JP4815587B2 - Judgment method of apple fruit harvest time - Google Patents

Description

  The present invention relates to a method for determining an appropriate harvest time of apple fruit simply and accurately.

  The apple fruit harvest time has a great influence on the fruit quality and shelf life (storability), and affects the commercial value. In general, when harvesting is too early, the taste is poor, and not only the sugar content is insufficient during storage, but also physiological disorders such as the occurrence of burn disease are likely to occur. On the other hand, when the harvest is too slow, it is easy to blur (aged), rot progresses, and browning due to aging is likely to occur. However, since the growth and ripening of fruits are based on multiple factors such as weather, region, flowering time, tree vigor, and cultivation management even during the same cultivar, it is extremely difficult to accurately grasp the harvest time. Therefore, in the present situation, the harvest time is determined empirically by grasping the number of days from the full bloom period and the maturation phenomenon, but the maturity level is different between the outer branch and the inner branch even in the same tree. It must be said that such a method is extremely rough.

  Then, an object of this invention is to provide the method of determining the harvest time of an appropriate apple fruit simply and accurately.

  In apples, it is known that ethylene synthesis rapidly increases as the fruit ripens, and at the same time, cell wall degrading enzymes and the like function and fruit tissue begins to soften. In order to elucidate a series of these physiological phenomena, analysis of related genes has been carried out in several research groups including our research group. MdACS3 (Rosenfield CL, Kiss E), one of the 1-aminocyclopropane-1-carboxylic acid synthase (ACS) genes involved in ethylene biosynthesis known to be specifically expressed in fruits , Hrazdina G (1996) MdACS2 (Accession No. U73815) and MdACS3 (Accession No. U73816): Two new 1-aminocyclopropane-1-carboxylate synthases in ripening apple fruit. Plant Physiol 112: 1735) However, if we find out that the fruit is expressed shortly before full-fledged ripening and use this phenomenon as an indicator of the functional expression timing of the promoter region of MdACS3a, it is simple and accurate. Harvest time It found that can be constant.

That is, the determination method of the harvest time of the apple of the invention, as claimed in claim 1 wherein, the MdACS3a an ACS gene apple, a promoter comprising at least part of the nucleotide sequence as shown in SEQ ID NO: 1 It is characterized by performing the activation timing as an index .

  ADVANTAGE OF THE INVENTION According to this invention, the method of determining the harvest time of an appropriate apple fruit simply and accurately is provided.

  The method for determining the harvest time of apple fruit according to the present invention is characterized in that it is performed using the function expression timing of the promoter region of MdACS3a, which is an ACS gene of apple, as an index. The base sequence of the promoter region of MdACS3a comprises at least part of the base sequence represented by SEQ ID NO: 1. The base sequence of the cDNA of MdACS3a is represented by SEQ ID NO: 2, which corresponds to the base sequence of the cDNA of MdACS3 disclosed in the above-mentioned document, but the above-mentioned document discloses the base sequence of the promoter region. Absent. Fig. 1 shows the base sequence in the upstream region of the translation codon including at least part of the promoter region of MdACS3a (the region without the underline is the region having the base sequence represented by SEQ ID NO: 1, the underline is the transcription region, and bold Is the starting point of translation). According to the study by the present inventors, MdACS3 has three types of subfamily genes, MdACS3a, MdACS3b, and MdACS3c, and MdACS3a and MdACS3c are proteins consisting of 446 amino acids disclosed in the above-mentioned literature, MdACS3b encodes a protein consisting of 445 amino acids, one less than that, but only MdACS3a is expressed throughout the fruit ripening process based on the expression analysis performed using CAPS in the third exon region of each gene. MdACS3b and MdACS3c are known not to be expressed. This may be because the MdACS3b and MdACS3c promoter regions contain a 333 bp insertion sequence that does not exist in the MdACS3a promoter region, and the expression of MdACS3b and MdACS3c is suppressed by the presence of this insertion sequence. It is guessed. Therefore, in the present invention, only the promoter function of MdACS3a can be used.

