Transcription factor involved in lignin synthesis, ejnac5-like, and applications thereof

Abstract

The application provides a transcription factor EjNAC5-like involved in lignin deposition and an application thereof, and the nucleotide sequence of the transcription factor EjNAC5-like is shown as SEQ ID NO. 1. The transcription factor EjNAC5-like can promote the expression of a key gene Ej4CL1 for monomer synthesis of pyracantha lignin and a polymerization gene EjPRX12 thereof, and further promote the accumulation of lignin. It is verified that the expression of the EjNAC5-like gene of the pyracantha fruit is positively correlated with the lignin content, the transient silencing of the EjNAC5-like gene can reduce the expression of the Ej4CL1 and EjPRX12 genes, and the lignin content of the pyracantha fruit is significantly reduced. The EjNAC5-like can be used as a candidate gene for pyracantha to carry out genetic engineering and improvement breeding, and is used for improving the texture quality of the fruit, and has potential application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant molecular biology and genetic engineering, and particularly relates to a transcription factor EjNAC5-like involved in lignin deposition of Eriobotrya japonica and application thereof. BACKGROUND

[0002] Eriobotrya japonica, a evergreen fruit tree originally from subtropical China, its fruit ripening period is in early summer, which is the off-season of fruits. Low temperature is often used for postharvest storage and transportation, but red-fleshed E. japonica varieties are cold-sensitive, and are prone to chilling injury during postharvest low temperature storage, showing lignin deposition (including synthesis and polymerization of lignin monomers) and woody tissue of fruit flesh, which results in increased hardness, difficult peeling, and flavor change, seriously affecting the edibility and commodity value. Therefore, it is of great significance to study the key genes of lignin regulation in E. japonica to reduce postharvest storage and transportation lignin deposition and improve the edibility and commodity value, and to accumulate gene resources for texture-oriented genetic improvement of E. japonica varieties.

[0003] The transcriptional regulatory network of lignin deposition in model plant Arabidopsis thaliana has been relatively clear, mainly involving MYB and NAC transcription factors. NAC is one of the largest transcription factor families in plants, with multiple functions, including plant secondary cell wall formation, biological and non-biological stress response. VASCULAR-RELATED NAC-DOMAIN 6 / 7 (VND6 / 7), NAC SECONDARY WALL THICKENING PROMOTING FACTOR 3 (NST3) / SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1 (SND1) and NST1, members of the NAC family in Arabidopsis, are the "master switch" of secondary cell wall formation and are conserved in vascular plants. However, the homologous genes in E. japonica fruit are almost not expressed, indicating that the lignification of fleshy fruit parenchyma cells is different from the regulatory mechanism of vascular tissue. Therefore, studying the lignin regulatory mechanism in E. japonica fruit can enrich the theoretical basis for lignification of fleshy fruit. SUMMARY

[0004] In order to solve the above problems, the present application aims to provide a transcription factor EjNAC5-like involved in lignin deposition of loquat, a transcription factor EjNAC5-like protein involved in lignin deposition of loquat and a coding gene thereof. The nucleotide sequence of the transcription factor EjNAC5-like is shown as SEQ ID NO. 1, the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2, the upstream primer nucleotide sequence of PCR amplification of the transcription factor EjNAC5-like is shown as SEQ ID NO. 3, and the downstream primer nucleotide sequence is shown as SEQ ID NO. 4.

[0005] The present application also provides a recombinant vector containing the transcription factor EjNAC5-like described in the above scheme, and the recombinant vector is EjNAC5-like-pHellsgate2.

[0006] Another object of the present application is to provide an application of the transcription factor EjNAC5-like in genetic engineering breeding for improving the texture quality of loquat fruits, and the application is to inhibit lignin deposition by silencing EjNAC5-like, and then to improve the eating quality and commodity of fruits.

[0007] The present application provides a transcription factor EjNAC5-like involved in lignin deposition of loquat fruits, belongs to the field of plant molecular biological technology and genetic engineering technology, and the EjNAC5-like has the nucleotide sequence shown as SEQ ID NO. 1. The accumulation of loquat lignin can be reduced by silencing the EjNAC5-like gene by using genetic transformation technology. Therefore, the EjNAC5-like gene can be used as a candidate gene for genetic engineering and improved breeding of loquat, and for improving the texture quality of fruits. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 is an electrophoresis photograph for verifying the coding region sequence of the loquat EjNAC5-like gene. In the photograph, M is DL2000 DNA marker, and the arrow indicates the band of the target gene amplified by PCR.

