CN117757816B - A functional gene PaPDS for regulating apricot fruit color and its application - Google Patents

Abstract

The present invention discloses a functional gene PaPDS for regulating the color of apricot fruit and its application. The sequence of the PaPDS gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the PaPDS gene is shown in SEQ ID NO.2. Transcriptomics analysis, overexpression experiments and gene silencing experiments confirm that the gene encoded by the PaPDS gene can regulate the content of β-carotene in apricot pulp, thereby affecting the color of apricot pulp. When the PaPDS gene is overexpressed, the color of the apricot pulp is darker. The present invention lays a foundation for the genetic improvement of the color of apricot pulp and the content of β-carotene in apricot pulp, and has potential value in the genetic engineering breeding of apricot trees.

Description

Functional gene PaPDS for regulating and controlling color of apricot fruits and application thereof

Technical Field

The invention relates to the technical field of genetic engineering. In particular to a functional gene PaPDS for regulating and controlling the color of apricot fruits and application thereof.

Background

The color is an important index for evaluating the maturity and quality of fruits. Previous studies have shown that chlorophyll, carotenoids, anthocyanins and betaines are the primary pigments affecting fruit color. Apricot (Prunus armeniaca) is one of the most important fruit crops in China, and is very popular due to its unique flavor, vivid color and rich nutrition. Different varieties of apricot fruits have different colors, mainly due to the different content of carotenoids, especially beta-carotene, in apricots. Carotenoids contribute to darkening of fruit color and improvement of nutritional value of apricots, most consumers prefer apricots with darker fruit color and higher nutritional value. However, during the production process, the apricot fruit color may be unstable, which may lead to a decrease in the profit of the fruit farmer.

Currently, the metabolic pathway of β -carotene in higher plants is well defined, which contains many essential enzymes, one of which is Phytoene Dehydrogenase (PDS). Many plant beta-carotene synthesis and regulation mechanisms have been explored, and these research results provide a good basis for the research of beta-carotene content in plants for which other beta-carotene synthesis and regulation mechanisms have not been explored. However, researches on the control of the synthesis of beta-carotene gene, which is the difference of the color of apricot fruits, are rarely reported at present, and the expression mode and the functional effect of the phytoene dehydrogenase gene involved in the synthesis of beta-carotene in apricot fruits are not clear. Therefore, the functional gene capable of regulating the color of the apricot fruits is ascertained, so that the application of the functional gene to the regulation of the color of the apricot fruits has great significance.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide the functional gene PaPDS for regulating and controlling the fruit color and the application thereof, so as to realize genetic improvement of the fruit color of the apricot tree.

In order to solve the technical problems, the invention provides the following technical scheme:

A functional gene PaPDS for regulating and controlling the color of apricot fruits has a sequence shown in SEQ ID NO.1, and an amino acid sequence of a protein coded by PaPDS genes is shown in SEQ ID NO. 2.

The application of the recombinant vector containing the coding region sequence of the functional gene PaPDS for regulating and controlling the color of the apricot fruits in regulating and controlling the color of the apricot fruits is that the coding region sequence of the PaPDS gene is the sequence shown as SEQ ID NO.1 or the sequence shown as SEQ ID NO. 3.

In the application, the original vector of the recombinant vector is pMD19-T, the PaPDS gene coding region sequence is positioned between BamHI and XbaI restriction enzyme sites of the pMD19-T vector, and the PaPDS gene coding region sequence is shown as SEQ ID NO. 1. After PaPDS genes are inserted into pMD19-T, when the PaPDS genes are required to be subjected to PCR cloning in the follow-up, the proper T vector can be used as a template for sequencing, and compared with the use of apricot cDNA as the template, the use of the proper T vector for sequencing as the template can reduce non-specific amplification during PCR and obtain purer amplification products.

In the application, the original vector of the recombinant vector is pBWA (V) HS-osgfp-ccdb-tnos, the sequence of the PaPDS gene coding region is inserted into the cleavage site of BsaI restriction endonuclease on the pBWA (V) HS-osgfp-ccdb-tnos vector, and the sequence of the PaPDS gene coding region is shown as SEQ ID NO. 1. The recombinant vector can be used for the overexpression of PaPDS genes to deepen the color of apricot fruits and the content of beta-carotene. In addition to the pBWA (V) HS-osgfp-ccdb-tnos vector, the original vector of the recombinant vector may also be a vector commonly used in the field of genetic engineering, such as viral and other plant expression vector plasmids, and the like.

In the application, the original vector of the recombinant vector is TRV II-GFP, the PaPDS gene coding region sequence is inserted between EcoRI and Xho I restriction enzyme sites of the TRV II-GFP vector, and the PaPDS gene coding region sequence is shown as SEQ ID NO. 3. The sequence shown in SEQ ID NO.3 is a part of PaPDS gene cDNA sequence, and after the part is connected with TRV II-GFP vector to form a recombinant vector, the corresponding sequence of mRNA transcribed from PaPDS gene in an individual transformed by the recombinant vector is degraded, and PaPDS gene cannot be expressed normally.

A method for deepening the color of apricot fruits comprises the step of transforming the functional gene PaPDS for regulating the color of the apricot fruits or a recombinant vector containing a coding region sequence of the functional gene PaPDS for regulating the color of the apricot fruits into apricot seedlings or apricot fruits.

In the method, the original vector of the recombinant vector is pBWA (V) HS-osgfp-ccdb-tnos, the sequence of a PaPDS gene coding region is inserted into the cleavage site of BsaI restriction endonuclease on the pBWA (V) HS-osgfp-ccdb-tnos vector, and the sequence of a PaPDS gene coding region is shown as SEQ ID NO. 1. The recombinant pBWA (V) HS-osgfp-ccdb-tnos vector mediates the overexpression of PaPDS genes in apricot fruits, improves the beta-carotene content of the apricot fruits, and deepens the color of the apricot fruits.