  In the present invention, the functional expression timing of the promoter region of MdACS3a, which is determined to be the harvest time of apple fruits, is known based on the expression of MdACS3a, which is the original function of the promoter of MdACS3a, by the method of the example described later. However, for example, the grape myb gene VlmybA1-2 (Science 14 May 2004; 304: 982) is used as a reporter gene, and this is expressed by a known method by using a promoter region of MdACS3a It can be prepared by designing an appropriate primer from the information of the base sequence represented by No. 1 and amplifying it by PCR using an apple-derived genomic DNA library as a template) and a reporter Appropriate terminator was added downstream of the gene, and Agrobacterium (EHA105 strain) binary vector (pBI 121) are introduced into apple cultivars by a method known per se and transformed (see, for example, Matsuda et al. Plant Cell Reports 24: 45-51 2005. if necessary). At least part of the fruit due to the accumulation of anthocyanins based on the expression of VlmybA1-2 in the fruit due to the expression of the promoter function just before the fruit fully begins ripeness, that is, immediately before the harvest period It is also possible to know based on the phenomenon of the deposition of (see Fig. 2). This method is particularly effective for yellow skin color varieties (for example, “Wang Lin” and “Golden Delicious”) in which red deposition is easy to confirm. It may be difficult to judge for red skin color varieties (eg “Fuji” or “Tsugaru”), but the red skin color varieties are yellow in the lower part of the fruit. Judgment is possible from deposition. When this method is applied to a red skin color variety, since the whole fruit is originally red, there is an advantage that there is no problem in appearance commercial value.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is limited to the following description and is not interpreted at all.

Reference examples: Preparation of genomic DNA library derived from apples and selection of genomic clones, sequence and structural analysis of MdACS3a (a) Using 3g of apple young leaves (variety: Golden Delicious) by the method of Varadarajan et al. (1991) Total DNA was extracted. 50 μg of the obtained DNA was partially decomposed with Sau3AI 0.8 unit, and size fractionated by 0.5% agarose gel electrophoresis. A region containing a 19-23 kb DNA fragment was excised, and the DNA fragment was extracted from the gel according to the GENECLEAN II protocol. These DNA fragments were incorporated into a λFIX II partial fill-in vector in the presence of Klenow enzyme. Ligation to the vector was performed using TAKARA DNA Ligation Kit Ver.2. The reaction was performed overnight at 16 ° C. Packaging was performed according to the protocol of Gigapack II Gold Packaging Extract (Stratagene).

(B) 1086-1573 bp of cDNA (Accession No. U73816) of MdACS3 was amplified by PCR and labeled with ³² P. Using this as a probe, the above genomic library 1 × 10 ⁵ plaques were screened under high stringency conditions (0.1 × SSC, 0.1% SDS, 68 ° C.), and three positive clones were taken out and purified. Subsequently, the phage DNA was purified and its genomic clone was excised. And the restriction enzyme map of each clone was produced. These clones were subcloned into pBluescript, and plasmids were extracted from E. coli using the alkaline SDS method and subjected to a sequencing reaction. For the analysis, a DNA sequencer “Model 40 Series Based Image IR software” of LI-COR was used. In addition, a base sequence that could not be analyzed once was subcloned again and analyzed using a sequence analysis service (MACROGEN, Korea) and ABI PRISM 310 Genetic Analyzer (ABI). By the above operation, the base sequence including the promoter region of MdACS3a was clarified (SEQ ID NO: 1 and SEQ ID NO: 2).

Example 1: Analysis of changes in expression of various genes from immediately after flowering to harvesting by RNA gel blotting (relationship with lateral diameter of fruit)
(experimental method)
(1) Experimental materials Five fruits of apple fruits (variety: Golden Delicious) were sampled every week (from May 6 to November 4) at Hirosaki University attached Fujisaki Farm. In the expression analysis of these samples, the whole florets were used until June 3rd, and the pulp part was used thereafter.

