Pear PyACS1 gene and application thereof in regulating maturity and hardness function

Abstract

The invention discloses a separated pear PyACS1 gene and application thereof. The PyACS1 gene for regulating and controlling fruit maturity and hardness in 'Max Red Bartlett' pear has a nucleotide sequence as shown in SEQ ID No.1, and an amino acid sequence coded by the PyACS1 gene is as shown in SEQ ID No.2 in a sequence table. Through heterologous stable overexpression in 'Micro-Tom' tomatoes, the PyACS1 is proved to be capable of promoting fruits to mature and soften in advance. Excavation of the PyACS1 gene not only provides a gene resource for improvement of fruit ripening and hardness of pears, but also provides valuable reference for fruit ripening and hardness quality improvement of other fruit trees.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and separates and clones a PyACS1 gene related to pear ripening and softening from Max Red Bartlett', and develops and applies a molecular marker. Background Art

[0002] Pear is a perennial woody plant of the Rosaceae family. It is the third largest cultivated fruit tree species in my country, with a long history of cultivation and a wide planting area. At present, pears are divided into two categories: one is the Oriental pear, which mainly includes white pear (P. bretschneideri), sand pear (P. pyrifolia), autumn pear (P. ussuriensis), Xinjiang pear (P. sinkiangensis); the other is the Western pear, mainly the Western pear (P. communis) (Bao et al., 2007; Wu et al., 2018). The process of pear fruit ripening involves complex changes, including color, flavor and fruit texture. Among them, fruit hardness is one of the important factors affecting quality. At the same time, fruit softening will also affect storage and transportation time and shorten shelf life, thereby affecting consumer choice (Braybrook and 2016). Ethylene is an important hormone involved in fruit ripening, softening and senescence. The level of ethylene release during fruit ripening affects the storage and shelf life of the fruit. According to whether there is a peak in the respiration intensity and ethylene release after the fruit matures, it can be divided into respiratory climacteric fruits and non-respiratory climacteric fruits. It is generally believed that Oriental pears are mainly non-respiratory climacteric fruits, while Western pears are respiratory climacteric fruits (Paul et al., 2012). Compared with Oriental pears, Western pears will have obvious ethylene and respiration peaks after harvest, the fruit hardness decreases rapidly, the shelf life is short, and the storage resistance is poor (Hiwasa et al., 2003). However, at present, there are few studies on the effects of ethylene release of Eastern and Western pears on fruit ripening, hardness and storage, which cannot effectively guide the genetic improvement and breeding of pear fruit ripening, storage and hardness. Summary of the invention

[0003] The purpose of the present invention is to provide a PyACS1 gene for regulating the ethylene release amount of pear fruit.

[0004] Another object of the present invention is to provide application of the gene.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A PyACS1 gene isolated from 'Max Red Bartlett' and having the function of affecting ethylene release and firmness of fruit, wherein the nucleotide sequence thereof is shown in SEQ ID No. 1 and comprises an open reading frame of 1422 bp.

[0007] The amino acid sequence of the protein encoded by the PyACS1 gene is shown in the sequence table SEQ ID No. 2.

[0008] A recombinant expression vector containing the PyACS1 gene of the present invention.

[0009] The recombinant expression vector uses pRI101-AN as the expression vector, and the insertion site of the PyACS1 gene is between SmaI and KpnI.

[0010] A host bacteria containing the PyACS1 gene of the present invention.

[0011] The primer pair for cloning the cDNA sequence of the PyACS1 gene of the present invention, the upstream primer PyACS1-F1 sequence is shown as SEQ ID No.3, and the downstream primer PyACS1-R1 sequence is shown as SEQ ID No.4.

[0012] The invention discloses an application of the PyACS1 gene in regulating the ripening period and firmness of pear fruits.

[0013] The invention discloses an application of the recombinant expression vector in regulating the ripening period and hardness of pear fruits.

[0014] The plants are preferably tomatoes and pears.

[0015] Beneficial Effects

[0016] 1. The discovery of the PyACS1 gene provides a new genetic resource for molecular breeding of pear trees, which is beneficial to improving the hardness and ripening period of pear fruit, extending the shelf life, reducing agricultural costs and achieving environmental friendliness.

