Use of ppsec61gamma in regulating chlorophyll degradation in plants

Abstract

The application discloses use of PpSec61gamma in regulating chlorophyll degradation in plants. The application discloses that PpSec61gamma has the function of regulating chlorophyll degradation in plants, and when the expression level of PpSec61gamma gene in plants is increased, the color of plant organs becomes yellow, the content of chlorophyll is significantly reduced, and the expression level of genes related to chlorophyll degradation is significantly increased. The PpSec61gamma of the application can be used as a target for regulating the chlorophyll degradation pathway or the organ color trait of plants, and is applied to plant breeding, and therefore has important scientific significance and application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant biotechnology, in particular to the use of PpSec61γ in regulating chlorophyll degradation in plants. BACKGROUND

[0002] Prunus persica (L.) Batsch is an important economic fruit tree originated in China, with very high nutritional and ornamental value, and is one of the most representative deciduous fruit trees in genetic research of fruit trees and forest trees. China is the main producer and consumer of peaches, with the largest planting area and yield in the world. However, the current peach industry still faces the bottleneck problems of insufficient high-quality varieties, unstable quality, and insufficient ornamental value, which seriously restricts the development of mechanization, standardization, and multifunctional utilization.

[0003] With the expansion of peach planting area, leaf yellowing problem is increasingly common. Leaf yellowing leads to a decrease in chlorophyll content, reducing photosynthetic efficiency, which not only affects the growth of fruit trees and fruit yield, but also affects fruit quality, fruit tree life span, and ecological landscape value, and has become one of the key obstacles restricting the development of the peach industry. Yellowing phenomenon widely exists in various crops and fruit trees, such as wheat, corn, soybean, grape, apple, citrus, and cherry. Chlorophyll degradation is the direct cause of plant leaf yellowing and is an important physiological process in the redistribution of photosynthetic products to other tissues, fruit development and maturation, and aging process. Studies have shown that this process is regulated by multiple enzymes (such as SGR, PPH, PAO, NYC1, etc.) and multiple hormone signaling pathways (such as ethylene, abscisic acid, salicylic acid, and jasmonic acid). Although there have been some studies in model plants such as Arabidopsis, rice, and tomato, and fruit and vegetable crops, but in peaches, there are still very limited reports on the regulatory factors of chlorophyll degradation, especially in the early yellowing of leaves and the formation of ornamental value, which is almost blank.

[0004] The Sec61 complex is a highly conserved protein transport channel on the endoplasmic reticulum, composed of α, β, and γ subunits. Among them, the Sec61γ subunit has only gene annotation and expression information in plants, and its specific function has not been systematically studied. In non-plant systems, the Sec61 complex is believed to be involved in protein transport between the endoplasmic reticulum and the cytoplasm, degradation of misfolded proteins, etc. Some literature reports that GmSec61 gene plays a key role in the defense response of soybean to H. glycines (soybean cyst nematode) in plants; under drought stress, transgenic Arabidopsis overexpressing HvSec61α grows more vigorously than wild-type plants.

[0005] Up to now, no literature has reported the functional relationship between Sec61 gamma and chlorophyll degradation in peach or other plants. In-depth study of the function of this gene is expected to reveal new mechanisms of peach leaf yellowing and chlorophyll degradation, which not only helps to regulate the nutrition and growth of fruit trees, but also serves the breeding of new varieties of ornamental fruit trees, and expands their application value in landscaping and ecological agriculture. SUMMARY

[0006] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide the use of PpSec61 gamma in regulating chlorophyll degradation in plants.

[0007] To achieve the above-mentioned objects and other related objects, the present application is obtained by the following technical solutions.

[0008] The first aspect of the present application protects the use of PpSec61 gamma protein or its regulator in at least one of A1) to A4),

[0009] A1) regulating chlorophyll degradation in plants;

[0010] A2) regulating plant organ color traits;

[0011] A3) regulating plant fruit ripening;

[0012] A4) cultivating transgenic plants.

[0013] In some embodiments, the chlorophyll comprises chlorophyll a or chlorophyll b.

[0014] In some embodiments, the organ is selected from a leaf or a flower.

[0015] In some embodiments, the regulator comprises a PpSec61 gamma promoter and a PpSec61 gamma inhibitor; the PpSec61 gamma inhibitor refers to a substance that can inhibit the transcription or translation of the PpSec61 gamma gene, or can inhibit the expression or activity of the PpSec61 gamma protein; the PpSec61 gamma promoter refers to a substance that can promote the transcription or translation of the PpSec61 gamma gene, or can promote the expression or activity of the PpSec61 gamma protein.

[0016] In some specific embodiments, the PpSec61 gamma promoter comprises a PpSec61 gamma protein or a biological material related to the PpSec61 gamma gene, and the biological material is selected from one or both of a recombinant expression vector or a bioengineering bacteria.

[0017] In some specific embodiments, the PpSec61 gamma inhibitor is selected from one or more of a nucleic acid molecule, a nucleic acid construct, a lentivirus, an adeno-associated virus, a gene editing system, and a genetically engineered bacteria.

[0018] In some more specific embodiments, the nucleotide of the PpSec61y gene comprises the sequence as set forth in SEQ ID NO. 1.

[0019] Another aspect of the present application protects a method for modulating chlorophyll degradation in a plant, comprising any one of the following:

[0020] D1) introducing into the plant the PpSec61y protein as described above;

[0021] D2) introducing into the plant the modulator as described above.

[0022] Another aspect of the present application protects a method for modulating organ color trait in a plant, comprising any one of the following:

[0023] D1) introducing into the plant the PpSec61y protein as described above;

[0024] D2) introducing into the plant the modulator as described above.

[0025] Another aspect of the present application protects a method for breeding a transgenic plant, comprising any one of the following:

[0026] D1) introducing into the plant the PpSec61y protein as described above;

[0027] D2) introducing into the plant the modulator as described above;

[0028] obtaining the transgenic plant.

[0029] In some embodiments, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0030] Another aspect of the present application protects the use of PpSec61y as a target in the preparation or screening of products for modulating chlorophyll degradation in a plant.

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] 1) The present application cloned PpSec61y protein from Malus prunifolia, which has a molecular weight of about 9.0KD and contains 70 amino acids. Through subcellular localization experiments, the present application found that PpSec61y protein is specifically localized in the endoplasmic reticulum, which suggests that it has the characteristics of Sec61 channel transmembrane protein and may be involved in protein transmembrane transfer process.

[0033] 2) The transient expression experiment of the present application in plant leaves shows that overexpression of PpSec61y can cause the plant organ color to turn yellow, the chlorophyll content to decrease significantly, and the expression level of genes related to chlorophyll degradation to increase significantly. These results prove that PpSec61y has the function of regulating plant chlorophyll degradation.

[0034] 3) The genetic transformation experiment of the present application in the model plant Arabidopsis thaliana shows that the leaf organ color of PpSec61y overexpressing Arabidopsis thaliana turns yellow, the chlorophyll content decreases significantly, and the expression level of genes related to chlorophyll degradation increases significantly. These results show that the expression level of PpSec61y is significantly positively correlated with chlorophyll degradation, and has the active function of promoting chlorophyll metabolism.

[0035] 4) The present application discloses that PpSec61y is a key regulatory factor for regulating plant chlorophyll degradation and organ color traits. This discovery provides a new target for further studying the mechanism of Sec61 channel related genes in chlorophyll metabolism and plant organ color traits, and has important scientific significance and application value. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The figure shows the subcellular localization image of PpSec61y protein in tobacco leaves in Example 1 of the present application.

