Application of pineapple AcPPO3 gene

Abstract

The invention discloses an application of a pineapple AcPPO3 gene in evaluation of pineapple pulp browning. The nucleotide sequence of the pineapple AcPPO3 gene is shown as SEQ ID NO: 1 in a sequence table. According to the invention, all four members of the PPO family of the Pineapple are cloned respectively, and the functions of the members are researched respectively, so that the AcPPO3 is determined to be possibly a key gene for regulating and controlling pulp browning after picking of pineapple varieties. By detecting the transcriptional level expression condition of the gene, the occurrence condition of pulp browning after pineapple picking can be judged.

Description

Technical Field

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

[0002] Pineapple Ananas comosus (L.) Merr], as one of the three major tropical fruits in the world, is not only the most characteristic fruit variety in the tropical and subtropical regions of China, but also an important part of the global tropical agricultural economy. Pineapples are mainly consumed fresh and are also widely used in the food processing and industrial fields, such as canning, concentrated fruit juice, extraction of bromelain, separation of high-quality fibers, and manufacture of animal feed, etc., forming a diversified industrial chain. Therefore, the pineapple industry has become one of the pillar industries of the rural economy in South China, which not only drives the increase of local farmers' income, but also injects strong impetus into the regional economic development.

[0003] Pineapple internal browning (IB) is caused by long-term low-temperature chilling injury in the field or after harvest, resulting in excessive reactive oxygen species (ROS: H2O2), causing membrane damage, and making the PPO and phenolic substrates that were originally compartmentalized come into contact and oxidize to produce quinone substances, which then polymerize to cause pulp browning. High PPO activity, production of phenols and H2O2 are three necessary conditions for the occurrence of IB. Research shows that PPO activity is positively correlated with the occurrence of pineapple IB, and the PPO activity in the browning pulp of pineapples is significantly greater than that in the non-browning pulp; low temperature induces PPO enzyme activity and enhances the occurrence of IB; after knocking out the PPO gene of 'Smooth Cayenne' pineapple, the tolerance to IB is significantly enhanced; the GARC element of the PPO promoter responds to gibberellin treatment, promotes expression, and significantly induces the occurrence of IB. Our previous research found that the pulp of 'Comte de Paris' pineapple begins to brown 3 days after harvest and gradually intensifies, while 'Golden Pineapple' does not have pulp browning, and the PPO expression and enzyme activity of 'Golden Pineapple' are also significantly lower than those of 'Comte de Paris' pineapple. The browning incidence rate, PPO expression and enzyme activity of the main cultivated 'Pattavia' pineapple in Thailand are significantly lower than those of 'Trat Sri Thong' pineapple. These results indicate that there are significant differences in the pulp browning of pineapples among different varieties after harvest. However, so far, there has not been a clear standard to judge and evaluate the pulp browning of pineapples among different varieties and at different storage stages. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of pineapple AcPPO3 genes in evaluating the browning of pineapple pulp.

[0005] Pineapple AcPPO3 The application of genes in evaluating the browning of pineapple pulp, wherein the nucleotide sequence of the pineapple AcPPO3 gene is as shown in Sequence Listing SEQ ID NO: 1.

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

[0007] The evaluation criterion is the AcPPO3 expression level of the gene in different pineapple varieties. The higher the expression level, the deeper the browning degree of pineapple pulp; the lower the expression level, the lighter the browning degree of pineapple pulp.

[0008] Advantages of the present invention: The present invention separately clones all 4 members of the PPO family of 'Balin' pineapple, studies their functions respectively, and clarifies that AcPPO3 may be the key gene regulating the occurrence of postharvest pulp browning among pineapple varieties. By detecting the expression level of this gene at the transcriptional level, the occurrence of postharvest pulp browning in pineapples can be judged. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 is AcPPOs the expression in different tissue parts of 'Balin' pineapple.

[0010] Figure 2 is AcPPOs the expression in different development stages of 'Balin' pineapple.

[0011] Figure 3 is the analysis of the incidence rate of postharvest pulp browning, PPO transcription and enzyme activity of highly resistant 'Golden Pineapple' (MD-2) and susceptible 'Balin' pineapple; A: The pulp of 'Golden Pineapple' does not turn brown after 15 days of storage; B: During the postharvest storage stage, AcPPOs both are continuously up-regulated, and the AcPPO3 expression level is the highest at each storage time point; C: During the postharvest storage stage, the AcPPOs expression level of 'Golden Pineapple' AcPPO3 significantly decreases,

[0012] Figure 4 is the subcellular localization of AcPPO1-4.

