Gene influencing morphogenesis of asparagus lettuce plant, application and regulation mechanism

Abstract

The invention provides a gene influencing morphogenesis of an asparagus lettuce plant as well as application and a regulation and control mechanism, and belongs to the technical field of gene engineering. The invention provides a gene LsGA20ox1 influencing morphogenesis of an asparagus lettuce plant, after overexpression of the gene LsGA20ox1, the stem length of the plant is significantly increased, the lignin content is increased, a hollow phenomenon occurs, the chlorophyll content in the stem is significantly increased, but the chlorophyll content of leaves is significantly reduced; the CRISPR knockout mutant shows a dwarfing phenotype, and the content of chlorophyll in leaves is increased; meanwhile, the content of endogenous gibberellin GA19 in overexpressed plants is remarkably reduced, and the content of GA20 is remarkably increased; in a gene knockout plant, the content of endogenous gibberellin GA19 is remarkably increased, and the content of GA20 is greatly reduced. The invention provides a mechanism for regulating and controlling the jointing and hollowing of the asparagus lettuce by a gibberellin metabolic pathway for the first time, and provides a theoretical support for establishing a genetic improvement strategy independent of exogenous hormone treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a gene affecting the morphological development of lettuce plants, its application and regulatory mechanism. Background Art

[0002] As an important leafy vegetable crop globally, the yield and quality of lettuce (Lactuca sativa L.) are directly affected by key agronomic traits such as plant type, leaf development, and flowering time. The special variety of Sichuan, Sanqing lettuce, is famous for its unique quality of "green peel, green flesh, and green leaves". However, its natural dwarfing characteristic leads to shortened internodes, and exogenous gibberellins (GAs) treatment is required to promote internode elongation to enhance commercial value. However, in field applications, the application effect of gibberellin is affected by spraying concentration, treatment period, and environmental factors, which easily leads to problems such as insufficient internode regulation or excessive elongation.

[0003] As a core hormone regulating plant cell division and elongation, GA20 oxidase (GA20ox) in its biosynthesis pathway is a rate-limiting enzyme, responsible for catalyzing the conversion of inactive precursors such as GA 12 and GA 53 into bioactive forms GA4 and GA1. Studies on model plants have shown that GA20ox family genes can significantly change phenotypes such as plant height, leaf morphology, and flowering time by regulating endogenous GA levels. However, the biological function of the GA20ox1 gene in lettuce has not been analyzed, and there is still a lack of direct evidence for its GA regulatory mechanism in lettuce morphological development. Summary of the Invention

[0004] The present invention provides a gene affecting the morphological development of lettuce plants, its application and regulatory mechanism. The gene has a regulatory effect on stem elongation and leaf development, and can provide a theoretical support for establishing a genetic improvement strategy independent of exogenous hormone treatment.

[0005] The present invention provides a gene affecting the morphological development of lettuce plants, and the gene includes the CDS sequence with the nucleotide sequence shown in SEQ ID No.1.

[0006] The present invention also provides the application of the above gene in regulating lettuce morphological development.

[0007] In a preferred embodiment of the present invention, the lettuce morphological development includes the stem and leaves of lettuce.

[0008] In a preferred embodiment of the present invention, the regulation includes that after overexpressing the gene, the endogenous GA content is reduced, the content of bioactive gibberellin GA1 is increased, the stem length is increased, the chlorophyll content in the stem is increased, and the chlorophyll content in the leaves is reduced;

[0009] After knocking out the expression of the gene, the endogenous GA content is increased, the content of active gibberellin GA1 is decreased, the stem length is dwarfed, the chlorophyll content in the stem is decreased, and the chlorophyll content in the leaves is increased.

[0010] The present invention also provides a biological material for regulating the above gene.

[0011] In a preferred embodiment of the present invention, the regulation includes overexpressing or knocking out the gene.

[0012] The present invention also provides the application of the above biological material in regulating the chlorophyll content of lettuce stems and / or leaves.

[0013] The present invention also provides an overexpression vector containing the CDS sequence of the above gene.

