Application of CmoPYR1 gene in regulating nutritional quality and photosynthetic performance of pumpkin

Abstract

The application discloses application of CmoPYR1 gene in regulating nutritional quality and photosynthetic performance of pumpkin, and belongs to the technical field of genetic engineering. The nucleotide sequence of the CmoPYR1 gene is shown as SEQ ID NO. 1. It is found by the application that the gene editing strain obtained by knocking out the CmoPYR1 gene of pumpkin has a significantly increased content of lutein, violaxanthin and total carotenoids in the fruit, and the nutritional quality of the fruit is significantly improved; the gene editing strain also shows improved photosynthetic physiological parameters such as increased net photosynthetic rate and reduced intercellular CO2 concentration, and is helpful to the growth of the pumpkin plant. The application provides a new gene resource for the cultivation and trait improvement of high-quality and high-photosynthetic-performance pumpkin new varieties, and provides technical support for accelerating the breeding process.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the CmoPYR1 gene in regulating the nutritional quality and photosynthetic performance of pumpkin. Background Technology

[0002] The Cucurbita genus of the Cucurbitaceae family is most notably represented by three cultivars: *Cucurbita pepo*, *Cucurbita maxima*, and *Cucurbita moschata*, which are widely cultivated globally. These three types of pumpkins can adapt to diverse climates and ecological conditions, making them important global vegetable crops with significant economic value. Carotenoids are crucial substances for the formation of pumpkin quality and its biological functions. Carotenoids are not only core components of the photosynthetic light-harvesting system and photoprotection mechanism, but also play a key role in human nutrition and health, including maintaining vision, preventing retinal damage, providing antioxidant and UV protection, and possessing potential anti-cancer and anti-aging health effects. Pumpkins and their closely related species are rich in carotenoids, and their flesh color can range from white to yellow and orange, mainly determined by α-carotene, β-carotene, and lutein. Furthermore, carotenoids are precursors to many key metabolites, including important aroma volatiles and plant hormones such as abscisic acid (ABA) and strigolactones (SLs). These signaling molecules play a central regulatory role in plant growth, development, and stress responses. Therefore, increasing the content of carotenoids is of great significance for the nutritional improvement and breeding of pumpkins.

[0003] Therefore, identifying and utilizing key genes that can enhance the carotenoid content and photosynthetic capacity of pumpkins is of great significance for improving the nutritional quality and yield potential of pumpkins. However, traditional breeding relies on natural variation and phenotypic selection, which is not only limited by the complexity of the genetic background but also suffers from problems such as long breeding cycles, low efficiency, and poor targeting, making it difficult to meet the demands of modern agriculture for high-quality pumpkin varieties. In recent years, the rapid development of molecular biology and omics technologies has accelerated the analysis of pumpkin functional genes, making it possible to find key regulatory genes from multiple dimensions such as metabolic regulation, hormone signaling, and environmental response. In particular, the advancement of genetic engineering technologies, such as transgenics and gene editing (CRISPR / Cas system), provides reliable means for precisely regulating carotenoid synthesis pathways and reshaping photosynthetic characteristics. Using these technologies, it is possible to achieve rapid and targeted modification of target genes, thereby cultivating new pumpkin varieties with high carotenoid content and high photosynthetic performance in a shorter time, providing new solutions for vegetable quality improvement and the construction of modern breeding systems. Summary of the Invention

[0004] The purpose of this invention is to provide an application of the CmoPYR1 gene in regulating the nutritional quality and photosynthetic performance of pumpkin, thereby addressing the problems existing in the prior art. This invention has found that gene-edited lines obtained by knocking out the CmoPYR1 gene in pumpkin exhibit significantly higher levels of lutein, apocynin, and total carotenoids in their fruits compared to the wild type, resulting in a marked improvement in fruit nutritional quality. These gene-edited lines also show improvements in photosynthetic physiological parameters, such as increased net photosynthetic rate and decreased intercellular CO2 concentration, which contribute to the growth of pumpkin plants.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of the CmoPYR1 gene in regulating the nutritional quality and photosynthetic performance of pumpkin, wherein the nutritional quality indicators include the content of aurantium, lutein, and total carotenoids in the fruit.

