Application of CmoPYR1 gene in regulation and control of nutritional quality and photosynthetic performance of pumpkin

Abstract

The invention discloses application of a CmoPYR1 gene to regulation and control of nutritional quality and photosynthetic performance of pumpkins, and belongs to the technical field of gene engineering. The nucleotide sequence of the CmoPYR1 gene is as shown in SEQ ID NO. 1. Researches find that the contents of xanthophyll, violaxanthin and total carotenoid in the fruit of a gene editing strain obtained by knocking out the pumpkin CmoPYR1 gene are remarkably increased compared with that of a wild type, and the nutritional quality of the fruit is remarkably improved; the gene edited strain also shows improvement of photosynthetic physiological parameters such as increase of net photosynthetic rate and reduction of intercellular CO2 concentration, and is beneficial to growth of pumpkin plants. A new gene resource is provided for cultivation and character improvement of a new pumpkin variety with high quality and high photosynthetic performance, and a technical support is provided for accelerating the breeding process.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, in particular to the application of CmoPYR1 gene in regulating the nutritional quality and photosynthetic performance of pumpkin. BACKGROUND

[0002] Cucurbita pepo, Cucurbita maxima and Cucurbita moschata are the most important cultivated species of Cucurbita L. in the Cucurbitaceae family, which are widely planted around the world. These three types of pumpkin can adapt to diverse climatic and ecological conditions, and are important vegetable crops worldwide, with significant economic value. Carotenoids are important substances for the formation of pumpkin quality and biological function. Carotenoids are not only the core components of the photosynthetic light-harvesting system and light protection mechanism, but also play a key role in human nutrition and health, including maintaining vision, preventing retinal damage, providing antioxidant and ultraviolet protection, and having potential health effects such as anti-cancer and anti-aging. Pumpkin and its close relatives are rich in carotenoids, and the flesh color can range from white to yellow, orange, mainly determined by α-carotene, β-carotene and lutein. In addition, carotenoids are also precursors of a variety of key metabolites, including important aroma volatile compounds and plant hormones such as abscisic acid (ABA) and strigolactones (SLs), which play a core regulatory role in plant growth and development and stress response. Therefore, increasing carotenoid content is of great significance for improving the nutritional quality and breeding of pumpkin.

[0003] Therefore, it is of great significance to mine and utilize key genes that can improve the carotenoid content and photosynthetic capacity of pumpkin to improve the nutritional quality and yield potential of pumpkin. However, traditional breeding relies on natural variation and phenotypic selection, which is limited by the complexity of genetic background, and has problems such as long breeding cycle, low efficiency, poor target specificity, etc., making it difficult to meet the demand for high-quality pumpkin varieties in modern agriculture. In recent years, the rapid development of molecular biology and omics technologies has accelerated the analysis of functional genes in pumpkin, making it possible to find key regulatory genes from multiple dimensions such as metabolic regulation, hormone signaling and environmental response. In particular, the progress of genetic engineering technologies such as transgenic and gene editing (CRISPR / Cas system) provides reliable means for precise regulation of carotenoid synthesis pathways and remodeling of photosynthetic characteristics. Using these technologies can achieve rapid and targeted modification of target genes, thereby cultivating new varieties of pumpkin with high carotenoid content and high photosynthetic performance in a shorter time, providing new solutions for vegetable quality improvement and modern breeding system construction. SUMMARY

[0004] The application aims to provide an application of CmoPYR1 gene in regulating nutritional quality and photosynthetic performance of pumpkin, so as to solve the problems in the prior art.

[0005] To achieve the above-mentioned purpose, the application provides the following solutions.

[0006] The application provides an application of CmoPYR1 gene in regulating nutritional quality and photosynthetic performance of pumpkin, wherein the indexes of the nutritional quality include the content of purple xanthophyll in the fruit, the content of lutein in the fruit and the content of total carotenoids in the fruit.

[0007] The photosynthetic performance includes the net photosynthetic rate of the leaf and the intercellular CO2 concentration of the leaf.

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

[0009] Further, the method for regulating is to knockout or silence the CmoPYR1 gene, so as to increase the content of purple xanthophyll, the content of lutein and the content of total carotenoids in the fruit of pumpkin, and increase the net photosynthetic rate of the leaf of pumpkin and reduce the intercellular CO2 concentration of the leaf of pumpkin.

