CN110577960B - Pear lignin synthesis gene PbMC1a/1b and application thereof in genetic improvement of fruit quality - Google Patents

Abstract

The invention discloses a pear lignin synthesis gene PbMC1a/1b and application thereof in fruit quality genetic improvement, wherein the PbMC1a/1b gene is a cysteine-like protease gene separated and cloned from Dangshan pear with high lignin content, and is named as PbMC1a/1b by an applicant, the sequence is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO. 2. The gene construction overexpression vector is introduced into arabidopsis thaliana, biological function verification is carried out on the obtained transgenic plant, and the cloned PbMC1a/1b gene can promote the remarkable thickening of fiber cell walls among a catheter, wood fibers and vascular bundles, promote the accumulation of lignin in stems, inhibit the growth of the plant, improve the expression quantity of genes related to lignin biosynthesis, provide new gene resources for fruit quality molecular design breeding, and is an important candidate gene for improving fruit quality breeding in future genetic engineering.

Description

Pear lignin synthesis gene PbMC1a/1b and application thereof in genetic improvement of fruit quality

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a pear lignin synthesis gene PbMC1a/1b and application thereof in genetic improvement of fruit quality.

Background

The pear is a fruit tree widely planted in the third largest of China, and the Dangshan pear is derived from the Dangshan county of China and is long-term used as a main variety of China. In recent years, due to factors such as reduced quality of varieties and poor field management, the content of stone cells in pear fruits increases, and the texture of pulp becomes rougher. These changes have an adverse effect on the flavour and quality of pears (Rogers et al, 2004). Stone cells in pear fruits, which are the key determinants of pear quality, exist in isolated or aggregated form in fruits and are rich in lignin and cellulose (Donaldson, 2001; Chang et al, 2006). Pear fruit stone cells are sclerenchyma cells formed by the secondary deposition of lignin on the first wall by parenchymal cells (Rogers and Campbell, 2004; Humphreys and Chapple,2002)), the process of formation of which may be associated with Programmed Cell Death (PCD). PCD is a developmentally and genetically controlled cell death process that falls into two broad categories: environmentally induced PCD and developmentally regulated PCD in plants (Wang et al, 2012; Fagundes et al, 2015; Lam et al, 2012). Environmentally induced PCD is primarily caused by external abiotic or biological signals, such as drought, hormones, heat shock, and pathogen stress (Li et al, 2012; Duan et al, 2010). In contrast, developmentally regulated PCD covers most organs and tissues of plants, such as the fruit, roots, stems, leaves, and xylem, that are caused by internal factors and occur in predictable locations and times (Wertman et al, 2012; Huang et al, 2014).

With the development of modern sequencing technologies, a number of lignin synthesis-related enzyme genes have been found in pear, including POD, CAD, and C4H, among others (Cao et al, 2016C; Cheng et al, 2017). Recent studies have focused mainly on cell wall lignification, which has been carried out around cloning, regulation, etc. of lignin synthesis structural genes based on pear genome and germplasm resources, and have made some progress (Cai et al, 2010; Tao et al 2015). It is noteworthy that plant body development and tissue renewal, including cell wall lignification processes such as TEs differentiation, xylem formation, are accompanied by the development of PCD (Conway and McCabe, 2018; Locato and De Gara, 2018; jangjie et al, 2012; pyro et al, 2007; Kwon et al, 2011; Escamez and Tuominen,2014), and that lignification of the cell wall, secondary wall thickening and PCD can be inhibited by the same inhibitor, further suggesting that PCD and lignification are closely related (Groover and Jones, 1999). We speculate that lithocyte formation by lignin deposition in pear cells is also two closely related events with PCD, but it is not clear whether proteins associated with PCD progression are involved in the occurrence of pear lithocyte.

Tsianiasiani et al (2011) have identified 9 MCs in Arabidopsis (AtMC1-AtMC 9). In UV-C and H ₂ O ₂ Under stress of (a), expression of AtMC8 was up-regulated in arabidopsis thaliana, accelerating the process of PCD in protoplasts (He et al, 2008). AtMC1 and AtMC2 were reported to antagonize pathogen-triggered PCD as positive and negative regulators, respectively (Coll et al, 2010). AtMCP2b/AtMC5 can activate apoptosis-like cell death during early senescence and during oxidative stress (Watanabe et al, 2005). AtMC9 is specifically expressed in differentiated xylem vessels and is involved in the PCD process of xylem vessel molecules: (

et al, 2012). In addition, it has been reported that nicotiana benthama NbMCA1, capsicum CaMC9, wheat TaMC4 play a role in stress and PCD (Hao et al, 2007).

However, previous researches mainly focus on the participation of model or herbaceous plant MCs in stress-induced cellular PCD processes, but whether the PCD processes initiated and executed by the MCs are involved in the synthesis of stone cells and lignin of pear fruits is not reported.

Disclosure of Invention

The invention aims to provide a pear lignin synthesis gene, which is a cysteine-like protease gene separated and cloned from Dangshan pear (Pyrus bretschneeri) with high lignin content, and the applicant names the gene as PbMC1a/1b, the sequence of the gene is shown as SEQ ID NO.1, and the corresponding amino acid sequence is shown as SEQ ID NO.2 of a sequence table. The discovery of the gene provides a new gene resource for molecular design breeding of fruit quality, and is an important candidate gene for improving fruit quality breeding in future genetic engineering.

