CN110387430B - Method for predicting apple fruit hardness based on variety of 225 th amino acid residue of apple transcription factor ERF4 - Google Patents
Method for predicting apple fruit hardness based on variety of 225 th amino acid residue of apple transcription factor ERF4 Download PDFInfo
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Abstract
The invention discloses a method for predicting apple fruit hardness based on the 225 th amino acid residue type from the N terminal of an apple transcription factor ERF 4. The hardness of the fruit of the apple to be tested, of which the variety of the 225 th amino acid residue from the N terminal of the apple transcription factor ERF4 is only proline residue, is higher than that of the apple to be tested, of which the variety of the 225 th amino acid residue from the N terminal of the apple transcription factor ERF4 is proline residue and alanine residue. Experiments prove that the variety of 225 th amino acid residues of the apple transcription factor ERF4 from the N terminal can be used as a detection object to predict the hardness of the apple fruit to be detected. The invention has great application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for predicting apple fruit hardness based on the type of 225 th amino acid residue from the N terminal of an apple transcription factor ERF 4.
Background
Besides the color change during the fruit ripening process, the texture change is also an important characteristic of fruit ripening. The texture of the fruits is a comprehensive concept, comprises the characteristics of hardness, looseness, brittleness, toughness, greasiness and the like of the fruits, and is an important index for evaluating the quality of the fruits in the processes of harvesting, storing and transporting. And the fruits with reduced hardness are easily subjected to physical and non-physical damage, such as mechanical damage, germ infection and the like.
Fruit firmness is closely related to the degradation of hemicellulose and pectin in the cell wall (Brummell and Harpster, 2001; Rose et al, 2004; Ng et al, 2013), and genes involved in the fruit softening process can be divided into two categories: one type is a structural gene, the protein encoded by which can be directly involved in cell wall degradation, such as polygalacturonase (PG; Atkinson et al, 2002; Wakasa et al, 2006; Quesada et al, 2009), pectin methylesterase (PME; Harriman et al, 1991; Carpita and Gibeaut, 1993); another class is regulatory genes, which encode proteins that regulate the level of transcription of structural genes.
Disclosure of Invention
The invention aims to solve the technical problem of predicting the hardness of the apple to be detected. The higher the hardness of the apple fruit, the easier it is to store and transport.
In order to solve the technical problems, the invention provides a method for predicting the hardness of an apple fruit to be detected.
The method for predicting the hardness of the apple fruit to be detected provided by the invention can be specifically a method I, and can comprise the following steps: detecting the 225 th amino acid residue type of the apple transcription factor ERF4 of the apple to be detected from the N terminal; the hardness of the fruit of the apple to be tested is higher than that of the apple to be tested, wherein the variety of the amino acid residue at the 225 th position from the N terminal of the apple transcription factor ERF4 is only proline residue.
The method for predicting the hardness of the apple fruit to be detected, which is provided by the invention, can be specifically a method II, and can comprise the following steps: detecting the nucleotide sequence of 225 th codon in the specific transcript of the total RNA of the apple to be detected; the specific transcript can be RNA obtained by transcription of a coding gene of an apple transcription factor ERF4, and the 1 st codon of the transcript is an initiation codon; the hardness of the fruit of the apple to be tested, which is obtained by only encoding proline in the nucleotide sequence of 225 th codon in the specific transcript, is higher than that of the apple to be tested, which is obtained by encoding proline and alanine in the nucleotide sequence of 225 th codon in the specific transcript.
The method for predicting the hardness of the apple fruit to be detected, which is provided by the invention, can be specifically a third method and comprises the following steps: detecting 673 rd nucleotide species of the coding gene of the apple transcription factor ERF4 in the total DNA of the apples to be detected from the 5' end; the hardness of the fruit of the apple to be tested with the 673 rd nucleotide species from the 5 'end of the coding gene of the apple transcription factor ERF4 being only C is higher than that of the apple to be tested with the 673 rd nucleotide species from the 5' end of the coding gene of the apple transcription factor ERF4 being C and G.
The method for predicting the hardness of the apple fruit to be detected provided by the invention can be specifically a fourth method, and can comprise the following steps: extracting total RNA of the apples to be detected and carrying out reverse transcription to obtain cDNA, then carrying out PCR amplification by adopting a specific primer pair, and detecting PCR amplification products; the hardness of the fruit of the apple to be detected, which has the DNA molecule shown in the sequence 7 of the sequence table and does not have the DNA molecule shown in the sequence 8 of the sequence table in the PCR amplification product, is higher than that of the apple to be detected, which has the DNA molecule shown in the sequence 7 of the sequence table and has the DNA molecule shown in the sequence 8 of the sequence table in the PCR amplification product.
In the fourth method, the specific primer pair may be composed of two primers for amplifying a specific DNA fragment. The specific DNA fragment has a target sequence of a primer pair consisting of a primer F1 and a primer R1 in the apple genome.