[0007] The photosynthetic performance includes the net photosynthetic rate of leaves and the intercellular CO2 concentration of leaves;

[0008] The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.1.

[0009] Furthermore, the regulation method involves knocking out or silencing the CmoPYR1 gene to increase the content of zeaxanthin, lutein, and total carotenoids in pumpkin fruits, as well as to increase the net photosynthetic rate of pumpkin leaves and reduce the intercellular CO2 concentration in pumpkin leaves.

[0010] This invention also provides the application of biomaterials with the CmoPYR1 gene knocked out in regulating the nutritional quality and photosynthetic performance of pumpkin, wherein the nutritional quality indicators include the content of aurantium, the content of lutein, and the content of total carotenoids in the fruit.

[0011] The photosynthetic performance includes the net photosynthetic rate of leaves and the intercellular CO2 concentration of leaves;

[0012] The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.1.

[0013] Optionally, the biological material includes sgRNA with the CmoPYR1 gene knocked out, a recombinant vector, or a recombinant microorganism.

[0014] Furthermore, the regulation method involves knocking out or silencing the CmoPYR1 gene to increase the content of zeaxanthin, lutein, and total carotenoids in pumpkin fruits, as well as to increase the net photosynthetic rate of pumpkin leaves and reduce the intercellular CO2 concentration in pumpkin leaves.

[0015] This invention also provides the application of biomaterials with the CmoPYR1 gene knocked out in the cultivation of high-nutritional-quality pumpkin lines, wherein the high-nutritional-quality pumpkin lines refer to pumpkin lines whose fruits are rich in apocynin, lutein and total carotenoids.

[0016] The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.1.

[0017] This invention also provides the application of biomaterials with the CmoPYR1 gene knocked out in the cultivation of high photosynthetic pumpkin lines, which are characterized by increased net photosynthetic rate of leaves and decreased intercellular CO2 concentration in leaves.

[0018] The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.1.

[0019] Optionally, the biological material includes sgRNA with the CmoPYR1 gene knocked out, a recombinant vector, or a recombinant microorganism.

[0020] The present invention also provides a method for cultivating high-nutritional-quality pumpkin lines, including the step of cultivating pumpkin lines whose fruits are rich in apocynin, lutein and total carotenoids by knocking out or silencing the CmoPYR1 gene in pumpkins.

[0021] The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.1.

[0022] The present invention also provides a method for cultivating high photosynthetic performance pumpkin lines, including the step of cultivating high photosynthetic performance pumpkin lines by knocking out or silencing the CmoPYR1 gene in pumpkin;

[0023] The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.1.

[0024] The present invention discloses the following technical effects:

[0025] This invention reveals that the CmoPYR1 gene in pumpkin can regulate the content of xanthophyll, lutein, and total carotenoids in pumpkin fruits; it can also regulate the photosynthetic performance of pumpkin leaves by increasing the net photosynthetic rate and decreasing the intercellular CO2 concentration. Experiments have confirmed that gene-edited lines obtained by knocking out the CmoPYR1 gene in pumpkin exhibit significantly higher levels of lutein, xanthophyll, and total carotenoids in their fruits compared to the wild type, resulting in a marked improvement in fruit nutritional quality. These gene-edited lines also show improvements in photosynthetic physiological parameters, such as increased net photosynthetic rate and decreased intercellular CO2 concentration, which are beneficial to pumpkin plant growth. This invention provides new gene resources for the breeding and trait improvement of high-quality, high-photosynthetic-performance pumpkin varieties, and provides technical support for accelerating the breeding process. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Manhattan diagram of candidate genes significantly associated with fruit carotenoid content; where a is xanthophyll, b is β-carotene, c is oxalool, and d is total carotenoids;

[0028] Figure 2 Figure 1 shows the characterization results of the CmoPYR1 gene; where a is the haplotype analysis results of the CmoPYR1 gene; and b is the differential expression analysis results of the CmoPYR1 gene in high-carotenoid and low-carotenoid materials.