The invention also aims to provide an application of the pear lignin synthesis gene PbMC1a/1b in lignin synthesis. The gene is constructed into an over-expression vector, and is introduced into arabidopsis thaliana through agrobacterium-mediated genetic transformation, and biological function verification shows that the cloned PbMC1a/1b gene has the function of regulating lignin synthesis.

The purpose of the invention is realized by the following technical scheme:

the applicant named a Metacapase gene which is separated and cloned from a Dangshan pear (Pyrus bretschneideri) with high lignin content based on a plant gene cloning technology and is named as PbMC1a/1b, and the sequence of the Metacapase gene is shown as SEQ ID NO. 1.

The invention also provides a protein coded by the gene PbMC1a/1b, and the corresponding amino acid sequence is shown as follows (SEQ ID NO. 2):

MALYWLVQGCQAGDSLFFHYSGHGSRQRNYNGDEVDGYDETLCPLDFETQGMIVDDE INAAIVRPI PPGAKLHAI I DACHSGTVLDLPFLCRMDRSGRYVWEDHRPRSGMWKGSGGGEVICFSGCDDDQTSADTAALSKITSTGAMTFCFIQAIERGQAGTYGS ILNSMRSTIRSTGTGGGGGGSLTSLLGGGGGGGGAVTSLVSMLVTGGSDTGGLKQEPQLTACEPFDVYAKPFSL*

wherein denotes a stop codon; open Reading Frame (ORF) predicts that the gene contains one ORF, is 717bp in length, encodes 239-amino acid protein, and has the molecular weight of 24.94kDa and the isoelectric point of 4.81.

Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the gene PbMC1a/1b also belong to the scope of protection of the present invention.

The existing plant expression vector can be used for constructing a recombinant expression vector containing the gene PbMC1a/1 b.

When the gene PbMC1a/1b is used for constructing a recombinant plant expression vector, a cauliflower mosaic virus (CAMV)35S strong promoter is added in front of a transcription initiation nucleotide; in addition, when the gene of the present invention is used to construct a plant expression vector, the ATG initiation codon is used, but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence.

In order to facilitate the identification and screening of transgenic plants, plant expression vectors are used which are processed by adding a gene encoding a luminescent compound (luciferase gene) and an antibiotic marker having resistance (kanamycin marker) expressed in plants. From the safety of transgenic plants, hygromycin can be directly used for screening transformed plants without adding any selective marker gene.

Primer pairs for amplifying the full length or any fragment of the gene PbMC1a/1b also belong to the protection scope of the invention.

In one example, the applicants designed a pair of primers and cloned the full-length cDNA sequence of the gene PbMC1a/1b using PCR.

The nucleotide sequences of the PCR primer pairs are shown below:

forward primer 1: 5'-gagaacacgggggactctagaATGGCATTATATTGGCTTGTACAAG-3', SEQ ID NO. 3;

reverse primer 1: 5'-gcccttgctcaccatggatccTAGGGAGAAGGGTTTTGCATACA-3', SEQ ID NO. 4.

The gene PbMC1a/1b, the protein, the recombinant expression vector, the transgenic cell line or the recombinant strain or the method can be applied to plant breeding.

In one embodiment of the invention, a vector for guiding an exogenous gene to express in a plant is utilized by pCAMBIA1300, and a gene for coding the protein is introduced into arabidopsis thaliana to obtain a transgenic arabidopsis thaliana plant. The expression vector carrying the gene can be transformed into Arabidopsis thaliana by using an Agrobacterium-mediated method (floral dip method), and the transformed Arabidopsis thaliana is harvested for seed.

The invention also provides application of at least one of the gene PbMC1a/1b, the protein, the recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant bacterium in plant breeding, in particular application in breeding plants with genetically improved fruit quality.

In one embodiment, the invention also provides application of at least one of the gene PbMC1a/1b, protein, recombinant expression vector, transgenic cell line or recombinant bacterium in breeding of plants with low lignin content.

The plant of the invention can be either a monocotyledon or a dicotyledon, such as: arabidopsis thaliana, pear, and the like.

The invention also relates to the application of the gene in the genetic improvement of fruit quality, the gene is overexpressed in arabidopsis thaliana, and the content of the obtained transgenic plant lignin is obviously improved. The plant overexpression vector of the PbMC1a/1b gene is constructed, the PbMC1a/1b gene is transformed by utilizing an agrobacterium-mediated genetic transformation method, and is introduced into arabidopsis thaliana through agrobacterium-mediated genetic transformation, and the obtained transgenic plant is proved to have the function of improving the lignin content through biological function verification.

In one embodiment, the invention also provides application of at least one of the gene PbMC1a/1b, protein, recombinant expression vector, transgenic cell line or recombinant bacterium in breeding pear with low stone cell content.