[0037] Figure 2 The figure shows the phenotype of plants and the expression of chlorophyll degradation related genes after transient overexpression of PpSec61y in the leaves of peaches in Example 2 of the present application.

[0038] Figure 3 The figure shows the phenotype of plants and the expression of chlorophyll degradation related genes after overexpression of PpSec61y in Arabidopsis thaliana in Example 3 of the present application. DETAILED DESCRIPTION

[0039] Foliage varieties refer to those plant varieties whose main ornamental value is the shape, color, texture, etc. of the leaves. The leaves of these plants usually have unique color, shape or texture, which can enrich the landscape color and level of garden landscapes such as parks, courtyards, street green spaces, etc., and can add beauty to indoor decorations such as living rooms, offices, balconies, etc. spaces, etc. For example, the ornamental plant Ginkgo biloba has very high ornamental value due to its unique leaf shape and golden yellow leaves in autumn; for example, the golden leaf elm, golden leaf locust tree and golden leaf metasequoia have bright colors and high ornamental and economic value.

[0040] The present application identifies and isolates a Sec61 gamma homolog gene significantly highly expressed in the naturally mutated leaf yellowing peach by transcriptome analysis of the peach and the naturally mutated leaf yellowing peach leaf, and names it PpSec61 gamma. Further, it is found by transient overexpression or genetic transformation that PpSec61 gamma expression is significantly up-regulated, and is significantly negatively correlated with chlorophyll content; its overexpression can reduce chlorophyll content, and presents an organ yellowing phenotype. It is preliminarily predicted that it can promote the transport of chlorophyll degradation related enzymes, accelerate the chlorophyll metabolic process, and thus cause the organ color to yellow early. Therefore, the gene and the protein coded thereby can be used for the research of the chlorophyll degradation pathway, and the improvement of the plant organ color trait; and can also be used for transgenic plant breeding and organ color trait marker breeding.

[0041] The first aspect of the present application protects the use of PpSec61 gamma protein or its modulator in at least one of A1) to A4),

[0042] A1) regulating plant chlorophyll degradation;

[0043] A2) regulating plant organ color trait;

[0044] A3) regulating plant fruit ripening;

[0045] A4) cultivating transgenic plants.

[0046] The amino acid sequence of the PpSec61 gamma protein comprises the sequence shown as SEQ ID NO. 2.

[0047] In some embodiments, the chlorophyll comprises chlorophyll a, chlorophyll b or carotenoid. It is found by overexpressing PpSec61 gamma in peach in the present application that the content of chlorophyll a, chlorophyll b and total chlorophyll in the plant can be significantly reduced; further, after overexpression in a model plant, the content of chlorophyll a, chlorophyll b, carotenoid and total chlorophyll in the model plant can also be significantly reduced. These results show that PpSec61 gamma plays an important role in regulating chlorophyll degradation.

[0048] In some embodiments, the organ comprises a leaf or a flower. It is found in the present application that after overexpressing PpSec61 gamma in peach, the organ can become yellow (yellowing); further, after overexpression in Arabidopsis, the Arabidopsis leaf can also become significantly yellow. It is shown that PpSec61 gamma has the function of regulating the plant organ color trait, and overexpression leads to organ yellowing, and inhibition of expression can possibly maintain organ stay green. The yellowing refers to the phenomenon that the plant leaf or other green tissues become yellow due to the decrease of chlorophyll content; the stay green refers to the phenomenon that the plant maintains green for a long time in the aging process due to slow or no degradation of chlorophyll.

[0049] The present application finds that overexpression of PpSec61y not only leads to a decrease in chlorophyll content, but also leads to a significant increase in the expression level of genes related to chlorophyll degradation. Therefore, PpSec61y can have a role in regulating fruit ripening, and overexpression of PpSec61y can lead to premature fruit ripening.

[0050] In some embodiments, the regulatory agent comprises a PpSec61y promoter and a PpSec61y inhibitor; the PpSec61y inhibitor refers to a substance that can inhibit the transcription or translation of the PpSec61y gene, or can inhibit the expression or activity of the PpSec61y protein; the PpSec61y promoter refers to a substance that can promote the transcription or translation of the PpSec61y gene, or can promote the expression or activity of the PpSec61y protein.

[0051] In some specific embodiments, the PpSec61y promoter comprises a PpSec61y protein.

[0052] In some specific embodiments, the PpSec61y promoter comprises a biological material related to the PpSec61y gene, and the biological material is selected from one or both of a recombinant expression vector or a bioengineered bacteria. The nucleotides of the PpSec61y gene comprise the sequence shown in SEQ ID NO. 1.

[0053] Further, the recombinant expression vector is formed after the PpSec61y gene is connected to a starting plasmid. The starting plasmid refers to a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vectors well known in the art. In general, any plasmid and vector can be used as long as it can replicate and be stable in the host. An important feature of the starting plasmid is that it usually contains a replication origin, a promoter, a marker gene, and a translation control element. The starting plasmid in the present application is not limited to the T-Vector pMD TM 19-PpSec61y, pHB-GFP. Methods well known to those skilled in the art can be used to construct an expression vector containing the nucleotide sequence encoding PpSec61y and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0054] Further, the bioengineered bacteria is obtained by transfecting a host with the recombinant expression vector described above. The bioengineered bacteria is cultured to allow it to express the PpSec61y protein. The host can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples include: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, etc. Not limited to the E. coli DH5a, Agrobacterium GV3101 used in the present application.

[0055] In some embodiments, the PpSec61y inhibitor is selected from one or more of a nucleic acid molecule, a nucleic acid construct, a lentivirus, an adeno-associated virus, a gene editing system, and a genetically engineered bacteria. The PpSec61y inhibitor inhibits or reduces PpSec61y expression to regulate plant chlorophyll reduction. For example, the PpSec61y can be partially inhibited, i.e., reducing the expression and / or function of PpSec61y; or can be completely inhibited, i.e., substantially completely eliminating the expression and / or function of PpSec61y.

[0056] Further, the inhibition or reduction of PpSec61y expression can be achieved by gene knockout, by gene silencing, or by homologous recombination. The gene knockout refers to inactivation of a specific target gene by changing the DNA sequence, and the knockout includes knocking out the PpSec61y gene using DNA homologous recombination technology, Cre / LoxP technology, or a CRISPR / Cas9 editing system. The gene silencing refers to a phenomenon of not expressing or lowly expressing a gene without damaging the original DNA. Gene silencing can occur at two levels, one is gene silencing at the transcription level caused by DNA methylation, heterochromatinization, and position effect, etc., and the other is post-transcriptional gene silencing, i.e., inactivating a gene by specifically inhibiting a target RNA after gene transcription, including antisense RNA, co-suppression, RNA interference (RNAi), and microRNA (miRNA)-mediated translation inhibition, etc.

[0057] Further, the PpSec61y inhibitor is a nucleic acid molecule, and the nucleic acid molecule is a nucleic acid molecule targeting the PpSec61y gene, such as siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0058] Further, the PpSec61y inhibitor is a nucleic acid construct comprising a nucleic acid molecule targeting PpSec61y gene, the sequence of which can be inserted into a vector to form a recombinant vector. The vector refers to a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus or other vectors well known in the art. In general, any plasmid and vector can be used as long as it can replicate and be stable in the host. The expression vector in the present application is not limited to CRISPR / Cas9 vector.

[0059] Further, the PpSec61y inhibitor is a gene editing system selected from one or more of the following: Cre-Loxp system, Flp-FRT system, Dre-ROX system, vCre-vloxp system, sCre-sloxp system, CRISPR / Cas9 system, CRISPR / Cas12 system, CRISPR / Cas derived single base editing system.

