Application of miR156k in regulating citrus fruit ripening

Abstract

The application firstly discovers and discloses the role of miR156k in regulating citrus fruit ripening, constructs the precursor sequence of miR156k into a plant expression vector and introduces into the poncirus to overexpress, carries out fruit ripening related physiological and biochemical detection on the obtained miR156k precursor sequence overexpression poncirus, and confirms that miR156k can significantly promote the ripening of the poncirus fruit, and meanwhile, the traits of small fruit and seed are produced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to application of miR156k in regulating citrus fruit ripening. BACKGROUND

[0002] microRNA (miRNA) is a long about 18-24 base endogenous non-coding single-stranded small molecule RNA synthesized by organisms. The biosynthesis of plant miRNA mainly includes three main steps of transcription, processing and assembly into RNA silencing complex. The transcription of plant miRNA occurs in the nucleus, and the miRNA gene is transcribed into a miRNA primary transcript (pri-miRNA) containing a stem loop structure under the catalysis of type II RNA polymerase. The pri-miRNA is cut by Dicer-like 1 enzyme (DCL1) to form a pre-miRNA with a stem loop structure, and DCL1 has the characteristics of RNsae III endonuclease. The pre-miRNA is cut again by DCL1 to form a miRNA / miRNA* double-stranded complex, which is methylated under the action of methyltransferase HEN1 (Hua Enhancer 1) to prevent the double-stranded from being degraded by small molecule exonuclease. Subsequently, it is transferred from the nucleus to the cytoplasm with the help of transport protein HST (HASTY). In the cytoplasm, miRNA forms an RNA induced silencing complex (RISC) with AGO (Argonaute) protein. The RISC complex guides miRNA to the target gene and inhibits the target gene through mRNA cleavage, translation inhibition or chromatin modification.

[0003] As the important role of miRNA in multiple biological processes of plants is gradually revealed, the research on the function and regulation mechanism of miRNA in many species including citrus has become a hot topic in recent ten years.

[0004] miR156 is one of the core members of the plant miRNA family and also one of the most conserved miRNAs in evolution. It has been widely studied in many plants. In Arabidopsis, there are 10 members of miR156 (miR156a-miR156j) from different precursor genes. Like model plants, the miR156 family generally contains multiple members, such as 10 members and their isomers in sweet orange; 9 members in grape; and 5 precursors forming 7 mature bodies in tomato genome.

[0005] The target gene of miR156 is a class of transcription factors SPL (Squamosa promoter Biding-like) family specific to plants, and the gene is found to have effects of changing leaf shape, regulating tillering or branching, participating in fruit shape building and maturation process, etc. miR156 can regulate the expression of the SPL family genes through two pathways of targeted mRNA cleavage and translation inhibition. In Arabidopsis, there are 16 SPL genes, 10 of which are target genes of miR156; in rice, there are 19 OsSPLs, only 11 of which are target genes of miR156. In addition, the target genes of miR156 in different species are different, thereby causing the functions of miR156 in different species to be different.

[0006] Based on the exploration of citrus miR156, a new miR156 gene, miR156k, is cloned from the late orange in Fengjie. The transgenic poncirus showing overexpression of the precursor sequence of miR156k displays accelerated fruit maturation process, and the transgenic material can be reasonably utilized to adjust the market time of citrus, serve the production, and the gene has not been reported at present. SUMMARY

[0007] One of the purposes of the present application is to provide a citrus fruit maturation gene miR156k, the full-length sequence of the transcript of which is shown as SEQ ID NO. 1, and the precursor sequence of which is shown as SEQ ID NO. 2.

[0008] Another purpose of the present application is to protect the application of the citrus fruit maturation gene miR156k in regulating the fruit maturation, fruit size and seed size of citrus.

[0009] In order to realize the above technical scheme, the present application adopts the following technical scheme:

[0010] The present application clones the precursor sequence (SEQ ID NO. 2) of miR156k gene from the late orange in Fengjie, and makes the precursor sequence fragment into the receptor poncirus for overexpression by using the agrobacterium-mediated method, records the time when the fruit reaches maturity, and detects the fruit maturation related indexes, and the results show that the fruit maturation period of the miR156k overexpression poncirus is significantly advanced, and the fruit and seed size are significantly smaller than those of the wild type.

