Application of pineapple AcPPO3 gene

Abstract

The invention discloses pineapple AcPPO3 Application of genes in evaluating pineapple flesh browning. AcPPO3 The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO: 1. The present invention cloned the 'Bali' pineapple PPO All four members of the family have their functions studied separately to clarify AcPPO3 This gene may be a key gene regulating the occurrence of post-harvest flesh browning in pineapple varieties. By detecting the expression of this gene at the transcriptional level, it is possible to determine the occurrence of post-harvest flesh browning in pineapples.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology, and specifically relates to pineapple AcPPO3 Application of genes in evaluating pineapple flesh browning. Background Art

[0002] Pineapple[ Ananas comosus As one of the world's three major tropical fruits, pineapple (L. Merr.) is not only the most distinctive fruit variety in my country's tropical and subtropical regions, but also a vital component of the global tropical agricultural economy. While primarily consumed fresh, pineapples are also widely used in food processing and industrial applications, such as canning, juice concentrate, bromelain extraction, high-quality fiber separation, and animal feed production, forming a diversified industrial chain.

[0003] Pineapple blackheart (IB) is caused by prolonged low temperatures in the field or after harvest, which leads to excessive reactive oxygen species (ROS: H₂O₂), causing membrane damage. This leads to the oxidation of previously compartmentalized PPO with phenolic substrates, producing quinones that polymerize and cause flesh browning. High PPO activity, phenolic compounds, and H₂O₂ production are essential for IB to develop. Studies have shown that PPO activity is positively correlated with IB in pineapples, with PPO activity significantly higher in browning pineapple flesh than in non-browning pineapple flesh. Low temperatures induce PPO activity, enhancing IB development. Knockout of the PPO gene in the 'Wuchikayin' pineapple significantly enhances IB tolerance. The GARC element in the PPO promoter responds to gibberellin treatment, promoting expression and significantly inducing IB development. Our previous research found that flesh browning in 'Bali' pineapples begins three days after harvest and gradually intensifies, while in 'Golden Pineapple', no flesh browning occurs. Furthermore, PPO expression and enzyme activity in 'Golden Pineapple' are significantly lower than in 'Bali'. The browning rate, PPO expression, and enzyme activity of the 'Pattavia' pineapple, a major Thai cultivar, were significantly lower than those of the 'Trat Sri Thong' pineapple. These results suggest that postharvest flesh browning varies significantly between pineapple varieties. However, to date, there is no clear standard for identifying and evaluating pineapple flesh browning between varieties and at different storage stages. Summary of the Invention

[0004] The object of the present invention is to provide pineapple AcPPO3 Application of genes in evaluating pineapple flesh browning.

[0005] Pineapple AcPPO3 Application of genes in evaluating pineapple flesh browning, the pineapple AcPPO3 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0006] described AcPPO3 The expressed protein sequence of the gene is shown in SEQ ID NO: 2.

[0007] The evaluation criteria are pineapple AcPPO3 The expression level of the gene in different pineapple varieties shows that high expression level indicates deep browning of pineapple flesh, while low expression level indicates shallow browning of pineapple flesh.

[0008] Beneficial effects of the present invention: The present invention clones the 'Bali' pineapple PPO All four members of the family have their functions studied separately to clarify AcPPO3 This gene may be a key gene regulating the occurrence of post-harvest flesh browning in pineapple varieties. By detecting the expression of this gene at the transcriptional level, it is possible to determine the occurrence of post-harvest flesh browning in pineapples. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 for AcPPOs Expression in different tissues of 'Bali' pineapple.

[0010] Figure 2 for AcPPOs Expression in different developmental stages of 'Bali' pineapple.

[0011] Figure 3 Analysis of the postharvest flesh browning rate, PPO transcription and enzyme activity in highly resistant 'Golden Pineapple' (MD-2) and susceptible 'Bali' pineapples; A: No browning occurred in 'Golden Pineapple' flesh after 15 days of storage; B: During postharvest storage, 'Bali' pineapples AcPPOs The expression of AcPPO3 The highest expression level; C: post-harvest storage stage, 'Golden Pineapple' AcPPOs The expression level was significantly reduced. AcPPO3 It first increased and then decreased rapidly; D: During the post-harvest storage stage, the PPO enzyme activity of 'Golden Pineapple' decreased significantly.

[0012] Figure 4 Subcellular localization of AcPPO1-4.