[0013] Figure 5 is AcPPO1-4 gene function verification; (A) Screening out overexpression lines of each significantly up-regulated AcPPOs gene; (B) Overexpression of AcPPO3 leads to browning of rice seeds, and the AcPPO3 gene of the browned seeds is extremely significantly up-regulated. (C) Overexpression of AcPPO3 leads to aggravated pulp browning. DETAILED DESCRIPTION OF THE INVENTION

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

[0015] Example 1 Different tissue parts and different development stages of pineapple AcPPOs Gene expression 1. Genomic RNA extraction (1) Tissue lysis: Take samples of different tissue parts of 'Bali' pineapple at the flowering stage and pineapple pulp at different development stages of the fruit, place them in a 2.0 mL centrifuge tube containing steel beads, add 1 mL of plant lysis buffer, and invert and mix 10 times; (2) RNA extraction: Add 200 μL of pre-cooled chloroform, invert and mix, and centrifuge at 10,000 g for 6 min at 4°C; (3) RNA adsorption: Pipette 600 μL of the upper aqueous phase containing RNA into a new RNase-free 1.5 mL centrifuge tube, add 1 / 2 volume of absolute ethanol, invert and mix, transfer to an adsorption column, and centrifuge at 12,000 g for 30 s at 4°C, discard the filtrate; (4) RNA washing: Add 500 μL of wash buffer with absolute ethanol added, centrifuge at 12,000 g for 30 s at 4°C, discard the filtrate, and repeat this step once; (5) Removal of wash buffer: Centrifuge the empty tube at 13,000 g for 2 min at 4°C, and place the adsorption column in a new enzyme-free 1.5 mL centrifuge tube; (6) RNA collection and preservation: Leave the lid open and place at room temperature for 2 min to evaporate the residual absolute ethanol; add 65 μL of RNase-free ddH2O to the center of the adsorption column, let stand at room temperature for 2 min, and then centrifuge at 12,000 g for 2 min at 4°C; after measuring the concentration, store at -80°C in the refrigerator for later use.

[0016] 2. Synthesis of the first strand of reverse transcribed cDNA Refer to the instruction manual of the reverse transcription kit HiScript III 1st Strand cDNA SynthesisKit (+gDNA wiper) of Novoprotein Scientific Inc. to perform the following operations: (1) Samples for real-time fluorescence quantitative PCR

[0017] Gently pipette and mix, and react at 42°C for 2 min (temperature controlled by the PCR instrument).

[0018]

[0019] Gently pipette to mix well.

[0020]

[0021] The product can be immediately used for real-time fluorescence quantitative PCR, or it can be aliquoted and stored at -80 °C for long-term preservation.

[0022] (2)Samples are used for gene cloning

[0023] Gently pipette to mix well, react at 65 °C for 5 min (temperature controlled by PCR instrument), and place on ice for 2 min.

[0024]

[0025] Gently pipette to mix well, react at 42 °C for 2 min (temperature controlled by PCR instrument).

[0026]

[0027] Gently pipette to mix well.

[0028]

[0029] The product can be immediately used for the cloning of gene CDS, or it can be aliquoted and stored at -80 °C for long-term preservation.

[0030] 3. Real-time fluorescence quantitative PCR Perform the following operations with reference to the instruction manual of the qPCR product Taq Pro Universal SYBR qPCR Master Mix from Novoprotein:

[0031]

[0032] ‘Bali’ pineapple AcPPOs The expression in different tissue parts of pineapple at the flowering stage is different. AcPPO1 and AcPPO2 The expression level is the highest in roots. AcPPO4 The expression level is the highest in flowers. However, the AcPPOs expression level in pulp is very low, especially AcPPO3 which is not expressed in different tissue parts; at different developmental stages of pineapple fruits after flowering, AcPPO1 、 AcPPO2 and AcPPO4 are rapidly up-regulated at 3 weeks after flowering and then rapidly decline, while AcPPO3 is lowly expressed at different developmental stages of fruits (Figure 1-2 ).

[0033] Example 2 The incidence of browning, PPO transcription, and enzyme activity of 'Golden Pineapple' were significantly lower than those of 'Bali' pineapple 1. Statistical analysis of postharvest flesh browning of pineapples: Select six - mature fruits without pests, diseases, mechanical damage, with uniform fruit size, incomplete - flat eyes, and a green peel area close to 100%. Soak the bottom of the fruits in 0.5 g·L-1 prochloraz for 1 min, and dry them in a cool and ventilated place for later use. Select 30 'Golden Pineapple' and 30 'Bali' pineapples each, place them in a perforated plastic box covered with a 0.02 - mm polyethylene film bag, and store them in a fruit storage warehouse at room temperature of 25°C and relative humidity of 90%. After 0 d, 3 d, 6 d, 9 d, 12 d, and 15 d of storage, cut the pineapple fruits transversely along the fruit axis, observe and count the postharvest flesh browning. The appearance of water - soaked and brown spots indicates flesh browning. Take the flesh near the pith of the pineapple fruits, use a part for measuring various indexes, and immediately freeze - store the other part in liquid nitrogen and place it in a - 80°C ultra - low - temperature freezer for later use.

