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CN-121992022-A - Product and method for improving output of breviscapine in plant body and construction method of tobacco synthesis biological chassis of breviscapine

CN121992022ACN 121992022 ACN121992022 ACN 121992022ACN-121992022-A

Abstract

The invention relates to the technical field of genetic engineering, in particular to a product and a method for improving the yield of breviscapine in plants and a construction method of a tobacco synthesis biological chassis of the breviscapine. The invention provides application of any one or more gene sequences shown in SEQ ID NO. 1-SEQ ID NO.6 in improving the yield of breviscapine in plants, wherein any one or more gene sequences shown in SEQ ID NO. 1-SEQ ID NO.6 are connected with a pCAMBIA1302 vector to obtain a recombinant expression vector, the recombinant expression vector is transformed into agrobacterium to obtain recombinant bacteria, the plants are infected by bacterial suspension of the recombinant bacteria to obtain plants with high yield of breviscapine, the obtained transgenic tobacco plants have the scutellarin yield of 42.09 mug/g DW, and a foundation is laid for efficient heterologous synthesis of the breviscapine in tobacco.

Inventors

  • YIN QUANYU
  • Jing Gege
  • YANG MENGQUAN

Assignees

  • 河南农业大学

Dates

Publication Date
20260508
Application Date
20260213

Claims (10)

  1. The application of 1.SEQ ID NO.1~SEQ ID NO.6 to the improvement of the yield of breviscapine in plants is characterized in that one or more gene sequences shown in SEQ ID NO. 1-SEQ ID NO.6 are connected with a pCAMBIA1302 vector to obtain a recombinant expression vector, the recombinant expression vector is transformed into agrobacterium to obtain recombinant bacteria, and the bacteria suspension of the recombinant bacteria is used for infecting plants to obtain the plants with high yield of breviscapine.
  2. 2. The use according to claim 1, wherein the gene sequence of SEQ ID No.1 is a erigeron breviscapus test ketone synthase gene; the gene sequence described by SEQ ID NO.2 is erigeron breviscapus ficus ketoisomerase gene; the gene sequence described by SEQ ID NO.3 is erigeron breviscapus flavone synthetase II gene; the gene sequence described by SEQ ID NO.4 is erigeron breviscapus flavonoid 7-O-glucuronic acid transferase gene; the gene sequence described by SEQ ID NO.5 is erigeron breviscapus uridine diphosphate glucose dehydrogenase gene; the gene sequence described by SEQ ID NO.6 is erigeron breviscapus flavone 6-hydroxylase gene.
  3. 3. The use according to claim 1, wherein the agrobacterium is agrobacterium GV3101; OD 600 of the bacterial suspension of the recombinant bacteria is 0.5-0.7.
  4. 4. The use according to claim 1, wherein the plant is tobacco.
  5. 5. An expression cassette for improving the yield of breviscapine in plants, which is characterized by comprising any one of the following components: (1) A recombinant expression vector; (2) Recombinant bacteria; The recombinant expression vector is constructed by connecting any one or more gene sequences shown in SEQ ID NO. 1-SEQ ID NO.6 with a pCAMBIA1302 vector; The construction method of the recombinant bacterium comprises the steps of transforming any one or more gene sequences shown in SEQ ID NO. 1-SEQ ID NO.6 into agrobacterium GV3101, or transforming the recombinant expression vector in (1) into agrobacterium GV 3101.
  6. 6. The use of the expression cassette of claim 5 for increasing the yield of breviscapine in plants.
  7. 7. A method for improving the yield of breviscapine in plants, which is characterized by comprising the following steps: Transferring any one or more gene sequences shown in SEQ ID No. 1-SEQ ID No.6 into a plant body, or transferring any one of the expression cassettes described in claim 5 into a plant body.
  8. 8. A construction method of a tobacco synthesis biological chassis of breviscapine, which is characterized by comprising the following steps: Connecting any one or more gene sequences shown in SEQ ID NO. 1-SEQ ID NO.6 with a pCAMBIA1302 vector to obtain a recombinant expression vector, transforming the recombinant expression vector into agrobacterium to obtain recombinant bacteria, and infecting tobacco with bacterial suspension of the recombinant bacteria.
  9. 9. The construction method according to claim 8, wherein the agrobacterium is agrobacterium GV3101; OD 600 of the bacterial suspension of the recombinant bacteria is 0.5-0.7.
  10. 10. The application of the primer pair for detecting the expression quantity of any one or more genes shown in SEQ ID NO. 1-SEQ ID NO.6 in screening of high-yield erigeron breviscapus varieties is characterized in that, The primer pair for detecting the expression quantity of the SEQ ID NO.1 gene is shown as SEQ ID NO. 31-SEQ ID NO. 32; The primer pair for detecting the expression quantity of the SEQ ID NO.2 gene is shown as SEQ ID NO. 33-SEQ ID NO. 34; The primer pair for detecting the expression quantity of the SEQ ID NO.3 gene is shown as SEQ ID NO. 35-SEQ ID NO. 36; The primer pair for detecting the expression quantity of the SEQ ID NO.4 gene is shown as SEQ ID NO. 37-SEQ ID NO. 38; The primer pair for detecting the expression quantity of the SEQ ID NO.5 gene is shown as SEQ ID NO. 39-SEQ ID NO. 40; The primer pair for detecting the expression quantity of the SEQ ID NO.6 gene is shown as SEQ ID NO. 41-SEQ ID NO. 42.