[0010] The application also provides an application of biological material for knocking out CmoPYR1 gene in regulating nutritional quality and photosynthetic performance of pumpkin, wherein the indexes of the nutritional quality include the content of purple xanthophyll in the fruit, the content of lutein in the fruit and the content of total carotenoids in the fruit.

[0011] The photosynthetic performance includes the net photosynthetic rate of the leaf and the intercellular CO2 concentration of the leaf.

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

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

[0014] Further, the method for regulating is to knockout or silence the CmoPYR1 gene, so as to increase the content of purple xanthophyll, the content of lutein and the content of total carotenoids in the fruit of pumpkin, and increase the net photosynthetic rate of the leaf of pumpkin and reduce the intercellular CO2 concentration of the leaf of pumpkin.

[0015] The application further provides application of the biological material with the CmoPYR1 gene knocked out in cultivating a high-nutrition-quality pumpkin strain, wherein the high-nutrition-quality pumpkin strain refers to a pumpkin strain with high contents of violaxanthin, lutein and total carotenoids in the fruit.

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

[0017] The application further provides application of the biological material with the CmoPYR1 gene knocked out in cultivating a high-photosynthetic-performance pumpkin strain, wherein the high-photosynthetic-performance pumpkin strain is characterized by increased net photosynthetic rate of leaves and decreased intercellular CO2 concentration of leaves.

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

[0019] Optionally, the biological material comprises sgRNA, a recombinant vector or a recombinant microorganism for knocking out the CmoPYR1 gene.

[0020] The application further provides a method for cultivating a high-nutrition-quality pumpkin strain, comprising the step of cultivating a pumpkin strain with high contents of violaxanthin, lutein and total carotenoids in the fruit by knocking out or silencing a CmoPYR1 gene in the pumpkin.

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

[0022] The application further provides a method for cultivating a high-photosynthetic-performance pumpkin strain, comprising the step of cultivating a high-photosynthetic-performance pumpkin strain by knocking out or silencing a CmoPYR1 gene in the pumpkin.

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

[0024] The application discloses the following technical effects:

[0025] The application researches and finds that the CmoPYR1 gene of pumpkin can regulate the content of violaxanthin, the content of lutein and the content of total carotenoids in pumpkin fruits; and can also regulate the photosynthetic performance of pumpkin leaves, and the regulation is realized by increasing the net photosynthetic rate of leaves and reducing the intercellular CO2 concentration. Experiments prove that the gene editing strain obtained by knocking out the CmoPYR1 gene of pumpkin has a significant increase in the content of lutein, violaxanthin and total carotenoids in the fruits compared with the wild type, and the nutritional quality of the fruits is obviously improved; the gene editing strain also shows improvements in the photosynthetic physiological parameters such as increased net photosynthetic rate and reduced intercellular CO2 concentration, which is helpful for the growth of pumpkin plants. 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. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0027] Figure 1 The Manhattan plot of the candidate gene significantly related to the carotenoid content of the fruit; wherein a is lutein, b is beta-carotene, c is violaxanthin, and d is total carotenoids;

[0028] Figure 2 The feature analysis result graph of the CmoPYR1 gene; wherein a is the haplotype analysis result graph of the CmoPYR1 gene; and b is the expression difference analysis result graph of the CmoPYR1 gene in high-carotenoid material and low-carotenoid material;

[0029] Figure 3 The sequencing result graph of the CmoPYR1 gene edited pumpkin strain;

[0030] Figure 4 The fruit nutritional quality detection result graph of the CmoPYR1 gene edited pumpkin strain and the wild type strain; wherein a is the violaxanthin content, b is the lutein content, c is the alpha-carotene content, d is the beta-carotene content, and e is the total carotenoid content;

[0031] Figure 5 The leaf photosynthesis physiological parameter detection result graph of the CmoPYR1 gene edited pumpkin strain and the wild type strain; wherein a is the net photosynthetic rate (Pn), b is the stomatal conductance (Gs), c is the intercellular CO2 concentration (Ci), d is the transpiration rate (Tr), and e is the chlorophyll content. DETAILED DESCRIPTION

[0032] The following detailed description of various example embodiments of the application will not be considered limiting of the application, but rather as a description of certain aspects, features and embodiments of the application.