In the method, the GV3101 agrobacterium containing the recombinant pBWA (V) HS-osgfp-ccdb-tnos vector is used to transform the functional gene PaPDS for regulating the color of apricot fruits into apricot seedlings or apricot fruits.

A method for reducing apricot fruit color comprises transforming apricot seedling or apricot fruit with recombinant engineering bacteria containing the recombinant TRV II-GFP vector and recombinant engineering bacteria containing TRV I vector. The TRV II-GFP vector and the TRV I vector are used together for gene silencing, so that mRNA transcribed from JTY-PaPDS gene is degraded, the synthesis of beta-carotene in apricot fruits is reduced, and finally the aim of reducing the color of the apricot fruits is fulfilled.

The method comprises the steps that the recombinant engineering bacteria containing the recombinant TRV II-GFP vector and the recombinant engineering bacteria containing the TRV I vector are GV3101 agrobacterium.

The invention provides PaPDS genes cloned from golden sun apricots, the protein coded by the genes can participate in the synthesis of beta-carotene in apricot fruits, and the color and the content of beta-carotene of the apricot fruits can be obviously deepened by over-expression of the genes in the apricot fruits, so that the genes can be used for genetic improvement of the color of apricot fruits.

The technical scheme of the invention has the following beneficial technical effects:

1. According to the content determination of beta-carotene and the analysis result of transcriptome, the full-length cDNA sequence of JTY-PaPDS gene capable of regulating and controlling the color of apricot flesh is obtained by reverse transcription and cloning from the total RNA of the flesh of golden sun apricot; furthermore, the invention utilizes genetic engineering technology to obtain a recombinant cloning vector and a recombinant over-expression vector containing JTY-PaPDS gene, and also obtains a VIGS silencing vector for silencing JTY-PaPDS gene. After the recombinant over-expression vector containing JTY-PaPDS gene and the VIGS silencing vector for silencing JTY-PaPDS gene are respectively transformed into apricot pulp cells, the content of beta-carotene in the apricot pulp cells is respectively increased and reduced, and further the color of the apricot pulp is respectively deepened and lightened.

2. The JTY-PaPDS gene, the recombinant over-expression vector and the VIGS silencing vector can be used for genetic improvement of the color of apricot flesh and the content of beta-carotene in the apricot flesh, and the recombinant cloning vector can be used for constructing other types of vectors containing the JTY-PaPDS gene so as to further study the functions of the JTY-PaPDS gene.

Drawings

FIG. 1 is a graph showing the measurement results of the content of beta-carotene in different varieties of oranges, gingko flesh and apricot fruits in the embodiment of the invention;

FIG. 2 is a graph showing the color contrast of mature fruits of the golden sun and X15 in the embodiment of the invention;

FIG. 3 is a schematic diagram of fruits at different stages of development of X15 and Jinsun in an embodiment of the invention;

FIG. 4 is a graph showing the results of measuring the beta-carotene content of X15 and Jintaiyang fruits at different development periods in the embodiment of the invention;

FIG. 5 is a graph showing the results of transcriptomic analysis of X15 and Jintaiyang fruits in the example of the present invention;

FIG. 6 is a diagram showing the comparison result of the gene sequences of X15 and Jinsun PaPDS in the embodiment of the present invention;

FIG. 7 is a diagram showing the result of comparing the coding protein sequences of the X15 gene and the Jinsun PaPDS gene in the embodiment of the invention;

FIG. 8 is a vector map of pBWA (V) HS-osgfp-ccdb-tnos in the example of the present invention;

FIG. 9 is a graph showing experimental results of transformation of apricot fruits with Agrobacterium containing an over-expression vector in the examples of the present invention;

FIG. 10 is a graph showing the results of determining the content of substances in transformed apricot fruits of Agrobacterium containing an overexpression vector according to the embodiment of the invention;

FIG. 11 is a graph of the results of a first PCR assay for constructing a VIGS silencing vector in an embodiment of the invention;

FIG. 12 is a graph showing the results of a second PCR assay for constructing a VIGS silencing vector in accordance with an embodiment of the present invention;

FIG. 13 is a graph showing the results of a third PCR assay for constructing a VIGS silencing vector in an embodiment of the invention;

FIG. 14 is a graph showing experimental results of transformation of apricot fruits with Agrobacterium containing the VIGS silencing vector in the example of the present invention;

FIG. 15 is a graph showing the results of determining the content of substances in transformed apricot fruits of Agrobacterium containing the VIGS silencing vector according to the example of the present invention;

FIG. 16 shows a TRVII-GFP vector map in the example of the present invention.

Detailed Description

The beta-carotene is known to be the main orange substance of apricot fruits, and on the basis of the orange substance, the technician collects different varieties of oranges and gingko flesh apricot fruits, and the content of the beta-carotene in the fruits is measured, and the result is shown in figure 1. Among all the harvested apricot fruits, a variety "X15" with the lowest content of beta-carotene and the whitest phenotypic observation was selected.

The content of beta-carotene in different development periods and transcriptome analysis are carried out by taking gingko pulp apricot "X15" and orange pulp apricot "Jinsun" (JTY or Sungold) as experimental objects respectively. The color pairs of the two apricot ripe fruits are shown in fig. 2, wherein the darker color is 'golden sun' and the lighter color is 'X15'. The photographs of the fruits at different stages of development are shown in fig. 3, the measurement results of the beta-carotene content of the fruits at different stages of development are shown in fig. 4, and the analysis results of transcriptome are shown in fig. 5. A candidate gene PaPDS was obtained by the skilled artisan through beta-carotene content determination and transcriptomic analysis.

