identified and obtained through amino acid sequence comparison with arabidopsis thaliana LUX. The poplar LUX1 gene is a protein for coding an amino acid sequence as shown in SEQ ID No.1. The LUX1 gene is knocked out through a CRISPR / Cas9 editing system, and the time that the poplar enters the dormancy period due to short sunlight in autumn is delayed, so that the poplar germplasm with long growth period and high biomass is obtained. After the LUX1 gene is knocked out, terminal bud growth stop and stem cambium activity reduction in the seasonal growth process of the poplar can be delayed, so that wood development is promoted. Deferring of the dormancy time under the autumn short-day condition has a great application prospect for prolonging the growth period of the trees so as to improve photosynthetic carbon sequestration and biomass accumulation.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to an application of knocking out a poplar LUX1 gene in delaying bud growth cessation or dormancy time under short-day conditions, and also to a method for maintaining cambium activity and xylem development under short-day conditions. Background Art

[0002] Poplar is one of the fastest-growing timber species with the largest cultivated area in the mid-latitude plains in the world. It has the characteristics of fast growth, high yield and easy renewal. It is widely used and planted in timber, papermaking, windbreak and sand fixation. Wood is one of the main components of the stems of vascular plants and an important source of renewable bioenergy. Therefore, cultivating new poplar varieties with long growth period and increased biomass is an important issue to be solved in the development of poplar industrialization, which is of great significance for increasing timber yield and improving timber quality.

[0003] Poplars are mainly used as timber and landscaping tree species in the northern plains of my country. Poplars have typical seasonal growth characteristics. Seasonal growth is mainly regulated by environmental factors such as photoperiod and temperature. Among them, short days in autumn are the main factor for poplars to enter dormancy. Vegetative growth stops in late summer or early autumn, and the apical meristem no longer produces new shoots, but forms tightly closed and hardened bud scales for protection (Maurya and Bhalerao, 2017). The process of the apical bud entering dormancy can be divided into two stages. The first is ecological dormancy, that is, the apical bud stops growing and stays in a state of growth stagnation under short-day conditions, but it maintains the ability to perceive external growth-promoting signals. The second is internal dormancy, in which the buds enter dormancy under continuous short-day and low-temperature conditions, lose the response to growth-promoting signals, and enhance the buds' low-temperature adaptability. The internal dormant buds continue to accumulate cold signals until they reach the required amount before breaking dormancy and then resuming growth under long-day and warming conditions (Singh et al., 2017). This cycle of growth and dormancy is critical to the ability of trees to adapt to different latitudes and altitudes. The higher the latitude or altitude, the earlier the trees stop growing and establish dormancy, and the later they sprout in the spring (Nilsson, 2022).

[0004] The vascular cambium activity and wood development in the stems of perennial trees also have significant periodic characteristics, and the formation of annual rings is the main manifestation of their seasonal growth. Annual rings are divided into earlywood and latewood according to seasonal changes. Earlywood (spring wood) formed at the beginning of the growing season (spring and summer) is characterized by large cell volume, thin cell walls, numerous vessels, and lighter color. Latewood (autumn wood) will be formed in the later stage of the growing season (autumn and winter). Compared with earlywood, latewood has the characteristics of small cell volume, thick cell walls, fewer vessels, and darker color. Wood with a higher earlywood rate has better performance and more wood volume for papermaking. Therefore, studying the mechanism of regulating the seasonal growth of poplar can not only improve the environmental adaptability of trees, but also has important significance for cultivating new poplar germplasms with long growth periods and high yields.

[0005] The biological clock is regarded as an important regulatory mechanism for organisms to synchronize with the rhythms of the external environment and is involved in regulating almost all processes of plant growth and development. The biological clock can help organisms anticipate the periodic changes in the environment and adjust their life activities in advance to enhance their environmental adaptability. The biological clock system can be simplified into three processes: the input pathway, the core oscillator, and the output pathway. The core oscillator of the plant biological clock is mainly composed of three interconnected transcriptional-translational feedback loops, namely the morning loop, the central loop, and the evening loop (Wei Hua, Wang Yan, Liu Baohui, Wang Lei, 2018). Among them, the evening loop is the EC evening complex composed of EARLY FLOWERING 3 (ELF3), ELF4, and LUX (LUX ARRHYTHMO), and its expression shows a typical rhythmic expression pattern and reaches its peak in the evening (Nusinow et al., 2011; Nakamichi, 2020). As a MYB-like transcription factor, LUX can recruit the EC complex to the promoters of target genes and inhibit the expression of these genes. There are few reports on the role of the biological clock gene LUX in poplar, and whether it plays an important role in regulating the seasonal growth of poplar remains to be further studied. Summary of the Invention

[0006] One of the purposes of the present invention is to provide an application of knocking out the LUX1 gene in poplar in delaying the cessation of bud growth or dormancy time under short-day conditions; another purpose of the present invention is to provide a method for maintaining cambium activity and xylem development under short-day conditions.