[0060] In some other embodiments, the PpSec61y inhibitor is a genetically engineered bacterium obtained by transfecting a host cell with the nucleic acid construct. The host cell can be a yeast, a bacterium, an algae or a fungus. The bacterium can be a gram-positive bacterium or a gram-negative bacterium. The gram-negative bacterium can be E. coli, Agrobacterium.

[0061] Another aspect of the present application protects a method for modulating chlorophyll degradation in a plant, comprising any one of the following:

[0062] D1) introducing into the plant a PpSec61y protein as described above;

[0063] D2) introducing into the plant a modulator as described above.

[0064] Another aspect of the present application protects a method for modulating organ color trait in a plant, comprising any one of the following:

[0065] D1) introducing into the plant a PpSec61y protein as described above;

[0066] D2) introducing into the plant a modulator as described above.

[0067] Another aspect of the present application protects a method for breeding a transgenic plant, comprising one of the following:

[0068] D1) introducing into the plant a PpSec61y protein as described above;

[0069] D2) introducing into the plant a modulator as described above;

[0070] obtaining the transgenic plant.

[0071] In some embodiments, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0072] Another aspect of the present application protects the use of PpSec61y as a target in the preparation or screening of products for regulating chlorophyll degradation in plants.

[0073] The use of PpSec61y as a target in the preparation or screening of products for regulating chlorophyll degradation in plants in the present application refers to using PpSec61y gene or PpSec61y protein as an object of action, screening products to find products that can promote or inhibit the expression level of PpSec61y gene, promote or inhibit the expression or activity of PpSec61y protein as products for regulating chlorophyll degradation in plants.

[0074] The products in the present application include molecules that can specifically promote the transcription or translation of PpSec61y gene, or can specifically promote the expression or activity of PpSec61y protein, so that the products can increase the expression level of PpSec61y gene in plants, reduce chlorophyll content, promote organ yellowing, and increase the expression level of chlorophyll degradation related genes.

[0075] The products in the present application include molecules that can specifically inhibit the transcription or translation of PpSec61y gene, or can specifically inhibit the expression or activity of PpSec61y protein, so that the products can reduce the expression level of PpSec61y gene in plants, increase chlorophyll content, promote organ stay green, and reduce the expression level of chlorophyll degradation related genes.

[0076] The effective components of the products in the present application can be only PpSec61y inhibitors or PpSec61y promoters, or can include other molecules that can have inhibitory or promoting effects. In the present application, the products can be single-component substances or multi-component substances. In the present application, the form of the products is not limited, and can be various forms of substances such as solids, liquids, gels, semi-liquids, and aerosols.

[0077] The products in the present application include but are not limited to nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins, or interfering lentiviruses. The form of the products is not limited, and can be various forms of substances such as solids, liquids, gels, semi-liquids, and aerosols.

[0078] In some embodiments, the plant is a dicotyledon or a monocotyledon. The dicotyledon includes but is not limited to peach (Prunus persica (L.) Batsch), apple (Malus domestica), pear (Pyrus spp.), plum (Prunus salicina), cherry (Prunus avium), grape (Vitis spp.), strawberry (Fragaria x ananassa), citrus (Citrus spp.), apricot (Prunus armeniaca), blueberry (Vaccinium spp.), Arabidopsis thaliana, alfalfa (Medicago sativa), petunia (Petunia spp.), cotton (Gossypium spp.), and castor-oil plant (Ricinus communis). The monocotyledon includes but is not limited to banana (Musa spp.), pineapple (Ananas comosus), mango (Mangifera indica), longan (Dimocarpus longan), litchi (Litchi chinensis), wheat (Triticum aestivum), corn (Zea mays), rice (Oryza sativa), and oat (Avena sativa).

[0079] In some specific embodiments, the plant is peach (Prunus persica (L.) Batsch). Peach and Prunus persica (L.) Batsch can be used interchangeably in the present application.

[0080] In the present application, the overexpression vector of PpSec61γ (pHB-PpSec61γ-Flag) is constructed by homologous recombination.

[0081] The overexpression vector pHB-PpSec61γ-Flag of the present application is obtained by homologous recombination. The enzyme cutting sites of BamHI are introduced into the primers, and the corresponding vectors are cut by these enzyme cutting sites. The obtained overexpression vector is obtained by transformation using homologous recombination enzyme.

[0082] A more specific embodiment of the present application is:

[0083] (1) Recombinant expression vector (intermediate vector) T-Vector pMD TM 19-Construction of PpSec61γ

[0084] a) The total RNA of the leaf of naturally mutated peach is extracted, and then the cDNA is obtained by reverse transcription;

[0085] b) Design primers PpSec61y-F1 and PpSec61y-R1, and perform PCR reaction with cDNA as template to obtain PpSec61y gene sequence;

[0086] PpSec61y-F1 primer: 5'-ATGGACGCCATAGAT-3' (SEQ ID NO. 3)

[0087] PpSec61y-R1 primer: 5'-AACAGATCCAACAAT-3' (SEQ ID NO. 4)

[0088] c) Connect PpSec61y with T-Vector pMD TM 19 (Simple) to obtain recombinant expression vector (intermediate vector) T-Vector pMD TM 19-PpSec61y.

[0089] (2) Construction of overexpression vector pHB-PpSec61y-Flag

[0090] Take recombinant expression vector (intermediate vector) T-Vector pMD TM 19-PpSec61y as template, use primers PpSec61y-F2 and PpSec61y-R2 to perform PCR amplification to obtain PpSec61y gene fragment with BamHI enzyme cutting site, use homologous recombination method to connect PpSec61y gene fragment with BamHI enzyme cutting site with pHB-Flag vector treated with BamHI enzyme to obtain overexpression vector pHB-PpSec61y-Flag.

[0091] PpSec61y-F2: 5'-ttcctgcagcccgggggatccATGGACGCCATAGAT-3' (SEQ ID NO: 5)

[0092] PpSec61y-R2: 5'-gataccgtcactagtggatccAACAGATCCAACAAT-3' (SEQ ID NO: 6)

[0093] In one embodiment of the present application, the overexpression vector pHB-PpSec61y-Flag was injected into the leaves of P. persica by Agrobacterium-mediated transformation, and the empty vector pHB-Flag was used as a control. On the sixth day, the contents of chlorophyll a, chlorophyll b and total chlorophyll in the treatment group and the control group were determined, and the relative expression levels of PpSec61y and key genes related to chlorophyll degradation were detected by qRT-PCR. Compared with the empty vector control, PpSec61y overexpression treatment significantly promoted chlorophyll degradation, manifested as a greener leaf color and an increased degree of yellowing; the contents of chlorophyll a and chlorophyll b in the overexpression group were significantly decreased, and the total chlorophyll content was significantly lower than that of the control; at the same time, the expression levels of key degradation genes such as AtNYC1, AtNOL, AtHCAR, AtCLH1, AtSGR1, AtPPH, AtPAO and AtRCCR were significantly up-regulated.

[0094] In one embodiment of the present application, the constructed overexpression vector pHB-PpSec61y-Flag was transformed into Agrobacterium GV3101, and then transformed into Arabidopsis (Col-0) by floral dip method, and a plurality of T2 generation transgenic plants were obtained by screening. The positive plants stably expressing PpSec61y were screened by RT-PCR and qRT-PCR detection, and were used for subsequent functional analysis. The results of phenotype observation and physiological determination of the T2 generation transgenic plants showed that, compared with the wild type, the leaves of the PpSec61y overexpression plants showed obvious yellowing phenotype; the contents of chlorophyll a, chlorophyll b and total chlorophyll in the transgenic plants were significantly decreased, which was consistent with the phenotype; the qRT-PCR results showed that the expression level of PpSec61y was significantly up-regulated, and the expression levels of key enzyme genes related to chlorophyll degradation were also significantly increased.