The PaPDS genes (designated JTY-PaPDS and X15-PaPDS, respectively) were cloned using the total DNA of JTY and X15 as templates, and comparing the sequences of the two, X15-PaPDS was found to have a 5bp deletion at exon 11, as compared to JTY-PaPDS, as shown in FIG. 6. This deletion results in premature termination of translation of X15-PaPDS, as shown in FIG. 7. Based on CDD domain predictions, the mutation occurs in PaPDS phytoene-desarturase functional domains.

And extracting JTY and X15 pulp total RNA, and cloning target genes or fragments by taking cDNA obtained by reverse transcription as a template. The cDNA sequences of JTY-PaPDS and X15-PaPDS are as follows:

JTY-PaPDS(SEQ ID NO.1)：

atgtctcagtgggcttgtgtctctgctgctaacttgagctgccaagctagcatcatcaacactcaaaagctacgaaacactcccagatgcgatgccttttcatttaaaggtagtgaatttatggctcaaagctgtagatttttaagcccacaagctatttatggaaggccgaggaatggtgcttgccctttgaaggtggtttgcgttgattatccaagaccagaccttgacaatactgctaatttcttagaagctgcatatttctcttccactttccgagcctctcctcgtccagctaagccgttgaaggtcgtgattgctggtgcaggtttggctggtcttgcaactgcaaaatatttggctgatgcaggtcataaacctatcttactggaagcaagagatgttctaggcggaaaggtggcagcatggaaagataaggatggagactggtacgaaacaggcctccatatcttctttggggcttatccgaatattcagaacctgtttggtgagcttggtattgatgatcgattgcagtggaaggagcattctatgatatttgcaatgccaaacaaaccaggagaattcagccggtttgatttccctgaagttttaccagcacccttaaatggaatatgggccatattgaagaacaatgagatgctgacttggccagagaaaataaagtttgcaattggactactgccagcaattcttggtgggcaggcttatgttgaagcccaagatggcttgagtgtaaaagattggatgaggaaacagggcataccggatcgagtgactactgaggtgtttattgccatgtcaaaggccctgaactttattaaccctgatgaactttcaatgcaatgcatattgattgctttgaaccgattccttcaggagaaacacggttccaagatggctttcttggatggtagtccccctgagagactctgtgcaccaattgttgatcatatccagtcattgggcggtgaagtccgaattaattcccgaatacagaaaattgagctaaataaagatgggaccgtgaagagttttgtactaaataatgggagcatgattgaagcagatgcctatgtatttgccactccagttgacatcctaaagcttctattgcctgataactggaaagagatcccatatttcaagaaattggagaaactaattggcgttccagttatcaatgttcacatatggtttgacagaaagctgaagaacacatatgatcatctactttttagcaggagtcctcttttaagtgtctatgctgacatgtctgtaacatgtaaggaatattataatccaaaccagtctatgctggagttggtttttgcaccagcagaagaatggatttcatgcagtgattcagaaattattgatgctacactcaaagaacttgcaaaactctttcctgatgagatagctgcagatcagagcaaagcaaagattttgaagtaccatgttgtgaaaacaccaaggtcggtttacaaaactgtaccagattgtgaaccttgccgtcccttgcaaagatctcccctagagggtttctatttagctggtgattacacaaaacaaaagtatttagcctcaatggaaggtgctgttctgtcagggaaactttgtgcacaagcaattgtacaggtaatttccatcaggactctggaatttatatggcatgatgaacacgcatgctcatctatgtctgaatttacttaa

X15-PaPDS：

atgtctcagtgggcttgtgtctctgctgctaacttgagctgccaagctagcatcatcaacactcaaaagctacgaaacactcccagatgcgatgccttttcatttaaaggtagtgaatttatggctcaaagctgtagatttttaagcccacaagctatttatggaaggccgaggaatggtgcttgccctttgaaggtggtttgcgttgattatccaagaccagaccttgacaatactgctaatttcttagaagctgcatatttctcttccactttccgagcctctcctcgtccagctaagccgttgaaggtcgtgattgctggtgcaggtttggctggtcttgcaactgcaaaatatttggctgatgcaggtcataaacctatcttactggaagcaagagatgttctaggcggaaaggtggcagcatggaaagataaggatggagactggtacgaaacaggcctccatatcttctttggggcttatccgaatattcagaacctgtttggtgagcttggtattgatgatcgattgcagtggaaggagcattctatgatatttgcaatgccaaacaaaccaggagaattcagccggtttgatttccctgaagttttaccagcacccttaaatggaatatgggccatattgaagaacaatgagatgctgacttggccagagaaaataaagtttgcaattggactactgccagcaattcttggtgggcaggcttatgttgaagcccaagatggcttgagtgtaaaagattggatgaggaaacagggcataccggatcgagtgactactgaggtgtttattgccatgtcaaaggccctgaactttattaaccctgatgaactttcaatgcaatgcatattgattgctttgaaccgattccttcaggagaaacacggttccaagatggctttcttggatggtagtccccctgagagactctgtgcaccaattgttgatcatatccagtcattgggcggtgaagtccgaattaattcccgaatacagaaaattgagctaaataaagatgggaccgtgaagagttttgtactaaataatgggagcatgattgaagcagatgcctatgtatttgccactccagttgacatcctaaagcttctattgcctgataactggaaagagatcccatatttcaagaaattggagaaactaattggcgttccagttatcaatgttcacatatggtttgacagaaagctgaagaacacatatgatcatctactttttagcaggagtcctcttttaagtgtctatgctgacatgtctgtaacatgaatattataatccaaaccagtctatgctggagttggtttttgcaccagcagaagaatggatttcatgcagtgattcagaaattattgatgctacactcaaagaacttgcaaaactctttcctgatgagatagctgcagatcagagcaaagcaaagattttgaagtaccatgttgtgaaaacaccaaggtcggtttacaaaactgtaccagattgtgaaccttgccgtcccttgcaaagatctcccctagagggtttctatttagctggtgattacacaaaacaaaagtatttagcctcaatggaaggtgctgttctgtcagggaaactttgtgcacaagcaattgtacaggtaatttccatcaggactctggaatttatatggcatgatgaacacgcatgctcatctatgtctgaatttacttaa

the amino acid sequences of the proteins coded by the two are respectively:

