Trichosanthes kirilowii Maxim resistance new variety cultivation method based on molecular breeding technology

Abstract

The invention relates to the technical field of trichosanthes kirilowii resistance new variety cultivation, and discloses a trichosanthes kirilowii resistance new variety cultivation method based on a molecular breeding technology, which comprises the following steps: (S1) screening trichosanthes kirilowii resistance genes, and utilizing a transcriptome sequencing technology and bioinformatics analysis to excavate differential expression genes related to trichosanthes kirilowii disease resistance; (S2) constructing an overexpression vector of the resistance gene, and inserting the resistance gene into a plant expression vector with a marker gene through a homologous recombination method; (S3) transforming the constructed vector into trichosanthes kirilowii maxim callus by using an agrobacterium tumefaciens-mediated method, and culturing a transgenic positive plant; (S4) carrying out a disease inoculation test on the positive plants, and screening plants with remarkable resistance; and (S5) breeding the screened resistant plants in a crossbreeding manner to obtain a new resistant variety with stable inheritance.

Description

Technical Field

[0001] The invention relates to the technical field of breeding new varieties of Trichosanthes kirilowii resistance, and specifically to a method for breeding new varieties of Trichosanthes kirilowii resistance based on molecular breeding technology. Background Art

[0002] As an important medicinal and edible plant, Trichosanthes kirilowii is widely used in traditional Chinese medicine and health food. The fruit, seeds, roots and vines of Trichosanthes kirilowii are rich in active ingredients, such as triterpenes, flavonoids and polysaccharide compounds, which have pharmacological effects such as clearing heat and resolving phlegm, moistening the lungs and relieving cough, and unblocking meridians and reducing swelling. Therefore, it has attracted much attention in the development of medicines and functional foods.

[0003] However, during the cultivation of Trichosanthes kirilowii, there are often serious threats from pests and diseases, especially vine blight, root-knot nematodes and anthracnose. These diseases not only lead to a significant decrease in the yield of Trichosanthes kirilowii, but also significantly affect its quality and economic benefits. Traditional disease prevention and control measures mainly rely on chemical pesticides and crop rotation, but these methods have many problems such as drug residues, environmental pollution and pathogen resistance. Therefore, the development of new varieties of Trichosanthes kirilowii with excellent disease resistance has become one of the key ways to improve the benefits of Trichosanthes kirilowii cultivation.

[0004] With the development of molecular breeding technology, transcriptome sequencing technology, gene editing and transgenic technology have become the cutting-edge means of modern agricultural breeding. These technologies can accurately discover key genes related to disease resistance and quickly achieve trait improvement through molecular means. Compared with traditional breeding methods, they have significant efficiency and precision advantages. However, in the field of resistance breeding of Trichosanthes kirilowii, research and application based on molecular breeding technology are still in the initial stage, lacking targeted and practical technical solutions.

[0005] The present invention utilizes transcriptome sequencing technology to mine differentially expressed genes related to disease resistance of Trichosanthes kirilowii, combines transgenic technology, Agrobacterium-mediated technology and molecular marker screening methods, and proposes a systematic and effective method for breeding new resistant varieties of Trichosanthes kirilowii. The present invention not only solves the problems of long traditional breeding cycle and low efficiency, but also provides an efficient technical path for resistance breeding of Trichosanthes kirilowii, thereby promoting variety improvement and industrial development of Trichosanthes kirilowii. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the deficiencies in the prior art, the present invention provides a method for breeding new resistant varieties of Trichosanthes kirilowii based on molecular breeding technology.

[0008] (II) Technical solution

[0009] To achieve the above object, the present invention provides the following technical solution: comprising the following steps:

[0010] (S1) Screening of resistance genes in Trichosanthes kirilowii, using transcriptome sequencing technology and bioinformatics analysis to mine differentially expressed genes related to disease resistance in Trichosanthes kirilowii;

[0011] (S2) constructing an overexpression vector of the resistance gene, and inserting the resistance gene into a plant expression vector carrying a marker gene by homologous recombination;

[0012] (S3) using an Agrobacterium-mediated method to transform the constructed vector into Trichosanthes kirilowii callus tissue and cultivate transgenic positive plants;

[0013] (S4) conducting a disease inoculation test on the positive plants to screen for plants with significant resistance;

[0014] (S5) The selected resistant plants are propagated through hybrid breeding to obtain new resistant varieties with stable inheritance.