[0095] These results collectively indicate that PpSec61y has the function of regulating plant chlorophyll degradation, and when the expression level of PpSec61y gene in plants is increased, the color of plant organs becomes yellow, the content of chlorophyll is significantly reduced, and the expression levels of genes related to chlorophyll degradation are significantly increased. The present application finds that PpSec61y can be used as a target for regulating plant chlorophyll degradation pathway or organ color trait, and has important scientific significance and application value in plant breeding.

[0096] The embodiments of the present application are described below by specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure of the specification. The present application can also be implemented or applied by different specific embodiments, and various modifications or changes can be made to the details in the specification without departing from the spirit of the present application.

[0097] Before particular embodiments of the present application are described, it is to be understood that the application is not limited to the particular specific embodiments described; it is also to be understood that the terminology used in the description is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present application. In this specification and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0098] When a numerical range is given in the embodiments, it is understood that, unless otherwise stated by the present application, both the end points of the numerical range and any number between the two end points can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, devices, materials used in the embodiments, any method, device and material of the prior art similar or equivalent to those described in the embodiments of the present application can also be used to implement the present application according to the knowledge of the prior art possessed by those skilled in the art and the description of the present application.

[0099] In the following embodiments of the present application, Prunus persica (L.) Batsch is the most widely used cultivated peach rootstock in China, which can be obtained commercially.

[0100] Example 1 Cloning of PpSec61y gene

[0101] In this embodiment, the cloning of PpSec61y gene is carried out, which includes the following:

[0102] 1.1 Obtaining of plant material

[0103] The present applicant found that when planting Prunus persica (L.) Batsch, there was a natural mutant strain with yellow leaf color, so 0.1 g of its tender leaves was placed in a 1.5 mL centrifuge tube, rapidly cooled in liquid nitrogen, and then ground into powder at low temperature for standby, which was used for RNA extraction.

[0104] 1.2 Extraction of RNA

[0105] The powder obtained in step 1.1 was used as the object, and the total RNA was extracted according to the operating instructions of the RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit (purchased from Aidley, item number: RN34).

[0106] 1.3 Reverse transcription of RNA to cDNA

[0107] The total RNA obtained according to step 1.2 was used as a template, and a First Strand cDNA Synthesis Kit (Cat: RN09) provided by Aidlab Biotechnologies Co., Ltd. was used to synthesize cDNA according to the instructions.

[0108] 1.4 Gene cloning

[0109] According to the nucleotide sequence of the PpSec61y gene of peach, the upstream primer and the downstream primer for amplifying the coding region were designed as follows:

[0110] PpSec61y-F1 primer: 5'-ATGGACGCCATAGAT-3' (SEQ ID NO: 3)

[0111] PpSec61y-R1 primer: 5'-AACAGATCCAACAAT-3' (SEQ ID NO: 4)

[0112] The cDNA obtained in step 1.3 was used as a template, and PCR amplification was performed according to the following reaction system and reaction procedure to obtain a PCR product.

[0113] The PCR reaction system was as follows: PrimeSTAR Max Premix 20 μL, primers PpSec61y-F1 and PpSec61y-R1 each 2 μL, 1 μL of cDNA diluted 20 times, and finally sterile distilled water was added to make up to 40 μL.

[0114] The PCR reaction procedure was as follows: 98°C pre-denaturation for 1 min; 98°C denaturation for 10 s, 60°C annealing for 30 s, 72°C extension for 10 s, a total of 32 cycles of denaturation to extension; 72°C for 10 min for sufficient extension, and 4°C storage.

[0115] 1.5 Vector construction

[0116] The PCR product obtained in step 1.4 was subjected to agarose gel electrophoresis. The size of the PCR amplification product was 210 bp, and the target band was cut and recovered according to the operation instructions of the gel recovery kit (Shenguo Bioengineering, Cat: B518131).

[0117] The recovered target fragment was ligated into T-Vector pMD TM 19 vector (TaKaRa) to obtain T-Vector pMD TM 19-PpSec61y intermediate vector, and then the T-Vector pMD TM19-PpSec61γ intermediate vector is transformed into E. coli competent cells DH5α by chemical transformation method, coated on LB plate (containing 100 mg / L ampicillin), and cultured at 37℃ overnight.

[0118] PCR identification is performed on the overnight colonies to obtain positive clones, plasmid of the positive clone is extracted, and the full-length gene sequence of PpSec61γ is obtained after sequencing, as shown in SEQ ID NO. 1; the amino acid sequence is shown in SEQ ID NO. 2.

[0119] The sequence shown in SEQ ID NO. 1 is specifically as follows:

[0120] ATGGACGCCATAGATTCAGTAGTGGATCCGCTCAGAGAGTTCTCTAAGGACAGCATTCGGCTCGTCAAGCGGTGCCACAAGCCAGATCGCAAAGAGTTCACGAAGGTCGCGCTCCGTACAGCGATCGGGTTCGTAGTGATGGGATTCGTTGGGTTCTTCGTGAAGCTGATCTTCATCCCCATCAACAACATCATTGTTGGATCTGTTTAG

[0121] The sequence shown in SEQ ID NO. 2 is specifically as follows:

[0122] MDAIDSVVDPLREFSKDSIRLVKRCHKPDRKEFTKVALRTAIGFVVMGFVGFFVKLIFIPINNIIVGSV*

[0123] The specific chemical transformation method is as follows:

[0124] The intermediate vector and E. coli competent cells are mixed and placed on ice for 30 min; 42℃ hot water bath for 60 s, ice bath for 2 min; 1 mL of LB liquid medium without antibiotics is added; placed at 37℃, 220 rpm shaking incubator for 30 min; centrifuged at 6,000 rpm for 2-3 min, the supernatant was discarded, mixed by sucking and beating, and coated on LB plate with corresponding antibiotics; inverted in a 37℃ incubator for 12-16 hours.

[0125] Example 2: Construction of overexpression vector pHB-PpSec61γ-Flag by homologous recombination method

[0126] In this embodiment, the overexpression vector pHB-PpSec61γ-Flag is constructed by homologous recombination method, including the following:

[0127] 2.1 Introducing enzyme cutting site in PpSec61γ gene

[0128] T-Vector pMD18-T containing PpSec61y gene obtained in step 1.5 of Example 1 TM 19- The intermediate vector of PpSec61y as a template, a primer for introducing a BamHI restriction site was designed (overexpression vector).

[0129] The primers were as follows:

[0130] PpSec61y-F2: 5'- ATGGACGCCATAGA-3' (SEQ ID NO. 5) ttcctgcagcccgggggatcc PpSec61y-R2: 5'- AACAGATCCAACAAT-3' (SEQ ID NO. 6)

[0131] PpSec61y-R2: 5'- AACAGATCCAACAAT-3' (SEQ ID NO. 6) gataccgtcactagtggatcc PpSec61y-R2: 5'- AACAGATCCAACAAT-3' (SEQ ID NO. 6)

[0132] Note: The lowercase base sequence in the primer sequence is the sequence containing the sequence on the vector and the restriction site (BamHI site), which is artificially introduced for the construction of the vector and does not belong to the sequence of PpSec61y gene.

[0133] Then, PCR amplification was performed according to the following PCR reaction system to obtain the PpSec61y gene product containing the restriction site.

[0134] The PCR reaction system was as follows: PrimeSTAR Max Premix 20 μL, primers PpSec61y-F2 and PpSec61y-R2 each 2 μL, template 1 μL, and finally sterile distilled water was added to make up to 40 μL.