The primer F1 can be h1) or h2) as follows:
h1) a single-stranded DNA molecule shown in sequence 5 of the sequence table;
h2) and (b) a DNA molecule which is obtained by replacing and/or deleting and/or adding one or more nucleotides in the sequence 5 and has the same function as the sequence 5.
The primer R1 can be i3) or i4) as follows:
i3) a single-stranded DNA molecule shown in sequence 6 of the sequence table;
i4) and (b) a DNA molecule which is obtained by replacing and/or deleting and/or adding one or more nucleotides in the sequence 6 and has the same function as the sequence 6.
In the fourth method, the specific primer pair may be composed of the primer F1 and the primer R1.
The method for predicting the hardness of the apple fruit to be detected, which is provided by the invention, can be specifically the fifth method and comprises the following steps: detecting whether the total DNA of the apples to be detected has a DNA molecule shown in a sequence 7 of a sequence table and a DNA molecule shown in a sequence 8 of the sequence table; the hardness of the fruit of the apple to be detected, which is provided with the DNA molecule shown in the sequence 7 of the sequence table and is not provided with the DNA molecule shown in the sequence 8 of the sequence table in the total DNA of the apple to be detected, is higher than that of the apple to be detected, which is provided with the DNA molecule shown in the sequence 7 of the sequence table and is provided with the DNA molecule shown in the sequence 8 of the sequence table in the total DNA of the apple to be detected.
In any of the above methods, the elevation may be statistically elevated.
The invention also protects the application of the substance A, the substance B or the substance C in predicting the hardness of the apple fruit to be detected.
In the above application, the substance A may be a substance for detecting the type of 225 th amino acid residue of the apple transcription factor ERF4 from the N-terminal.
In the above application, the substance B may be a substance for detecting the nucleotide sequence of the 225 th codon in a specific transcript; the specific transcript can be RNA obtained by transcription of a coding gene of an apple transcription factor ERF4, and the 1 st codon of the RNA is an initiation codon.
In the above application, the substance C may be a substance for detecting the 673 rd nucleotide species from the 5' end of the coding gene of the apple transcription factor ERF 4.
In the above application, the substance C may be specifically the specific primer pair.
The invention also protects the application of the complete set of product A, the complete set of product B or the complete set of product C in predicting the hardness of the apple fruit to be detected.
In the application, the kit A can be the substance A and a carrier recorded with the method A; the method A can be as follows: the hardness of the fruit of the apple to be tested is higher than that of the apple to be tested, wherein the variety of the amino acid residue at the 225 th position from the N terminal of the apple transcription factor ERF4 is only proline residue.
In the application, the kit B can be the substance B and a carrier for recording the method B; the method B can be as follows: the hardness of the fruit of the apple to be tested, which is obtained by only encoding proline in the nucleotide sequence of 225 th codon in the specific transcript, is higher than that of the apple to be tested, which is obtained by encoding proline and alanine in the nucleotide sequence of 225 th codon in the specific transcript.
In the application, the kit C can be the substance C and a carrier recorded with the method C; the method can be as follows: the hardness of the fruit of the apple to be tested with the 673 rd nucleotide species from the 5 'end of the coding gene of the apple transcription factor ERF4 being only C is higher than that of the apple to be tested with the 673 rd nucleotide species from the 5' end of the coding gene of the apple transcription factor ERF4 being C and G.
The following A1) or A2) or A3) also belong to the scope of protection of the invention:
A1) the application of the 225 th amino acid residue variety of the apple transcription factor ERF4 from the N terminal as a detection object in predicting the hardness of an apple fruit to be detected;
A2) the application of the nucleotide sequence of the 225 th codon in the specific transcript as a detection object in predicting the hardness of the apple fruit to be detected; the specific transcript is RNA obtained by transcription of an encoding gene of an apple transcription factor ERF4, and the 1 st codon of the transcript is an initiation codon;
A3) application of 673 rd nucleotide species from 5' terminal of coding gene of apple transcription factor ERF4 as detection object in predicting hardness of apple fruit to be detected.
The invention also protects any one specific primer pair. The specific primer pair is used for predicting the hardness of the apple fruit to be detected.
As above, the fruit firmness may be peel firmness and/or pulp firmness.
As above, the apple transcription factor ERF4(MDP0000324718) is located on chromosome 3 of the apple genome at position chr 3: 27617815-: 15777-16518. The apple transcription factor ERF4 contains two conserved domains, namely an AP2 conserved domain and an EAR suppression domain.
As mentioned above, the apple transcription factor ERF4 can be a protein, and can be composed of segment I, segment II and segment III in sequence from N-terminal to C-terminal. The segment II may be specifically an amino acid residue. The one amino acid residue is a proline residue or an alanine residue. The segment iii may consist of four amino acid residues. The four amino acid residues are PSEV in sequence from N end to C end.