[0014] The present invention also provides the application of the above overexpression vector in increasing the stem length of lettuce.

[0015] The present invention also provides a group of sgRNAs for knocking out the expression level of the above gene, including sgRNA1 with the nucleotide sequence shown in SEQ ID No. 3 and sgRNA2 with the nucleotide sequence shown in SEQ ID No. 4.

[0016] The present invention also provides the application of the above gene in creating lettuce germplasm.

[0017] Beneficial effects: The present invention provides a gene LsGA20ox1 that affects the morphological development of lettuce plants. In the examples, Sanqing lettuce is used as an example for verification. After overexpressing the gene LsGA20ox1, the stem length of the plants is significantly increased, and the chlorophyll content in the stem is significantly increased, but the chlorophyll content in the leaves is significantly decreased; while the CRISPR knockout mutants show a dwarfed phenotype and increase the chlorophyll content in the leaves. In the examples of the present invention, the regulation mechanism of the gene LsGA20ox1 is also deeply studied. For example, in overexpressing plants, the endogenous gibberellin GA 19 content is significantly decreased, and the GA 20 content is significantly increased; in gene knockout plants, the endogenous gibberellin GA 19 content is significantly increased, and the GA 20 content is greatly decreased, confirming that the GA20ox enzyme encoded by LsGA20ox1 is mainly responsible for catalyzing the conversion process of GA 19 to GA 20 ; at the same time, the gene expression level during the conversion process is measured, and the overexpression of LsGA20ox1 promotes the increase in the expression of the downstream LsGA2ox1 gene, thereby promoting the increase in the content of active gibberellin GA1, suggesting that LsGA20ox1 affects cell elongation by regulating GA synthesis.

[0018] Through morphological analysis, it was found that the plant height of the "Sanqing" lettuce with overexpression of the gene GA20ox1 in the present invention increased significantly, the lignin content increased, but the structure of the apical meristem was abnormal, and there was a serious hollow phenomenon in the stem. Section analysis of the hollow tissue showed that the cells in the hollow part were dispersed and the cell walls were damaged. It is very likely that the hollow was caused by cell wall degradation. Based on this, the present invention first proposed the mechanism of gibberellin metabolic pathway regulating the jointing and hollowness of lettuce, providing valuable research materials for subsequent studies on the role of gibberellin regulation pathway in the formation of hollow stem in lettuce, and providing theoretical support for establishing a genetic improvement strategy independent of exogenous hormone treatment. Description of the Drawings

[0019] Figure 1 It is a schematic diagram for the construction of gene-edited and overexpressed plants of the LsGA20ox1 gene in "Sanqing" lettuce. In the figure, A: Schematic diagram of the gene-editing site of LsGA20ox1; B: Protein sequence analysis of the gene-edited plants of LsGA20ox1; C: PCR detection results of the overexpressed plants of LsGA20ox1; D: Relative expression levels of the LsGA20ox1 gene and downstream genes.

[0020] Figure 2 It is a functional analysis diagram of the LsGA20ox1 gene in "Sanqing" lettuce. In the figure, A: Phenotype of lettuce plants grown in the greenhouse for 56 days; B: Measurement results of the length of lettuce stems; C: Measurement results of the internode distance of lettuce, and the internode distance is the stem length divided by the number of leaves; D: Measurement results of the chlorophyll content of lettuce leaves; The "*" above the bar chart represents significant difference compared with the wild-type plants (P<0.05, n = 10), scale bar = 5 cm.

[0021] Figure 3 It is a diagram showing the measurement results of the structure of the apical meristem and the gibberellin content of overexpressed and knockout mutant plants of the LsGA20ox1 gene; In the figure, A-C: Lettuce plants grown in the artificial climate chamber for 28 days, which are wild-type "Sanqing" lettuce WT (A), LsGA20ox1 overexpressed mutant OE#1 (B) and knockout mutant plant KO#1 (C); D-F: Paraffin sections of the shoot apical meristem of lettuce, which are WT (D), OE#1 (E) and KO#1 (F); G: Gibberellin content in young leaves of lettuce plants.