The invention utilizes qRT-PCR technology to analyze the space-time expression of PbMC1a/1b gene in different development stages of pear fruit, and analyzes the contents of lignin and stone cells in the development of pear fruit, and the analysis result shows that the PbMC1a/1b gene has high expression quantity in the early development stage of fruit and is consistent with the change trend of stone cells and lignin. The cloned gene PbMC1a/1b of pear cysteine protease has important significance for the research of pear stone cell breeding reduction.

Drawings

FIG. 1 is a technical flowchart of example 5.

FIG. 2 shows the change in the contents of stone cells and lignin in the pulp during the development of pear fruit. Fig. 2A is stone cell content change and fig. 2B is lignin content change, different lower case letters on the column indicate very different significance (P < 0.05).

FIG. 3 is the expression pattern of PbMC1a/1b during fruit development, with different lower case letters on the bar indicating significant heterosis (P < 0.05).

FIG. 4 shows the subcellular localization of the PbMC1a/1b gene. Wherein: FIG. 4A shows the GFP gene (control) under Ultraviolet (UV) light, FIG. 4B shows the image under bright field, and FIG. 4C shows the image after superposition; FIG. 4D shows the Ultraviolet (UV) light exposure of the PbMC1a/1b gene, FIG. 4E shows the bright field image, and FIG. 4F shows the superimposed image.

FIG. 5 is the identification of positive seedlings of Arabidopsis thaliana transformed with PbMC1a/1b gene. Wherein: FIG. 5A shows the detection of positive seedlings at T1 generation, FIG. 5B shows the relative expression of PbMC1a/1B in the leaves of different positive seedlings at T1 generation, and FIG. 5C shows the identification of positive seedlings of transgenic lines OE9, OE10 and OE15 at T2 generation (p < 0.01).

FIG. 6 shows the determination of the biomass of Wild Type (WT) and PbMC1a/1b transgenic Arabidopsis thaliana. FIGS. 6A-C show WT and transgenic plants grown in the long daylight cycle for 7d (A), 6 weeks (B) and 8 weeks (C); FIG. 6D is the root length after germination for 7D; FIG. 6E shows the plant height at 5 weeks of germination; FIG. 6F shows the plant height at 8 weeks of germination; (xp < 0.01).

FIG. 7 shows the analysis of lignin and cell wall thickness in WT and PbMC1a/1b transgenic Arabidopsis. FIG. 7A is the diameter of the inflorescence stems 8 weeks after germination; FIG. 7B is a statistical analysis of lignin content in nascent inflorescence stems; FIG. 7C is the relative expression pattern of PbMC1a/1b in the WT and transgenic plant stems; FIG. 7D is a statistical analysis of secondary cell wall thickness of fibers between a catheter, wood fibers, and bundles of fibers; (xp < 0.01).

FIG. 8 shows the change in Arabidopsis cell wall over-expressing PbMC1a/1 b. FIGS. 8A-P are Arabidopsis thaliana toluidine blue stain (I-L: xylem; M-P: fasciculate fiber); if: inter-bundle fibers; xy: a xylem; ve: a conduit; xf: wood fiber.

FIG. 9 shows the change in the expression level of a lignin anabolic gene in stems of PbMC1a/1b transgenic Arabidopsis plants.

Detailed Description

The present invention will be described in detail with reference to specific examples. From the following description and examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

The following examples are experimental methods without specifying specific conditions, and generally follow the methods known in the art. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1 cloning of the full-Length cDNA of the Pear PbMC1a/1b Gene

The Arabidopsis AtMC1 is used as a keyword to search a pear gene database, 11 nucleotide sequences which are most similar and have the highest score are obtained, and the nucleotide sequence is named as PbMC1a/1 b. Based on the sequence of the PbMC1a/1b gene, a specific Primer pair for amplifying the sequence was designed using Primer Premier 5.0.

The method comprises the following specific steps:

taking Dangshan pear cDNA as a template, adopting high-fidelity enzyme for amplification, wherein an amplification system is shown in a table 1, an amplification program is shown in a table 2, and an amplification primer sequence is as follows:

PbMC1a/1b-F：5’-gagaacacgggggactctagaATGGCATTATATTGGCTTGTACAAG-3’

PbMC1a/1b-R：5’-gcccttgctcaccatggatccTAGGGAGAAGGGTTTTGCATACA-3’

by using

And purifying and recovering the amplified product by using a DNA gel recovery kit (Axygene, USA). pCAMBIA1300 vector (TransGen, China) was subjected to double digestion with XbaI and BamHI restriction enzymes (the. sup. m.Scientific, China), as shown in Table 3, at 37 ℃ for 2 hours and then subjected to reaction

The digested vector was purified and recovered by using a DNA gel recovery kit (Axygene, USA). The purified product and the vector after double enzyme digestion are connected by using a Cloneexpress II One Step Cloning Kit non-ligase dependent single fragment rapid Cloning Kit (Vazyme, China) to construct an expression vector p1300-PbMC1a/1b, wherein the connection system is shown in Table 4, and after incubation for 30min at 37 ℃, Escherichia coli competence Trans5 alpha is transformed. The transformation method of Escherichia coli is as follows:

(1) adding 20. mu.L of the ligation product into 50. mu.L of Escherichia coli competent Trans5 alpha (TransGen, China) cells melted in ice bath, gently mixing, and standing on ice for 30 min;