The segment I may be a1) or a2) or a3) as follows:
a1) the amino acid sequence is a polypeptide shown in 1 st to 224 th positions from the N tail end of a sequence 3 in a sequence table;
a2) the amino acid sequence is a polypeptide shown in 1 st to 224 th positions from the N terminal of a sequence 4 in a sequence table;
a3) the polypeptide with the same function is obtained by replacing one or more amino acid residues of the polypeptide shown in a1) or a 2).
a4) Protein which has 80% or more than 80% of identity with the polypeptide shown in a1) or a2) or a3), is derived from apple and has the same function.
The term "identity" as used in a4) above, refers to sequence similarity to the native amino acid sequence. "identity" includes an amino acid sequence having 80%, or 85% or more, or 90% or more, or 95% or more identity to the amino acid sequence shown at positions 1 to 224 from the N-terminus of sequence 3 or sequence 4 in the sequence listing of the present invention.
Any one of the above apple transcription factors ERF4 may be d1) or d2) or d3) or d4) or d 5):
d1) the amino acid sequence is protein shown as a sequence 3 in a sequence table;
d2) the amino acid sequence is protein shown as a sequence 4 in a sequence table;
d3) protein with the same function obtained by replacing and/or deleting and/or adding one or more amino acid residues of the protein shown in d1) or d 2);
d4) protein which has 80% or more than 80% of identity with the protein shown by d1) or d2) or d3), is derived from apple and has the function of the apple transcription factor ERF 4.
The term "identity" as used in d4) above refers to sequence similarity to the native amino acid sequence. "identity" includes amino acid sequences that are 80%, or 85% or more, or 90% or more, or 95% or more identical to the amino acid sequence encoding the apple transcription factor ERF 4.
The coding gene of the apple transcription factor ERF4 can be a DNA molecule shown in the following x1) or x2) or x3) or x4) or x5) or x6) or x7) or x 8):
x1) the coding region is shown as a DNA molecule in a sequence 1 in a sequence table;
x2) the nucleotide sequence is a DNA molecule shown in a sequence 1 in a sequence table;
x3) the coding region is shown as a DNA molecule in a sequence 2 in a sequence table;
x4) is a DNA molecule shown in a sequence 2 in the sequence table;
x5) has 75% or more than 75% of identity with the nucleotide sequence defined by x1) or x2) or x3) or x4) and encodes the apple transcription factor ERF 4;
x6) hybridizes with a nucleotide sequence defined by x1) or x2) or x3) or x4) under strict conditions and encodes the apple transcription factor ERF 4.
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
The term "identity" as used in x5) above refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75%, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity to a nucleotide sequence of the protein consisting of the amino acid sequence represented by sequence 3 in the sequence listing of the present invention, or a nucleotide sequence of the protein consisting of the amino acid sequence represented by sequence 4 in the sequence listing of the present invention.
The sequence 1 in the sequence table consists of 693 nucleotides, and the nucleotide of the sequence 1 in the sequence table encodes an amino acid sequence shown as a sequence 3 in the sequence table. The sequence 2 in the sequence table consists of 693 nucleotides, and the nucleotide of the sequence 2 in the sequence table encodes an amino acid sequence shown as a sequence 4 in the sequence table.
Above, identity can be evaluated with the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
In one embodiment of the invention, the apple transcription factor ERF4 of Fuji can be an apple transcription factor ERF4-TC shown in a sequence 3 in a sequence table and an apple transcription factor ERF4-AG shown in a sequence 4 in the sequence table. The gene for coding the apple transcription factor ERF4-TC is shown as a sequence 1 in a sequence table. The gene for coding the apple transcription factor ERF4-AG is shown as a sequence 2 in the sequence table.
Experiments prove that the variety of 225 th amino acid residues of the apple transcription factor ERF4 from the N terminal can be used as a detection object to predict the hardness of the apple fruit to be detected. The invention has great application value.
Drawings
FIG. 1 is a genetic linkage map (LOD >2.8) of the progeny population of the zisengzhu and red Fuji cross. The red mark is the QTLs position of the apple fruit hardness.
FIG. 2 shows the alignment of the amino acid sequences of apple transcription factor ERF4-TC and apple transcription factor ERF 4-AG.
FIG. 3 shows the nucleotide sequence alignment of ERF4-TC gene and ERF4-AG gene.
FIG. 4 is an analysis of the peel color phenotype, peel firmness and pulp firmness of red Fuji and Ziseneming.
FIG. 5 is a chart of the pericarp coloration phenotype of hybrid progeny lines of Fuji and Zisenzhu.
FIG. 6 is a pericarp hardness analysis of hybrid progeny lines of Fuji and Zisenzhu.
FIG. 7 is a flesh firmness analysis of hybrid progeny lines of red Fuji and Zisening pearl.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative experiments in the following examples, three replicates were set up and the results averaged.