[0029] Figure 3 Sequencing results of the CmoPYR1 gene-edited pumpkin lines;

[0030] Figure 4 Figure 1 shows the results of fruit nutritional quality testing for CmoPYR1 gene-edited pumpkin lines and wild-type lines; where a represents the content of xanthophyll, b represents the content of lutein, c represents the content of α-carotene, d represents the content of β-carotene, and e represents the content of total carotenoids.

[0031] Figure 5 Figure 1 shows the results of detecting physiological parameters of photosynthesis in leaves of CmoPYR1 gene-edited pumpkin lines and wild-type lines; where a is net photosynthetic rate (Pn), b is stomatal conductance (Gs), c is intercellular CO2 concentration (Ci), d is transpiration rate (Tr), and e is chlorophyll content. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. The pumpkin materials involved in the following examples were all provided by the Vegetable Research Institute of Guangdong Academy of Agricultural Sciences.

[0038] Example 1: Acquisition and Characterization of the CmoPYR1 Gene

[0039] This invention performed whole-genome resequencing on 261 natural pumpkin populations, obtaining a total of 1,435,928 high-quality SNP loci. Based on these SNPs, genome-wide association studies (GWAS) were conducted to determine the contents of lutein, xanthophyll, α-carotene, β-carotene, and total carotenoids. The analysis identified 225 loci significantly associated with carotenoid accumulation, including an important pleiotropic locus on chromosome 2. This locus was significantly associated with the contents of lutein, xanthophyll, β-carotene, and total carotenoids, indicating its crucial role in regulating the accumulation of multiple carotenoids. Figure 1 This pleiotropic locus spans approximately 0.33 Mb of the genome. Further haplotype analysis and expression analysis of high- and low-carotenoid pumpkin populations identified CmoPYR1 (gene number CmoCh02G016560) as the candidate gene. The results showed a significant negative correlation between CmoPYR1 gene expression and fruit carotenoid content, suggesting that this gene may play a negative regulatory role in carotenoid accumulation. Figure 2 ).

[0040] The CDS sequence of the CmoPYR1 gene is 684 bp in length and encodes 227 amino acids. The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the CmoPYR1 gene is shown in SEQ ID NO.2.

[0041] SEQ ID NO.1:

[0042] ATGGAAAAGGGCGAACAGTCAGAATTCGGCCACCACCATCACGACTCTGCCACTGCTGCCGCCGCCACCACCGCCACTTCCCACCACCTAGCCTTCCCAAACGGATTTTCTCAGCACGAGTTCGATGACCTGAAGCACATCATCTTGCAGTTCCACTCCTACGAACTACGGCCGGGCCAATGCTCCTCCCTCCTCTCTCAACTCATCCGTGCGCCGCGCGACGTCGTTTGGTCTGTCGTTCGCCGCTTTGATAAACCTCAGACTTACAAGCACTTCATCAAGAGCTGTACCGTCGCTGAAGGCTTCACAATGACCGTAGGATGCACCAGAGACGTTAATGTAATCTCTGGCCTTCCGGCGGCGACGAGTACAGAGCGGCTCGATATACTCGATGACGATCGGTATGTGACTGGTTTCAGTATCACAGGCGGCGAGCATCGGTTGAGGAACTACCGGTCTGTGACGACGGTGCATGAGATGGAGCGCGATGGTCAGATCTGGACGGTGGTTTTGGAATCGTACATCGTGGATGTGCCGGAAGGGAATACGGAAGAGGATACGCGTCTATTTGCAGATACAGTTGTGAAATTGAATCTGCAGAAGCTTACGTCCGTCACTGAAGGAATGGCTCGCGCCGTTGATGCTTCTGCTGCAGGTACTTCTACATCTAAATCAACACTCTGA。

[0043] SEQ ID NO.2:

[0044] MEKGEQSEFGHHHHDSATAAAATTATSHHLAFPNGFSQHEFDDLKHIILQFHSYELRPGQCSSLLSQLIRAPRDVVWSVVRRFDKPQTYKHFIKSCTVAEGFTMTVGCTRDVNVISGLPAATSTERLDILDDDRYVTGFSITGGEHRLRNYRSVTTVHEMERDGQIWTVVLESYIVDVPEGNTEEDTRLFADTVVKLNLQKLTSVTEGMARAVDASAAGTSTSKSTL。