[0013] Figure 5 for AcPPO1-4 Gene function verification; (A) Screening of significantly upregulated AcPPOs gene overexpression lines; (B) AcPPO3 overexpression causes rice seed browning, with AcPPO3 expression highly significantly upregulated in browning seeds. (C) AcPPO3 overexpression exacerbates fruit flesh browning. DETAILED DESCRIPTION

[0014] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0015] Example 1 Different tissue parts and different developmental stages of pineapple AcPPOs Gene expression

[0016] 1. Genomic RNA Extraction

[0017] (1) Tissue lysis: Take pineapple pulp samples from different tissue parts during the flowering period and different fruit development stages of the 'Bali' pineapple, place them in a 2.0 mL centrifuge tube containing steel balls, add 1 mL of plant lysis solution, and mix thoroughly by inverting 10 times;

[0018] (2) RNA extraction: Add 200 μL of pre-chilled chloroform, mix thoroughly by inversion, and centrifuge at 10,000 g for 6 min at 4°C;

[0019] (3) RNA adsorption: aspirate 600 μL of the upper aqueous phase containing RNA, place it in a new RNase-free 1.5 mL centrifuge tube, add 1 / 2 volume of anhydrous ethanol, mix thoroughly by inversion, transfer to the adsorption column, centrifuge at 12,000 g for 30 s at 4°C, and discard the filtrate;

[0020] (4) RNA cleaning: add 500 μL of rinsing solution with anhydrous ethanol, centrifuge at 12,000 g for 30 s at 4°C, discard the filtrate, and repeat this step once;

[0021] (5) Remove the rinse solution: Centrifuge the empty tube at 13,000 g for 2 min at 4°C and place the adsorption column in a new 1.5 mL centrifuge tube without enzyme.

[0022] (6) RNA collection and storage: After opening the lid, place at room temperature for 2 minutes to evaporate the residual anhydrous ethanol; add 65 μL RNase-free ddH2O to the center of the adsorption column, let it stand at room temperature for 2 minutes, and then centrifuge at 12,000 g at 4°C for 2 minutes; after measuring the concentration, store in a refrigerator at -80°C for future use.

[0023] 2. Synthesis of the First Strand of Reverse Transcribed cDNA

[0024] Refer to the instructions of the Novozymes reverse transcription kit HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) and perform the following operations:

[0025] (1) Samples used for real-time fluorescence quantitative PCR

[0026]

[0027] Mix gently by pipetting and react at 42°C for 2 min (temperature controlled by PCR instrument).

[0028]

[0029] Mix by gently pipetting.

[0030]

[0031] The product can be used immediately for real-time fluorescence quantitative PCR or stored in separate devices at -80°C for a long term.

[0032] (2) Samples used for gene cloning

[0033]

[0034] Mix gently by pipetting, react at 65°C for 5 min (temperature controlled by PCR instrument), and place on ice for 2 min.

[0035]

[0036] Mix gently by pipetting and react at 42°C for 2 min (temperature controlled by PCR instrument).

[0037]

[0038] Mix by gently pipetting.

[0039]

[0040] The product can be used immediately for cloning gene CDS, or it can be stored in a separate container at -80℃ for a long time.

[0041] 3. Real-time Fluorescence Quantitative PCR

[0042] Refer to the instructions for the Taq Pro Universal SYBR qPCR Master Mix from Novozymes and perform the following steps:

[0043]

[0044]

[0045] 'Bali' pineapple AcPPOs The expression of different tissues in pineapple is different during flowering. AcPPO1 and AcPPO2 The highest expression level was in roots. AcPPO4 The highest expression level is in flowers. AcPPOs The expression levels in the pulp were very low, especially AcPPO3 , which is not expressed in different tissues; at different developmental stages of pineapple fruit after flowering, AcPPO1 、 AcPPO2 and AcPPO4 The expression of β-actin was rapidly increased 3 weeks after flowering and then decreased rapidly. AcPPO3 Low expression was observed at different stages of fruit development ( Figure 1-2 ).