The cultivation of apples ' Red Fuji ' (Malus domestica Borkh cv. ' Red Fuji ') and Malus asiatica Pearl ' (Malus asiatica Nakai. cv. ' Zisai Pearl ') are described in the following documents: jingxiaokang, Lusong, Liu Xin Ying, Wang Ying, Wu Ting, Zhang Xin Zhong, Han Zheng Hai, Li Tianhong, Malus asiatica x M.domestica interspecies hybrid apple alternaria QTL location of susceptibility, Chinese university of agriculture report 2014, 19 (6): 140-147; zhengfeixue, Zhangxinzhong, Queenyi, Han Zhenhai, apple seedling tree in different stages of ontogeny and development2O2Changes in content and related enzyme activities, fruit tree bulletins, 2013, 30 (5): 759 contains 764, publicly available from the university of agriculture. The cultivated apple 'Red Fuji' (Malus domestica Borkh cv. 'Red Fuji') is hereinafter abbreviated as Red Fuji. The crabapple 'Zisai Pearl' (Malus asiatica nakai. cv. 'Zisai Pearl') is hereinafter referred to simply as Zisai Pearl.
Both the pEasy-T1 vector and the HIFI Mix are products of Beijing Quanyujin Biotechnology, Inc.
Example 1 discovery of apple transcription factor ERF4 and cloning of Gene encoding apple transcription factor ERF4
Discovery of apple transcription factor ERF4
In 2014 and 2015, respectively, the fruit hardness of filial generations of Fuji and Ziseng pearl is subjected to genetic linkage analysis. The results of the analysis are shown in FIG. 1: the LOD scores all peaked at the position marked by the linkage group CTG1058426, No. 3, red fuji. Through a large number of experiments, the inventor of the invention found the apple transcription factor ERF4(MDP0000324718) at the position, which is located on chromosome 3 of the apple genome, and the position is chr 3: 27617815-: 15777-16518. The apple transcription factor ERF4 contains two conserved domains, namely an AP2 conserved domain and an EAR suppression domain.
Cloning of gene (namely ERF4 gene) coding apple transcription factor ERF4
For the convenience of describing the structure of the ERF4 gene, the position of A in the ATG of the initiation codon of the ERF4 gene is designated as "+ 1", "-" represents the 5 'direction of the ATG of the initiation codon of the ERF4 gene, and "+" represents the 3' direction of the ATG of the initiation codon of the ERF4 gene.
1. Extracting total RNA of red Fuji pericarp, and performing reverse transcription to obtain first strand cDNA.
2. And (3) carrying out PCR amplification by using the cDNA obtained in the step (1) as a template and adopting a primer pair consisting of a primer F1 and a primer R1 to obtain a PCR amplification product.
Primer F1: 5'-CTCGCATCTTTCTCCAAGCAGC-3' (SEQ ID NO: 5 in the sequence Listing).
Primer R1: 5'-CCGGAAAGAAAGAAAAGCGGTGATC-3' (SEQ ID NO: 6 in the sequence Listing).
Reaction system (50 μ L): consists of 25. mu.L of HIFI Mix, 2. mu.L of primer F1 aqueous solution (10. mu.M), 2. mu.L of primer R1 aqueous solution (10. mu.M), 2. mu.L of cDNA (containing 10-50ng of cDNA), and 19. mu.LH2And (C) O.
Reaction procedure: 5min at 95 ℃; 30sec at 94 ℃, 30sec at 58 ℃, 1min at 72 ℃ and 34 cycles; 7min at 72 ℃; storing at 4 ℃.
3. And (3) connecting the PCR amplification product obtained in the step (2) with a pEasy-T1 vector to obtain a recombinant plasmid A.
Sequencing the recombinant plasmid A obtained in the step 3 by using the Huada gene. Sequencing results show that the recombinant plasmid A has two types, one type contains a DNA molecule shown as a sequence 1 in a sequence table and is named as recombinant plasmid A-1; the other DNA molecule containing the sequence 2 in the sequence table is named as recombinant plasmid A-2.
The nucleotide sequence shown in the sequence 1 in the sequence table encodes an apple transcription factor ERF4-TC shown in the sequence 3 in the sequence table. The gene (namely the nucleotide sequence shown as the sequence 1 in the sequence table) for coding the apple transcription factor ERF4-TC is named as ERF4-TC gene.
The nucleotide sequence shown in the sequence 2 in the sequence table encodes an apple transcription factor ERF4-AG shown in the sequence 4 in the sequence table. The gene (namely the nucleotide sequence shown as the sequence 2 in the sequence table) for coding the apple transcription factor ERF4-AG is named as ERF4-AG gene.