JTY-PaPDS(SEQ ID NO.2)：

MSQWACVSAANLSCQASIINTQKLRNTPRCDAFSFKGSEFMAQSCRFLSPQAIYGRPRNGACPLKVVCVDYPRPDLDNTANFLEAAYFSSTFRASPRPAKPLKVVIAGAGLAGLATAKYLADAGHKPILLEARDVLGGKVAAWKDKDGDWYETGLHIFFGAYPNIQNLFGELGIDDRLQWKEHSMIFAMPNKPGEFSRFDFPEVLPAPLNGIWAILKNNEMLTWPEKIKFAIGLLPAILGGQAYVEAQDGLSVKDWMRKQGIPDRVTTEVFIAMSKALNFINPDELSMQCILIALNRFLQEKHGSKMAFLDGSPPERLCAPIVDHIQSLGGEVRINSRIQKIELNKDGTVKSFVLNNGSMIEADAYVFATPVDILKLLLPDNWKEIPYFKKLEKLIGVPVINVHIWFDRKLKNTYDHLLFSRSPLLSVYADMSVTCKEYYNPNQSMLELVFAPAEEWISCSDSEIIDATLKELAKLFPDEIAADQSKAKILKYHVVKTPRSVYKTVPDCEPCRPLQRSPLEGFYLAGDYTKQKYLASMEGAVLSGKLCAQAIVQVISIRTLEFIWHDEHACSSMSEFT

X15-PaPDS：

MSQWACVSAANLSCQASIINTQKLRNTPRCDAFSFKGSEFMAQSCRFLSPQAIYGRPRNGACPLKVVCVDYPRPDLDNTANFLEAAYFSSTFRASPRPAKPLKVVIAGAGLAGLATAKYLADAGHKPILLEARDVLGGKVAAWKDKDGDWYETGLHIFFGAYPNIQNLFGELGIDDRLQWKEHSMIFAMPNKPGEFSRFDFPEVLPAPLNGIWAILKNNEMLTWPEKIKFAIGLLPAILGGQAYVEAQDGLSVKDWMRKQGIPDRVTTEVFIAMSKALNFINPDELSMQCILIALNRFLQEKHGSKMAFLDGSPPERLCAPIVDHIQSLGGEVRINSRIQKIELNKDGTVKSFVLNNGSMIEADAYVFATPVDILKLLLPDNWKEIPYFKKLEKLIGVPVINVHIWFDRKLKNTYDHLLFSRSPLLSVYADMSVT

When cloning the target gene (PaPDS genes), proper enzyme cutting sites and sequences required by homologous recombination are added to corresponding specific primers for subsequent construction of expression vectors by homologous recombination. Wherein the restriction enzyme site is selected according to the sequence of the target gene and the sequence of the required expression vector, and the BamHI restriction enzyme site is added upstream of the start codon and the XbaI restriction enzyme site is added downstream of the stop codon. In this example, the cDNA of both PaPDS genes can be amplified using the same primers. The PaPDS gene clone amplification primers used finally are:

forward amplification primer:

5'-CTCTCTCTCAAGCTTGGATCCATGTCTCAGTGGGCTTGTGTCTC-3'

Reverse amplification primer:

5'-GATACGAACGAAAGCTCTAGATTAAGTAAATTCAGACATAGATGAGCATG-3'

PCR cloning is carried out on PaPDS genes of two apricots by using the primers according to the conventional technical means in the field, then agarose gel electrophoresis is carried out to detect the band size of the PCR products, and the band with correct size is subjected to gel cutting recovery, so that a first recovery product (containing X15-PaPDS sequences) and a second recovery product (containing JTY-PaPDS sequences) are respectively obtained. The first recovered product and the second recovered product are respectively connected with cloning vector pMD19-T (using a T-carried kit pClone007, 007 Blunt Simple Vector Kit of the Optimago family) to obtain two recombinant cloning vectors containing target genes.

Two recombinant cloning vectors are used to transform colibacillus competence, and after being coated on a flat plate (ampicillin resistance), 10-20 monoclonals are picked for PCR identification. During cloning, randomly picking colonies on a flat plate by using a sterilized pipette tip in an ultra-clean workbench, putting into 10-20 mu L of sterile water for blowing for several times to obtain bacteria solutions to be tested, taking 2 mu L of each bacteria solution to be tested as a template, performing bacterial solution PCR, adding 600 mu L of liquid LB culture medium into the remained bacteria solutions to be tested, and continuously culturing for a period of time at 37 ℃ and 220rpm to obtain seed retaining bacteria solutions.

Positive clones were selected according to agarose gel electrophoresis results of the PCR products and sequenced. The positive clone sequencing primer is (the recombination cloning vector containing two sequences is universal):

M13F：5'-TGTAAAACGACGGCCAGT-3'

M13R：5'-CAGGAAACAGCTATGACC-3'

after the sequencing is finished, the seed-retaining bacterial liquid of the clone with correct sequencing is selected for use.

For PaPDS transient overexpression analysis, two PaPDS overexpression vectors containing the X15-PaPDS and JTY-PaPDS genes, respectively, were constructed. In this example, the sequence of the X15-PaPDS gene and the JTY-PaPDS gene sequence were obtained by PCR using the correctly sequenced bacterial liquid as a template, and the primers used in PCR were as follows. Each pair of primers is added with a sequence required for constructing a vector by a homologous recombination method.