[0022] Figure 4The figure shows the results of the effect of LsGA20ox1 on the hollowness of the stems of "Sanqing" lettuce. In the figure, A: longitudinal sections of the stems of the overexpressing mutant plants of the LsGA20ox1 gene (left) and wild-type "Sanqing" lettuce (right) 28 days after transplantation; B: internal cross-sections of the stems of the overexpressing mutant plants of the LsGA20ox1 gene and wild-type "Sanqing" lettuce 28 days after transplantation; C: chlorophyll content in the stem tissues of the overexpressing mutant plants of the LsGA20ox1 gene and wild-type "Sanqing" lettuce 28 days after transplantation; D: longitudinal sections of the stems of the overexpressing mutant plants of the LsGA20ox1 gene (left) and wild-type "Sanqing" lettuce (right) 56 days after transplantation; "*" above the bar graph indicates significant difference compared with the wild-type plants (P < 0.05, n = 5), scale bar = 2 cm;

[0023] Figure 5 The figure shows the results of the effect of LsGA20ox1 on the lignin content of the stems of "Sanqing" lettuce. In the figure, A and B represent the cross-sections of the hollow stems of wild-type and overexpressing mutant plants of the LsGA20ox1 gene respectively; C and D represent the lignin staining diagrams of wild-type and overexpressing mutant plants of the LsGA20ox1 gene respectively; E: differentially expressed genes in the lignin synthesis pathway in lettuce plants 28 days after transplantation; HCT, hydroxycinnamoyl-CoA transferase; CCR, cinnamoyl-CoA reductase; POD, peroxidase; F: gibberellin content in lettuce stems 28 days and 56 days after transplantation; "*" above the bar graph indicates significant difference compared with the wild-type plants (P < 0.05, n = 3), scale bar = 2 cm; Detailed implementation mode

[0024] The present invention provides a gene that affects the morphological development of lettuce plants, and the gene includes the CDS sequence with the nucleotide sequence shown in SEQ ID No.1.

[0025] As used in the present invention, morphological development refers to the process in the development of a plant body where different cells gradually differentiate in different directions, thereby forming cells, tissues, and organs with various special structures and functions, and ultimately forming an individual with phenotypic characteristics. This process is called morphological development.

[0026] The nucleotide sequence of the CDS sequence of the gene LsGA20ox1 of the present invention is shown in SEQ ID No.1.

[0027] The gene LsGA20ox1 of the present invention is a homologous gene of the rice OsGA20ox2 gene and has a similar function in the stem length phenotype, that is, overexpression significantly increases the stem length, and knockout shows a dwarf phenotype. At the same time, the gene LsGA20ox1 of the present invention also shows a new function, that is, overexpression increases the chlorophyll content in the stem, and knockout reduces the chlorophyll content.

[0028] The present invention also provides the application of the above gene in regulating the morphological development of lettuce.

[0029] The morphological development of lettuce described in the present invention includes the stems and leaves of lettuce. By regulating the expression level of the gene LsGA20ox1, the traits of the stems and leaves of lettuce can be adjusted. The present invention also proposes a mechanism for regulating the jointing and hollowness of lettuce by the gibberellin metabolic pathway. Specifically, after overexpressing the gene, the endogenous GA content is reduced, the content of active gibberellin GA1 is increased, the stem length is increased, the chlorophyll content in the stem is increased, and the chlorophyll content in the leaves is reduced; after knocking out the expression of the gene, the endogenous GA content is increased, the content of active gibberellin GA1 is reduced, the stem length is dwarfed, the chlorophyll content in the stem is reduced, and the chlorophyll content in the leaves is increased. At the same time, through morphological analysis, the plant height of the Sanqing lettuce overexpressing the gene GA20ox1 in the present invention is significantly increased, but the structure of the apical meristem is abnormal, and serious hollowness appears in the stem. Section analysis of the hollow tissue found that the cells in the hollow part were dispersed and the cell walls were damaged, most likely due to the degradation of the cell walls causing hollowness. Based on this, the present invention first proposes a mechanism for regulating the jointing and hollowness of lettuce by the gibberellin metabolic pathway, providing valuable research materials for subsequent research on the role of the gibberellin regulation pathway in the formation of hollowness in lettuce stems, and providing theoretical support for establishing a genetic improvement strategy independent of exogenous hormone treatment.