The amino acid sequences shown in the sequence 3 and the sequence 4 are aligned and analyzed by using DNAman and NCBI, and the experimental result is shown in figure 2. The nucleotide sequences shown in the sequence 1 and the sequence 2 are compared and analyzed by using DNAman and NCBI, and the experimental result is shown in figure 3. The result shows that the apple transcription factor ERF4 of Fuji has natural mutation in the conserved domain (+171bp, +174bp) of AP2 and the EAR inhibition domain (+672bp and +673bp), but the mutation in the nucleotide base of +673bp only causes the mutation in the amino acid residue; the 225 th position of the apple transcription factor ERF4-TC from the N terminal is a proline residue, and the 225 th position of the apple transcription factor ERF4-AG from the N terminal is an alanine residue. From this, it can be seen that there are two kinds of amino acid residues at position 225 from the N-terminus of the apple transcription factor ERF4 of red fuji, one is a proline residue, and the other is an alanine residue.
Replacing red Fuji with zisaiming pearl according to the steps 1 to 3, and obtaining the recombinant plasmid B without changing other steps. Sequencing the recombinant plasmid B by the Huada gene. And judging the type of the 225 th amino acid residue of the apple transcription factor ERF4 from the N end according to the sequencing result. The result shows that only one kind of the 225 th amino acid residue of the apple transcription factor ERF4 of the zisaimingzhu is proline residue from the N terminal.
Example 2 analysis of peel color phenotype, peel firmness and pulp firmness of red Fuji and Ziseneming
The apple to be tested is red Fuji or Zisaiming pearl.
1. Planting the tree seedlings (3 pieces) of the apples to be tested in the Zhuang eight-mouth test base in Chang plain area of Beijing city.
2. After the step 1 is completed, in the sixth year, After the apples to be tested are planted and flowers are contained (DAFB), at the 50 th, 80 th, 110 th, 140 th or 170 th time, 12 fruits are collected from each sapling.
3. And (4) taking the fruits collected in the step (2), and recording the color of the peel (photographing).
4. And (3) taking the fruits collected in the step (2), respectively measuring the peel hardness and the pulp hardness by adopting a TA.XT-21 texture analyzer (Stable Micro Systems Ltd., Godalming, Surrey, UK), and then taking an average value.
The results of peel color are shown in FIG. 4A. The peel color results were as follows: the fruit grows at 50 th after the flowers are contained in the Fuji pot, the color of the peel keeps green at 80 th to 140 th after the flowers are contained, and the peel begins to be colored red at 170 th after the flowers are contained; the 140 th pericarp of zisaiming pearl after flourishing the flower begins to be colored red, and the 170 th pericarp of zisaiming pearl after flourishing the flower is all red.
The measurement result of peel hardness is shown in B in FIG. 4. The pulp firmness test results are shown in fig. 4C. The results show that as the fruit ripens, the hardness of the peel and the hardness of the pulp both gradually decrease, specifically: the hardness of the peel and the pulp of the Fuji is highest when the Fuji grows young fruits (50 th after full bloom), the hardness of the peel and the pulp of the Fuji grow lower rapidly from 80 th to 110 th after full bloom, and the hardness of the peel and the pulp of the Fuji grow lower slowly from 110 th to 170 th after full bloom; the change trend of the pericarp hardness and the pulp hardness of the zisaiminzhu is basically similar to that of the red Fuji, but the level of the pericarp hardness and the pulp hardness is higher as a whole.
Example 3 analysis of peel color phenotype, peel firmness and flesh firmness of hybrid progeny lines of Fuji and Zisenzhu
Analysis of pericarp color phenotype, pericarp hardness and pulp hardness of hybrid progeny strains of red Fuji and Ziseng pearl
1. Hybridizing the red Fushi (male parent) and the Zisaiming pearl (female parent) to obtain hybrid F1 generation seeds.
2. After completion of step 1, hybrid F1 seeds were sown to give a hybrid population (consisting of seedlings of hybrid F1 seeds). 30 hybrid lines (respectively named 02-152, 04-012, 04-153, 04-174, 06-105, 08-201, 09-119, 09-129, 11-132, 14-016, 15-112, 16-040, 16-113, 16-122, 17-133, 01-072, 01-105, 02-112, 04-034, 04-185, 06-174, 06-181, 07-130, 09-134, 09-177, 11-018, 11-139, 12-013, 15-004 and 17-050) with obvious difference in peel hardness and pulp hardness are used as the lines to be tested.
3. And (3) planting the seedlings of the 30 hybrid lines obtained in the step (2) in the Zhuang eight-mouth test base in Chang's plain area of Beijing city.
4. After completing step 3, 12 fruits per seedling were harvested at 50d, 80d, 110d, 140d or 170d After the hybrid plant flowers (DAFB) in the sixth year of planting, respectively.
5. And (4) respectively measuring the peel hardness and the pulp hardness of the fruits collected in the step (4) by adopting a TA.XT-21 texture analyzer (Stable Micro Systems Ltd., Godalming, Surrey, UK), and then averaging.
The 30 hybrid lines are sorted from high to low according to the hardness of the peel, the first 15 hybrid lines (with higher peel hardness) form a first group, and the last 15 hybrid lines (with lower peel hardness) form a second group. The first group comprises 02-152, 04-012, 04-153, 04-174, 06-105, 08-201, 09-119, 09-129, 11-132, 14-016, 15-112, 16-040, 16-113, 16-122 and 17-133. The second group comprises 01-072, 01-105, 02-112, 04-034, 04-185, 06-174, 06-181, 07-130, 09-134, 09-177, 11-018, 11-139, 12-013, 15-004 and 17-050.