(2) after heat shock in 42 deg.C water bath for 45s, placing in ice for 2min without shaking the centrifuge tube;

(3) adding 500 μ L LB liquid culture medium without antibiotic, shaking table at 37 deg.C, culturing at 200rpm for 1h to allow bacteria to recover;

(4) uniformly coating 100-200 mu L of recovered competent cells on an LB solid culture medium containing corresponding antibiotics, inversely placing the culture dish in a constant-temperature culture box at 37 ℃, and culturing overnight;

TABLE 1 Gene amplification System

TABLE 2 Gene amplification PCR procedure

And (3) selecting a single clone on a flat plate in a 2.0mL centrifuge tube 12-16h after transformation, adding an LB liquid culture medium containing corresponding antibiotics, shaking and culturing in a shaking table at 37 ℃ until the bacterial liquid is turbid, and then carrying out positive identification. The reagents used were 2 XTSINGKE Master Mix (Tsingke, China), the reaction system is shown in Table 5, and the PCR program is shown in Table 6. After obtaining the positive clone, the positive clone is sent to the engine company for sequencing, and according to the sequencing result, the full length of the gene and the DNA sequence of the PbMC1a/1b are obtained.

TABLE 3pCAMBIA1300 vector double enzyme digestion System

TABLE 4pCAMBIA1300 vector ligation System

TABLE 5 Positive identification reaction System

TABLE 6 Gene amplification PCR procedure

The gene sequence of the gene in pear is obtained by cloning, and after sequencing, a 717bp sequence is obtained by separation, the sequence is SEQ ID NO.1, the length is 717bp, and the protein with 239 amino acids is coded, and the sequence is as follows: (SEQ ID NO. 2):

MALYWLVQGCQAGDSLFFHYSGHGSRQRNYNGDEVDGYDETLCPLDFETQGMIVDDEINAAIVRPIPPGAKLHAIIDACHSGTVLDLPFLCRMDRSGRYVWEDHRPRSGMWKGSGGGEVICFSGCDDDQTSADTAALSKITSTGAMTFCFIQAIERGQAGTYGSILNSMRSTIRSTGTGGGGGGSLTSLLGGGGGGGGAVTSLVSMLVTGGSDTGGLKQEPQLTACEPFDVYAKPFSL*

wherein denotes a stop codon, the protein has a molecular weight of 24.94kDa and an isoelectric point of 4.81.

Example 2 analysis of changes in the Stone cell and Lignin content of pulp during fruit development

The content of stone cells in the pulp was determined by cryo-fractionation (Syros et al, 2004). Taking three fruits (more young fruits) with the same size, removing pericarp, taking edible part of the fruit according to quartering method, weighing 100g, placing in a refrigerator at-20 deg.C for 24h, taking out, thawing at room temperature, adding 200ml distilled water, and mashing with tissue masher (1000-1500 r. min.) ^-1 ) Mashing for 5 min. Transferring the homogenate to a 1000ml beaker, stirring with a glass rod for 1min, standing for 5min to precipitate stone cells at the bottom of the beaker, pouring out the suspension on the upper layer, suspending the precipitate in 0.5M hydrochloric acid solution for 30min, stirring once every 5min during the period, removing floating substances, rinsing with distilled water for 5-6 times, collecting the first few suspensions, and rinsing. Mixing the obtained stone cells, filtering with coarse filter paper, separating to obtain pure stone cells, drying to constant weight, and weighing.

Accurately weighing 0.01g pulp powder sample with ten-thousandth balance, grinding with 95% ethanol to homogenate, metering to 5ml, centrifuging at 12000g for 2min, discarding supernatant, washing with 95% ethanol for 3 times, and further using ethanol: n-hexane was washed 3 times at a ratio of 1:2(V/V), and dried in a hood. Then 2mL of 25% bromoacetoacetic acid solution was added, the reaction was stopped by incubating in a 70 ℃ water bath for 30min, 0.9mL of 2M NaOH solution was added, 5mL of acetic acid and 0.1mL of 7.5M hydroxylamine chloride solution were added, the volume was adjusted to 10mL with glacial acetic acid, the absorbance was measured at 280nm, and finally the lignin content was determined by a lignin standard (Sigma-Aldrich, USA) curve (Syros et al, 2004).

The experiment researches the change of contents of stone cells and lignin in the fruit development process, and the contents of the stone cells and the lignin in the pear fruit are measured 15 to 75 Days After Flowering (DAF) (figure 2). During the 15 to 45DAF period, stone cell content in the fruit increased rapidly and reached a maximum at 45DAF, with a content of 13.78% (fig. 2A). During the 45 to 75DAF period, the lignin content in the pear fruit increased rapidly during the 15 to 37DAF period, reaching a maximum of 1.38% at 37DAF and decreased after 37DAF (fig. 2B). The results show that when the pear fruit developed to 37DAF, the lignin content reached a maximum, the stone cell content reached a maximum at 45DAF, and the peak in lignin content appeared before the stone cells.