[0030] The present invention also provides a biological material for regulating the above gene.

[0031] In a preferred embodiment of the present invention, the regulation includes overexpressing or knocking out the gene. In an embodiment of the present invention, according to the lettuce genome database (NCBI accession number: LOC111904022), specific primers GA20Fw / GA20Rv (Table 1) were designed to amplify the full-length CDS of GA20ox1, and then the GA20ox1 CDS was inserted into the pCAMBIA1300-35S vector (HindIII / XbaI sites) to construct an overexpression vector 35S::GA20ox1 recombinant plasmid; the overexpression vector was transferred into Agrobacterium tumefaciens GV3101, and then an overexpressing plant was constructed by genetic transformation. Among them, the PCR program used for amplification includes the following cycling program: 98°C for 3 min; 98°C for 10 s, 58°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 10 min, and stored at 4°C.

[0032] In an embodiment of the present invention, two gRNA sequences (Table 1) were designed according to the first exon of GA20ox1 and constructed into the pKSE401 vector (U6 promoter-driven sgRNA) to obtain a gene knockout vector. The gene knockout vector was transferred into Agrobacterium tumefaciens GV3101, and then a gene knockout plant was constructed by genetic transformation.

[0033] Table 1 Primer information used in the present invention

[0034]

[0035]

[0036] The present invention also provides an overexpression vector containing the CDS sequence of the above gene.

[0037] Based on the forward primer GA20Fw of the overexpression vector and the reverse primer GA20Rv of the overexpression vector, the full-length CDS sequence of the LsGA20ox1 gene described in the present invention was amplified. The amplification procedure includes: 98°C for 3 min; 98°C for 10 s, 58°C for 30 s, 72°C for 1 min, for 30 cycles; 72°C for 10 min, stored at 4°C.

[0038] The present invention inserted the amplified LsGA20ox1 CDS sequence into the HindIII / XbaI site of the pCAMBIA1300-35S vector to construct a 35S::GA20ox1 recombinant plasmid as the overexpression vector described in the present invention.

[0039] The present invention also provides the application of the above overexpression vector in increasing the stem length of lettuce.

[0040] In the examples of the present invention, Sanqing lettuce was used for verification, and it was proved that overexpressing plants were constructed using the 35S::GA20ox1 recombinant plasmid. The stem length of the overexpressing plants was significantly increased, 4 times higher than that of the wild type, but the structure of the apical meristem was abnormal and the stem showed serious hollowness. Section analysis of the hollow tissue found that the cells in the hollow part were dispersed and the cell walls were damaged, most likely due to the degradation of the cell walls causing the hollowness.

[0041] The present invention also provides the application of the above biological material in regulating the chlorophyll content of lettuce stems and / or leaves.

[0042] In the examples of the present invention, it was confirmed that after overexpressing the gene, the chlorophyll content in the stems could be increased and the chlorophyll content in the leaves could be decreased; after knocking out the gene, the chlorophyll content in the stems was decreased and the chlorophyll content in the leaves was increased.

[0043] The present invention also provides a group of sgRNAs for knocking out the expression level of the above gene, including sgRNA1 with the nucleotide sequence shown in SEQ ID No. 3 and sgRNA2 with the nucleotide sequence shown in SEQ ID No. 4.

[0044] The present invention uses the sgRNA1 as gene editing target 1 and the sgRNA2 as gene editing target 2, anneals the two gene editing targets and ligates them into a gene editing Cas9 vector, such as the pKSE401 vector, and uses the U6 promoter to drive the sgRNA. The present invention constructs a gene knockout mutant by means of genetic transformation. The knockout mutant exhibits a dwarf phenotype, and its stem length is reduced by approximately 17% compared to the wild type.

[0045] The present invention also provides the application of the above gene in creating lettuce germplasm.