[0015] Preferably, the transcriptome sequencing in step (S1) is performed using the DNBSEQ platform. After quality control of the sequencing data, GO functional enrichment analysis and KEGG pathway enrichment analysis are performed using the DAVID database to determine candidate genes related to disease resistance.

[0016] Preferably, the overexpression vector in step (S2) is constructed using a pMDC202 vector with an mGFP tag for screening positive plants; the CDS region of the resistance gene is amplified by PCR and inserted into the multiple cloning site of the vector using homologous recombination technology.

[0017] Preferably, in the step (S3), Agrobacterium is used to infect the Trichosanthes kirilowii callus, and after the infection, the callus is transferred to a culture medium containing a selective antibiotic for screening until resistance-positive plants are obtained.

[0018] Preferably, the disease inoculation test in step (S4) comprises:

[0019] (S41) inoculating with vine blight pathogen, root knot nematode and anthracnose pathogen respectively;

[0020] (S42) observing the occurrence of diseases and recording resistance scores;

[0021] (S43) qRT-PCR technology was used to detect the expression level of resistance genes and verify their correlation with disease resistance.

[0022] Preferably, the hybrid breeding method in step (S5) includes the following process:

[0023] (S51) using the selected resistant plants as parents and artificially pollinating to obtain F1 generation;

[0024] (S52) screening the F1 generation plants for disease resistance, and selecting plants with stable resistance performance;

[0025] (S53) Further hybridizing the plants with stable resistance until a new resistant variety of the F2 generation is obtained.

[0026] Preferably, the resistance verification of the transgenic Trichosanthes kirilowii positive plants includes detecting the expression of the mGFP tag using a fluorescence microscope, combined with PCR technology to verify the integration of the resistance gene.

[0027] Preferably, the obtained new resistant variety of Trichosanthes kirilowii is subjected to field trials, including:

[0028] (a) Plant new resistant varieties of Trichosanthes kirilowii in multiple suitable areas and conduct comparative studies on their disease resistance, yield and quality;

[0029] (b) The control varieties were selected as local common Trichosanthes kirilowii varieties, and the growth cycle, disease occurrence and economic trait data were recorded;

[0030] (c) Apply statistical methods to test the significance of test results and evaluate the overall performance of new varieties.

[0031] Preferably, the interaction between the key resistance gene and its downstream target gene is further verified by chromatin immunoprecipitation sequencing (ChIP-seq) and gel retardation experiment.

[0032] (III) Beneficial technical effects

[0033] (1) Improving the disease resistance of Trichosanthes kirilowii: By utilizing molecular breeding technology, the present invention accurately screens and introduces genes closely related to the disease resistance of Trichosanthes kirilowii, significantly enhancing the resistance of Trichosanthes kirilowii to diseases such as vine blight, root-knot nematodes and anthracnose, and reducing the negative impact of diseases on the growth and yield of Trichosanthes kirilowii.

[0034] (2) Accelerate the breeding process: The present invention combines modern breeding methods such as transcriptome sequencing, gene editing, and transgenic technology. Compared with traditional hybrid breeding methods, it significantly shortens the breeding cycle of new varieties and improves breeding efficiency.

[0035] (3) Ensure the genetic stability of resistance: Resistant plants obtained through transgenic technology can maintain stable resistance traits after multiple generations of reproduction, ensuring the reliability and consistency of new varieties during large-scale planting.

[0036] (4) Improving the benefits of Trichosanthes kirilowii cultivation: New resistant varieties have shown excellent disease resistance and yield advantages in field trials, effectively reducing the use of pesticides and lowering production costs. At the same time, it has improved the quality and market competitiveness of Trichosanthes kirilowii, bringing significant economic benefits to growers.