(2) Targeted genes
In addition to MdACS3a, MdACS1 (Oraguzie NC, Iwanami H, Soejima J, Harada T, Hall A: Inheritance of Md-ACS1 gene and its relationship to fruit softening in apple (Malus X domestica Borkh.). Theor Appl Genet 108: 1526-1533 (2004)), MdACO1 (Ross GS, Knighton ML, Lay-Yee M (1992) An ethylene-related cDNA from ripening apple. Plant Mol Biol 19: 231-238), MdPG1 (Atkinson RG (1994) A cDNA clone for endopolygalacturonase from apple. Plant Physiol 105: 1437-1438), MdERF2 (Research on Breeding, Vol. 6, Annex 2, page 182, 2004) was the target gene.
(3) RNA extraction
Total RNA was extracted from apple flesh tissue by improving the method of Dong et al. (1991). That is, 5 g of pulp was powdered using liquid nitrogen, and 1/10 amount of polyvinyl pyrrolidone of the sample was added and mixed. Add 20ml Lysis buffer (4.0M guanidine thiocyanate, 10mM EDTA, 300mM Tris-HCl, pH7.5, 1% 2-mercaptoethanol, 0.5% sodium lauroyl sarcosine) Left for 2 hours. This was centrifuged at 4 ° C. and 8000 rpm for 15 minutes, and the supernatant was collected by filtration through Miracloth. The pellet obtained by equilibrium density gradient centrifugation with CsCl was dissolved in sterilized water. After phenol / chloroform treatment, the ethanol-precipitated RNA fraction was dissolved in sterilized water.

(4) RNA gel blot analysis
5 μg of total RNA was dissolved in buffer (1 × MOPS, 50% formamide, 2.2 M formaldehyde, 10 mM EDTA), denatured at 65 ° C. for 15 minutes, and then rapidly cooled on ice. This was electrophoresed on a 1.2% agarose gel (1 × MOPS, 0.66M formaldehyde) at 40 V for 3 hours. The gel was washed twice with 10 × SSC for 20 minutes and then transferred onto a nylon membrane (Magna Nylon Transfer Membrane, MSI) with 20 × SSC. After fixation with a UV crosslinker (UV Stratalinker 2400, STRATAGENE), prehybridization is performed in a buffer containing 50% formamide, 5 × Denhardt's solution, 0.5% SDS, 5 × SSPE, 20 μg / ml salmon sperm DNA. ³² Probe DNA labeled with P was added, and hybridization was performed at 42 ° C. overnight. Each probe was prepared by purifying a PCR product using each cDNA clone as a template with a PCR purification Kit (QIAGEN). The probe region of each gene and the primers used are as follows. MdACS3a (Accession No. U73816 (MdACS3), 1086-1573bp, 5'-GACAAATAGAAAGGACTGAGACG-3 '(SEQ ID NO: 3) and 5'-CCATCGATTATACAAACTGATTGTG-3' (SEQ ID NO: 4)), MdACS1 (Accession No. U89156, 4056- 4153bp, 5'-GATGAAGGTACCTGTCTGA-3 '(SEQ ID NO: 5) and 5'-TACACTAATCACATTGTAG-3' (SEQ ID NO: 6)), MdACO1 (Accession No. AF030859, 3558-3780bp, 5'-TGAAATCCAAGCCAAGGAG-3 '(SEQ ID NO: 7) and 5'-TTCAACTACCAAACAGAGTGG-3 '(SEQ ID NO: 8)), MdPG1 (Accession No. L27743, 433-1650bp, 5'-AGGTCATGGAATTGATCAGGCC-3' (SEQ ID NO: 9) and 5'-ATGCCATGCCCATAATTATGACCC-3 '(sequence) Number 10)). For MdERF2, see Tournier et al. (2003) degenerate primer (5'-CCRTGGGGRAAATKKGCGGCK-3 '(SEQ ID NO: 11) and 5'-CATAAGCVAVAKBGCRGCTTCYTC-3' (SEQ ID NO: 12)), ripe golden delicious fruits harvested at the appropriate time for 6 days RT-PCR was performed using Gene Amp RNA PCR Kit (Perkin-Elmer) using total RNA extracted from the processed pulp as a template, and the resulting cDNA was sequenced to confirm that it was MdERF2. Furthermore, 3 'untranslated region was cloned by 3'RACE method, and this region was amplified with primers (5'-TATGCTGGCAATTGGCGAGC-3' (SEQ ID NO: 13) and 5'-ATGACCAATCCCGCACTCAC-3 '(SEQ ID NO: 14)). Used as a probe.