[0046] In the examples of the present invention, through metabolite detection, it is confirmed that LsGA20ox1 is the key rate-limiting enzyme regulating GA conversion in the gibberellin biosynthesis pathway, and abnormal expression levels thereof will affect the proliferation and differentiation patterns of meristem cells by changing gibberellin homeostasis. The gene expression results also show that overexpression of LsGA20ox1 promotes the increased expression of the downstream LsGA2ox1 gene, thereby promoting the increase in the content of the active gibberellin GA1, thus proposing a mechanism for regulating the jointing and hollowness of lettuce by the gibberellin metabolic pathway, providing a theoretical support for establishing a genetic improvement strategy independent of exogenous hormone treatment. 19 To GA 20 transformation, and abnormal expression levels thereof will affect the proliferation and differentiation patterns of meristem cells by changing gibberellin homeostasis. The gene expression results also show that overexpression of LsGA20ox1 promotes the increased expression of the downstream LsGA2ox1 gene, thereby promoting the increase in the content of the active gibberellin GA1, thus proposing a mechanism for regulating the jointing and hollowness of lettuce by the gibberellin metabolic pathway, providing a theoretical support for establishing a genetic improvement strategy independent of exogenous hormone treatment.

[0047] To further illustrate the present invention, the following examples are used to describe in detail a gene, its application and regulatory mechanism affecting the morphological development of lettuce plants provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0048] In the examples of the present invention, all data were analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism 9, and the significance of differences was tested by Duncan's test (p < 0.05).

[0049] Example 1

[0050] 1. Plant materials

[0051] Seeds of Sanqing lettuce (Lactuca sativa var. sanqing) were purchased from Sichuan Maidehao Agricultural Technology Co., Ltd.

[0052] 2. Cloning of GA20ox1 gene and vector construction

[0053] According to the lettuce genome database (NCBI accession number: LOC111904022), specific primers GA20Fw / GA20Rv were designed to amplify the full-length CDS of GA20ox1. The GA20ox1 CDS was inserted into the pCAMBIA1300-35S vector (HindIII / XbaI sites) to construct a 35S::GA20ox1 recombinant plasmid.

[0054] Two gRNA sequences (sgRNA1 and sgRNA2) were designed for the first exon of GA20ox1 on the gene editing design website (http: / / crispr.hzau.edu.cn / CRISPR2 / ) and constructed into the pKSE401 vector (sgRNA driven by the U6 promoter).

[0055] 3. Genetic transformation and plant screening

[0056] The overexpression vector and the gene editing vector were transferred into Agrobacterium tumefaciens GV3101. Lettuce was transformed by the leaf disc method (refer to 116445535A, an Agrobacterium-mediated lettuce genetic transformation method and its application), and the infection solution was the GV3101 bacterial solution with OD 600 = 0.4 (containing 50 μM AS). After co-culturing for 48 h, it was transferred to a selection medium containing 0.1 mg L -1 6-BA, 0.05 mg L -1 NAA, 50 mg L -1 kanamycin and 200 mg L -1 ticarcillin. The medium was changed every 12 days. When the callus differentiated into buds and grew to 3 - 5 cm, it was transferred to a rooting medium containing 50 mg L -1 kanamycin and 200 mg L -1 ticarcillin until roots grew, and then it was transferred to a seedling raising substrate to continue growing to obtain T0 generation plants. DNA of T1 generation plants was extracted. For gene editing plants, after amplification with primers GA20cFw / GA20cRv, second-generation sequencing was performed to detect whether the gene editing sites were successfully edited; for gene overexpression plants, after amplification with primers d35SFw / GA20cRv, agarose gel electrophoresis was performed to detect positive plants. Seeds were harvested (OE#1, OE#2) for subsequent index determination.

[0057] To construct the "Sanqing" lettuce LsGA20ox1 knockout mutant plants, two knockout sites were designed in the first exon region of this gene ( Figure 1 A in). The sequencing results of gene editing plants showed that two independent lines (KO#1 and KO#2) both successfully achieved the knockout of the LsGA20ox1 gene. Specifically, the KO#1 mutant inserted one base at the first knockout site and deleted 3 bases at the second knockout site, resulting in a frameshift mutation and an early appearance of a terminator; the KO#2 mutant was cleaved at both knockout sites, with a 50-base deletion in the middle region, a change in the open reading frame, and an early appearance of a stop codon ( Figure 1 B in).