[0037] (5) Reducing environmental pollution: By reducing the use of chemical pesticides, the present invention is environmentally friendly, helps to achieve sustainable agricultural development, and meets the requirements of green planting and ecological agriculture.

[0038] (6) Promote the industrial development of Trichosanthes kirilowii: The present invention provides a systematic and scientific technical path for the disease-resistant breeding of Trichosanthes kirilowii, fills the technical gap in the field of molecular breeding of Trichosanthes kirilowii, provides technical support for the improvement of Trichosanthes kirilowii varieties and large-scale promotion, and promotes the rapid development of Trichosanthes kirilowii-related industries. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below.

[0040] Example 1

[0041] The present invention provides a method for breeding a new variety of Trichosanthes kirilowii resistance based on molecular breeding technology, comprising the following steps:

[0042] (S1) Screening of resistance genes in Trichosanthes kirilowii, using transcriptome sequencing technology and bioinformatics analysis to mine differentially expressed genes related to disease resistance in Trichosanthes kirilowii;

[0043] (S2) constructing an overexpression vector of the resistance gene, and inserting the resistance gene into a plant expression vector carrying a marker gene by homologous recombination;

[0044] (S3) using an Agrobacterium-mediated method to transform the constructed vector into Trichosanthes kirilowii callus tissue and cultivate transgenic positive plants;

[0045] (S4) conducting a disease inoculation test on the positive plants to screen for plants with significant resistance;

[0046] (S5) The selected resistant plants are propagated through hybrid breeding to obtain new resistant varieties with stable inheritance.

[0047] In another embodiment provided by the present invention, the transcriptome sequencing in the step (S1) is performed using the DNBSEQ platform. After quality control of the sequencing data, GO functional enrichment analysis and KEGG pathway enrichment analysis are performed using the DAVID database to determine candidate genes related to disease resistance.

[0048] The overexpression vector in step (S2) is constructed using the pMDC202 vector, which has an mGFP tag for screening positive plants; the CDS region of the resistance gene is amplified by PCR and inserted into the multiple cloning site of the vector using homologous recombination technology.

[0049] In the step (S3), Agrobacterium is used to infect the Trichosanthes kirilowii callus, and after the infection, the callus is transferred to a culture medium containing a selective antibiotic for screening until resistance-positive plants are obtained.

[0050] The disease inoculation test in the step (S4) comprises:

[0051] (S41) inoculating with vine blight pathogen, root knot nematode and anthracnose pathogen respectively;

[0052] (S42) observing the occurrence of diseases and recording resistance scores;

[0053] (S43) qRT-PCR technology was used to detect the expression level of resistance genes and verify their correlation with disease resistance.

[0054] The hybrid breeding method in the step (S5) comprises the following process:

[0055] (S51) using the selected resistant plants as parents and artificially pollinating to obtain F1 generation;

[0056] (S52) screening the F1 generation plants for disease resistance, and selecting plants with stable resistance performance;

[0057] (S53) Further hybridizing the plants with stable resistance until a new resistant variety of the F2 generation is obtained.

[0058] In another embodiment provided by the present invention, the resistance verification of the transgenic Trichosanthes kirilowii positive plants includes detecting the expression of the mGFP tag using a fluorescence microscope, combined with PCR technology to verify the integration of the resistance gene.

[0059] Conduct field trials on the new resistant varieties of Trichosanthes kirilowii, including:

[0060] (a) Plant new resistant varieties of Trichosanthes kirilowii in multiple suitable areas and conduct comparative studies on their disease resistance, yield and quality;

[0061] (b) The control varieties were selected as local common Trichosanthes kirilowii varieties, and the growth cycle, disease occurrence and economic trait data were recorded;

[0062] (c) Apply statistical methods to test the significance of test results and evaluate the overall performance of new varieties.

[0063] Chromatin immunoprecipitation sequencing (ChIP-seq) and gel retardation experiments were used to further verify the interaction between key resistance genes and their downstream target genes.