[0011] The present application provides a new method for regulating the ripening process of citrus fruits, and the transgenic plants cultivated by genetic engineering can overcome the shortcomings of traditional breeding and shorten the breeding cycle. Overexpression of miR156k gene in citrus can significantly accelerate the ripening process of fruits, so that the fruit market time can be artificially controlled. Compared with artificial hormone treatment of fruits, it is more green and environmentally friendly, and the effect is more stable, so it has obvious advantages and irreplaceable importance. At the same time, the trait of smaller fruit may be beneficial to reverse explore the mechanism of fruit enlargement, thereby guiding production, improving yield, and bringing better economic benefits to agriculture. In summary, the present application can provide a convenient way for large-scale production of citrus fruits, can greatly reduce the economic losses caused by fruit softening and rotting, can save costs and improve management level for agricultural production, and therefore the present application has a broad market application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 is the enzyme digestion site map of the overexpression vector PK7WG2D used in the present application.

[0013] Figure 2 is the expression analysis (A), comparison analysis of fruit ripening period (B) and comparison analysis of fruit size (C) of miR156k overexpression Citrus aurantium strains (#16, #17) and wild type Citrus aurantium (WT).

[0014] Figure 3 is the seed phenotype diagram of wild type Citrus aurantium (WT) and miR156k overexpression Citrus aurantium strains (#16, #17). DETAILED DESCRIPTION

[0015] The present application will be further described below in combination with specific examples, and the following examples are only specific embodiments of the present application, but the protection scope of the present application is not limited thereto. The methods used in the present application are all conventional methods in the art unless otherwise specified.

[0016] Example 1: Cloning of miR156k precursor sequence

[0017] After the leaves of Fengjie late orange in the growing period were ground into powder with liquid nitrogen, the powder was transferred into a centrifuge tube, and total RNA was extracted by using the Trizol method. The HiScript III All-in-one RT SuperMix Perfect for qPCR was used to synthesize the first strand of cDNA with total RNA as a template, and the reaction system and operation process were as follows: 5x All-in-one qRT SuperMix 4 μL, Enzyme Mix 1 μL, template RNA 1 μL, a total of 20 μL system (the above mixture was prepared with an RNase-free centrifuge tube, and a pipette gun was gently sucked and hit 8-10 times to fully mix, and centrifuged briefly to collect at the bottom of the tube); the reaction program was 50°C for 15 min, 85°C for 5 sec. After the first strand of cDNA was synthesized, it was stored at -20°C for standby use.

[0018] The synthesized first strand of cDNA was used as a template to amplify the miR156k precursor sequence fragment (the primer sequences were 5'-ACATTGAAATTGACAGAAGAGAGTG-3' and 5'-GTACTGGAAGCTGACAGAAAGAGCA-3'). The purpose gene was amplified by using the high-fidelity DNA polymerase Phanta Max Super-Fidelity DNA polymerase of Nuoyuan Biology, and the PCR reaction conditions were as follows: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 60 s, 34 cycles; 72°C for 5 min. The reaction system (50 μL) was 2 μL cDNA, 25 μL 2x Phanta Max Buffer, 1 μL dNTP (10 mM each), 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 1 μL Phanta Max Super-Fidelity DNA polymerase, and 17 μL ddH2O; after the PCR was completed, 5 μL was used for agarose gel electrophoresis to detect the amplified fragment of the desired size, and the miR156k precursor sequence was shown as SEQ ID NO. 2.

[0019] Example 2: Construction of plant overexpression vector

[0020] After the PCR product of the miR156k precursor sequence fragment was recovered, BP reaction was performed. The reaction system included 50-100 ng of the PCR gel recovery product, 1 μL of the pDONR207 vector, 0.5 μL of the BP clonase, and about 2.5 μL of the reaction system. The reaction system was incubated at 25°C overnight, and the next day, it could be placed on ice, and it could be directly used for E. coli transformation without terminating the reaction. After transformation, it was cultured at 37°C until single colonies were grown. After a single colony was picked and PCR identification was positive, sequencing was performed (the required primers for sequencing were: PDONR-For: 5'-TCGCGTTAAC GCTAGCATGGATCTC-3'; PDONR-Rev: 5'-GTAACATCAGAGATTTTGAGA CAC-3').