6. And (4) taking the fruits collected in the step (4), and recording the color of the peel (photographing).
The results of the partial peel color are shown in FIG. 5(A for the first group and B for the second group). The 50 th hybrid line of the first group produces young fruits after flourishing, the peel color of the 80 th to 140 th hybrid lines keeps green all the time after flourishing, and the 170 th hybrid lines begin to be colored and turn red after flourishing; the 140 th peel of each hybrid line in the second group begins to be colored and red after full bloom, and the 170 th peel is all red after full bloom.
The results of peel hardness measurements are shown in FIG. 6(A for the first group and B for the second group). The results of the pulp firmness measurements are shown in FIG. 7(A for the first group and B for the second group). The result shows that the peel hardness and the pulp hardness of the 50 th to 110 th hybrid lines after full bloom have no obvious difference; at 110d-170d after the blooming of 30 hybrid lines, the peel hardness and pulp hardness of each hybrid line in the first group are obviously higher than those in the second group.
Secondly, detecting the type of 225 th amino acid residue of the apple transcription factor ERF4 from the N end of the hybrid progeny strain of red Fuji and Zisening pearl
And (3) the plant to be detected: 02-152 strain plants, 04-012 strain plants, 04-153 strain plants, 04-174 strain plants, 06-105 strain plants, 08-201 strain plants, 09-119 strain plants, 09-129 strain plants, 11-132 strain plants, 14-016 strain plants, 15-112 strain plants, 16-040 strain plants, 16-113 strain plants, 16-122 strain plants, 17-133 strain plants, 01-072 strain plants, 01-105 strain plants, 02-112 strain plants, 04-034 strain plants, 04-185 strain plants, 06-174 strain plants, 06-181 strain plants, 07-130 strain plants, 09-134 strain plants, 09-177 strain plants, 11-018 strain plants, Plants of line 11-139, plants of line 12-013, plants of line 15-004 or plants of line 17-050.
1. Extracting total RNA of the pericarp of the plant to be detected, and then carrying out reverse transcription to obtain first-strand cDNA.
2. The cDNA obtained in step 1 was used as a template, and PCR amplification was carried out using a primer pair consisting of the primer F1 and the primer R1 in step two 1 in example 1, to obtain a PCR amplification product.
The reaction system was the same as that of the reaction system of the step two 1 (2) in example 1.
The reaction sequence was the same as that of (2) in step two 1 of example 1.
3. And connecting the PCR amplification product with a pEasy-T1 vector to obtain a recombinant plasmid.
4. Sequencing the recombinant plasmid obtained in the step 3 by using the Huada gene. And judging the type of the 225 th amino acid residue of the apple transcription factor ERF4 from the N end according to the sequencing result.
The result shows that when the plant to be detected is a 02-152 strain plant, a 04-012 strain plant, a 04-153 strain plant, a 04-174 strain plant, a 06-105 strain plant, a 08-201 strain plant, a 09-119 strain plant, a 09-129 strain plant, a 11-132 strain plant, a 14-016 strain plant, a 15-112 strain plant, a 16-040 strain plant, a 16-113 strain plant, a 16-122 strain plant or a 17-133 strain plant, only one kind of amino acid residue at the 225 th position from the N end of the apple transcription factor ERF4 is selected as the proline residue; when the plant to be tested is a 01-072 strain plant, a 01-105 strain plant, a 02-112 strain plant, a 04-034 strain plant, a 04-185 strain plant, a 06-174 strain plant, a 06-181 strain plant, a 07-130 strain plant, a 09-134 strain plant, a 09-177 strain plant, a 11-018 strain plant, a 11-139 strain plant, a 12-013 strain plant, a 15-004 strain plant or a 17-050 strain plant, the types of the 225 th amino acid residues of the apple transcription factor ERF4 from the N end are two, one is a proline residue, and the other is an alanine residue.
The results show that according to the variety of the 225 th amino acid residue from the N end of the apple transcription factor ERF4 of the apple to be detected, the peel hardness and the pulp hardness of the apple to be detected can be predicted.
<110> university of agriculture in China
<120> method for predicting hardness of apple fruits based on variety of 225 th amino acid residue of apple transcription factor ERF4
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Claims (9)
1. A method for predicting the hardness of an apple fruit to be tested comprises the following steps: detecting the 225 th amino acid residue type of the apple transcription factor ERF4 of the apple to be detected from the N terminal;
the hardness of the fruit of the apple to be tested is higher than that of the apple to be tested, wherein the variety of the amino acid residue at the 225 th position from the N terminal of the apple transcription factor ERF4 is only proline residue;
the apple transcription factor ERF4 is composed of a segment I, a segment II and a segment III in sequence from the N end to the C end;
the segment I is polypeptide with an amino acid sequence shown in 1 st to 224 th positions from the N terminal of a sequence 3 in a sequence table;
the section II is proline residue or alanine residue;
the section III is sequentially provided with PSEV from the N end to the C end;
the apple to be detected is red Fuji, zisaiming pearl and/or filial generation of the red Fuji and the zisaiming pearl.