[0135] The PCR reaction program was as follows: 98°C pre-denaturation for 1 min; 98°C denaturation for 10 s, 60°C annealing for 30 s, 72°C extension for 10 s, a total of 32 cycles from denaturation to extension; 72°C for 10 min for sufficient extension, and 4°C for storage.

[0136] The PCR amplification product was purified to recover the PpSec61y gene fragment containing the restriction site, and then subjected to restriction enzyme digestion to obtain the PpSec61y gene fragment.

[0137] 2.2 Enzyme digestion reaction

[0138] The pHB-Flag (the pHB-Flag vector is obtained by removing the FvCMB1L gene from pHB-FvCMB1L-Flag by enzyme digestion, and the pHB-FvCMB1L-Flag is described in the literature: XIAO K. ANAC transcription factor and a MADS-box protein antagonistically regulate sucrose accumulation in strawberry receptacles. Plant Physiology. 2025, the expression vector is digested with restriction enzyme BamHI.

[0139] The enzyme digestion reaction system is as follows: 10x Buffer 5 μL, pHB-Flag plasmid 4 μL, BamHI and XhoI each 1 μL, ddH2O to 50 μL, reaction conditions: 37°C enzyme digestion for 2 h, 85°C enzyme inactivation for 10 min, ready for use.

[0140] 2.3 Connection reaction

[0141] The PpSec61γ gene fragment of step 2.1 in this example and the digested vector of step 2.2 are then connected according to the following system:

[0142] Table 1. Connection reaction system

[0143]

[0144]

[0145] After mixing the above components, place them at 37°C for 45 min of ligation, and obtain the recombinant expression vector by ligation.

[0146] 2.4 Screening of positive clone strains and sequencing

[0147] The recombinant expression vector obtained in step 2.3 is transformed into E. coli competent cells JM107 using the same transformation method as in Example 1, and plated on LB plates containing 50 mg / L kanamycin and incubated at 37°C overnight.

[0148] The colonies after overnight culture are identified by PCR to obtain positive clones, the positive clones are shaken, and the plasmid is extracted and then verified by sequencing to confirm the correctness of the gene sequence, and the overexpression vector pHB-PpSec61γ-Flag is obtained.

[0149] Example 3 Subcellular localization of PpSec61γ protein

[0150] The expression vector used in the present embodiment is pMDC83, which is used to construct a C-terminal fusion expression vector of PpSec61 γ and green fluorescent protein (GFP) for subcellular localization research. The construction method of the vector is as follows: using the intermediate vector T-Vector pMDTM19-PpSec61 γ constructed in Embodiment 1 as a template, the primers are as follows:

[0151] PpSec61 γ-SpeI-F3: 5'-ACTAGTATGGACGCCATAGATTC-3' (SEQ ID NO. 39)

[0152] PpSec61 γ-AscI-R3: 5'-GGCGCGCCTAACAGATCCAACAAT-3' (SEQ ID NO. 40)

[0153] After the target gene fragment is obtained by PCR amplification, it is cut with SpeI and AscI respectively, and is connected with the expression vector pMDC83 which is also treated with the same enzymes, to construct the fusion expression vector pMDC83-PpSec61 γ-GFP. The recombinant plasmid is transformed into E. coli DH5α, resistance screening and sequencing verification are performed, and after confirming that the sequence is correct, it is used for subsequent subcellular localization experiments. The overexpression vector pMDC83-PpSec61 γ-GFP which is correct after sequencing is transformed into Agrobacterium GV3101, and further used for subcellular localization research in the leaves of Nicotiana benthamiana, including the following: 3.1 Transformation of overexpression vector into Agrobacterium

[0154] Take 50 μL of Agrobacterium GV3101 competent cells in one branch on ice, and after it is dissolved, add 7 μL of the overexpression vector pMDC83-PpSec61 γ-GFP which is correct after sequencing in step 2.4 of Embodiment 2, mix gently with a pipette, and then place it on ice for 5 min, then freeze it in liquid nitrogen for 5 min, and then thaw it at 37°C for 5 min, and then place it on ice for 5 min. Subsequently, 1 mL of YEP liquid medium without antibiotics is added, and the culture is incubated at 28°C on a shaker at 220 rpm for 3 h, and then centrifuged at 6000 rpm for 5 min at room temperature, and then the bacterial pellet is resuspended by pipetting after removing 900 μL of supernatant. Finally, 100 μL of bacterial solution is spread on YEP solid medium containing 50 mg / L Rif (rifampicin) and 50 mg / L Kan (kanamycin), and incubated at 30°C for 3 d, and single colonies are picked for PCR detection, and positive clones are selected for shaking, to obtain Agrobacterium GV3101 containing the overexpression vector.

[0155] 3.2 Subcellular localization of PpSec61 γ protein

[0156] The Agrobacterium GV3101 containing the overexpression vector was cultured in YEP + 50 mg / L Kan (kanamycin) + 50 mg / L Rif (rifampicin) medium, and shaken to OD 600 = 0.6-0.8, 28℃6000rpm, 20min centrifugal collection of bacterial bodies, resuspend the bacterial bodies with the invasion buffer containing 10mM 2-morpholinoethanesulfonic acid (MES), 100pM acetyl-syringone (AS) and 10mM MgCl2 (PH 5.6), activate the bacterial bodies at 28℃ in the dark for 2h, obtain the Agrobacterium liquid containing the overexpression vector, marked as P35S::PpSec61γ-GFP. The endoplasmic reticulum localization fluorescent marker vector uses pCAMBIA1300-35S-ER-mCherry-HDEL.

[0157] Meanwhile, pMDC83::GFP was used as a negative control.

[0158] Then, P35S::PpSec61γ-GFP or pMDC83::GFP and P35S::ER-mCherry were respectively infiltrated into the leaves of N. benthamiana using a syringe without a needle. After incubation in the dark for 36-40 hours, the tobacco leaf samples were examined by confocal microscopy (Leica TCS SP5II) to detect the expression of various fluorescent proteins.

[0159] Figure 1 The confocal microscope image of PpSec61γ protein in this example is shown in the figure. Among them, GFP is the green fluorescent signal image; mCherry is the red fluorescent signal image; BF is the bright field image; Merge is the superimposed image of the green and red fluorescent images and the bright field image.

[0160] From Figure 1 It can be seen that the PpSec61γ-GFP fusion protein is located in the endoplasmic reticulum, supporting its basic function of participating in protein transport.

[0161] Example 4 Agrobacterium-mediated transient injection of PpSec61γ gene into peach leaves

[0162] In this example, the overexpression vector pHB-PpSec61γ-Flag with correct sequencing in Example 2 was transformed into Agrobacterium GV3101, and then further injected into the leaves of peach by transient injection. The leaves were photographed and observed, and the physiological indicators and chlorophyll degradation gene expression of the leaves were detected, including the following:

[0163] 4.1 Transient injection of peach leaves

[0164] The agrobacterium strain containing overexpression vector pHB-PpSec61y-Flag was activated and added to liquid YEP medium containing 50 mg / L Rif (rifampicin) and 50 mg / L Kan (kanamycin), and shaken to OD 600 = 0.6-0.8, centrifuged at room temperature at 6000 rpm for 15 min to collect the bacteria, resuspended the bacteria in MS (MS 4.43 g / L, sucrose 30 g / L) buffer containing 100 mM / L AS (pH 5.6), and activated the bacteria at 28°C in the dark for 2 h to obtain the infection bacteria liquid containing overexpression vector pHB-PpSec61y-Flag.