[0034] 2. Determination of PPO enzyme activity during postharvest storage: Use a pulverizer to grind the flesh frozen in liquid nitrogen into powder, and put it into a 50 - mL centrifuge tube for the determination of the content of various substances. Use the polyphenol oxidase (PPO) activity assay kit from Jiangsu Keming Biotechnology Co., Ltd. to determine the change in polyphenol oxidase (PPO) activity.

[0035] Flesh browning of 'Bali' pineapple started to occur and gradually intensified 3 days after harvest, while 'Golden Pineapple' did not show flesh browning ( Figure 3 A). During the postharvest storage stage of 'Bali' pineapple, AcPPOs genes were continuously up - regulated, especially AcPPO3 , with extremely significant up - regulation; however, for 'Golden Pineapple', AcPPOs gene expression was significantly lower than that of 'Bali' pineapple, and AcPPO1 , AcPPO2 and AcPPO4 showed no significant difference , AcPPO3 significantly up - regulated at 3 d of storage and then rapidly down - regulated, with no significant difference afterwards ( Figure 3 B, C). In addition, at each postharvest storage time point, the PPO enzyme activity of 'Bali' pineapple was significantly higher than that of 'Golden Pineapple' ( Figure 3 D). This result indicates that there are varietal differences in postharvest flesh browning of pineapples.

[0036] Example 3 AcPPOs Subcellular localization Four genes of the PPO family of 'Bali' pineapple were cloned, and pBE::AcPPO1-4::eGFP vectors were constructed respectively. After transformation into Agrobacterium and infection of tobacco, microscopic observation found that AcPPO1 and AcPPO2 were localized on the cell membrane, while AcPPO3 and AcPPO4 were localized in peroxisomes ( Figure 4 ).

[0037] Example 4 AcPPOs Functional verification Phenol color reaction: Twenty seeds of each positive strain were selected and placed in a 5 mL test tube, and 2 mL of 1% (v / v) phenol solution was added. The tube was quickly capped. After soaking for 5 days, it was dried in a fume hood, and the color change of the seeds was observed. The seeds after the phenol color reaction were quickly frozen in liquid nitrogen and stored at -80 °C for later use.

[0038] Construct four AcPPO gene overexpression vectors of pOx2:: AcPPO1~4 and heterologously transform Nipponbare (NIP) rice by Agrobacterium infection, with Guangluai (GLA) rice as the control. Positive overexpression strains were screened, and the transcription levels of each overexpression strain AcPPO were extremely significantly up-regulated ( Figure 5 A); further, through the phenol color reaction, it was found that only overexpression of AcPPO3 led to browning of rice seeds, and the expression level in the browned seeds was AcPPO3 extremely significantly up-regulated ( Figure 5 B). By constructing pCAMBIA2300:: AcPPO3 It was further confirmed in pineapple fruits that AcPPO3 overexpression induced browning of postharvest pineapple pulp ( Figure 5 C), indicating that AcPPO3 is a key gene regulating postharvest browning of pineapple pulp.

[0039] The above embodiments only represent several implementation manners of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. Pineapple AcPPO3 The application of the gene in evaluating the browning of pineapple pulp, characterized in that, The pineapple described above AcPPO3 The nucleotide sequence of the gene is shown in Sequence Listing SEQ ID NO:

1.

2. The application of the gene in evaluating the browning of pineapple pulp according to claim 1, characterized in that, AcPPO3 The application of the gene in evaluating the browning of pineapple pulp, characterized in that, The AcPPO3 expression protein sequence of the gene is shown in Sequence Listing SEQ ID NO:

2.

3. The application of the gene in evaluating the browning of pineapple pulp according to claim 1, characterized in that, AcPPO3 The application of the gene in evaluating the browning of pineapple pulp, characterized in that, The evaluation criterion is pineapple AcPPO3 The expression level of the gene in different pineapple varieties. A higher expression level indicates a deeper browning degree of pineapple pulp, while a lower expression level indicates a shallower browning degree of pineapple pulp.

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