2. A method for predicting the hardness of an apple fruit to be tested comprises the following steps: detecting the nucleotide sequence of 225 th codon in the specific transcript of the total RNA of the apple to be detected; the specific transcript is RNA obtained by transcription of an encoding gene of an apple transcription factor ERF4, and the 1 st codon of the transcript is an initiation codon;
the hardness of the fruit of the apple to be tested, which is obtained by only encoding proline in the nucleotide sequence of the 225 th codon in the specific transcript, is higher than that of the apple to be tested, which is obtained by only encoding proline in the nucleotide sequence of the 225 th codon in the specific transcript, in the fruit to be tested;
the apple transcription factor ERF4 is composed of a segment I, a segment II and a segment III in sequence from the N end to the C end;
the segment I is polypeptide with an amino acid sequence shown in 1 st to 224 th positions from the N terminal of a sequence 3 in a sequence table;
the section II is proline residue or alanine residue;
the section III is sequentially provided with PSEV from the N end to the C end;
the apple to be detected is red Fuji, zisaiming pearl and/or filial generation of the red Fuji and the zisaiming pearl.
3. A method for predicting the hardness of an apple fruit to be tested comprises the following steps: detecting 673 rd nucleotide species of the coding gene of the apple transcription factor ERF4 in the total DNA of the apples to be detected from the 5' end;
the hardness of the fruit of the apple to be detected with the 673 rd nucleotide species from the 5 'end of the coding gene of the apple transcription factor ERF4 being only C is higher than that of the apple to be detected with the 673 rd nucleotide species from the 5' end of the coding gene of the apple transcription factor ERF4 being C and G;
the coding gene of the apple transcription factor ERF4 is x1) or x2) as follows:
x1) the nucleotide sequence is a DNA molecule shown in the sequence 1 in the sequence table;
x2) the nucleotide sequence of which is a DNA molecule shown in the sequence 2 in the sequence table;
the apple to be detected is red Fuji, zisaiming pearl and/or filial generation of the red Fuji and the zisaiming pearl.
4. A method for predicting the hardness of an apple fruit to be tested comprises the following steps: extracting total RNA of the apples to be detected and carrying out reverse transcription to obtain cDNA, then carrying out PCR amplification by adopting a specific primer pair, and detecting PCR amplification products;
the hardness of the fruit of the apple to be detected, which is provided with the DNA molecule shown in the sequence 1 of the sequence table and is not provided with the DNA molecule shown in the sequence 2 of the sequence table in the PCR amplification product, is higher than that of the apple to be detected, which is provided with the DNA molecule shown in the sequence 1 of the sequence table and is provided with the DNA molecule shown in the sequence 2 of the sequence table in the PCR amplification product;
the specific primer pair consists of two primers for amplifying specific DNA fragments; the specific DNA fragment has a target sequence of a primer pair consisting of a primer F1 and a primer R1 in the apple genome;
the primer F1 is a single-stranded DNA molecule shown in a sequence 5 in a sequence table;
the primer R1 is a single-stranded DNA molecule shown in a sequence 6 in a sequence table;
the apple to be detected is red Fuji, zisaiming pearl and/or filial generation of the red Fuji and the zisaiming pearl.
5. A method for predicting the hardness of an apple fruit to be tested comprises the following steps: detecting whether the total DNA of the apples to be detected has a DNA molecule shown in a sequence 1 of a sequence table and a DNA molecule shown in a sequence 2 of the sequence table;
the hardness of the fruit of the apple to be detected, which is provided with the DNA molecule shown in the sequence 1 of the sequence table and is not provided with the DNA molecule shown in the sequence 2 of the sequence table in the total DNA of the apple to be detected, is higher than that of the apple to be detected, which is provided with the DNA molecule shown in the sequence 1 of the sequence table and is provided with the DNA molecule shown in the sequence 2 of the sequence table in the total DNA of the apple to be detected;
the apple to be detected is red Fuji, zisaiming pearl and/or filial generation of the red Fuji and the zisaiming pearl.