Example 3 analysis of expression patterns of PbMC1a/1b during fruit development

Analyzing the expression mode of the PbMC1a/1b gene by adopting a real-time fluorescent quantitative PCR (qRT-PCR) method, wherein the quantitative reagent is QuantiNova ^TM SYBRGreen PC (QIGEN, Germany), the procedure is described in the description, and the reaction system is shown in Table 7.

TABLE 7 quantitative PCR reaction System

Tublin in pear is used as an internal reference gene, and cDNA obtained by reverse transcription is used as a template. Each sample was replicated three times and the reaction procedure is shown in table 8. After the reaction is complete, use 2 ^-ΔΔCt The algorithm calculates gene expression. The primers of the reference gene qRT-PCR are as follows:

Tublin-F：5’-TGGGCTTTGCTCCTCTTAC-3’

Tublin-R：5’-CCTTCGTGCTCATCTTACC-3’

TABLE 8 quantitative PCR reaction procedure

The expression pattern of PbMC1a/1b in fruit development process was analyzed in this experiment, and the result showed that PbMC1a/1b showed a significant increase in DAF expression from 15 to 22 and a decrease in DAF expression from 22 to 75 (FIG. 3). PbMC1a/1b was maintained at high expression levels of 22 to 37DAF, and was consistent with a trend toward changes in stone cells and lignin content.

Example 4 subcellular localization of the PbMC1a/1b Gene

The ORF region of PbMC1a/1b (without stop codon) was amplified and a p35S-PbMC1a/1b-GFP vector was constructed, and PbMC1a/1b-GFP and control GFP were transiently transformed into leaf epidermal cells of Nicotiana benthamiana, respectively. The agrobacterium infection of tobacco mesophyll cells is carried out according to the following method: (1) selecting Agrobacterium from fresh culture medium, inoculating Agrobacterium on 3ml LB/Kan/Rif liquid culture medium (containing 50mg/L kanamycin and 50mg/L rifampicin), culturing at 28 deg.C for 1-2d at 220rpm/min, and activating bacteria liquid; (2) inoculating activated bacteria liquid into 50ml LB/Kan/Rif liquid culture medium (containing 50mg/L kanamycin and 50mg/L rifampicin) at a ratio of 50:1, culturing at 28 ℃ for 8-12 h at 220rpm/min, and detecting the bacteria liquid OD600 between 0.6 and 0.8 during culture; (3) transferring the bacterial liquid into a 50ml centrifugal tube, centrifuging at 5000rpm/min for 5min, and removing the supernatant; (4) 10ml of wash solution (10mM MES +10mM MgCl) was added ₂ ) Resuspending thallus, centrifuging at 4000rpm/min for 5min, removing supernatant, repeating with 5ml of cleaning solution once, adding 3ml of cleaning solution, and fully sucking, beating and mixing; (5) absorbing the heavy suspension bacteria liquid, adding the heavy suspension bacteria liquid into the cleaning liquid at a ratio of 19:1, and measuring OD 600; (6) adjusting the final concentration OD600 of the two bacterial liquids to be about 0.8, adding 5 mu l of Acetosyringone (AS), uniformly mixing, standing at normal temperature for 4h, and waiting for injection; (7) infection liquidLoading into an injector (5ml), injecting 2-3 leaves in a combined manner, randomly selecting and distributing the leaves on different plants, erecting the leaves with proper size, and pressing the injector to inject liquid into the lower epidermis of the tobacco leaves; (8) after the injection is finished, culturing the tobacco at 25 ℃ for 48-72 h; (9) the reporter gene localization was then observed by confocal laser microscopy, which showed that the control vector transformed cells fluoresced throughout the cells, as shown in FIGS. 4A-C, whereas the recombinant vector transformed cells fluoresced only in the cytoplasm, as shown in FIGS. 4D-E, indicating that PbMC1a/1b is a cytoplasmic-localized protein.

Example 5 transformation of Arabidopsis

As shown in fig. 1:

(1) PCR-verified correct PbMC1a/1b Agrobacterium strain, streaked on LB solid medium (containing 50. mu.g/mL kanamycin and 50. mu.g/mL rifampicin), incubated at 28 ℃ for 2-3d in an incubator;

(2) scraping the monoclonal strain in a superclean bench with a gun head (for sterilization), and oscillating in a shaking table at 28 ℃ and 200r/min for 16 h;

(3) collecting the bacterial liquid at room temperature for 20min at the speed of 5000 g;

(4) the thalli is suspended in a transformation medium (2.25g/L MS culture medium, 5g/L sucrose, 10 mu g/L6-BA, pH value is adjusted to 5.7 by KOH) with the same volume;

(5) cutting off horn and bloomed flowers of the generational transformed arabidopsis (bolting 10 cm);

(6) swillt-77 was added to the transformation medium to a final concentration of 0.025%;

(7) soaking the flower of Arabidopsis thaliana in the bacterial liquid, vacuumizing to 0.6-0.8KPa, and soaking for 5 min;

(8) dark culturing at 22 deg.c for 24 hr, taking out plant, culturing normally and harvesting seed for screening.