Description

Product and method for improving output of breviscapine in plant body and construction method of tobacco synthesis biological chassis of breviscapine Technical Field The invention relates to the technical field of genetic engineering, in particular to a product and a method for improving the yield of breviscapine in plants and a construction method of a tobacco synthesis biological chassis of the breviscapine. Background Erigeron breviscapus, also known as erigeron breviscapus, is a dried whole herb of erigeron breviscapus Erigeron breviscapus (vant.) hand. The erigeron breviscapus has a plurality of active ingredients, and at present, approximately 200 compounds are separated from the erigeron breviscapus, wherein flavone and glycoside, polysaccharide, caffeoyl and other compounds are main active ingredients of the erigeron breviscapus. The erigeron breviscapus is a mixture of flavonoids extracted from herba Erigerontis. Scutellarin (scutellarin, SCU) is the main active substance of scutellarin, has abundant pharmacological effects, and can dilate blood vessel, protect nerve, inhibit platelet aggregation, etc. Has good therapeutic effect on cerebral ischemia injury, tumor, osteoarthritis, atherosclerosis, alzheimer disease and other diseases. Especially in the aspect of tumor treatment, scutellarin has good inhibition effect on breast cancer, lung cancer, liver cancer, prostate cancer, gastric cancer, melanoma and the like. The extraction sources of the breviscapine are limited, and the market demands cannot be met only by relying on the traditional plant extraction mode. To solve this problem, studies have been continuously conducted on the synthetic route of calendula. The complete synthesis approach of breviscapine is established in Saccharomyces cerevisiae by genomics and synthetic biology methods, key enzymes of flavone-7-O-glucuronyl transferase (EbF GAT) and flavone-6-hydroxylase (EbF H) are identified from the erigeron breviscapus, and the engineering strain can directly produce main active components of scutellarin and apigenin-7-O-glucuronide of the erigeron breviscapus from glucose through metabolic engineering optimization, and the yields reach 108 mg/L - 1 and 185 mg/L - 1 respectively. In yarrowia lipolytica, the prior art uses CRISPR/Cas9 technology for multiple rounds of gene integration, allowing scutellarin production to reach 346 mg/L in the fermenter. Subsequently, one improved oxygen supply by screening for a highly active P450 enzyme combination (SbF 6H-ATR 2) and introducing heterologous hemoglobin, and finally achieved the highest yield of 703 mg/L in shake flasks. In addition, there have been studies on the production of breviscapine using plants as a chassis, by identifying its naturally contained precursor of breviscapine in Artemisia annua and heterologously expressing flavone synthase II (EbFNSII), flavone-6-hydroxylase (EbF H) and flavone-7-O-glucuronic acid transferase (EbF-GAT) from erigeron breviscapus, the synthesis of breviscapine in Artemisia annua was successful. Further use of SbF6H from Scutellaria baicalensis significantly improved the yield (up to 0.64 mg/g dry weight) without affecting artemisinin synthesis. Provides a new strategy for the rapid and large-scale production of natural plant products. Tobacco is a commonly used synthetic biological chassis plant, has unique advantages in the synthesis of flavonoids, is itself rich in abundant phenylpropane precursor substances, such as naringenin and apigenin, and provides a solid metabolic basis for the biosynthesis of flavonoids. Moreover, the Nicotiana benthamiana and the common Nicotiana in the tobacco have the characteristics of higher economic value, relatively shorter growth cycle, suitability for genetic transformation operation, good disease resistance and stress resistance, large-scale planting and the like, and are widely applied to experimental operation of various synthetic biology at present. The synthesis of breviscapine by using tobacco as a biological chassis plant has important biological significance, and the invention is put forward based on the important biological significance. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a product and a method for improving the output of breviscapine in plants and a construction method of a tobacco synthesis biological chassis of the breviscapine. In order to achieve the above object, the present invention provides the following technical solutions: The invention provides application of any one or more gene sequences shown in SEQ ID No. 1-SEQ ID No.6 in improving the yield of breviscapine in plants, wherein any one or more gene sequences shown in SEQ ID No. 1-SEQ ID No.6 are connected with a pCAMBIA1302 vector to obtain a recombinant expression vector, the recombinant expression vector is transformed into agrobacterium to obtain recombinant bacteria, and bacteria suspension of the recombinant bacteria is utilized to infe