[0058] For the overexpression strains, the present invention uses vector-specific primers and specific primers for the inserted gene to perform PCR identification, and positive plants are screened and obtained ( Figure 1 in C). The quantitative PCR results show ( Figure 1 in D) that in the LsGA20ox1 overexpression plants, the expression level of this gene is significantly up-regulated by 20 times compared with the wild type, and at the same time, the downstream genes LsGA2ox1 and LsGA2ox2 in the synthesis pathway are significantly up-regulated.

[0059] Table 2 Quantitative PCR primer information

[0060]

[0061] 4. Determination of phenotype and chlorophyll content

[0062] The plant type and chlorophyll content of lettuce plants grown in the greenhouse of the Horticultural Research Institute of Sichuan Academy of Agricultural Sciences for 56 days were measured.

[0063] The plant type measurement includes stem length (from the base to the growth point), stem diameter (measured with a vernier caliper), and the number of leaves.

[0064] The chlorophyll content of lettuce leaves was determined by the ethanol extraction method. Weigh 0.2 g of fresh sample, add 20 ml of 95% ethanol, and soak and extract for 48 h under dark conditions. Use a UV spectrophotometer (UV-1800, Shimadzu, Japan) to measure the absorbance values at 665 and 649 nm. The chlorophyll content is calculated using the following formula.

[0065] Chlorophyll a concentration (mg·L -1 ) = 13.95A665 - 6.88A649;

[0066] Chlorophyll b concentration (mg·L -1 ) = 24.96A649 - 7.32A665;

[0067] Total chlorophyll concentration (mg·L -1 ) = 18.08A649 + 6.63A665.

[0068] Pigment content (mg·g -1 ) = pigment concentration (mg·L -1 ) × extraction solution volume (L) × dilution factor / fresh sample mass (g).

[0069] The plant height, internode distance, and chlorophyll content of lettuce plants grown for 56 days were compared and analyzed. The results are as Figure 2As shown, under conventional cultivation conditions, the internode distance of "Sanqing" lettuce is relatively dense, the stem length is 8.2 cm, and the average internode distance is only 0.39 cm. Overexpression of LsGA20ox1 significantly increased the plant height of lettuce, the stem length reached 35 cm, and the internode distance increased to 1.8 cm; while the LsGA20ox1 knockout mutant plants were further dwarfed, with a plant height of only 6.8 cm and an internode distance of 0.28 cm( Figure 2 in B and C). At the same time, the chlorophyll content in the leaves decreased significantly in the overexpressing plants, from 1.65 mg g -1 in the wild type to 1.49 mg g -1 ; the chlorophyll content in the knockout mutant plants increased significantly, reaching 1.82 mg g -1 ( Figure 2 in D).

[0070] 5. Analysis of the structure of the apical meristem and gibberellin content in LsGA20ox1 overexpressing and knockout mutant plants

[0071] Microscopic structural analysis of the apical meristem of lettuce grown in the greenhouse for 28 days( Figure 3 in A-C, plant phenotypes) showed that the meristems of the wild type and the LsGA20ox1 knockout mutant were both smooth spherical, and the cells in the pith region were closely arranged and evenly distributed( Figure 3 in D and F); while the morphology of the meristem of the LsGA20ox1 overexpressing plants changed significantly, the area of the basic meristem region increased significantly, but a lamellar structure gradually appeared in the pith region, and the cell gap increased significantly( Figure 3 in E).