[0009] Figure 2 is the change of lignin content of loquat fruits under postharvest low-temperature treatment.

[0010] Figure 3 is the gene expression level of EjNAC5-like of loquat fruits under postharvest low-temperature treatment.

[0011] Figure 4 is the correlation analysis of lignin content and gene expression level of EjNAC5-like of loquat fruits under postharvest low-temperature treatment.

[0012] Figure 5 is NAC phylogenetic analysis.

[0013] Figure 6 is gene expression analysis of transiently silenced EjNAC5-like in E. japonica. EV represents empty vector, and R-EjNAC5L represents silenced EjNAC5-like.

[0014] Figure 7 is gene expression analysis of Ej4CL1 (A) and EjPRX12 (B) of transiently silenced EjNAC5-like in E. japonica. EV represents empty vector, and R-EjNAC5L represents silenced EjNAC5-like.

[0015] Figure 8 is lignin content analysis of transiently silenced EjNAC5-like in E. japonica. EV represents empty vector, and R-EjNAC5L represents silenced EjNAC5-like. DETAILED DESCRIPTION

[0016] The following examples are intended to illustrate the present application but not to limit the scope of protection of the present application. If not specifically indicated, the examples are carried out according to the conventional experimental conditions, or according to the conditions suggested by the manufacturer's instructions. The following examples are provided to illustrate the present application in detail.

[0017] Example 1 EjNAC5-like in E. japonica is positively correlated with lignin content

[0018] (I) Experimental methods

[0019] 1. Experimental materials

[0020] The present application takes mature fruits of red-fleshed E. japonica cultivar ‘Dahongpao’ as the material, which are collected in Jinhua, Zhejiang Province. The fruits with uniform size and maturity, and free of diseases, pests and mechanical damage are selected for experimental treatment. The treatment is divided into two groups, 0 ℃ and 5 ℃, and each group of fruits is set up 3 biological replicates, and each replicate has 3 fruits. The pulp is sampled at 0, 0.5, 1 and 2 d after treatment. The peel and core are removed during sampling, and the pulp is fully chopped, frozen with liquid nitrogen in time, and then stored in a -80 ℃ refrigerator.

[0021] 2. Lignin content determination

[0022] About 1.5 g of fresh loquat pulp sample was weighed, 4 mL of lignin washing solution (100 mM phosphate buffer, 0.5% Triton X-100, 0.5% PVP, pH 7.8) was added, and ultrasonic treatment was performed for 10 min. Centrifugation was performed at 3500 rpm for 10 min, the supernatant was removed, and the above step was repeated once. Subsequently, methanol washing was performed, and the above steps were repeated 4 times, and the precipitate was dried in an oven at 65°C overnight. 0.2 mL of mercaptoacetic acid (aladdin, China) and 2 mL of hydrochloric acid (2M) were added, and reaction was performed in a water bath at 98°C for 8 h, then cooled on ice to terminate the reaction. After washing with water twice, it was placed in a 65°C oven overnight to dry. 2 mL of sodium hydroxide solution (1M) was added, and the precipitate was dissolved by shaking for 18 h, then centrifuged at 3500 rpm for 20 min. 0.5 mL of supernatant was taken into a 1.5 mL centrifuge tube, 100 μL of concentrated hydrochloric acid was added, and it was placed at 4°C for 4 h to precipitate, then centrifuged at 12000 rpm at 4°C for 20 min, the supernatant was removed, and the precipitate was dissolved in 1 mL of sodium hydroxide solution (1M). 200 μL of supernatant was taken into a UV plate (3635, Corning, USA), 1M sodium hydroxide solution was used as a blank control, and the absorbance at 280 nm was measured using a Synergy H1 enzyme marker (BioTek, USA). Standard curve was prepared using 0, 5, 10, 15, 20, 30, 40 and 50 mg of lignin (Macklin, China), and the lignin content was calculated according to the standard curve.