[0033] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of "including," "comprising," "having," "containing," and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless otherwise indicated, the use of the approximately or about symbol (e.g., ~) is meant to encompass ± 10% of the recited value.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials which are related to the present application. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0035] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof, and it is to be understood that all such modifications and variations warrant the patentable subject matter under the patent laws. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0036] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

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

[0038] Example 1 Obtaining and Characterizing CmoPYR1 Gene

[0039] The present application resequences whole genomes of 261 pumpkin natural population materials, and obtains a total of 1,435,928 high-quality SNP sites. Based on these SNPs, genome-wide association analysis (GWAS) of lutein, violaxanthin, alpha-carotene, beta-carotene and total carotenoid content is carried out. The analysis result identifies 225 loci significantly associated with carotenoid accumulation, wherein an important pleiotropic locus is found on chromosome 2, which is significantly related to lutein, violaxanthin, beta-carotene and total carotenoid content, indicating that it plays a key role in regulating the accumulation of multiple carotenoids Figure 1 ). The pleiotropic locus spans a genomic region of about 0.33 Mb. Further haplotype analysis and expression analysis of high and low carotenoid pumpkin material populations determine that the candidate gene is CmoPYR1 (gene number CmoCh02G016560). The analysis result shows that the expression amount of CmoPYR1 gene is significantly negatively correlated with the carotenoid content of fruits, suggesting that the gene may play a negative regulatory role in the process of carotenoid accumulation Figure 2 ).

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

[0041] SEQ ID NO. 1:

[0042] ATGGAAAAGGGCGAACAGTCAGAATTCGGCCACCACCATCACGACTCTGCCACTGCTGCCGCCGCCACCACCGCCACTTCCCACCACCTAGCCTTCCCAAACGGATTTTCTCAGCACGAGTTCGATGACCTGAAGCACATCATCTTGCAGTTCCACTCCTACGAACTACGGCCGGGCCAATGCTCCTCCCTCCTCTCTCAACTCATCCGTGCGCCGCGCGACGTCGTTTGGTCTGTCGTTCGCCGCTTTGATAAACCTCAGACTTACAAGCACTTCATCAAGAGCTGTACCGTCGCTGAAGGCTTCACAATGACCGTAGGATGCACCAGAGACGTTAATGTAATCTCTGGCCTTCCGGCGGCGACGAGTACAGAGCGGCTCGATATACTCGATGACGATCGGTATGTGACTGGTTTCAGTATCACAGGCGGCGAGCATCGGTTGAGGAACTACCGGTCTGTGACGACGGTGCATGAGATGGAGCGCGATGGTCAGATCTGGACGGTGGTTTTGGAATCGTACATCGTGGATGTGCCGGAAGGGAATACGGAAGAGGATACGCGTCTATTTGCAGATACAGTTGTGAAATTGAATCTGCAGAAGCTTACGTCCGTCACTGAAGGAATGGCTCGCGCCGTTGATGCTTCTGCTGCAGGTACTTCTACATCTAAATCAACACTCTGA.

[0043] SEQ ID NO. 2:

[0044] MEKGEQSEFGHHHHDSATAAAATTATSHHLAFPNGFSQHEFDDLKHIILQFHSYELRPGQCSSLLSQLIRAPRDVVWSVVRRFDKPQTYKHFIKSCTVAEGFTMTVGCTRDVNVISGLPAATSTERLDILDDDRYVTGFSITGGEHRLRNYRSVTTVHEMERDGQIWTVVLESYIVDVPEGNTEEDTRLFADTVVKLNLQKLTSVTEGMARAVDASAAGTSTSKSTL.

[0045] Construction of CmoPYR1 gene editing vector

[0046] 1. Design of sgRNA target

[0047] CRISPR / Cas9 double target was designed for the conserved sequence of CmoPYR1 gene, and the sgRNA target sequence was 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 vector

[0051] Four-primer PCR amplification was performed with pCBC-DT1T2 vector as template. The sequences of the primers used are shown in Table 1, the PCR reaction system is shown in Table 2, and the PCR reaction program is 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; 4℃ storage.

[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 then connected to pKSE402 vector by T4 ligase to obtain the gene editing vector pKSE402-CmoPYR1. The enzyme digestion-connection system is shown in Table 3, and the reaction conditions are as follows: 37℃ incubation for 5 h, 50℃ standing for 5 min, and 80℃ standing for 10 min.