[0072] The gibberellin synthesis pathway is as Figure 3 shown in G, and the GA 20 oxidase (GA20ox) encoded by LsGA20ox1 specifically catalyzes the conversion of GA 19 to GA 20 . Metabolite detection showed that the content of GA 20 increased significantly in the LsGA20ox1 overexpressing mutant, which was 5.4 times that of the wild type (P<0.01), and the downstream active gibberellin GA1 accumulated synchronously, which was 1.8 times that of the wild type; a large amount of GA 19 was retained in the LsGA20ox1 knockout mutant, which was 5.27 times that of the wild type (P<0.001), while the content of GA 20 decreased to 28.4% of the wild type, indicating that the gene deletion blocked the metabolic flow of GA 19 →GA 20 . It was confirmed that LsGA20ox1 is a regulator of GA 19 to GA 20The key rate-limiting enzyme in the conversion, abnormal expression levels of which can affect the proliferation and differentiation patterns of meristematic cells by altering gibberellin homeostasis.

[0073] 6. Hollow phenotypes appear in LsGA20ox1 overexpressing lettuce plants

[0074] Experimental steps for phloroglucinol-hydrochloric acid lignin staining: Place lettuce stem tissues in FAA fixative (a mixture of formaldehyde - glacial acetic acid - ethanol) for more than 24 hours, embed in paraffin after gradient dehydration, and use a microtome to prepare paraffin sections with a thickness of 8 - 10 μm. The sections are dewaxed in xylene I and II for 10 minutes each, and rehydrated to distilled water through gradient ethanol (100% - 50%). Subsequently, perform acid hydrolysis treatment: Immerse the sections in 1% concentrated hydrochloric acid (HCl) solution for 2 minutes to open the lignin guaiacyl structure. After pouring off the hydrochloric acid, immediately add freshly prepared 1% phloroglucinol ethanol solution (dissolved in 95% ethanol) and stain for 3 minutes, rinse with 50% ethanol to remove the floating color, and finally mount with neutral balsam and observe under an optical microscope. Lignified cell walls (such as vessels, fibers) show purple-red after specific binding with the dye, and the staining results can be recorded through a microscopic imaging system.

[0075] Studying the LsGA20ox1 overexpressing lettuce stems at 28 days and 56 days after transplantation, it was found that after LsGA20ox1 overexpression, the stems showed hollowing at 28 days, and the hollow part accounted for about 1 / 5 of the whole ( Figure 4 in A), and the inside of the stems was completely hollow after 56 days ( Figure 4 in D). At the same time, it was found that the chlorophyll content in the overexpressing lettuce stems was twice that of the wild type lettuce ( Figure 4 in B and C). Through paraffin section analysis of the stems, it was found that the central cells of the hollow stems were fragmented ( Figure 5 in A and B). Qualitative analysis of the lignin content showed that the area of the lignin part in the overexpressing plants was larger than that of the wild type plants ( Figure 5 in C and D). Through transcriptome sequencing, it was found that the expression levels of lignin synthesis genes (LsHCT1, LsHCT2, LsCCR1, and LsPOD1) were significantly upregulated in the overexpressing plants ( Figure 5 in E). After detection, the lignin content increased by 10.2% in the overexpressing plant stems at 28 days after transplantation and by 21% at 56 days ( Figure 5 in F), indicating an association between lignin content and stem hollowness.

[0076] Table 3 Quantitative PCR primers

[0077]

[0078] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A gene that affects the morphology of lettuce plants, characterized in that: The gene includes a CDS sequence as shown in SEQ ID No.

1.

2. Use of the gene according to claim 1 in regulating lettuce morphogenesis.

3. The application according to claim 2, characterized in that: The lettuce morphological structure includes the stem and leaves of lettuce.

4. A biological material for regulating the gene according to claim 1.

5. The biomaterial according to claim 4, characterized in that: The regulation includes overexpression or knockout of the gene.

6. Use of the biological material according to claim 4 or 5 in regulating the chlorophyll content in lettuce stems and / or leaves.

7. An overexpression vector comprising the CDS sequence of the gene according to claim 1.

8. Use of the overexpression vector according to claim 7 in increasing the stem length of lettuce.

9. A set of sgRNAs for knocking out the expression of the gene according to claim 1, characterized in that: It includes sgRNA1 with nucleotide sequences as shown in SEQ ID No.3 and sgRNA2 with nucleotide sequences as shown in SEQ ID No.

4.

10. Use of the gene according to claim 1 in creating lettuce germplasm.

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