[0021] After sequencing was correct, it was used for LR reaction. The reaction system included 50-100 ng of the above-mentioned plasmid, 1 μL of the PK7WG2D vector, 0.5 μL of the LR clonase, and about 2.5 μL of the reaction system. The reaction system was incubated at 25°C overnight, and the next day, it could be placed on ice, and it could be directly used for E. coli transformation without terminating the reaction. After transformation, it was cultured at 37°C until single colonies were grown. After PCR identification was positive, a single colony was picked and expanded, and plasmid enzyme digestion verification or sequencing verification (the required primers for sequencing were CaMV 35S) was performed, and at the same time, the bacterial liquid was preserved in 50% glycerol at the same volume.

[0022] The constructed plant expression vector was transformed into the prepared Agrobacterium GV3101 competent cells by using the liquid nitrogen freeze-thaw method. The operation steps were as follows: 2 μg of the expression vector plasmid was added into a centrifuge tube containing 100 μL of the competent cells, and after being mixed gently, it was placed on ice for 5 min, then it was frozen in liquid nitrogen for 5 min, and then it was quickly placed in a 37°C water bath for 5 min, and then it was immediately placed in an ice bath for 5 min, 700 μL of antibiotic-free LB liquid medium was added, and it was cultured at 28°C for 2-3 h. After the activated Agrobacterium was centrifuged at 6000 rpm for one minute, the bacterial body was collected, about 100 μL of it was resuspended and spread on LB solid medium containing 100 mg / L spectinomycin (Spe), and it was cultured at 28°C; a single colony was selected and shaken, and bacterial liquid PCR was performed using the primers for amplifying the miR156k precursor sequence fragment to detect whether the expression vector was transformed into the Agrobacterium; for the positive clone, glycerol was added and it was stored at -80°C for later use.

[0023] Example 3: Poncirus trifoliate genetic transformation

[0024] The genetic transformation system adopted is Agrobacterium-mediated genetic transformation method, and the Agrobacterium strain is GV3101, and the transformation receptor material is Qinglei wild species of Citrus aurantium. The seeds are taken out from fresh Citrus aurantium fruits, and the pulp adhered thereto is washed away, and washed for five times, and the floating seeds are discarded, and the seeds are dried indoors (not more than half a day, and a piece of soaked filter paper is covered on the seeds to prevent the seeds from being dried too much; auxiliary tools such as mesh or sieve can be used to wash the seeds). The seeds after drying are soaked in 1 mol / L NaOH (8 g NaOH, 200 mL distilled water or sterile water) and stirred for 15 minutes (stirring for 5 min, standing for 5 min, and stirring for 5 min); the NaOH solution and floating seeds are poured out, and the upper floating matter is washed away, and the seeds are washed with clean water for 4-5 times; the seeds are transferred into 2% NaClO (50 ml 5.2% NaClO, 50 ml sterile water), and soaked on the clean bench for 15 minutes, and shaken several times in the middle; the NaClO solution is poured out, and the seeds are washed with sterile water for 4-5 times. Sowing to MT test tube culture medium, 3-4 per tube (using forceps or spoon). Sealing dark room (temperature 25°C) culture for one month to one and a half months. Taking the second activated Agrobacterium GV3101 plate containing plant expression vector, scraping all the bacteria on the clean bench with sterile scalpel blade, and placing in MT suspension culture medium (MT basic medium + malt extract powder 0.5 g / L + glutamine 1.5 g / L, without agar, completely dissolving pH=5.88, and high-temperature high-pressure sterilization), and placing in 220 rpm, 28°C constant temperature incubator for 40 min-1 h, so as to disperse the bacteria. At 40 min, the concentration of suspended bacteria solution is measured by ultraviolet spectrophotometer, and the OD 600between 0.6-0.8. Cut the seedlings during the shaking. Add AS (a kind of chemotactic inducer, which makes Agrobacterium to move to the wounded part of the plant tissue (chemotaxis) and activates the Vir region (induction) gene of Agrobacterium, so that T-DNA is transferred and inserted into the chromosomal DNA, generally prepare a stock solution of 50 mg / ml, use 1 μL AS stock solution: 1 ml of infection solution, ready to use) in the infection solution at a volume ratio of 1:1000. Cut the seedlings of Poncirus trifoliate, which have been green for 10 days, into 1 cm or so trapezoidal stem segments on sterile filter paper with a new blade sterilized by burning on a clean bench (the faster the better), and put them into MT suspension medium to prevent the stem segment incision from drying out and affecting the regeneration rate and transformation rate. After all the stem segments are cut, pour out the MT suspension medium, and use tweezers to block the stem segments to prevent waste. Burn a few circles of the bottle opening with an alcohol lamp, pour the prepared Agrobacterium solution into the bottle, and let it stand on the clean bench for 20 min, shaking appropriately in the middle. After 20 min, pour out the bacterial solution, use sterile filter paper to absorb the residual Agrobacterium solution on the surface of the Poncirus trifoliate stem segments, and place the stem segments with the incision upwards on the CM medium (MT bud culture medium + AS 50 mg / L, wherein the MT bud culture medium formula is MT basic medium + BA 0.5 mg / L + NAA 0.1 mg / L + KT 0.5 mg / L, completely dissolve and adjust pH = 5.88, then add agar 8 g / L to each, high-temperature high-pressure sterilization), 21°C constant temperature box dark culture for 3 days. Move the Poncirus trifoliate stem segments co-cultured for 3 days into a triangular flask containing sterile water, wash the stem segments 3-5 times with sterile water, shake the triangular flask vigorously in the middle, take out a part, and use sterile filter paper to absorb the moisture on the surface of the stem segments. Place the stem segments on the screening medium (MT bud culture medium + 50 mg / L Kana and 400 mg / L Cef), and dark culture at 26°C ± 2°C for 6 days. Transfer to light culture conditions, 16 h light / 8 h dark, 20-30 d subculture once. Observe whether there is Agrobacterium contamination at intervals, and remove the completely yellow stem segments in time. Take a photo of the positive bud under fluorescence, and then cut the positive bud with a blade and insert it into the rooting medium (rooting medium formula: 1 / 2 MT (half of the amount of macronutrients, the rest unchanged) + NAA 0.5 mg / L + IBA 0.1 mg / L + activated carbon 0.5 mg / L + agar 8 g / L, without antibiotics). Culture for 2-3 months, and the resistant seedlings with root systems reaching more than 3 cm are washed to remove the medium on the roots, placed in water for 10 days for seedling training, transplanted into the substrate, preferably a long black plastic pot, and watered thoroughly every 3 days. If there is no wilting phenomenon of the transgenic seedlings after two weeks, the transgenic citrus transformation is successful.