6. The method of any of claims 1 to 5, wherein: the fruit hardness is the peel hardness and/or the pulp hardness.
7. The application of the substance A, the substance B or the substance C in predicting the hardness of the apple fruit to be detected;
the substance A is used for detecting the type of 225 th amino acid residue of the apple transcription factor ERF4 from the N terminal; the apple transcription factor ERF4 is composed of a segment I, a segment II and a segment III in sequence from the N end to the C end; the segment I is polypeptide with an amino acid sequence shown in 1 st to 224 th positions from the N terminal of a sequence 3 in a sequence table; the section II is proline residue or alanine residue; the section III is sequentially provided with PSEV from the N end to the C end; the hardness of the fruit of the apple to be tested is higher than that of the apple to be tested, wherein the variety of the amino acid residue at the 225 th position from the N terminal of the apple transcription factor ERF4 is only proline residue;
the substance B is a substance for detecting the nucleotide sequence of the 225 th codon in the specific transcript; the specific transcript is RNA obtained by transcription of an encoding gene of an apple transcription factor ERF4, and the 1 st codon of the transcript is an initiation codon; the apple transcription factor ERF4 is composed of a segment I, a segment II and a segment III in sequence from the N end to the C end; the segment I is polypeptide with an amino acid sequence shown in 1 st to 224 th positions from the N terminal of a sequence 3 in a sequence table; the section II is proline residue or alanine residue; the section III is sequentially provided with PSEV from the N end to the C end; the hardness of the fruit of the apple to be tested, which is obtained by only encoding proline in the nucleotide sequence of the 225 th codon in the specific transcript, is higher than that of the apple to be tested, which is obtained by only encoding proline in the nucleotide sequence of the 225 th codon in the specific transcript, in the fruit to be tested;
the substance C is used for detecting the 673 rd nucleotide species of the coding gene of the apple transcription factor ERF4 from the 5' end; the coding gene of the apple transcription factor ERF4 is x1) or x2) as follows: x1) the nucleotide sequence is a DNA molecule shown in the sequence 1 in the sequence table; x2) the nucleotide sequence of which is a DNA molecule shown in the sequence 2 in the sequence table; the hardness of the fruit of the apple to be detected with the 673 rd nucleotide species from the 5 'end of the coding gene of the apple transcription factor ERF4 being only C is higher than that of the apple to be detected with the 673 rd nucleotide species from the 5' end of the coding gene of the apple transcription factor ERF4 being C and G;
the apple to be detected is red Fuji, zisaiming pearl and/or filial generation of the red Fuji and the zisaiming pearl.
8, a1) or a2) or A3):
A1) the application of the 225 th amino acid residue variety of the apple transcription factor ERF4 from the N terminal as a detection object in predicting the hardness of an apple fruit to be detected; the apple transcription factor ERF4 is composed of a segment I, a segment II and a segment III in sequence from the N end to the C end; the segment I is polypeptide with an amino acid sequence shown in 1 st to 224 th positions from the N terminal of a sequence 3 in a sequence table; the section II is proline residue or alanine residue; the section III is sequentially provided with PSEV from the N end to the C end; the hardness of the fruit of the apple to be tested is higher than that of the apple to be tested, wherein the variety of the amino acid residue at the 225 th position from the N terminal of the apple transcription factor ERF4 is only proline residue; the apple to be detected is red Fuji, zisaiming pearl and/or filial generation of the red Fuji and the zisaiming pearl;
A2) the application of the nucleotide sequence of the 225 th codon in the specific transcript as a detection object in predicting the hardness of the apple fruit to be detected; the specific transcript is RNA obtained by transcription of an encoding gene of an apple transcription factor ERF4, and the 1 st codon of the transcript is an initiation codon; the apple transcription factor ERF4 is composed of a segment I, a segment II and a segment III in sequence from the N end to the C end; the segment I is polypeptide with an amino acid sequence shown in 1 st to 224 th positions from the N terminal of a sequence 3 in a sequence table; the section II is proline residue or alanine residue; the section III is sequentially provided with PSEV from the N end to the C end; the hardness of the fruit of the apple to be tested, which is obtained by only encoding proline in the nucleotide sequence of the 225 th codon in the specific transcript, is higher than that of the apple to be tested, which is obtained by only encoding proline in the nucleotide sequence of the 225 th codon in the specific transcript, in the fruit to be tested; the apple to be detected is red Fuji, zisaiming pearl and/or filial generation of the red Fuji and the zisaiming pearl;
A3) the application of the 673 rd nucleotide species from the 5' end of the coding gene of the apple transcription factor ERF4 as a detection object in predicting the hardness of the apple fruit to be detected; the coding gene of the apple transcription factor ERF4 is x1) or x2) as follows: x1) the nucleotide sequence is a DNA molecule shown in the sequence 1 in the sequence table; x2) the nucleotide sequence of which is a DNA molecule shown in the sequence 2 in the sequence table; the hardness of the fruit of the apple to be detected with the 673 rd nucleotide species from the 5 'end of the coding gene of the apple transcription factor ERF4 being only C is higher than that of the apple to be detected with the 673 rd nucleotide species from the 5' end of the coding gene of the apple transcription factor ERF4 being C and G; the apple to be detected is red Fuji, zisaiming pearl and/or filial generation of the red Fuji and the zisaiming pearl.
9. Use according to claim 7 or 8, characterized in that: the fruit hardness is the peel hardness and/or the pulp hardness.
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