[0165] The leaves of peach with the same growth environment, similar attachment position, and about 10-15 days after leaf expansion were selected for transient injection and labeled as PpSec61y-OE group. Specifically:

[0166] A 1 mL syringe was used to inject the leaves of peach on the back of the needle, and the leaves were immersed to obtain the leaves into which the overexpression vector pHB-PpSec61y-Flag was transferred.

[0167] At the same time, the EV-OE group was set up, and the empty vector pHB-Flag bacteria liquid was injected into the leaves of peach as a negative control (or empty control group), and the operation was the same as above.

[0168] 4.2 Observation of peach leaf phenotype and determination of chlorophyll and carotenoid content after transient injection

[0169] 0.2 g of leaf sample was weighed, cut into pieces and placed in a mortar, and a small amount of quartz sand and calcium carbonate powder was added. 10 mL of 95% ethanol was added and ground until the leaves were completely homogenized. The homogenate was allowed to stand for 3-5 minutes. The extract was filtered through filter paper into a 25 mL conical flask, and 95% ethanol was added to a final volume of 25 mL to ensure that the filtrate was clear and free of impurities. The absorbance values were measured at 470 nm, 649 nm and 665 nm wavelengths using a 1 cm path length cuvette, with 95% ethanol as the blank control. The concentrations of chlorophyll a (ChI a), chlorophyll b (ChI b), total chlorophyll (Total ChI) and carotenoid (Carotemoid) in the extract were calculated according to the following formula:

[0170] Chlorophyll a (mg / L) = 13.95 x A 665 -6.88 x A 649

[0171] Chlorophyll b (mg / L) = 24.96 x A 649 -7.32 x A 665

[0172] Total chlorophyll (mg / L) = chlorophyll a + chlorophyll b

[0173] Carotenoid (mg / L) = (1000 x A 470 -2.05 x chlorophyll a - 114.8 x chlorophyll b) / 245

[0174] The content of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid in the extraction solution was calculated according to the following formula to obtain the content in the leaf sample (mg.g -1 FW) respectively.

[0175] Content (mg / g) = (C x V x N) / 1000 x W

[0176] In the formula: C is the concentration of chlorophyll a, chlorophyll b, total chlorophyll or carotenoid in the extraction solution (mg / L); V is the volume of the extraction solution (mL); N is the dilution multiple; W is the mass of the leaf sample (g).

[0177] 4.3 Study on genes related to chlorophyll degradation in peach leaves after transient injection

[0178] 1) Extraction of RNA

[0179] According to the operating instructions of the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (purchased from Aidlab, item number: RN34), total RNA was extracted.

[0180] 2) Obtaining of cDNA

[0181] Using the total RNA obtained in step 4.3 1) as a template, the First Strand cDNA Synthesis Kit (item number: RN09) provided by Aidlab Biotechnologies Co., Ltd. was used to obtain cDNA according to the instructions.

[0182] 3) Fluorescent quantitative PCR analysis

[0183] Specific primers were designed to analyze the relative expression of genes related to chlorophyll degradation in peach leaves, such as PpNYC1, PpHCAR, PpCLH1, PpSGR, PpPPH, PpPAO and PpRCCR.

[0184] According to the nucleotide sequences of chlorophyll degradation related genes PpNYC1, PpHCAR, PpCLH1, PpSGR, PpPPH, PpPAO and PpRCCR, the Primer premier v6.0 primer design software was used to design specific primers for quantitative analysis in Real-time PCR.

[0185] Primers for PpNYC1 gene are as follows:

[0186] Forward primer PpNYC1-F: 5'-AGCTGGTTCTGACGGAAGAC-3' (SEQ ID NO. 7)

[0187] Reverse primer PpNYC1-R: 5'-ACTTCGCCTGCTTCGTAGTT-3' (SEQ ID NO. 8)

[0188] Primers for PpHCAR gene are as follows:

[0189] Forward primer PpHCAR-F: 5'-CGTGGAAGAGAGATGCTCAGT-3' (SEQ ID NO. 9)

[0190] Reverse primer PpHCAR-R: 5'-AACTTTGGGGCAGGTTCAGG-3' (SEQ ID NO. 10)

[0191] Primers for PpCLH1 gene are as follows:

[0192] Forward primer PpCLH1-F: 5'-CCTCCAATCCCTGCTACCTG-3' (SEQ ID NO. 11)

[0193] Reverse primer PpCLH1-R: 5'-GCCACAGGGTCAATGCCTAT-3' (SEQ ID NO. 12)

[0194] Primers for PpSGR gene are as follows:

[0195] Forward primer PpSGR-F: 5'-GGCTATTTGGGCCAGCCATA-3' (SEQ ID NO. 13)

[0196] Reverse primer PpSGR-R: 5'-ACCACCCTTGCAACTGAGAG-3' (SEQ ID NO. 14)

[0197] Primers for PpPPH gene are as follows:

[0198] Forward primer PpPPH-F: 5'-CTTGTTGTGCAGCAAAGGCA-3' (SEQ ID NO. 15)

[0199] Reverse primer PpPPH-R: 5'-AATGGAGGCACCGAAACCAT-3' (SEQ ID NO. 16)

[0200] The primers of PpPAO gene are as follows:

[0201] Forward primer PpPAO-F: 5'-ACAGAGGTGAAAGGGTCGTG-3' (SEQ ID NO. 17)

[0202] Reverse primer PpPAO-R: 5'-GCAACGATCCTCGTAACAGC-3' (SEQ ID NO. 18)

[0203] The primers of PpRCCR gene are as follows:

[0204] Forward primer PpRCCR-F: 5'-GTCCTCCACCCTGACTACCT-3' (SEQ ID NO. 19)

[0205] Reverse primer PpRCCR-R: 5'-TCAGCCTCTGCTTCAATCCG-3' (SEQ ID NO. 20)

[0206] The reference gene is PpActin gene, and the primers are as follows:

[0207] PpActin-F: 5'-GTTATTCTTCATCGGCGTCTTCG-3' (SEQ ID NO. 21)

[0208] PpActin-R: 5'-CTTCACCATTCCAGTTCCATTGTC-3' (SEQ ID NO. 22)

[0209] Fluorescence quantitative analysis: The cDNA synthesized in 2) in step 4.3 is used as a template, and the specific primers of genes PpNYC1, PpHCAR, PpCLH1, PpSGR, PpPPH, PpPAO, PpRCCR and the reference gene PpActin are used for amplification for fluorescence quantitative PCR analysis. The Real-time PCR reaction is performed on a CFX Connect "Optics Module real-time quantitative instrument. The reaction system is 20 μL, and the reaction adopts two-step method, 95°C denaturation for 2 min, followed by 40 cycles: 95°C for 30 s; 60°C for 15 s. After the reaction, the data is exported to an Excel table, and the relative expression of each gene is calculated by using the formula 2 -△△Ct -ΔCt.

[0210] Figure 2 The figure of the phenotype and the expression of chlorophyll degradation metabolism related genes after transient overexpression of PpSec61γ gene in the leaves of peaches in this embodiment.

[0211] Figure 2Fig. 5A is a chart showing the morphological phenotype of peach leaf injected with PpSec61y overexpression treatment group (PpSec61y-OE) and empty vector control group (EV-OE) after 6 days.

[0212] From Fig. 5A, it can be seen that the leaves of the PpSec61y-OE group are obviously yellow, and the leaves of the EV-OE group remain green, indicating that overexpression of PpSec61y can induce chlorophyll degradation. Figure 2

[0213] Fig. 5B is a chart showing the changes in pigment content in the leaves of peach after transient overexpression of PpSec61y gene. Figure 2 From Fig. 5B, it can be seen that the contents of chlorophyll a, chlorophyll b and total chlorophyll in the PpSec61y-OE group are significantly lower than those in the EV-OE group, while the content of carotenoid does not change significantly.