The harvested seeds of the T0 generation were screened in a screening medium (containing MS medium, 30g/L sucrose, 0.75% agar, 20mg/L hygromycin, 100mg/L timentin and 100mg/L carboxychange), the emerging seedlings were transferred to plastic pots with a mixture of vermiculite and soil (1:2), cultivated in a greenhouse with 16h light/8 h dark and 40% relative humidity, and seeds were harvested after they had matured. Extracting DNA from T1 transgenic line leaves, wherein the DNA extraction steps are as follows:

(1) placing a small amount of tobacco leaves into a 1.5mL centrifuge tube, grinding the tobacco leaves into powder by using liquid nitrogen, adding 600 mu L of CATB extracting solution, wherein the preparation method of CTAB extracting solution is shown in Table 9;

(2) mixing completely, placing into 65 deg.C water bath, water bathing for 90min, and mixing by reversing every 30 min;

(3) after the water bath is finished, 700 mu L of 24:1 (chloroform: isoamylol) mixed extract is added, the mixture is violently inverted and uniformly mixed, the mixture is centrifuged for 15min at 12000r/min at normal temperature, and supernatant (about 500 mu L) is absorbed and transferred into a new centrifugal tube of 1.5 mL;

(4) adding precooled isopropanol with the same volume as the supernatant, reversing the upper part and the lower part, uniformly mixing, and putting the mixture in a refrigerator at the temperature of minus 20 ℃ for precipitation (the precipitation time can be prolonged);

(5) taking out after the precipitation is finished, and centrifuging at 12000r/min for 10 min. Pouring off the supernatant, adding 1mL of precooled 75% ethanol, washing for 2-3 times, removing the ethanol, and air-drying in a fume hood;

(6) add 20-30. mu.L of ddH per tube ₂ O dissolving DNA, and storing the dissolved DNA in a refrigerator at the temperature of-20 ℃.

And (3) identifying the positive plant of the transgenic arabidopsis, detecting by using the extracted DNA as a template and two pairs of primers, adding a 35S promoter forward primer and a gene reverse primer and a PbMC1a/1b specific primer, and carrying out agarose gel electrophoresis detection. The primer sequences are as follows:

35S-F:5’-TCCTCGGATTCCATTGCCCAGC-3’

forward primer 1: 5'-GGCGATGAAGTTGATGGATATG-3', respectively;

reverse primer 1: 5'-TATCGTCCACTCCTGTCCATTC-3' are provided.

The results showed that 6 positive seedlings were identified in the T1 generation by DNA extraction, 0E9, OE10, OE12, OE14, OE15 and OE16, respectively (FIG. 5A). And (3) extracting RNA from the detected positive seedling leaves, and detecting the expression quantity by qRT-PCR. Through qRT-PCR verification, the relative expression amount of PbMC1a/1B in leaves of OE14 expression line is remarkably improved (P <0.01) except that the relative expression amount of PbMC1a/1B in leaves of OE14 expression line is low, wherein the relative expression amount of OE9, OE10 and OE15 is more than 15 times at most (FIG. 5B).

TABLE 9CTAB extractive solution formula

And (3) generating 3 lines of T1 with the highest expression quantity, namely, OE9, OE10 and OE15 seeds on a screening culture medium to form T2 generation plants, extracting DNA from 7 seedlings of each line to perform positive seedling identification, and collecting seeds. If the germination rate of seeds of the T1 generation is close to 100 percent, the plants of the T2 generation are considered to be homozygote-transformed PbMC1a/1b gene plants. The results show that the seed germination rate of each line is close to 100%, which indicates that T2 generation plants are homozygote-transformed PbMC1a/1b gene plants, 7 seedlings of each line are selected to extract DNA, and the results show that the seedlings are positive seedlings (figure 5C).

Example 6 physiological measurement of transgenic Arabidopsis thaliana

T2 generation seeds and wild type seeds were grown in MS medium to form T3 generation transgenic line plants for physiological quantity determination. Wild type and T3 generation transgenic line arabidopsis seeds were germinated in petri dishes for one week before root length was determined using a ruler (50 replicates) and transplanted into plastic pots with a mixture of vermiculite and soil (1:2) and cultured in a greenhouse with a photoperiod of 16h light/8 h dark and a relative humidity of 40%. The plant height of the aerial parts of the plants was determined at the flowering stage (5 weeks of age) and photographed (50 replicates). And (3) measuring the height of the overground part of the plant during the mature period, taking a picture, drying the stem of the nascent inflorescence to constant weight, grinding the stem into powder by using a sample grinder, and measuring the content of lignin.

After the arabidopsis is cultured for 8 weeks, 3T 3 generation transgenic lines and wild arabidopsis are randomly selected, the stem of the nascent inflorescence which is 2cm away from the base is fixed for more than 7 days at 4 ℃ by FAA fixing solution, and a paraffin section is prepared according to the following steps:

(1) and (3) dehydrating: dehydrating with 85%, 95%, 100%, and 100% ethanol for 2 hr, and adding anhydrous ethanol: xylene 1:1(V/V) eluting ethanol for 2 h;

(2) and (3) transparency: soaking in xylene for 2h, and repeating for 1 time;

(3) wax infiltration: adding xylene with half volume into a fixed bottle, then adding paraffin with half volume, heating in an oven to 70 ℃ to dissolve the paraffin, then taking a cover, infiltrating the paraffin with the molten paraffin for 2 hours after 2 hours, and repeating once;