X15-PaPDS：

D3669_0S1(+):5'-ACGGCATGGACGAGCTCTACATGTCTCAGTGGGCTTGTGTCTCTG-3'

D3669_0S1(-):5'-TGAAGACAGAGCTAGTTACATTAAGTAAATTCAGACATAGATGAGCATGCGTGTTC-3'

JTY-PaPDS：

D3668_0S1(+):5'-ACGGCATGGACGAGCTCTACATGTCTCAGTGGGCTTGTGTCTCTG-3'

D3668_0S1(-):5'-TGAAGACAGAGCTAGTTACATTAAGTAAATTCAGACATAGATGAGCATGCGTGTTC-3'

The reaction system and the procedure used in PCR are shown in Table 1 and Table 2, respectively.

TABLE 1PCR reaction System

Composition of the components	Volume of
		Nuclease-free water	20uL
Biorun Pfu PCR Mix	25uL
		Primer (+)	2uL
Primer (-)	2uL
		Template	1uL
Total volume of	50uL

TABLE 2PCR procedure

Temperature (temperature)	Time of	Cycle number
			94℃	For 5 minutes	1
94℃	30 Seconds	30
			50℃	45 Seconds	30
72℃	104 Seconds	30
			72℃	For 10 minutes	1
16℃	30 Minutes	1

After the PCR reaction, the reaction product was subjected to agarose gel electrophoresis to recover the target fragment, thereby obtaining a third recovered product (containing the sequence of X15-PaPDS) and a fourth recovered product (containing the sequence of JTY-PaPDS), respectively. For the third and fourth recovered products, a portion was removed for sequencing, respectively. The primers used in the sequencing were:

JTY-PaPDS：

Forward primer 5'-CCATTTAAGGGTGCTGGTAAAAC-3'

Reverse primer 5'-CACTTAAAAGAGGACTCCTGCTA-3'

X15-PaPDS：

Forward primer 5'-CCATTTAAGGGTGCTGGTAAAAC-3'

Reverse primer 5'-CACTTAAAAGAGGACTCCTGCTA-3'

The template used in the PCR is the seed-preserving bacterial liquid of the positive clone with correct sequencing, and further sequencing identification is needed after the recombinant overexpression vector is constructed, so that the sequencing is not needed, and the recovered strip is only needed to be determined to be correct through sequencing. After sequencing is completed, the correct recovered product is left for use.

The overexpression vector pBWA (V) HS-osgfp-ccdb-tnos is then digested to linearize it, and the linearized vector can be ligated with the third recovered product and the fourth recovered product, respectively, to construct a recombinant overexpression vector. The cleavage reaction of pBWA (V) HS-osgfp-ccdb-tnos was carried out at 37℃for 1 hour, the reaction system is shown in Table 3, and the map of the recombinant overexpression vector pBWA (V) HS-osgfp-ccdb-tnos is shown in FIG. 8.

TABLE 3pBWA (V) HS-osgfp-ccdb-tnos cleavage reaction system

Composition of the components	Volume of
		Nuclease-free water	13μL
10 Xrestriction enzyme buffer	2μL
		BsaⅠ/Eco31Ⅰ	1μL
pBWA(V)HS-osgfp-ccdb-tnos	4μL
		Totals to	20μL

The pBWA (V) HS-osgfp-ccdb-tnos vector will form two blunt ends after single cleavage by BsaI enzyme. The linearized vector after cleavage was purified with a PCR purification kit for use in homologous recombination reactions. In this example, in vitro homologous recombination reactions are performed, and in other examples, homologous recombination reactions in E.coli may be selected. The temperature conditions for the homologous recombination reaction were 37℃and the incubation time was 30 hours, and the reaction system was as shown in Table 4.

TABLE 4 recombination reaction System

Composition of the components	Volume of
		Nuclease-free water	0uL
Biorun 2×EasyClone Mix	10uL
		Third or fourth recovered product	5uL
PBWA (V) HS-osgfp-ccdb-tnos (linearized)	5uL
		Totals to	20uL

Taking 10uL of a recombination reaction product of the third recovery product and the linearization vector (namely X15-PaPDS recombination over-expression vector pBWA-X15) and 10uL of a homologous recombination reaction product of the fourth recovery product and the linearization vector (namely JTY-PaPDS recombination over-expression vector pBWA-JTY), respectively transforming escherichia coli competence, coating a kanamycin resistance plate after transformation, culturing for 12 hours at 37 ℃, and carrying out plaque PCR identification. When the bacterial plaque is identified, 10 clones are selected and simultaneously subjected to 1.5mL EP tube joint bacteria and PCR identification, and the specific operation method is the same as that of the recombinant cloning vector selected cloning and PCR identification method. The two primers used in sequencing were designed based on the sequence of pBWA (V) HS-osgfp-ccdb-tnos, and were located upstream and downstream of the recombinant fragment on the vector, respectively.

Identification primer X15-PaPDS PCR:

Forward primer 5'-ttcatttggagagaacacgggggac-3'

Reverse primer 5'-ccatttaagggtgctggtaaaac-3'

JTY-PaPDS PCR identification primers:

Forward primer 5'-ttcatttggagagaacacgggggac-3'

Reverse primer 5'-ccatttaagggtgctggtaaaac-3'

After the sequencing is finished, two recombinant over-expression vectors are respectively selected to select a clone with correct sequencing, and the clone is subjected to fungus shaking, and then plasmids are extracted.