[0046] Example 2 The browning incidence, PPO transcription and enzyme activity of 'Golden Pineapple' were significantly lower than those of 'Bali' pineapple

[0047] 1. Postharvest pineapple flesh browning statistics: Selected pineapple fruits that were free of pests and diseases, mechanical damage, uniform in size, with eyes not completely flattened, and with nearly 100% green peel area, were half-ripe. The bottoms of the fruits were soaked in 0.5 g·L⁻¹ prochloraz for 1 minute and air-dried in a cool, ventilated place for later use. Thirty pineapples each of the 'Golden Pineapple' and 'Bali' varieties were placed in hollow plastic frames lined with 0.02 mm polyethylene film bags and stored in a fruit storage facility at 25°C and 90% relative humidity. After 0, 3, 6, 9, 12, and 15 days of storage, the fruits were cross-sectioned along the axis and observed for postharvest flesh browning. The presence of water stains and brown spots was considered browning. Pineapple flesh near the pith was sampled; a portion was used to measure various parameters, while a portion was immediately frozen in liquid nitrogen and placed in a -80°C ultra-low temperature freezer for later use.

[0048] 2. PPO enzyme activity determination during postharvest storage: Pulp frozen in liquid nitrogen was ground into powder using a grinder and placed in a 50 mL centrifuge tube for determination of various substances. Changes in polyphenol oxidase (PPO) activity were determined using a polyphenol oxidase (PPO) activity assay kit from Jiangsu Keming Biotechnology Co., Ltd.

[0049] The flesh of the 'Bali' pineapple began to brown 3 days after harvest and gradually intensified, while the flesh of the 'Golden Pineapple' did not brown ( Figure 3 A). Post-harvest storage stage of 'Bali' pineapple, AcPPOs Gene expression is continuously upregulated, especially AcPPO3 , which was significantly up-regulated; however, 'Golden Pineapple' AcPPOs The expression of the gene was significantly lower than that of the 'Bali' pineapple, and AcPPO1 、 AcPPO2 and AcPPO4 No significant difference , AcPPO3 It was significantly up-regulated on the 3rd day of storage and then rapidly down-regulated, and there was no significant difference thereafter ( Figure 3 B, C). In addition, the PPO enzyme activity of 'Bali' pineapple was significantly higher than that of 'Golden Pineapple' at each postharvest storage time point ( Figure 3 D). This result indicates that there are varietal differences in the browning of pineapple flesh after harvest.

[0050] Example 3 AcPPOs Subcellular localization

[0051] All four genes of the PPO family of 'Bali' pineapple were cloned and the pBE::AcPPO1-4::eGFP vectors were created. After transformation with Agrobacterium, tobacco was infected. Microscopic observation revealed that AcPPO1 and AcPPO2 were localized in the cell membrane, while AcPPO3 and AcPPO4 were localized in the peroxisome ( Figure 4 ).

[0052] Example 4 AcPPOs Functional Verification

[0053] Phenol color reaction: 20 seeds of each positive strain were selected and placed in a 5 mL test tube. 2 mL of 1% (v / v) phenol solution was added and the tube was quickly covered. After soaking for 5 days, the tube was placed in a fume hood to dry. The color change of the seeds was observed and the seeds were quickly frozen in liquid nitrogen after the phenol color reaction and stored at -80°C for later use.

[0054] Build 4 AcPPO pOx2:: AcPPO1~4 The overexpression vector was transformed into Nipponbare (NIP) rice by Agrobacterium infection, and Guanglu Avian (GLA) rice was used as the control. AcPPO Gene transcription levels were significantly upregulated ( Figure 5 A); further phenol color reaction revealed that only overexpression AcPPO3 Causes rice seeds to turn brown, and the horizontal seeds after browning AcPPO3 Very significantly upregulated expression ( Figure 5 B). By constructing pCAMBIA2300:: AcPPO3 This was further confirmed in pineapple fruit. AcPPO3 Overexpression induces postharvest browning of pineapple flesh ( Figure 5 C), indicating AcPPO3 It is a key gene that regulates the browning of pineapple flesh after harvest.

[0055] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. Detecting pineapples AcPPO3 The use of a reagent for measuring the expression level of a gene in evaluating the difference in browning of pineapple flesh after harvest is characterized in that: The pineapple AcPPO3 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; AcPPO3 The expressed protein sequence of the gene is shown in SEQ ID NO:

2.

2. Detection pineapple according to claim 1 AcPPO3 The use of a reagent for measuring the expression level of a gene in evaluating the difference in browning of pineapple flesh after harvest is characterized in that: The evaluation criteria are pineapple AcPPO3 The expression level of the gene in different pineapple varieties shows that high expression level indicates deep browning of pineapple flesh, while low expression level indicates shallow browning of pineapple flesh.

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