(4) embedding: embedding the wax-infiltrated material in a carton with a molten pure wax solution;

(5) slicing: the samples were cut to a thickness of 5 μm using an ultramicrotome (Leica RM 2015, Leica mikrosystem, Germany);

(6) unfolding the slices: picking up the cut sample by using a brush pen, putting the sample into a water bath (at 35 ℃) for spreading, picking up a slide glass for slicing after spreading, and putting the slide glass into an oven (at 37 ℃) for drying;

(7) dewaxing: washing the sample slices with pure xylene for 10min (repeating once), absolute ethyl alcohol for 4min (repeating once), 95% ethyl alcohol for 4min, 85% ethyl alcohol for 4min, and 70% ethyl alcohol for 2min in sequence;

(8) toluidine blue staining: staining the sample slices with toluidine blue solution (boric acid 1:1 (W/V)) for 5min, then washing the slices with 95% ethanol for 2min, absolute ethanol for 3min (repeated once), xylene for 10min, and xylene for 5min, finally covering the slices with neutral gum, and drying in an oven at 37 ℃.

Images of toluidine blue stained sections were visualized by a confocal spectroscopic microscope (Leica TCs SP 2). And measuring the diameter of the stem, the cell wall thickness of the fiber cells among the catheter, the wood fiber and the vascular bundle by imageJ.

As a result, it was found that the transgenic lines arabidopsis root length was significantly shortened compared to wild type arabidopsis (3.1cm), with three transgenic lines reduced in root length by an average of 1.17cm (. p. <0.01) (fig. 6A and D). Then the transgenic line and the wild arabidopsis thaliana grow under the long-day condition, when the flowering period (5 weeks), the transgenic line plant obviously grows slowly, and the plant height at the moment is measured: wild type 25.00cm, OE9 14.48cm, OE10 14.09cm and OE15 13.81cm (. P <0.01) (fig. 6B and E). By the time the arabidopsis silique matured (8 weeks), the plant height of the transgenic lines was significantly lower than that of the wild type, and 3 transgenic lines were reduced by an average of 2.9cm by 8.7% (. about.p <0.01) (fig. 6C and F). By measuring root length and plant height, it was shown that the inflorescence stems of wild type Arabidopsis grow faster and that overexpression of PbMC1a/1b results in a dwarf phenotype of the transgenic Arabidopsis plants.

Intertidal fibers and xylem cells are the main stem tissues that support the erect growth of inflorescences and to further observe changes in intertidal fibers and xylem ductal cells in arabidopsis stems we used toluidine blue for section staining. Transgenic lines plants OE9, OE10 and OE15 had stem diameters significantly smaller than wild type by paraffin sectioning (fig. 7A and fig. 8A-D). Cross-sectional staining of arabidopsis thaliana stems showed deeper staining of xylem and fiber between vascular bundles in transgenic plants compared to wild type, indicating that PbMC1a/1b might promote lignification of cells and accumulation of lignin in transgenic arabidopsis thaliana stems. To verify the above speculation, the lignin content in the primary inflorescence stems of the transgenic lines and wild-type plants was further determined. The results show that the lignin content in the nascent inflorescence stems of the transgenic lines is significantly higher than that of the wild type, with the highest lignin content of OE15 being 36.23%, which is increased by 16.57% compared to the wild type (FIG. 7B). Meanwhile, the relative expression amount of PbMC1a/1b in OE9, OE10 and OE15 stems was shown to be more than 30-fold using qRT-PCR analysis (FIG. 7C). No change in vessel and lignocellulosic cell morphology was observed in the over-expression lines by sectioning (fig. 8E-H), but the cell wall thickness of the vessels, the wood fibers and the fibers between bundles was found to be significantly thicker by measurement compared to the wild type (fig. 8I-P), and the cell wall thickness of the three types increased by 28.73%, 44% and 36% on average, respectively (fig. 7D). The functions of promoting cell lignification and lignin accumulation of PbMC1a/1b are shown by dyeing, measuring the lignin content and measuring the thickness of cell walls.

Example 7 expression analysis of Lignin biosynthesis genes in transgenic Arabidopsis

Wild type and T3 generation transgenic Arabidopsis thaliana primary inflorescence stalks are taken to be placed in a 2mL EP tube, frozen by liquid nitrogen and stored in a refrigerator at minus 80 ℃, RNA is extracted and inverted into cDNA, the RNA is extracted by using a polysaccharide polyphenol plant RNA extraction Kit of Chengdu Fujian biotechnology limited company, and the RNA is subjected to reverse transcription by using a reverse transcription Kit HiScript II1st Strand cDNA Synthesis Kit (+ gDNA wiper) of Nanjing Novozan biotechnology limited company, and the method refers to the instruction. The variation of the expression level of genes participating in lignin biosynthesis in Arabidopsis was determined by qRT-PCR using AtActin as an internal reference, and the primer information is detailed in Table 10.