(Experimental result)
Fig. 3 shows the relationship between changes in the lateral diameter of fruits (average of 5 fruits) and changes in gene expression. As is clear from FIG. 3, it was found that MdACS3a was expressed from around October 7 immediately before ripeness preceding other genes.

Example 2: Analysis of changes in the expression of various genes from just before harvesting of apple fruits by RNA gel blotting (relationship with ethylene production)
(experimental method)
Apple fruits (variety: Golden Delicious) were sampled every 5 days from October 1 to November 4 at Fujisaki Farm, Hirosaki University, and the amount of ethylene produced was measured within 1 hour after harvest. The amount of ethylene produced was measured by sealing the sampled fruit in a 1.3m ³ container and leaving it in a room at 24 ° C for 1 hour, then collecting 1ml of the gas in the container with a syringe and measuring 3mm in diameter and 2m in length. It was performed using FID-gas chromatography (GC-8A, Shimadzu, Japan) on an alumina column. After measuring the amount of ethylene produced, the pulp was immediately sliced and frozen in liquid nitrogen. For the purpose of reducing variation among fruit individuals, 15 samples for 3 days were collected and used as one sample represented by an intermediate day. That is, the result of October 3 was expressed by the average value of the measured amount of ethylene on October 1, 3 and 5. In addition, gel blotting was performed in the same manner as in Example 1 using RNA extracted from a material obtained by uniformly mixing these cryopreserved samples, and gene expression fluctuations were analyzed.

(Experimental result)
Fig. 4 shows the relationship between fluctuations in ethylene production and gene expression. As is clear from FIG. 4, MdACS3a was found to precede other genes just before the start of full-scale ethylene synthesis (October 3) accompanying fruit ripening.

  From the results of Example 1 and Example 2, it was revealed that MdACS3a is expressed not just after the apple fruit is fully ripe, but just before that. In addition to the “Golden Delicious” varieties used in the above example, this phenomenon also occurs in 13 varieties such as “Fuji”, “Himekami”, “Kitarou”, and “Kotarou”. Confirmed with. Therefore, for example, the function of the promoter region of MdACS3a, which can be known by cutting out a few pericarps of unripe fruit and examining the presence or absence of MdACS3a expression, or by examining the presence or absence of expression using a reporter gene Using the expression timing as an index, it was found that the harvest time of apple fruits can be determined easily, accurately and appropriately.

  INDUSTRIAL APPLICABILITY The present invention has industrial applicability in that it can provide a method for easily and accurately determining an appropriate harvest time for apple fruits.

This is a nucleotide sequence upstream of the translation codon including at least part of the promoter region of MdACS3a. It is a conceptual diagram of the determination method of the harvest time of the apple fruit performed using a reporter gene. It is a figure which shows the relationship of the fluctuation | variation (average value of 5 fruits) of the lateral diameter of the fruit in an Example, and the expression fluctuation | variation of a gene. It is a figure which shows the relationship between the fluctuation | variation of ethylene production amount and a gene expression fluctuation | variation similarly.

Claims (1)

Using MdACS3a, which is a 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene of apple , as an index, the activation timing of a promoter comprising at least a part of the base sequence represented by SEQ ID NO: 1 method for determining the harvest time of the apple, which comprises carrying out.