For each recombinant overexpression vector, 1 mu L of plasmid is added into 50 mu L of GV3101 agrobacterium competent cells, the mixture is fully and uniformly mixed and then is sucked into an electric rotating cup, 1mL of LB liquid culture medium is added after electric rotation, the mixture is fully and uniformly mixed and then is sucked into a 1.5mL centrifuge tube, and the mixture is subjected to shaking culture at 180rpm for 30min at the temperature of shaking table 30 ℃ for carrying out the sexual activation. Then 50 mu L of activated agrobacterium liquid is sucked and inoculated on LB solid medium, and dark culture is carried out for 48 hours at 30 ℃. In addition, 1 mu L pBWA (V) HS-osgfp-ccdb-tnos empty vector (pBWA) was transformed with the same Agrobacterium in the same manner, and the Agrobacterium transformed into the empty vector was used for treatment of the control group.

After the dark culture, single colonies on LB solid medium were identified by PCR. For single colonies to be identified, half of them were picked as PCR templates and the other half were left on the plate for further culture. The identification primer is the PCR identification primer of the colibacillus colony transformed by the recombinant over-expression vector. After the identification, colonies of positive clones left on the plate were picked, placed in 3mL of LB medium (containing 100mg/mL kanamycin+50 mg/mL rifampicin), and cultured with shaking at 200rpm on a shaker at 28℃for 24 hours. After 24 hours, the 3mL of the bacterial liquid is inoculated into 200mL of LB culture medium (containing 100mg/mL kanamycin and 50mg/mL rifampicin), and the bacterial liquid is subjected to shaking culture for 10-12 hours to OD ₆₀₀ = 0.6-0.8 at the rotation speed of a shaking table 200rpm at the temperature of 28 ℃, the rotation speed of a centrifugal machine is set to 5000rpm, the bacterial liquid is centrifuged for 5 minutes at room temperature, and the supernatant is discarded, so that bacterial cells are collected. For the collected thalli, firstly rinsing the thalli with 1mL of penetration buffer solution, centrifuging for 5min at the room temperature of 5000rpm, discarding the liquid, and then re-suspending the thalli to OD ₆₀₀ = 0.6-0.8 by using new penetration buffer solution to obtain the bacterial liquid to be injected.

The formulation of the permeation buffer is shown in table 5.

Table 5 permeation buffer formulation

Reagent(s)	Volume of use	Final concentration
			100mmol/L AS	75μL	150μmol/L
1mol/L MES	500μL	10mmol/L
			1mol/L MgCl2	500μL	10mmol/L
ddH2O	Constant volume to 50mL

The preparation method of 100mmol/L Acetosyringone (AS) comprises the following steps: 0.3924g of acetosyringone powder is weighed and directly dissolved in 20mL of DMSO, and the powder is split charging after filter membrane sterilization and frozen at the temperature of minus 20 ℃.

The preparation method of the 1mol/L MES comprises the following steps: 4.265g of MES powder was weighed and dissolved in 20mL of ultrapure water. The pH is adjusted to 5.6-5.7 by 1mol/L NaOH.

The preparation method of 1mmol/L MgCl ₂ comprises the following steps: 1.9042g of MgCl ₂ powder were weighed out and dissolved in 20mL of ultrapure water.

In this example, the bacterial solutions to be injected were divided into 3 species depending on the types of vectors contained in Agrobacterium (pBWA-JTY, pBWA-X15, and empty vector pBWA, respectively). 3 bacteria liquid to be injected are placed for 2-3 hours at room temperature, apricot fruits to be changed are injected, 4-6 different positions of each fruit are selected for injection, an injection part is circled by a marker pen, and phenotypes are observed and sampled after 5-7 days.

FIG. 9 is a physical image of the fruits developed to maturity after injection of 3 agrobacteria, the variety being silvery white. As is apparent from the figure, fruits containing pBWA-JTY Agrobacterium were injected, the pulp near the pinhole thereof became significantly red, and fruits containing two other agrobacteria were injected, the pulp near the pinhole thereof had no significant color change, thus indicating that PaPDS controlled the color of apricot fruits to be darker and lighter, and when the gene expression level was increased, the color of apricot fruits became darker. The skilled person further measured the content of beta-carotene etc. in the fruit and the results are shown in fig. 10. As can be seen from FIG. 10, injection of Agrobacterium suspension containing pBWA-JTY promotes the expression of PaPDS and accumulation of beta-carotene and its upstream metabolites in apricot fruits, compared to controls pBWA-X15 and pBWA.

To further verify the function of the X15-PaPDS and JTY-PaPDS genes, a VIGS silencing vector was constructed and transient VIGS silencing analysis was performed on PaPDS. The VIGS silencing vector will degrade the mRNA transcribed from the gene of interest and the mRNA will not translate normally.

Firstly, cloning a target gene, cloning the target gene by using the escherichia coli bacterial liquid containing the JTY-PAPDS recombinant cloning vector as a template through a PCR method, wherein the primers are as follows:

TRVⅡ-GFP-F：5'-TGAGTAAGGTTACCGAATTCTCCCGAATACAGAAAATTGAGCTA-3'

TRVⅡ-GFP-R：5'-GGACATGCCCGGGCCTCGAGACATGTTACAGACATGTCAGCA-3'

wherein, the TRV II-GFP-F primer adds the homologous recombination sequence and EcoRI recognition sequence to one end of the target fragment, and the TRV II-GFP-R primer adds the homologous recombination sequence and Xho I recognition sequence to the other end of the target fragment, so that the recombinant plasmid can be constructed by using the homologous recombination method subsequently. The PCR reaction system used is shown in Table 6 and the PCR procedure is shown in Table 7.