TABLE 10 primer sequences for expression analysis

To elucidate the molecular mechanism of PbMC1a/1b in lignin synthesis, the expression patterns of 14 lignin synthesis genes in the nascent inflorescence stems of wild-type and transgenic lines were compared by qRT-PCR. These genes include: 4CL1, C3H1, C4H, CAD4, CAD9, CCoAOMT1, CCR1, COMT, F5H1, HCT, LAC4, LAC11, LAC17, and PAL 1. As shown in fig. 9, the relative expression amounts of these genes in the transgenic lines were significantly higher than the wild type, and LAC4, LAC11, and LAC17 had the highest expression levels in the transgenic lines. These genes may act synergistically to promote the synthesis of lignin in transgenic arabidopsis stems, where laccases may play a key role.

Sequence listing

<110> Nanjing university of agriculture

<120> pear lignin synthesis gene PbMC1a/1b and application thereof in genetic improvement of fruit quality

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 717

<212> DNA

<213> Dangshan pear (Pyrus bretschneideri)

<400> 1

atggcattat attggcttgt acaaggctgt caagcaggcg actccctctt ttttcattac 60

tctggccatg gttcacggca gaggaattat aatggcgatg aagttgatgg atatgatgaa 120

accctttgtc cccttgactt cgaaactcag ggtatgattg ttgatgatga gataaatgca 180

gcaattgtga gacccattcc acccggggct aagcttcatg caataataga tgcttgtcat 240

agtggcactg tactggattt gccattcctt tgcagaatgg acaggagtgg acgatatgta 300

tgggaggatc atcgccctcg atcaggcatg tggaaaggat caggcggtgg agaagtcatt 360

tgcttcagtg gttgtgatga tgatcaaacg tctgctgaca cagcagctct atcaaagatc 420

acatcaacag gagccatgac tttctgcttc atccaagcaa tcgagcgtgg acaagcgggc 480

acctatggaa gcatactcaa ttctatgcgc tctaccattc ggagtacagg tacgggtggt 540

ggtggtggtg gcagtttaac atctcttctt ggtggtggtg gtggtggtgg tggtgctgtg 600

acatccctag tcagcatgct tgtgacagga ggcagtgata ctggcgggct aaaacaggaa 660

ccgcaattaa ctgcctgtga gccatttgat gtgtatgcaa aacccttctc cctatga 717

<210> 2

<211> 238

<212> PRT

<213> Dangshan pear (Pyrus bretschneideri)

<400> 2

Met Ala Leu Tyr Trp Leu Val Gln Gly Cys Gln Ala Gly Asp Ser Leu

1 5 10 15

Phe Phe His Tyr Ser Gly His Gly Ser Arg Gln Arg Asn Tyr Asn Gly

20 25 30

Asp Glu Val Asp Gly Tyr Asp Glu Thr Leu Cys Pro Leu Asp Phe Glu

35 40 45

Thr Gln Gly Met Ile Val Asp Asp Glu Ile Asn Ala Ala Ile Val Arg

50 55 60

Pro Ile Pro Pro Gly Ala Lys Leu His Ala Ile Ile Asp Ala Cys His

65 70 75 80

Ser Gly Thr Val Leu Asp Leu Pro Phe Leu Cys Arg Met Asp Arg Ser

85 90 95

Gly Arg Tyr Val Trp Glu Asp His Arg Pro Arg Ser Gly Met Trp Lys

100 105 110

Gly Ser Gly Gly Gly Glu Val Ile Cys Phe Ser Gly Cys Asp Asp Asp

115 120 125

Gln Thr Ser Ala Asp Thr Ala Ala Leu Ser Lys Ile Thr Ser Thr Gly

130 135 140

Ala Met Thr Phe Cys Phe Ile Gln Ala Ile Glu Arg Gly Gln Ala Gly

145 150 155 160

Thr Tyr Gly Ser Ile Leu Asn Ser Met Arg Ser Thr Ile Arg Ser Thr

165 170 175

Gly Thr Gly Gly Gly Gly Gly Gly Ser Leu Thr Ser Leu Leu Gly Gly

180 185 190

Gly Gly Gly Gly Gly Gly Ala Val Thr Ser Leu Val Ser Met Leu Val

195 200 205

Thr Gly Gly Ser Asp Thr Gly Gly Leu Lys Gln Glu Pro Gln Leu Thr

210 215 220

Ala Cys Glu Pro Phe Asp Val Tyr Ala Lys Pro Phe Ser Leu

225 230 235

<210> 3

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gagaacacgg gggactctag aatggcatta tattggcttg tacaag 46

<210> 4

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gcccttgctc accatggatc ctagggagaa gggttttgca taca 44

Claims (2)

1, SEQ ID NO.1PbMC1a/1bAnd the protein shown in SEQ ID NO.2 or the gene containing the protein shown in SEQ ID NO.1PbMC1a/1bThe recombinant expression vector or the recombinant strain is applied to cultivation of pear varieties with different fruit qualities; the fruit quality is related to the lignin content and/or the stone cell content.

2, SEQ ID NO.1PbMC1a/1bAnd the protein shown in SEQ ID NO.2 or the gene containing the protein shown in SEQ ID NO.1PbMC1a/1bThe recombinant expression vector or the recombinant strain of (1) is applied to the cultivation of arabidopsis thaliana with improved lignin content.

Images