[0045] Example 2 Construction of CmoPYR1 gene editing vector

[0046] 1. Design of sgRNA targets

[0047] A CRISPR / Cas9 dual-target design was created targeting the conserved sequence of the CmoPYR1 gene. The sgRNA target sequence is as follows:

[0048] sgRNA1: 5'- TCACGACTCTGCCACTGCTG-3' (SEQ ID NO.3);

[0049] sgRNA2: 5'-CCACTCCTACGAACTACGGC-3' (SEQ ID NO. 4).

[0050] 2. Construction of gene editing vectors

[0051] Four-primer PCR amplification was performed using the pCBC-DT1T2 vector as a template. The sequences of the primers used are shown in Table 1, and the PCR reaction system is shown in Table 2. The PCR reaction program was as follows: 98℃ for 2 min; 98℃ for 10 s, 58℃ for 10 s, 72℃ for 5 s, 25-35 cycles; 72℃ for 1 min; and stored at 4℃.

[0052] Table 1 Primer Sequences

[0053]

[0054] Table 2 PCR reaction system

[0055]

[0056] The PCR product was purified and recovered, digested with BsaI, and ligated into the pKSE402 vector using T4 ligase to obtain the gene editing vector pKSE402-CmoPYR1. The enzyme digestion-ligation system is shown in Table 3. The reaction conditions were: incubation at 37℃ for 5 h, standing at 50℃ for 5 min, and standing at 80℃ for 10 min.

[0057] Table 3 Enzyme digestion-ligation system

[0058]

[0059] The gene-editing vector pKSE402-CmoPYR1 contains two editing sites, sgRNA1 and sgRNA2, targeting the CmoPYR1 gene, as well as the coding sequence for the Cas9 protein. After the gene-editing vector pKSE402-CmoPYR1 is introduced into recipient plant cells, the two guide RNAs (sgRNAs) transcribed under vector-driven conditions recognize the target sequence near the PAM sequence in the CmoPYR1 genome through base complementarity. Guided by the sgRNAs, the Cas9 protein induces a double-strand break at the target site. Subsequently, the plant's own DNA damage repair mechanisms (primarily non-homologous end joining, NHEJ) repair the break. During this process, base insertions, deletions, or frameshift mutations occur in the target sequence, leading to frameshift mutations or premature termination in the CmoPYR1 coding region, thereby achieving functional knockout of the CmoPYR1 gene.

[0060] Example 3: Obtaining CmoPYR1 gene-edited pumpkin lines

[0061] 1. Genetic transformation of pumpkin

[0062] Chinese pumpkin was selected as the recipient material, and isolated cotyledons were used as explants. The gene-editing vector pKSE402-CmoPYR1 was introduced into the explant cells using Agrobacterium-mediated transformation. During transformation, U-shaped wounds were created on the explants using nanobrushes, and vacuum pressure was applied using a syringe to improve Agrobacterium infection efficiency. After transformation, the explants were sequentially placed on co-culture, differentiation, and rooting media to allow the transformed cells to gradually differentiate and regenerate, ultimately obtaining preliminary transformed lines.

[0063] 2. Identification of gene-edited strains

[0064] First, GFP fluorescent labeling on the vector was used to observe the fluorescence expression of explants or transformed lines under a fluorescence microscope to rapidly screen positive transformants. Then, PCR technology was used to further test the screened GFP-positive lines to confirm the integration of the gene editing vector pKSE402-CmoPYR1 into the plants. Further sequencing analysis of the CmoPYR1 target sequence was performed to identify base insertions, deletions, or frameshift mutations. The results showed that only the first target site produced an editing mutation, and two homozygous edited plants were ultimately obtained, with mutation types of single-base insertion and double-base deletion. Figure 3 This result demonstrates that the functional knockout of the CmoPYR1 gene was successful, and the CmoPYR1 gene-edited pumpkin lines were successfully constructed. The two homozygous edited plants were named Cmopyr1#1 and Cmopyr1#2, respectively.