TABLE 6 PCR reaction System for cloning silencing vector

Reagent(s)	Dosage of
		2 XP 515 enzyme	20μL
TRVⅡ-GFP-F(10μM)	1μL
		TRVⅡ-GFP-R(10μM)	1μL
Template	1uL
		Sterilizing ddH 2 O	17μL

TABLE 7 PCR reaction procedure for cloning silencing vectors

Temperature (temperature)	Time of	Cycle number
			95℃	For 5 minutes	1
95℃	30 Seconds	33
			56℃	30 Seconds	33
72℃	120 Seconds	33
			72℃	For 10 minutes	1
4℃	30 Minutes	1

The final amplified PCR product contained JTY-PaPDS with the coding region sequence (SEQ ID NO. 3):

tcccgaatacagaaaattgagctaaataaagatgggaccgtgaagagttttgtactaaataatgggagcatgattgaagcagatgcctatgtatttgccactccagttgacatcctaaagcttctattgcctgataactggaaagagatcccatatttcaagaaattggagaaactaattggcgttccagttatcaatgttcacatatggtttgacagaaagctgaagaacacatatgatcatctactttttagcaggagtcctcttttaagtgtctatgctgacatgtctgtaacatgt

For the obtained PCR product, separation was performed using 1% agarose gel electrophoresis. And adding the PCR product into a sample application hole of the gel, and carrying out electrophoresis under the voltage of 60-100V, wherein DNA in the sample moves from a negative electrode to a positive electrode. Electrophoresis was stopped when bromophenol blue moved to about 1cm from the lower edge of the gel plate. When the DNA is observed under an ultraviolet lamp, the DNA shows a fluorescent band when the DNA exists, and the DNA is photographed and stored by a gel imaging system. The electrophoresis result is shown in FIG. 11, wherein the left side of the figure is Marker, and the brightest right side is the band of the target fragment. Rapidly cutting gel containing the target strip under an ultraviolet lamp, transferring into a 2.0mL centrifuge tube, recovering the target fragment according to the specification of Magen gel recovery kit (HiPure Gel Pure DNA MINI KIT), obtaining a fifth recovery product, and storing at-20deg.C for later use.

Next, the TRVII-GFP vector was linearized using EcoRI and XhoI restriction enzymes, the reaction system is shown in Table 8, and the TRVII-GFP vector is shown in FIG. 16. During enzyme digestion, incubating for 40min at 37 ℃; after the incubation was completed, the reaction system was left at 65℃for 20min to inactivate the restriction enzymes.

Table 8TRV II-GFP vector double cleavage reaction System

Reaction system	Usage amount
		5×Tango buffer	4.0μl
EcoRⅠ	2μL
		XhoⅠ	1μL
Plasmid (TRV II-GFP)	8μg
		ddH2O	Constant volume to 40. Mu.L

And (3) after enzyme digestion, carrying out agarose gel electrophoresis on enzyme digestion products, cutting gel of a band corresponding to the linearized TRV II-GFP carrier large fragment, recovering the linearized TRV II-GFP carrier large fragment according to the instruction of Magen gel recovery kit, and placing the recovered product at the temperature of minus 20 ℃ for standby. The short fragments excised from the vector together with EcoRI and XhoI are small fragments which cannot be recovered.

The fifth recovered product was then ligated to the linearized TRVIII-GFP vector. In this embodiment, a homologous recombination method is used to construct a recombinant vector, and the method is as follows: a reaction buffer was added to the PCR tube, 2. Mu.L of the target DNA fragment (fifth recovered product) and 3. Mu.L of the linearized vector were added to the reaction buffer, and finally the homologous recombination enzyme was added. And (3) uniformly mixing, and then preserving the temperature at 37 ℃ for 30min to obtain the recombinant TRV II-GFP vector.

Coli was transformed using a recombinant TRVII-GFP vector as follows:

a. a tube of 50uL of competent cells of the large intestine was thawed on ice and the walls of the tube were flicked to re-suspend the cells. 10uL of recombinant TRV II-GFP vector was added to competent cells, and incubated on ice for 30min at the flick number.

And b, rapidly putting the mixture on ice for 2min after heat shock in a water bath at 42 ℃ for 50 s.

C. 700uL of LB liquid medium is added and incubated for 60min at 37 ℃ in a shaking table.

D. In the ultra clean bench to get 400uL bacterial liquid even coating plate (kanamycin resistance), after the plate dry 37 ℃ inverted overnight.

After the inverted culture at 37 ℃ overnight, 10 single clones are selected, positive clone identification is carried out by using a PCR method, and the method is the same as the PCR identification method of the escherichia coli containing the recombinant cloning vector. The primers used in PCR were:

pTRV2-seq-F:5'-GAGTCCCACATATTCGCACG-3'

pTRV2-seq-R:5'-CCCCCCAACAATCTCTTAGC-3'

The result of agarose gel electrophoresis of the PCR product is shown in FIG. 12, the right band in the figure is the target band, 3 positive clones are selected for sequencing, one clone with correct sequencing is selected for shaking, and PLASMID MINI KIT I (OMEGA, cat# D6943-01) is used for extracting the plasmid, and the extracted plasmid is the recombinant TRV II-GFP vector.

The recombinant TRVII-GFP vector and the matched TRVII vector are used for respectively transforming the agrobacterium, and the TRVII-GFP empty vector is additionally used for transforming the agrobacterium. The method for transforming the agrobacterium is as follows:

a. GV3101 competent cells 50uL were removed and thawed on ice;

b. Adding 5uL plasmid, slightly mixing, and ice-bathing for 15min;

c. Quick-freezing with liquid nitrogen for 5min;

d, water bath at 37 ℃ for 5min;

e. Ice bath for 5min;

f. adding 700uL of non-resistant LB liquid medium, and shaking for 2 hours at 28 ℃;

g. 400uL of bacterial liquid is coated on a flat plate (kanamycin and rifampicin are added in the flat plate, the final concentration of the two antibiotics is 50 mg/L), and the flat plate is cultivated in an inverted way at 28 ℃;

h. After single colonies were grown, 5 single colonies were selected for colony PCR verification using the primers pTRV2-seq-F and pTRV2-seq-R. Verification PCR results as shown in fig. 13, one clone verified to be correct was selected for shaking for each plasmid for collection of agrobacterium.