[0023] 3. RNA extraction

[0024] The total RNA of loquat fruit was extracted by CTAB method. 0.2 g of loquat fruit sample ground with liquid nitrogen was weighed, 4 mL of 65°C preheated CTAB and 80 μL of β-mercaptoethanol were added, vortex mixed, and 65°C water bath was performed for 10 min. 4 mL of chloroform:isopropyl alcohol (24:1) was added and vortex mixed. 8000 rpm centrifugation was performed at 15°C for 10 min, and the supernatant (about 3 mL) was taken to a new 10 mL centrifuge tube, and the above steps were repeated. The supernatant was taken to a new centrifuge tube, 1 / 5 volume of 12 mol / L LiCl was added, and it was placed at 4°C overnight. The next day, 8000 rpm centrifugation was performed at 4°C for 30 min, and the supernatant was removed. 400 μL of 65°C preheated SSTE was added to dissolve the precipitate. 400 μL of chloroform:isopropyl alcohol (24:1) was added and vortex mixed. It was transferred to a 1.5 mL centrifuge tube and centrifuged at 15°C at 10000 rpm for 15 min. The supernatant was taken to a new centrifuge tube, 2 volumes of -20°C pre-cooled anhydrous ethanol was added, and it was mixed by inverting up and down. It was placed at -80°C for 1 h. 10000 rpm centrifugation was performed at 4°C for 20 min, the supernatant was removed, and the residual liquid was taken out after brief centrifugation. The precipitate was dried in a fume hood. 20 μL of DEPC water was added to dissolve the precipitate, and the total RNA was obtained. The quality of the RNA was determined by agarose gel electrophoresis experiment, and the concentration of the RNA was determined by using NanoDrop microspectrophotometer (Thermo, USA).

[0025] 4. Reverse transcription and real-time fluorescent quantitative polymerase chain reaction (RT-qPCR)

[0026] 1 μg of RNA was reverse transcribed to synthesize cDNA. The synthesis of cDNA was performed using HiScript III RT SuperMix for qPCR with gDNA wiper (Vazyme, China) and according to the instructions. The RT-qPCR reaction system was performed according to the instructions of ChamQ SYBR qPCR Master Mix (Vazyme, China). The detection was performed using CFX96 Tough Real-Time PCR Detection System (Bio-Rad, USA) instrument. The reaction program was performed according to the instructions of the reaction system. The reaction program was set as 95°C, 30 s, 35 cycles of 95°C, 10 s, and 60°C, 30 s, and finally the melting curve analysis program was run. EjACT (GenBank no. JN004223) was used as an internal reference gene, the primers were SEQ ID NO. 7 and SEQ ID NO. 8, and the relative expression amount was calculated using 2 –ΔCT method.

[0027] 5. Gene cloning and phylogenetic tree analysis

[0028] Based on loquat genome data, gene amplification primers (SEQ ID NO. 3 and SEQ ID NO. 4) were designed at both ends of the full-length coding region of the EjNAC5-like gene. The reaction conditions were: 95℃ for 5 min; 95℃ for 15 s, 56℃ for 15 s, 72℃ for 30 s, for 35 cycles; 72℃ for 10 min. After the PCR reaction, the target band was excised after detection by 1% agarose gel electrophoresis. Figure 1 The PCR products were recovered using an agarose gel DNA recovery kit. The DNA was ligated into the pGEM-Teasy vector (Promega, USA), transformed into competent *E. coli* cells, and single clones were selected for sequencing. Sequence alignment was performed using DNAMAN software to obtain the EjNAC5-like CDS sequence (SEQ ID NO.1). The CDS sequence of the loquat EjNAC5-like gene was translated using MEGA-X software to obtain the protein sequence (SEQ ID NO.2). The sequences of Arabidopsis NAC transcription factor family proteins were obtained from the TAIR database (https: / / www.arabidopsis.org / ); the sequences of PbrNSC (QF157763.1) and NAC (XP_015902286.1) were downloaded from NCBI (https: / / www.ncbi.nlm.nih.gov / ); and the sequence of CgNAC043 (Cg5g000340.1) protein was obtained from the Citrus Pan-genome to Breeding Database (http: / / citrus.hzau.edu.cn / ). Phylogenetic trees were constructed using MEGA-X with the neighbor-joining (NJ) method, and the results were visualized in the Interactive Tree of Life database (https: / / itol.embl.de / ).

[0029] (II) Experimental Results

[0030] When loquat fruits are stored at 0℃ after harvest, they undergo chilling injury and lignification, resulting in increased lignin content. Treatment at 5℃ can delay the increase in lignin content. Figure 2 The expression level of EjNAC5-like proteins significantly increased during storage at 0°C, while storage at 5°C delayed this increase. Figure 3 This is consistent with the change in lignin content and shows a positive correlation. Figure 4 It is hypothesized that EjNAC5-like positively regulates the lignification process of loquat fruit under chilling injury. Phylogenetic analysis showed that EjNAC5-like did not cluster with the SND, NST, and VND subgroups involved in secondary cell wall synthesis in the model plant. Figure 5This suggests that EjNAC5-like may be a novel transcription factor independent of the known secondary cell wall metabolic network.