[0214] Figure 2 Fig. 5C is a chart showing the expression level of chlorophyll degradation related genes in the leaves of peach after transient overexpression of PpSec61y gene.

[0215] Figure 2 From Fig. 5C, it can be seen that overexpression of PpSec61y significantly up-regulates the expression levels of PpNYC1, PpHCAR, PpCLH1, PpSGR, PpPPH, PpPAO and PpRCCR genes, further indicating that PpSec61y gene mediates the degradation of chlorophyll by regulating the degradation pathway.

[0216] Figure 2

[0217] Example 5 Stable expression and functional verification of PpSec61y gene in Arabidopsis thaliana

[0218] In this embodiment, Arabidopsis thaliana is used as a model plant to express and verify the function of PpSec61y gene. The following is included:

[0219] 5.1 Obtaining of transgenic Arabidopsis thaliana plants

[0220] The overexpression vector pHB-PpSec61y-Flag constructed and verified by sequencing in Example 2 is transformed into the competent cells of Agrobacterium GV3101.

[0221] ​​​The wild type Col-0 seed of Arabidopsis thaliana was surface sterilized, and then sowed on 1 / 2MS medium after vernalization for 3 days at 4℃. The seedlings were then transplanted into pots (nutrient soil: vermiculite = 1:1) under constant light intensity of 1500 lx, 16 h light, 22℃, and 85% relative humidity. When the plants grew to 3 cm in height, the apical inflorescences were removed to stimulate the growth of the leaf inflorescences. The plants were used for transformation 5-7 days after the apical inflorescences were removed. The plants were watered thoroughly the day before transformation, and the siliques and open flowers were removed. The Agrobacterium containing the plant expression vector was inoculated into 5 mL YEP (containing Kana, Rif, TET) liquid medium, and cultured at 28℃ for 24-36 h. The culture was then transferred into 200 mL YEP medium at a ratio of 1:50, and shaken until the OD 600 was 1.2-1.6. The bacteria were collected by centrifugation at 6000 rpm for 15 min, and the precipitate was suspended in an appropriate amount of infiltration medium (sucrose 5%, Silwet L-770 0.02%) to an OD 600 of 0.6-0.8. The Agrobacterium suspension was poured into a beaker, and the flowers of Arabidopsis were immersed in the suspension for 3-5 min, with the part above the rosette submerged in the liquid. The plants were placed horizontally in the dark for 16-24 h, and then placed normally after being taken out. The loose flower buds were tied. Normal watering was performed until the plants matured. The seeds were collected after the seeds matured. After obtaining the T0 generation seeds, single plants were screened on 1 / 2MS medium containing 50 mg / L kanamycin at a segregation ratio of 3:1. The surviving seedlings were transplanted into soil for further culture, and the T1 generation seeds were obtained by single plant seed collection. The T1 generation seeds were again screened as described above at a segregation ratio of 3:1, and the T2 generation homozygous seeds were obtained by single plant seed collection.

[0222] The T2 generation seeds after sterilization were uniformly sowed on the medium (4.43 g / L MS + 20 g / L sucrose + 8 g / L agar, pH = 5.8), and then placed in a refrigerator at 4℃ for vernalization for 3 d. The seedlings were transplanted into soil and placed in a light incubator (light intensity 30%, temperature 23℃, humidity 60%) for further growth. Finally, the L2 (OE-2), L5 (OE-5), and L9 (OE-9) lines were selected for detailed phenotype recording, chlorophyll and carotenoid content determination, and qRT-PCR analysis.

[0223] Meanwhile, wild type Arabidopsis Col-0 was used as a control group (WT).

[0224] 5.2 Observation of Arabidopsis leaf phenotype and determination of chlorophyll and carotenoid content after Arabidopsis transformation

[0225] Take 0.2 g leaf sample, cut into small pieces and put into a mortar, add a small amount of quartz sand and calcium carbonate powder. Add 10 mL 95% ethanol, grind until the leaves are completely homogenized. Let the homogenate stand for 3-5 minutes. Filter the extract through filter paper into a 25 mL conical flask, add 95% ethanol to 25 mL, make sure the filtrate is clear and free of impurities. Use a 1 cm light path cuvette to measure the absorbance at wavelengths of 470 nm, 649 nm and 665 nm, respectively, with 95% ethanol as a blank control. Calculate the concentrations of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids, as in step 4.2 of Example 4.

[0226] 5.3 Study of chlorophyll degradation-related genes in Arabidopsis thaliana leaves after transformation

[0227] 1) Extraction of RNA

[0228] Total RNA was extracted according to the instructions of the Aidlab RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (purchased from Aidlab, Catalog No.: RN34).

[0229] 2) Obtaining of cDNA

[0230] Using the total RNA obtained in step 5.3, 1) as a template, the First Strand cDNA Synthesis Kit (Catalog No.: RN09) provided by Aidlab Biotechnologies Co., Ltd. was used to obtain cDNA according to the instructions.

[0231] 3) Fluorescent quantitative PCR analysis

[0232] Specific primers were designed to analyze the relative expression of chlorophyll degradation-related genes such as AtNYC1, AtNOL, AtHCAR, AtCLH1, AtSGR1, AtPPH, AtPAO and AtRCCR in Arabidopsis thaliana leaves.

[0233] According to the nucleotide sequences of chlorophyll degradation-related genes AtNYC1, AtNOL, AtHCAR, AtCLH1, AtSGR1, AtPPH, AtPAO and AtRCCR, specific primers for quantitative analysis in Real-time PCR were designed using Primer premier v6.0 primer design software.

[0234] The primers for AtNYC1 gene are as follows:

[0235] Forward primer AtNYC1-F: 5'-TGACGATGACAAGTACTGGAGC-3' (SEQ ID NO. 23)

[0236] Reverse primer AtNYC1-R: 5'-TCCCCTAGTGCTTCCGGTTA-3' (SEQ ID NO. 24)

[0237] Primers for the AtNOL gene are as follows:

[0238] Forward primer AtNOL-F: 5'-TTTGGCAGAGGCCTCAGATG-3' (SEQ ID NO. 25)

[0239] Reverse primer AtNOL-R: 5'-GCAGCAAATCTGGGTGTTGG-3' (SEQ ID NO. 26)

[0240] Primers for the AtHCAR gene are as follows:

[0241] Forward primer AtHCAR-F: 5'-CGGTTACATGGGTGTTCCGA-3' (SEQ ID NO. 27)

[0242] Reverse primer AtHCAR-R: 5'-CTTAGCAGCATCGTCTGCCT-3' (SEQ ID NO. 28)

[0243] Primers for the AtCLH1 gene are as follows:

[0244] Forward primer AtCLH1-F: 5'-AGCGGAGATTCGATTGATTG-3' (SEQ ID NO. 29)

[0245] Reverse primer AtCLH1-R: 5'-CATGCAACTTAGGACATGAACA-3' (SEQ ID NO. 30)

[0246] Primers for the AtPPH gene are as follows:

[0247] Forward primer AtPPH-F: 5'-GGAGAGCTATCTTTCTCCGAAGC-3' (SEQ ID NO. 31)

[0248] Reverse primer AtPPH-R: 5'-AGTTCACCACCTCAGGGACT-3' (SEQ ID NO. 32)

[0249] Primers for the AtPAO gene are as follows:

[0250] Forward primer AtPAO-F: 5'-CACCCCGTCTAACCAACCTC-3' (SEQ ID NO. 33)

[0251] Reverse primer AtPAO-R: 5'-ATCAGAAGGAACACCAGCCG-3' (SEQ ID NO. 34)