[0025] Example 4: Transgenic citrus phenotype analysis

[0026] After the miR156k transgenic Poncirus fruiting lines stably flowered and fruited, the fruit phenotype statistics analysis of two growth cycles was completed. In the miR156k Poncirus overexpression lines, by quantitative analysis of the expression of miR156k and its three target genes (FhSPL2, FhSPL6, FhSPL13) in Poncirus fruit, it was found that the expression of the three target genes in the overexpression lines was significantly down-regulated, indicating that the three target genes were significantly regulated by miR156k in the overexpression lines Figure 2 A); through the phenotype observation and data statistics of two overexpression lines (#16 and #17) in two fruiting periods, we found that the overexpression of miR156k could significantly promote the maturity of citrus fruit (the mature period was shortened by more than 30-35 days) Figure 2 B), while significantly reducing the size of fruit (reduced by 35-50%) Figure 2 C) and seed Figure 3 ). Therefore, miR156k has the function of regulating the size of citrus fruit and maturity and the size of seeds.

Claims

1. Use of miR156k in promoting citrus fruit ripening, characterized in that, A citrus plant overexpressing a precursor sequence of miR156k, said precursor sequence of miR156k being as set forth in SEQ ID NO.

2. A citrus plant overexpressing a precursor sequence of miR156k, said precursor sequence of miR156k being as set forth in SEQ ID NO.

2.

2. Use of miR156k in reducing citrus fruit size, characterized in that, A citrus plant overexpressing a precursor sequence of miR156k, said precursor sequence of miR156k being as set forth in SEQ ID NO.

2. A citrus plant overexpressing a precursor sequence of miR156k, said precursor sequence of miR156k being as set forth in SEQ ID NO.

2.

3. Use of miR156k in reducing seed size in citrus, characterized in that, A citrus plant overexpressing a precursor sequence of miR156k, said precursor sequence of miR156k being as set forth in SEQ ID NO.

2. A citrus plant overexpressing a precursor sequence of miR156k, said precursor sequence of miR156k being as set forth in SEQ ID NO.

2.

4. A method of promoting ripening of citrus fruit, characterized in that, ​ ​

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