[0031] Example 2: Transient silencing of EjNAC5-like molecules reduced the lignin content of loquat fruit.

[0032] (I) Experimental Methods

[0033] 1. Construction of recombinant plasmids

[0034] A CDS sequence specific to the EjNAC5-like gene was selected, and upstream primer SEQ ID NO.5 and downstream primer SEQ ID NO.6 were designed. PCR products of the EjNAC5-like silenced fragment were obtained, and the products were recovered by electrophoresis using a gateway system (…). BP Clonase TM II. Using Enzyme Mix (invitrogen), the PCR fragment was inserted into pHellsgate 2 containing recombination sites attP1 and attP2 to obtain EjNAC5-like-pHellsgate 2. The ligation product was transformed into E. coli DH5α, and positive colonies were picked and sequenced to verify the recombinant plasmid. The recombinant plasmid was then transformed into Agrobacterium GV3101 and stored in glycerol at -80℃ for later use.

[0035] 2. Transient silencing experiment of loquat fruit

[0036] The preparation of Agrobacterium silencing bacterial suspension was consistent with that used in the dual-luciferase experiment. Two loquat fruits of similar size and maturity from the same branch were selected as a control and a treatment group, respectively. Each fruit was injected with 2 mL of bacterial suspension; one fruit was injected with the silenced gene EjNAC5-like, and the other with the empty vector pHellsgate 2. The experiment was repeated three times. After 7 days, the fruits were harvested, peeled, pitted, and frozen in liquid nitrogen at -80℃ for later use.

[0037] 3. Determination of lignin content

[0038] Weigh 0.5g of loquat powder for the determination of lignin content, using the same method as described in Example 1.

[0039] 4. Analysis of EjNAC5-like expression and downstream structural gene expression in loquat fruit after transient silencing.

[0040] Total RNA was extracted using the CTAB method. 1 μg of RNA was reverse transcribed to synthesize cDNA. cDNA synthesis was performed using a HiScriptIII RT SuperMix for qPCR with gDNA wiper (Vazyme, China) according to the manufacturer's instructions. The RT-qPCR reaction system followed the instructions for the ChamQ SYBR qPCR Master Mix (Vazyme, China). Detection was performed using a CFX96 ToughReal-Time PCR Detection System (Bio-Rad, USA), following the manufacturer's instructions for the reaction system and procedure: 95℃ for 30 s, 35 cycles of 95℃ for 10 s, and 60℃ for 30 s. Finally, a melting curve analysis program was run. EjACT (GenBank no. JN004223) was used as the loquat internal reference gene, with primer sequences SEQ ID NO.7 and SEQ ID NO.8, EjNAC5-like primer sequences SEQ ID NO.9 and SEQ ID NO.10, Ej4CL1 primer sequences SEQ ID NO.11 and SEQ ID NO.12, and EjPRX12 primer sequences SEQ ID NO.13 and SEQ ID NO.14. Relative expression levels were calculated using 2... –ΔCT Law.

[0041] (II) Experimental Results

[0042] Compared to the control fruit, transient silencing of the EjNAC5-like gene ( Figure 6 The expression levels of lignin monomer synthesis gene Ej4CL1 and lignin monomer polymerization gene EjPRX12 in loquat fruit decreased. Figure 7 (A and B), lignin content decreased ( Figure 8 Therefore, EjNAC5-like can serve as a candidate gene for improving the texture of loquat.

[0043] As can be seen from the above embodiments, the present invention provides a transcription factor EjNAC5-like that participates in the regulation of lignin deposition in loquat and its application. EjNAC5-like can promote lignin accumulation by activating the expression of Ej4CL1 and EjPRX12 through transcription. Silencing EjNAC5-like can reduce the expression of Ej4CL1 and EjPRX12, reduce lignin content, and improve fruit quality.

[0044] This invention has been described in detail with general descriptions and specific embodiments. However, some improvements and modifications can be made to it based on the invention. All such modifications or improvements made without departing from the spirit of the invention are within the scope of protection claimed by the invention.

Claims

1. The use of a transcription factor EjNAC5-like silencing nucleotide sequence as shown in SEQ ID NO. 1 in improving the quality of loquat fruits, characterized in that, The application is to reduce the lignin content and improve the fruit quality.

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