[0064] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and describes these embodiments in detail to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is only limited by the claims and their full scope and equivalents. The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and describes these embodiments in detail to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A method for breeding new resistant varieties of Trichosanthes kirilowii based on molecular breeding technology, characterized in that: The following steps are involved: (S1) Screening of resistance genes in Trichosanthes kirilowii, using transcriptome sequencing technology and bioinformatics analysis to mine differentially expressed genes related to disease resistance in Trichosanthes kirilowii; (S2) constructing an overexpression vector of the resistance gene, and inserting the resistance gene into a plant expression vector carrying a marker gene by homologous recombination; (S3) using an Agrobacterium-mediated method to transform the constructed vector into Trichosanthes kirilowii callus tissue and cultivate transgenic positive plants; (S4) conducting a disease inoculation test on the positive plants to screen for plants with significant resistance; (S5) The selected resistant plants are propagated through hybrid breeding to obtain new resistant varieties with stable inheritance.

2. The method for breeding new resistant varieties of Trichosanthes kirilowii based on molecular breeding technology according to claim 1, characterized in that: The transcriptome sequencing in step (S1) is completed using the DNBSEQ platform. After the sequencing data is quality controlled, GO functional enrichment analysis and KEGG pathway enrichment analysis are performed using the DAVID database to determine candidate genes related to disease resistance.

3. The method for breeding new resistant varieties of Trichosanthes kirilowii based on molecular breeding technology according to claim 1, characterized in that: The overexpression vector in step (S2) is constructed using the pMDC202 vector, which has an mGFP tag for screening positive plants; the CDS region of the resistance gene is amplified by PCR and inserted into the multiple cloning site of the vector using homologous recombination technology.

4. The method for breeding new resistant varieties of Trichosanthes kirilowii based on molecular breeding technology according to claim 1, characterized in that: In the step (S3), Agrobacterium is used to infect the Trichosanthes kirilowii callus, and after the infection, the callus is transferred to a culture medium containing a selective antibiotic for screening until resistance-positive plants are obtained.

5. The method for breeding new resistant varieties of Trichosanthes kirilowii based on molecular breeding technology according to claim 1, characterized in that: The disease inoculation test in the step (S4) comprises: (S41) inoculating with vine blight pathogen, root knot nematode and anthracnose pathogen respectively; (S42) observing the occurrence of diseases and recording resistance scores; (S43) qRT-PCR technology was used to detect the expression level of resistance genes and verify their correlation with disease resistance.

6. The method for breeding new resistant varieties of Trichosanthes kirilowii based on molecular breeding technology according to claim 1, characterized in that: The hybrid breeding method in the step (S5) comprises the following process: (S51) using the selected resistant plants as parents and artificially pollinating to obtain F1 generation; (S52) screening the F1 generation plants for disease resistance, and selecting plants with stable resistance performance; (S53) Further hybridizing the plants with stable resistance until a new resistant variety of the F2 generation is obtained.

7. The method for breeding new resistant varieties of Trichosanthes kirilowii based on molecular breeding technology according to claim 1, characterized in that: The resistance verification of transgenic Trichosanthes kirilowii positive plants includes detecting the expression of mGFP tag by fluorescence microscopy and verifying the integration of resistance gene by PCR technology.

8. The method for breeding new resistant varieties of Trichosanthes kirilowii based on molecular breeding technology according to claim 1, characterized in that: Conduct field trials on the new resistant varieties of Trichosanthes kirilowii, including: (a) Plant new resistant varieties of Trichosanthes kirilowii in multiple suitable areas and conduct comparative studies on their disease resistance, yield and quality; (b) The control varieties were selected as local common Trichosanthes kirilowii varieties, and the growth cycle, disease occurrence and economic trait data were recorded; (c) Apply statistical methods to test the significance of test results and evaluate the overall performance of new varieties.

9. The method for breeding new resistant varieties of Trichosanthes kirilowii based on molecular breeding technology according to claim 1, characterized in that: Chromatin immunoprecipitation sequencing (ChIP-seq) and gel retardation experiments were used to further verify the interaction between key resistance genes and their downstream target genes.