According to the method for collecting the agrobacterium containing the recombinant overexpression vector, the agrobacterium transferred into the TRV I vector, the agrobacterium transferred into the recombinant TRV II-GFP vector and the agrobacterium transferred into the TRV II-GFP empty load are respectively collected, and suspended by about 30mL of penetration buffer solution, so that the OD600 value of the final concentration is 0.6-0.8. Uniformly mixing the agrobacteria suspension transferred into TRV I with the agrobacteria suspension transferred into TRV II-GFP in equal volume, and infecting fruits of a control group; the agrobacteria suspension transferred into TRV I and the agrobacteria suspension transferred into recombinant TRV II-GFP vector are uniformly mixed in equal volume for infecting fruits of experimental groups. The method for infecting fruits is the same as the method for infecting fruits by the recombinant overexpression vector. A photograph of the infested fruit (apple apricot variety) is shown in FIG. 14. In the figure, TRV1+TRV2 is a fruit of a control group, and the color of a region of the fruit of the control group, which is injected with agrobacterium, is normal, and TRV1+TRV2 is as follows: paPDS is the fruits of the experimental group, the orange color of the area of the fruits of the experimental group injected with agrobacterium is obviously lighter than that of the area not injected with agrobacterium, which indicates that after PaPDS gene in the fruits of the experimental group is silenced, the orange color of the fruits becomes lighter, namely PaPDS gene controls the orange color of apricot fruits. The content of beta-carotene and other substances in the fruits was further measured by the skilled person, and the results are shown in FIG. 15. As can be seen from FIG. 15, the expression of PaPDS and the accumulation of β -carotene and its upstream metabolites were inhibited in apricot fruits of the experimental group (TRV1+TRV2: paPDS) compared to the control (TRV1+TRV2).

In this embodiment, the recombinant overexpression vector and the recombinant TRV II-GFP vector are used directly to transform apricot fruit cells, and in other embodiments, the two vectors can be used to transform apricot seedlings and to culture the seedlings into complete individuals to change the color of fruits produced by the seedlings. In addition, the cDNA of JTY-PaPDS gene and other plant expression vectors can be formed into recombinant vector, and the recombinant vector can be transformed into apricot seedling or apricot fruit to deepen the color of apricot fruit.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While the obvious variations or modifications which are extended therefrom remain within the scope of the claims of this patent application.

Claims (10)

1. A functional gene PaPDS for regulating and controlling the color of apricot fruits is characterized in that the sequence of PaPDS gene is shown in SEQ ID NO.1, and the amino acid sequence of protein coded by PaPDS gene is shown in SEQ ID NO. 2.

2. The application of the over-expression recombinant vector containing the coding region sequence of the functional gene PaPDS for regulating and controlling the color of apricot fruits in darkening the color of apricot fruits is characterized in that the coding region sequence of the PaPDS gene is the sequence shown as SEQ ID NO. 1.

3. The use according to claim 2, wherein the original vector of the recombinant vector is pMD19-T, the PaPDS gene coding region sequence is located between SalI and XbaI restriction enzyme sites of the pMD19-T vector, and the PaPDS gene coding region sequence is the sequence shown in SEQ ID NO. 1.

4. The use according to claim 2, wherein the original vector of the recombinant vector is pBWA (V) HS-osgfp-ccdb-tnos, the PaPDS gene coding region sequence is inserted at the cleavage site of BsaI restriction enzyme on the pBWA (V) HS-osgfp-ccdb-tnos vector, and the PaPDS gene coding region sequence is as shown in SEQ ID NO. 1.

5. The application of the recombinant vector containing the coding region sequence of the functional gene PaPDS for regulating and controlling the color of apricot fruits in reducing the color of apricot fruits is characterized in that the original vector of the recombinant vector is TRV II-GFP, the coding region sequence of the PaPDS gene is inserted between EcoRI and Xho I restriction enzyme sites of the TRV II-GFP vector, and the coding region sequence of the PaPDS gene is shown as SEQ ID NO. 3.

6. A method for darkening apricot fruit color, characterized in that the over-expression recombinant vector of the functional gene PaPDS for regulating apricot fruit color as described in claim 1 is transformed into apricot seedling or apricot fruit.

7. The method according to claim 6, wherein the original vector of the recombinant vector is pBWA (V) HS-osgfp-ccdb-tnos, the PaPDS gene coding region sequence is inserted at the cleavage site of BsaI restriction enzyme on the pBWA (V) HS-osgfp-ccdb-tnos vector, and the PaPDS gene coding region sequence is as shown in SEQ ID NO. 1.

8. The method according to claim 7, wherein the functional gene PaPDS regulating the color of apricot fruits is transformed into apricot seedlings or apricot fruits using GV3101 agrobacterium containing the recombinant pBWA (V) HS-osgfp-ccdb-tnos vector.

9. A method for lightening apricot fruit color is characterized in that recombinant engineering bacteria containing a recombinant vector and recombinant engineering bacteria containing a TRV I vector are used for jointly transforming apricot seedlings or apricot fruits, the recombinant vector is TRV II-GFP, paPDS gene coding region sequences are inserted between EcoRI and Xho I restriction enzyme sites of the TRV II-GFP vector, and PaPDS gene coding region sequences are shown as SEQ ID NO. 3.

10. The method of claim 9, wherein the recombinant engineering bacteria comprising the recombinant TRV II-GFP vector and the recombinant engineering bacteria comprising the TRV I vector are both GV3101 Agrobacterium.