[0007] To achieve the above purposes, the present invention provides the following technical solutions:

[0008] 1. Application of knocking out Populus LUX1 gene in delaying growth cessation or dormancy time under short-day conditions, wherein the Populus LUX1 gene is a gene encoding a protein with the amino acid sequence shown in SEQ ID No. 1.

[0009] Preferably, in the present invention, the knocking out of the Populus LUX1 gene is achieved by knocking out the LUX1 gene through the CRISPR / Cas9 editing system.

[0010] Preferably, in the present invention, the target sequences of the CRISPR / Cas9 editing system are as shown in SEQ ID No. 3 and SEQ ID No. 4. Preferably, knocking out the Populus LUX1 gene delays growth cessation under short-day conditions, enabling Populus to grow more leaf numbers and internode numbers; meanwhile, the vascular cambium in the stem continues to maintain division activity, resulting in the production of more xylem cells and inhibiting short-day-mediated latewood development of xylem.

[0011] 2. A method for extending the growth period of Populus under short-day conditions. The specific method is to transform Populus tomentosa with the LUX1 gene through

[0012] a CRISPR / Cas9 gene editing vector to obtain transgenic plants with LUX1 gene-edited mutations; the

[0013] nucleotide sequence of LUX1 is as shown in SEQ ID NO. 2.

[0014] Preferably, in the present invention, the method for transforming Populus tomentosa is mediated by Agrobacterium.

[0015] Preferably, in the present invention, the Agrobacterium is Agrobacterium tumefaciens GV3101.

[0016] Compared with wild-type plants, the lux1 mutant plants obtained by the method of the present invention have significantly increased numbers of lateral branches. After cultivating Populus tomentosa wild-type WT and lux1 mutants under long-day conditions (16 h light / 8 h darkness) for 2 months, a part of the materials were transferred to short-day conditions (12 h light / 12 h darkness) for treatment, and another part of the materials (control group) continued to be cultivated under long-day conditions. After approximately 3 weeks of short-day treatment, it was found that the growth cessation and dormancy time of the apical buds of the lux1 mutant materials were postponed compared with those of WT poplar. The apical buds of the mutant plants still maintained a relatively high growth activity and grew more leaves under short-day conditions, and the above-ground biomass of the lux1 mutants decreased less than that of WT under short-day conditions; by observing the stem sections, it was found that compared with wild-type plants, the vascular cambium activity of the lux1 mutants was higher under short-day conditions, and more secondary xylem (wood) could be formed. This indicates that the sensitivity of the lux1 mutant plants to short days is reduced, and they can still maintain the growth of the apical meristem (apical bud) and lateral meristem (cambium) under short-day conditions, improving the photosynthetic carbon fixation and biomass accumulation of trees.

[0017] The beneficial effects of the present invention are as follows: The present invention shows that after knocking out the LUX1 gene, the growth cessation of the apical buds and the decrease in cambium activity in the stem during the seasonal growth of poplar can be delayed, thereby promoting wood development. The postponement of the dormancy time under short-day conditions in autumn has great application prospects for extending the growth period of trees and improving photosynthetic carbon fixation and biomass accumulation. Description of the Drawings

[0018] To make the objectives, technical solutions, and beneficial effects of the present invention clearer, the following drawings are provided for illustration:

[0019] Figure 1 Creation of Populus tomentosa lux1 mutant materials (a: Design of the LUX1 gene structure and target positions (T1, T2) of Populus tomentosa; b: Genotype sequencing results of Populus tomentosa lux1 mutant materials (two lines L1 and L2)).

[0020] Figure 2 Phenotype observation of Populus tomentosa lux1 mutant materials (a: Phenotypes of WT lines and lux1 mutant lines of Populus tomentosa wild-type and lux1 mutant materials after being transferred from long-day (LD, 16 h light / 8 h darkness) to short-day (SD, 12 h light / 12 h darkness) treatment for 3 weeks; b: Statistical analysis of plant heights of Populus tomentosa wild-type and lux1 mutant materials; c: Statistical analysis of fresh weights of above-ground parts of Populus tomentosa wild-type and lux1 mutant lines).

[0021] Figure 3Short-day delays the growth arrest of lux1 mutant plants (a: Phenotypes of the apical buds of the WT line and the lux1 mutant line after 3 weeks of transfer from long-day (16 h light / 8 h dark) to short-day (12 h light / 12 h dark) treatment; b: Number of newly grown leaves of the WT line and the lux1 mutant line after transfer to short-day).