[0252] Primers for AtRCCR gene are as follows:

[0253] Forward primer AtRCCR-F: 5'-CCATGGAAGACCACGACGAT-3' (SEQ ID NO. 35)

[0254] Reverse primer AtRCCR-R: 5'-GGCGGAACCGTTAGGGTTAT-3' (SEQ ID NO. 36)

[0255] Reference gene is AtActin gene, and primers are as follows:

[0256] AtActin-F: 5'-CTACGAGGGGTATGCTCTTCC-3' (SEQ ID NO. 37)

[0257] AtActin-R: 5'-TCAGCTCCGATGGTTATGACTT-3' (SEQ ID NO. 38)

[0258] Fluorescent quantitative analysis: cDNA synthesized in 2) in step 5.3 is used as a template, and specific primers of genes AtNYC1, AtNOL, AtHCAR, AtCLH1, AtSGR1, AtPPH, AtPAO and AtRCCR, and reference gene AtActin are used for amplification for fluorescent quantitative PCR analysis. Real-time PCR reaction is performed on a CFX Connect "Optics Module real-time quantitative instrument. The reaction system is 20 μL, and the reaction adopts two-step method, 95°C denaturation for 2 min, followed by 40 cycles: 95°C for 15 s; 60°C for 30 s.

[0259] After the reaction, the data is exported to an Excel table, and the relative expression amount of each gene is calculated by using 2 -△△Ct formula.

[0260] Figure 3 Phenotype and expression of chlorophyll degradation metabolism related genes after overexpression of Ppsec61γ gene in Arabidopsis thaliana in this embodiment.

[0261] Figure 3 Figure A is a morphological phenotype image of T2 generation plants (OE-2, OE-5, OE-9) after overexpression of Ppsec61γ gene in Arabidopsis thaliana. WT is wild type Arabidopsis thaliana Col-0.

[0262] From Figure 3From Table A, the overexpression lines all showed obvious leaf yellowing and other phenotypic characteristics.

[0263] Figure 3 Table B is the relative expression level of PpSec61y in T2 generation plants (OE-2, OE-5, OE-9) of Arabidopsis thaliana overexpressing PpSec61y gene.

[0264] From Figure 3 From Table B, the expression level of PpSec61y in the overexpression lines was significantly higher than that in WT (p<0.05).

[0265] Figure 3 Table C is a statistical chart of the changes in chlorophyll a, chlorophyll b and total chlorophyll content (unit: mg.g -1 FW) in the leaves of T2 generation plants (OE-2, OE-5, OE-9) of Arabidopsis thaliana overexpressing PpSec61y gene.

[0266] From Figure 3 From Table C, the contents of chlorophyll a, chlorophyll b and total chlorophyll in the overexpression lines were significantly lower than those in WT (p<0.05), indicating that overexpression of PpSec61y can promote chlorophyll degradation.

[0267] Figure 3 Table D is a statistical chart of the changes in carotenoid content (unit: mg.g -1 FW) in T2 generation plants (OE-2, OE-5, OE-9) of Arabidopsis thaliana overexpressing PpSec61y gene.

[0268] From Figure 3 From Table D, the carotenoid content in the overexpression lines was significantly lower than that in WT (p<0.05).

[0269] Figure 3 Table E is a statistical chart of the relative expression amount of multiple genes (AtNYC1, AtNOL, AtHCAR, AtCLH1, AtSGR1, AtPPH, AtPAO, AtRCCR) related to chlorophyll degradation in T2 generation plants (OE-2, OE-5, OE-9) of Arabidopsis thaliana overexpressing PpSec61y gene.

[0270] From Figure 3 From Table E, the expression of genes related to chlorophyll degradation in the overexpression lines was significantly up-regulated (p<0.05), further indicating that PpSec61y can regulate chlorophyll degradation by activating the degradation pathway.

[0271] The application successfully identifies and separates PpSec61gamma gene by deeply analyzing the leaf transcriptome of Mao peach natural mutant (leaf yellowing phenotype). Then, the function of PpSec61gamma gene is deeply researched by using transient overexpression and genetic transformation technology, and the results show that the up-regulation of PpSec61gamma gene expression level is significantly negatively correlated with the content of chlorophyll, and causes the yellowing phenotype of plant organs, and PpSec61gamma gene may accelerate the metabolic process of chlorophyll by regulating the transport pathway of chlorophyll degradation related enzyme, and finally causes the yellowing of plant organs. In view of the achievement of the application, PpSec61gamma gene and the protein coded by it have important value in the basic research of chlorophyll degradation pathway, and can be used for in-depth exploration of the molecular mechanism of chlorophyll metabolism. At the same time, PpSec61gamma has wide application potential in the improvement of plant organ color traits, can provide new targets and strategies for the genetic improvement of plant color traits, and is expected to play an important role in the fields of transgenic plant breeding and marker breeding based on organ color traits, so as to promote the development of plant breeding technology and create more plant varieties meeting market demand and consumer preference.

[0272] The above examples are intended to illustrate the embodiments disclosed in the present application, and should not be understood as a limitation of the present application. In addition, various modifications listed herein and changes in the method and composition of the application are obvious to those skilled in the art without departing from the scope and spirit of the present application. Although the present application has been specifically described in conjunction with various specific preferred embodiments thereof, it should be understood that the present application should not be limited to these specific embodiments. In fact, various modifications as described above to obtain the application which are obvious to those skilled in the art should be included in the scope of the present application.

Claims

1. Use of a PpSec61 gamma protein or a PpSec61 gamma promoter in at least one of A1) - A2), A1) promoting degradation of chlorophyll in a plant; A2) modulating a plant organ color trait; the amino acid sequence of the PpSec61 gamma protein is shown as SEQ ID NO. 2; the modulating a plant organ color trait means yellowing a plant organ, the organ comprising a leaf or a flower; The PpSec61γ promoter refers to a substance capable of promoting the transcription or translation of a gene, or capable of promoting the expression or activity of a PpSec61γ protein. PpSec61 gamma The nucleotide sequence of the gene is shown in SEQ ID NO.

1. PpSec61 gamma The nucleotide sequence of the gene is shown in SEQ ID NO.

1. the plant is peach or Arabidopsis.

2. Use according to claim 1, characterized in that, the chlorophyll comprises chlorophyll a or chlorophyll b.

3. Use according to claim 1, characterized in that, The PpSec61y promoter comprises a PpSec61y protein or a biological material related to the gene selected from one or both of a recombinant expression vector or a bioengineered bacteria. PpSec61 gamma The PpSec61y promoter comprises a PpSec61y protein or a biological material related to the gene selected from one or both of a recombinant expression vector or a bioengineered bacteria.

4. A method of promoting degradation of chlorophyll in a plant, characterized by, is any one of the following: D1) introducing into a plant the PpSec61 gamma protein as described in claim 1; D2) introducing into a plant the promoter as described in claim 1; the amino acid sequence of the PpSec61 gamma protein is shown as SEQ ID NO. 2; the plant is peach or Arabidopsis.

5. A method of modulating a plant organ color trait, comprising, is any one of the following: D1) introducing into a plant the PpSec61 gamma protein as described in claim 1; D2) introducing into a plant the promoter as described in claim 1; the amino acid sequence of the PpSec61 gamma protein is shown as SEQ ID NO. 2; the modulating a plant organ color trait means yellowing a plant organ, the organ comprising a leaf or a flower, the plant is peach or Arabidopsis.

6. Use of PpSec61 gamma as a target in preparing or screening a product for promoting degradation of chlorophyll in a plant, the amino acid sequence of the PpSec61 gamma protein is shown as SEQ ID NO. 2, the plant is peach or Arabidopsis.

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