[0017] 2. The PyACS1 gene was functionally verified in 'Micro-Tom' tomatoes by Agrobacterium-mediated genetic transformation. The results showed that the PyACS1 gene cloned in the present invention has the function of regulating fruit ripening and hardness, providing a more efficient approach for molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1Analysis of the expression level of PyACS1 gene in different developmental stages of 'Max Red Bartlett' fruit. Note: DAFB: Days after full bloom; the expression level of PyACS1 gene in different fruit developmental stages. H2: 2 days after fruit harvest; H4: 4 days after fruit harvest; H8: 8 days after fruit harvest.

[0019] Figure 2 This is a schematic diagram of the carrier of Example 2 of the present invention.

[0020] Figure 3 This is the functional analysis of the stable transformation of 'Micro-Tom' with the PyACS1 gene of the present invention.

[0021] Among them, A: fruit development and tomato color change of PyACS1 transgenic 'Micro-Tom' tomatoes and wild-type 'Micro-Tom' (WT) tomatoes; BD: color change period, ethylene release and hardness of mature fruits of PyACS1 transgenic 'Micro-Tom' tomatoes and WT. DETAILED DESCRIPTION

[0022] The present invention is described in detail below in conjunction with specific embodiments. Based on the following description and these embodiments, those skilled in the art can determine the essential features of the present invention, and can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention to make it suitable for various uses and conditions.

[0023] Example 1 Analysis of PyACS1 gene expression pattern

[0024] Fruit samples of Max Red Bartlett at different stages were collected from Yantai Academy of Agricultural Sciences (2021). Fruit samples of Max Red Bartlett at 30, 60, 90, 120, 135 days (maturity period) after flowering, 2, 4 and 8 days after harvest were selected as experimental materials, and transcriptome (RNA-seq) analysis was performed with three biological replicates at each developmental stage. RNA was extracted from this material and sequenced by Illumina high-throughput sequencing, and the FPKM value of the PyACS1 gene in each sample was calculated to measure its expression level.

[0025] Example 2 PyACS1 gene cloning and construction of overexpression vector

[0026] 1 μg of 'Max Red Bartlett' pulp RNA was taken and reverse transcribed using one-step gDNA removal and cDNA synthesis kit (Transgen, China), according to the instructions. Primers SEQ ID No. 3 and SEQ ID No. 4 were designed using snapgene software according to the general principle of primer design. The 50 μL reaction system included 200 ng cDNA, 1× buffer (TransStart Fast Pfu Buffer), 10 mM dNTP, 1 U Taq polymerase (TransStart Fast Pfu DNA Polymerase) (the aforementioned buffer and Taq polymerase were purchased from TRANS), and 500 nM of the above primers. The PCR reaction was completed on an eppendorf amplification instrument according to the following program: 95°C, pre-denaturation for 3 minutes; 95°C denaturation for 15 seconds, 60°C annealing for 15 seconds, 72°C extension for 1 minute and 30 seconds, 35 thermal cycles; 72°C extension for 10 minutes; 4°C storage. A single PCR product was generated. After the PCR product was recovered and purified, the full-length sequence of PyACS1 was inserted into the pRI101-AN vector using homologous recombination technology. The Escherichia coli DH5α was transformed by heat shock method and then incubated with 100 μg mL -1 K + The cells were cultured in LB solid medium, positive clones were screened, propagated and sequenced. The sequencing results showed that the full length of the PyACS1 gene was 1422 bp, the nucleotide sequence of which was shown in SEQ ID No.1, and it encoded a protein of 473 amino acid residues, the sequence of which was shown in SEQ ID No.2. The plasmid with the correct sequencing was transformed into the final recombinant vector into the Agrobacterium strain GV3101 by freeze-thaw method, and then incubated in 50 μg.mL -1 K + , 100 μg mL -1 Rifampicin + ) in LB medium, expand and propagate the correctly identified Agrobacterium strain in a 10 mL sterile centrifuge tube, and store it at -80°C for later use.

[0027] Example 3 Stable transformation of 'Micro-Tom' tomato with PyACS1 and detection of firmness and fruit maturity

[0028] (1) Stable transformation of 'Micro-Tom' tomato

[0029] The 'Micro-Tom' tomato was infected using the Agrobacterium-mediated method. The sterile cultured tomato cotyledons were scratched, incubated in the dark for 2 days, placed in the Agrobacterium resuspension, shaken for 5-10 minutes, and then the bacterial solution was blotted dry on sterile filter paper. The tomato was placed on a co-culture medium and incubated in the dark for 3 days. The tomato was then transferred to a screening plate and incubated under light conditions until new shoots grew. The plants were subcultured every 2 weeks and transferred to a rooting medium when they grew to about 10 cm. After rooting, the plants were transplanted into nutrient pots.