[0022] Figure 4 The lux1 mutant line alleviates the inhibition of short-day on secondary development (a: Number of internode growths of the WT line and the lux1 mutant line after 3 weeks of short-day (12 h light / 12 h dark) treatment, where m is the marked internode, u2 is the second internode above the mark, and u4 is the fourth internode above the mark; b: Observation of the vascular development (proportion of xylem) of the marked internode and the second and fourth internodes above it in the WT and lux1 mutant lines treated with short-day by toluidine blue staining; c: Number of cell layers in the cambium of the marked internode and the second and fourth internodes above it in the WT and lux1 mutant lines treated with short-day; d: Statistics of the number of cell layers in the cambium of the marked internode; e: Observation of the developing xylem cells of the marked internode and the second and fourth internodes above it in the WT and lux1 mutant lines treated with short-day by phloroglucinol staining; f: Data statistics of the vessel cells). Detailed implementation mode

[0023] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited do not limit the present invention.

[0024] Example 1: Construction of the CRISPR / Cas9 knockout vector of the LUX1 gene

[0025] First, download the Populus LUX1 gene (gene sequence as SEQ ID No.2, encoded protein as SEQ ID No.1) from the Phytozome database. Use the SnapGene Viewer software to design specific target sequences for the LUX1 gene (design sgRNA). To ensure gene editing efficiency, 2 optimal target sequences were designed for each gene (Table 1). Design primers through targetDesign, amplify by PCR, and ligate these target sequences into the pYLCRISPR_Cas9P35S-N vector by homologous recombination, transform Escherichia coli DH5a, perform PCR bacterial inspection, pick single colonies for shaking culture and extract plasmids. After verifying the vector by sequencing, transform Agrobacterium tumefaciens GV3101 for Populus tomentosa genetic transformation.

[0026] >LUX1 protein sequence (SEQ ID No.1):

[0027] MGEEVKMSEYEINDGEDNINGDDERVAVWEIGLPTPDDLTPLSQTLIPPELASAFSIFPEPHRTPLDVNRACQTTLSNLRGQLNALSSINFKSFNETTGQTHDPIVVDLDDKTGAVDRDGSGSEARKLRRVDSEEEDSALRAENSAEDPSSAAARNLKRPRLVWTPQLHKRFVDVVGHLGIKNVVPKTIMQLMNVEGLTRENVASHLQKYRLYLKRMQGLSSEGPSASDQLFASTPLPQSFPESSGGGGNGNFGIPIPMPYHHPATAGGMMPMPVYGHMGMQMGNNNGHNNNSSDNHQHHQVSINGHQNGYNGNVAHGHMLQQRDWNGNHYGSYTHHPHQVAPNDNM

[0028] >LUX1 gene sequence

[0029]

[0030] Table 1 Designed sgRNA Sequences

[0031]

[0032] Example 2. Genetic Transformation and Genotype Identification of Poplar

[0033] Using Populus tomentosa as the receptor material, the successfully constructed CRISPR / Cas9 vector was introduced into poplar leaves by the Agrobacterium-mediated leaf disc method, and positive transgenic plants with corresponding resistance were screened through poplar tissue culture.

[0034] First, the successfully constructed CRISPR / Cas9 vector was transferred into Agrobacterium tumefaciens GV3101 and cultured on YEP solid medium (containing 50 mg / L Kan + 40 mg / L Rif) in an incubator at 28°C for 2 days. Then, single colonies were picked and inoculated into YEP liquid medium (containing 50 mg / L Kan + 40 mg / L Rif), and cultured overnight on a shaker at 28°C until the OD600 reached 0.8 - 1.0. The overnight-cultured Agrobacterium was inoculated into YEP liquid medium (containing 50 mg / L Kan + 40 mg / L Rif) at a ratio of 1:50 and cultured with shaking at 28°C until the OD reached 0.4 - 0.6. Subsequently, centrifugation was carried out in a low-temperature centrifuge at 4°C at 4000 rpm for 10 min to collect the bacterial cells. The bacterial cells were resuspended with a WPM resuspension containing AS and the volume was adjusted to 50 mL, and then cultured with shaking at 28°C for 40 min for transformation. Sterile and well-growing poplar leaves were selected, cut into 4 - 6 mm leaf discs in a laminar flow hood, placed in the shaken culture solution for infection for 7 - 10 min, shaken 3 times during this period, then the surface bacterial solution of the leaf discs was blotted dry with sterile paper, and then placed on the co-culture medium for dark culture for 2 days. After 2 days, it was transferred to a medium with kanamycin resistance for dark culture for about 4 weeks until callus grew and was transferred to a shoot induction medium with kanamycin resistance. When the adventitious buds grew to about 2 cm, they were cut and transferred to a rooting medium containing kanamycin to induce rooting.