[0057] Table 3 Enzyme digestion-connection system

[0058]

[0059] The gene editing vector pKSE402-CmoPYR1 comprises two editing sites sgRNA1 and sgRNA2 targeting CmoPYR1 gene and the coding sequence of Cas9 protein. After the gene editing vector pKSE402-CmoPYR1 is introduced into the recipient plant cells, the two guide RNAs (sgRNA) transcribed under the driving of the vector can recognize the target sequence near the PAM sequence in the CmoPYR1 genome through base complementarity. The Cas9 protein produces a DNA double-strand break at the target site under the guidance of sgRNA. Subsequently, the plant's own DNA damage repair mechanism (mainly non-homologous end joining, NHEJ) repairs the break, and in this process, base insertion, deletion or frameshift mutation occurs in the target sequence, resulting in frameshift mutation or premature termination in the CmoPYR1 coding region, thereby achieving functional knockout of the CmoPYR1 gene.

[0060] Example 3 Obtaining of CmoPYR1 gene editing pumpkin lines

[0061] 1. Genetic transformation of pumpkin

[0062] Chinese pumpkin was selected as the recipient material, and detached cotyledons were used as explants. The gene editing vector pKSE402-CmoPYR1 was introduced into the explant cells by Agrobacterium-mediated transformation method. During the transformation process, the explant U-shaped wound was treated with nanobrush and combined with a syringe to apply vacuum negative pressure to improve the efficiency of Agrobacterium infection. After transformation, the explants were cultured on co-culture, differentiation and rooting culture media in turn to allow the transformed cells to gradually differentiate and regenerate, and finally obtain the preliminary transformed lines.

[0063] 2. Identification of gene editing lines

[0064] Firstly, the GFP fluorescent marker on the vector was used to observe the fluorescence expression of the explants or transformed lines under a fluorescence microscope to quickly screen positive transformed lines. Then, PCR technology was used to further detect the GFP positive lines screened to confirm the integration of the gene editing vector pKSE402-CmoPYR1 in the plants. Further sequencing analysis of the CmoPYR1 target sequence was performed to identify base insertion, deletion or frameshift mutation. The results showed that only the first target site produced editing mutation, and finally two homozygous editing plants were obtained, with single base insertion and double base deletion (TAT) as the mutation type. Figure 3 The results showed that the functional knockout of CmoPYR1 gene was successful, and CmoPYR1 gene editing pumpkin lines were successfully constructed. The two homozygous editing plants were named Cmopyr1#1 and Cmopyr1#2, respectively.

[0065] 3. Phenotype and function detection of CmoPYR1 gene editing pumpkin lines

[0066] The obtained CmoPYR1 gene editing strains are planted, and the fruit carotenoid content (lutein, violaxanthin, a-carotene, b-carotene and total carotenoids) and leaf photosynthetic physiological indicators (net photosynthetic rate Pn, intercellular CO2 concentration Ci, stomatal conductance Gs, transpiration rate Tr, chlorophyll a and chlorophyll b) are detected. The results show that, compared with the wild type control plants, the CmoPYR1 gene editing strains show significant improvement in fruit nutritional quality and key photosynthetic physiological indicators. Specifically, knocking out CmoPYR1 gene significantly improves the content of fruit violaxanthin, lutein and total carotenoids Figure 4 ); knocking out CmoPYR1 gene also improves the net photosynthetic rate of plant leaves, reduces the intercellular CO2 concentration, and can enhance the carbon fixation efficiency and photosynthetic capacity of plants, which is helpful for the growth and development of plants Figure 5 .

[0067] The above-described embodiments are only descriptions of the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. The application of the CmoPYR1 gene in regulating the nutritional quality and photosynthetic performance of pumpkin, characterized by, The nutritional quality indicators include the content of aurantium, lutein, and total carotenoids in the fruit. The photosynthetic performance includes the net photosynthetic rate of leaves and the intercellular CO2 concentration of leaves; The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, 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.

3. 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 include the content of aurantium, lutein, and total carotenoids in the fruit. The photosynthetic performance includes the net photosynthetic rate of leaves and the intercellular CO2 concentration of leaves; The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.

1.

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

5. The application according to claim 3, characterized in that, 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.

6. 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.

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

1.

8. The application according to claim 6 or 7, characterized in that, The biological material includes sgRNA with the CmoPYR1 gene knocked out, recombinant vectors, or recombinant microorganisms.

9. 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 or silencing the CmoPYR1 gene in pumpkins; The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.

1.

10. 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 or silencing the CmoPYR1 gene in pumpkins; The nucleotide sequence of the CmoPYR1 gene is shown in SEQ ID NO.1.

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