[0030] (2) Detection of ethylene release in transgenic plants

[0031] Fruits of WT and PyACS1 transgenic plants were collected 47 days after anthesis and stored in 300 ml open glass bottles for 2 h to minimize the wound ethylene effect caused by picking. The glass bottles were then sealed and incubated at room temperature for 24 h. Ethylene production was detected using a PLT-BX-C2H4 (Pulitong, Shenzhen, China) instrument.

[0032] (3) Transgenic plant hardness test

[0033] The fruits of WT and PyACS1 transgenic plants were collected 47 days after anthesis and the hardness of the fruits was measured using a Brookfield CT3 texture analyzer with the following parameters: disc-shaped probe, trigger 4 g, depth 6 mm, speed 1.0 mm / s; the results were recorded in kg / cm2.

[0034] The results of functional analysis of PyACS1 transgenic 'Micro-Tom' tomatoes are shown in Figure 3 ,right Figure 3 It can be seen that the fruits of PyACS1 transgenic plants mature earlier than those of WT plants, and the fruit ethylene release is significantly higher than that of WT, while the fruit hardness is significantly lower than that of WT. The results show that the PyACS1 gene has the function of regulating fruit ripening period, ethylene release and fruit hardness, providing genetic resources for molecular breeding of fruit trees.

[0035] Main references

[0036] Bao,L.,Chen,K.,Zhang,D.,Cao,Y.,Yamamoto,T.,and Teng,Y.(2007).Geneticdiversity and similarity of pear(Pyrus L.)cultivars native to East Asiarevealed by SSR(simple sequence repeat)markers.Genetic Resources and CropEvolution 54,959-971.

[0037] Braybrook,S.A.,and H.(2016).Shifting foundations:themechanical cell wall and development.Curr Opin Plant Biol 29,115-120.

[0038] Hiwasa,K.,Kinugasa,Y.,Amano,S.,Hashimoto,A.,Nakano,R.,Inaba,A.,andKubo,Y.(2003).Ethylene is required for both the initiation and progression ofsoftening in pear(Pyrus L.)fruit.Journal of Experimental Botany 54,771-779.

[0039] Paul,V.,Pandey,R.,and Srivastava,G.C.(2012).The fading distinctionsbetween classical patterns of ripening in climacteric and non-climactericfruit and the ubiquity of ethylene-An overview.J Food Sci Tech Mys 49,1-21.

[0040] Wu,J.,Wang,Y.,Xu,J.,Korban,S.S.,Fei,Z.,Tao,S.,Ming,R.,Tai,S.,Khan,A.M.,Postman,J.D.,et al.(2018).Diversification and independent domesticationofAsian and European pears.Genome Biol 19,77.

Claims

1. A PyACS1 gene isolated from 'Max Red Bartlett' pear that regulates fruit ripening and softening, characterized in that: The nucleotide sequence is shown in SEQ ID No.

1.

2. The protein encoded by the PyACS1 gene according to claim 1, characterized in that The amino acid sequence is shown as SEQ ID No.

2.

3. A primer pair for cloning the gene sequence according to claim 1, characterized in that: The sequence of the upstream primer PyACS1-F1 is shown in SEQ ID No.3, and the sequence of the downstream primer PyACS1-R1 is shown in SEQ ID No.

4.

4. A recombinant expression vector containing the PyACS1 gene according to claim 1.

5. The recombinant expression vector according to claim 4, characterized in that pRI101-AN was used as the overexpression vector.

6. Use of the PyACS1 gene according to claim 1 in promoting fruit ripening and softening.

7. Use of the PyACS1 gene according to claim 1 in constructing plants with early-maturing fruits.

8. The use according to claim 7, characterized in that The plant is tomato.

9. Use of the recombinant expression vector according to claim 4 or 5 in promoting fruit ripening and softening.

10. Use of the recombinant expression vector according to claim 4 or 5 in constructing a plant with early-maturing fruits.

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