[0035] After obtaining the plants, plant DNA was extracted from the plant leaves using the CTAB method. To identify its genotype, specific primers for LUX1 and the gene containing the target sequence were used to amplify the gene fragment, and accurate gene editing information was obtained through PCR sequencing. The specific results were as follows: In the lux1 mutant L1, there was a 3-bp base deletion at the T1 target of the LUX1 gene and a 1-bp base insertion at the T2 target. In the lux1 mutant L2, there was a 264-bp base deletion from the T1 target to the T2 target of the LUX1 gene, as Figure 1 shown.

[0036] Example 3. Biological Functions of Transgenic Plants

[0037] After cultivating the wild-type Populus tomentosa WT and lux1 mutant materials under long-day conditions (16 h light / 8 h dark) for 2 months, a part of the materials was transferred to short-day conditions (12 h light / 12 h dark) for treatment, and another part of the materials (control group) continued to be cultivated under long-day conditions. Under the normal long-day cultivation conditions, compared with the wild-type plants, the number of lateral branches of the mutant plants increased significantly, and the results are as shown in Figure 2 . According to the scoring criteria for the growth status of apical buds in previous studies, the changes in the growth status of apical buds of poplars with different genetic backgrounds were observed, recorded, and evaluated after short-day treatment. After about 4 weeks (4W) of short-day treatment, the above-ground biomass of WT poplars decreased by 38%, while the above-ground biomass of the lux1 mutant decreased by about 13%-18% ( Figure 2 ). Under short-day conditions, the growth cessation and dormancy time of the apical buds of the lux1 mutant were postponed compared with those of WT poplars, that is, the apical buds of the mutant plants still maintained a high growth activity under short-day conditions, thus growing more new leaves (number of internodes) ( Figure 3 , a, b). These results indicate that knocking out the LUX1 gene can weaken the sensitivity of poplars to short-day conditions and promote biomass accumulation by extending the growth period of the trees.

[0038] In addition, through sectioning and toluidine blue / phloroglucinol staining of the internodes above the marker and observing, it was found that under short-day conditions, the number of growing internodes increased (a, b in Figure 4 ), the xylem cells of the WT stem quickly lignified and showed the characteristics of late wood, while the stem of the lux1 mutant continued to grow during the same period, forming significantly more layers than the WT, and the xylem did not show the characteristics of late wood ( Figure 4 c, d in

[0039] ). The xylem of the lux1 mutant stem is more than that of the WT, and the number of vessel cells is large and has a larger lumen area ( Figure 4 e, f in

[0039] ). The present invention shows that after knocking out the LUX1 gene, the xylem phenology in the seasonal growth process of poplars can be changed. Maintaining the cambium activity of the lux1 mutant under short-day conditions in autumn has great application prospects for extending the growth period of trees to improve photosynthetic carbon fixation and wood yield. The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Use of knocking out the Populus LUX1 gene in delaying the growth cessation or dormancy time under short-day conditions, characterized in that: The poplar LUX1 gene is a gene encoding a protein with the amino acid sequence shown in SEQ ID No.

1.

2. The application according to claim 1, wherein: The knockout of the poplar LUX1 gene is achieved by knocking out the LUX1 gene through the CRISPR / Cas9 editing system.

3. The application according to claim 1, wherein: The target sequences of the CRISPR / Cas9 editing system are shown in SEQ ID No. 3 and SEQ ID No.

4.

4. The application according to claim 1, characterized in that: The knockout of the poplar LUX1 gene delays the growth cessation under short-day conditions, resulting in the poplar growing more leaf numbers and internode numbers; meanwhile, the vascular cambium in the stem continues to maintain the division activity, leading to the production of more xylem cells and inhibiting the development of latewood in the xylem mediated by short-day.

5. A method for extending the growth period of poplar under short-day conditions, characterized in that, The specific method is to transform Populus tomentosa with the LUX1 gene through a CRISPR / Cas9 gene editing vector to obtain transgenic plants with LUX1 gene-edited mutations; the nucleotide sequence of LUX1 is shown in SEQ ID NO.

2.

6. The method for extending the growth period of poplar under short-day conditions according to claim 5, wherein: The method for transforming Populus tomentosa is mediated by Agrobacterium.

7. The method for extending the growth period of poplar under short-day conditions according to claim 5, characterized in that: The Agrobacterium is Agrobacterium tumefaciens GV3101.

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