[0065] 3. Phenotypic and functional detection of CmoPYR1 gene-edited pumpkin lines

[0066] The obtained CmoPYR1 gene-edited lines were planted, and the content of carotenoids in the fruit (lutein, xanthophyll, α-carotene, β-carotene, and total carotenoids) and photosynthetic physiological indicators in the leaves (net photosynthetic rate Pn, intercellular CO2 concentration Ci, stomatal conductance Gs, transpiration rate Tr, chlorophyll a, and chlorophyll b) were systematically measured. The results showed that compared with wild-type control plants, the CmoPYR1 gene-edited lines exhibited significant improvements in fruit nutritional quality and key photosynthetic physiological indicators. Specifically, knocking out the CmoPYR1 gene significantly increased the content of xanthophyll, lutein, and total carotenoids in the fruit. Figure 4 Knocking out the CmoPYR1 gene also increased the net photosynthetic rate of plant leaves, reduced intercellular CO2 concentration, and enhanced the plant's carbon fixation efficiency and photosynthetic capacity, which is beneficial to the plant's growth and development. Figure 5 ).

[0067] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of CmoPYR1 gene knockout in regulating the nutritional quality and photosynthetic performance of pumpkin, characterized by, The nutritional quality indicators are the content of aurantium, lutein, and total carotenoids in the fruit. The photosynthetic performance refers to the net photosynthetic rate of the leaf and the intercellular CO2 concentration of the leaf. The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.1; The regulation of pumpkin's nutritional quality and photosynthetic performance is achieved by knocking out the CmoPYR1 gene, which increases the content of zeaxanthin, lutein, and total carotenoids in pumpkin fruit, increases the net photosynthetic rate of pumpkin leaves, and reduces the intercellular CO2 concentration in pumpkin leaves.

2. Application of biomaterials with the CmoPYR1 gene knocked out in regulating the nutritional quality and photosynthetic performance of pumpkin, characterized in that, The nutritional quality indicators are the content of aurantium, lutein, and total carotenoids in the fruit. The photosynthetic performance refers to the net photosynthetic rate of the leaf and the intercellular CO2 concentration of the leaf. The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.1; The regulation of pumpkin's nutritional quality and photosynthetic performance is achieved by knocking out the CmoPYR1 gene, which increases the content of zeaxanthin, lutein, and total carotenoids in pumpkin fruit, increases the net photosynthetic rate of pumpkin leaves, and reduces the intercellular CO2 concentration in pumpkin leaves.

3. The application according to claim 2, characterized in that, The biological material includes sgRNA with the CmoPYR1 gene knocked out, recombinant vectors, or recombinant microorganisms.

4. Application of biomaterials with the CmoPYR1 gene knocked out in the cultivation of high-nutritional-quality pumpkin lines, characterized in that... The high-nutritional-quality pumpkin strains refer to pumpkin strains whose fruits are rich in apocynin, lutein, and total carotenoids. The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.

1.

5. Application of biomaterials with the CmoPYR1 gene knocked out in the cultivation of high-photosynthetic pumpkin lines, characterized in that... The high photosynthetic performance pumpkin lines are those with increased net photosynthetic rate of pumpkin leaves and decreased intercellular CO2 concentration in leaves. The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.

1.

6. The application according to claim 4 or 5, characterized in that, The biological material includes sgRNA with the CmoPYR1 gene knocked out, recombinant vectors, or recombinant microorganisms.

7. A method for cultivating high-nutritional-quality pumpkin strains, characterized in that, This includes the steps of cultivating pumpkin lines whose fruits are rich in azadirachtin, lutein, and total carotenoids by knocking out the CmoPYR1 gene in pumpkins; The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.

1.

8. A method for cultivating pumpkin lines with high photosynthetic performance, characterized in that, This includes the steps of cultivating high-photosynthetic-performance pumpkin lines by knocking out the CmoPYR1 gene in pumpkins; The high photosynthetic performance pumpkin lines are those with increased net photosynthetic rate of pumpkin leaves and decreased intercellular CO2 concentration in leaves. The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.1.

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