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CN-121975879-A - Preparation method of diisopropylpeptide and application of diisopropylpeptide in agriculture

CN121975879ACN 121975879 ACN121975879 ACN 121975879ACN-121975879-A

Abstract

The invention discloses a preparation method of diisopropylpeptide and application thereof in agriculture, and relates to the technical field of agriculture, comprising the following steps of knocking out a common penicillium murA gene by gene editing, introducing an ilvBN-ilvA expression cassette, and constructing engineering bacteria BA-VV01; after fermentation, the cyclic (valine-valine) dipeptide, namely the diisopropylpeptide, is finally obtained through solid-liquid separation, resin enrichment, silica gel column and ODS chromatographic preliminary purification, HPLC preparation and NMR identification, and the preparation method of the diisopropylpeptide and the application thereof in agriculture, through knocking out mu rA and introducing an exogenous gene expression cassette (ilvBN-ilvA), the precursor supply and cyclization steps are directionally strengthened, the fermentation yield of the diisopropylpeptide is obviously improved, the defect of a chemical synthesis method is overcome, the inherent nutritional quality and the appearance commodity of crops can be simultaneously improved by the diisopropylpeptide, and the requirements of modern agriculture on green and high-efficiency input products are met.

Inventors

  • XIE JIALI
  • CHEN DAYIN
  • SHI LEI
  • WANG HONGFENG
  • ZHANG XIAOYING
  • DAI LILI
  • GENG QUANZHENG

Assignees

  • 山东蓬勃生物科技有限公司
  • 泰安蓬勃生物化工有限公司

Dates

Publication Date
20260505
Application Date
20260120

Claims (10)

  1. 1. The preparation method of the diisopropylpeptide is characterized by comprising the following steps of; S1, firstly constructing a knockout primer of the MurA gene of penicillium simplicissimum, obtaining a homologous arm fragment through PCR amplification, cloning the homologous arm fragment to pCas vectors, constructing knockout plasmid pCas-delta MurA, converting the knockout plasmid into host bacteria, and screening to obtain a MurA gene knockout strain; S2, connecting the ilvBN gene and the ilvA gene in series to construct an expression cassette, cloning the expression cassette into an expression vector pHT01 to obtain a recombinant expression plasmid, and transforming the recombinant expression plasmid into a mu rA gene knockout strain to obtain a recombinant engineering bacterium BA-VV01; S3, fermenting and culturing recombinant engineering bacteria BA-VV01 to obtain fermentation liquor; S4, carrying out solid-liquid separation on the fermentation liquor, and collecting supernatant, and carrying out enrichment extraction on the diisopropylpeptide in the supernatant by using macroporous adsorption resin or an organic solvent to obtain a crude product; S5, taking the crude product, performing primary separation by 200-300 mesh silica gel column chromatography and ODS (oxide dispersion medium) compression chromatography, combining with TLC (thin layer chromatography), and performing HPLC (high performance liquid chromatography) purification and NMR (nuclear magnetic resonance) analysis to obtain the cyclic (valine-valine) dipeptide, namely the diisopropylpeptide 。
  2. 2. The method for preparing the diisopropylpeptide according to claim 1, wherein the nucleotide sequence of the knockout primer of S1 is shown as SEQ ID NO.1 and SEQ ID NO.2, and the common Penicillium is preserved in China general microbiological culture Collection center with a preservation number of CGMCC NO.42330.
  3. 3. The method for producing a diisopropylpeptide according to claim 1, wherein the conditions for the fermentation culture of S3 are that the control temperature is 33-35℃in the early stage of fermentation, 28-30℃in the middle and later stages of fermentation, and the total fermentation time is 72 hours.
  4. 4. The method for producing a diisopropylpeptide according to claim 1, wherein the OD 600 of the recombinant engineering bacterium BA-VV01 is 2.8 and the inoculation volume of the recombinant engineering bacterium BA-VV01 is 10% of the volume of the fermentation medium in the fermentation culture of S3.
  5. 5. Use of a diisopropylpeptide prepared by the method of any one of claims 1-4 in agriculture.
  6. 6. The use according to claim 5, characterized in that the use in agriculture is the use in improving crop quality.
  7. 7. The use of claim 6, wherein the improvement in crop quality comprises an increase in at least one of vitamin content, soluble sugar content, single fruit weight, commodity rate, storage endurance of the crop, and further comprising a decrease in at least one of nitrate content, malformed fruit rate, post harvest rotten fruit rate.
  8. 8. The use according to claim 7, wherein the crop is fruit and vegetable and leaf vegetables.
  9. 9. Use according to claim 8, wherein said fruit vegetables comprise tomatoes and cucumbers and said leaf vegetables comprise lettuce.
  10. 10. The use according to claim 5, wherein the diisopropylpeptide is used at a concentration of 50-200ng/mL.

Description

Preparation method of diisopropylpeptide and application of diisopropylpeptide in agriculture Technical Field The invention relates to agricultural technology, in particular to a preparation method of diisopropylpeptide and application thereof in agriculture. Background The diisopropylpeptide, i.e., the cyclo (valine-valine) dipeptide, is a natural dipeptide having a unique cyclic structure. However, its extremely low content in nature, complicated chemical synthesis steps, low yields and the possibility of using harmful reagents, have limited its in-depth research and scale application. Therefore, the development of a preparation method of the diisopropylpeptide which is efficient, green and suitable for large-scale production has important significance. In agricultural production, plant growth regulators are often used to increase crop yield and quality in order to meet increasing quality and safety requirements. However, the chemical synthesis regulator commonly used at present has the problems of residual risk, single function, unfriendly environment and the like. Searching for biologically active substances with natural sources, safety, high efficiency and multiple benefits becomes a research hotspot in the agricultural field. If the diisopropylpeptide obtained by biological fermentation can be applied to agriculture, the diisopropylpeptide is expected to provide a novel and green solution for improving the quality of crops. Disclosure of Invention The invention aims to provide a preparation method of diisopropyl peptide and application thereof in agriculture, so as to solve the problems of complex preparation method, low yield, single function of a plant growth regulator and no green. In order to achieve the aim, the invention provides the following technical scheme that the preparation method of the diisopropylpeptide comprises the following steps of; S1, firstly constructing a knockout primer of the MurA gene aiming at penicillium vulgaris, obtaining a homologous arm fragment through PCR amplification, cloning the homologous arm fragment to pCas vectors, constructing a knockout plasmid pCas-delta mu rA, converting the knockout plasmid into host bacteria, and screening to obtain a MurA gene knockout strain; S2, connecting the ilvBN gene and the ilvA gene in series to construct an expression cassette, cloning the expression cassette into an expression vector pHT01 to obtain a recombinant expression plasmid, and transforming the recombinant expression plasmid into a mu rA gene knockout strain to obtain a recombinant engineering bacterium BA-VV01; S3, fermenting and culturing recombinant engineering bacteria BA-VV01 to obtain fermentation liquor; S4, carrying out solid-liquid separation on the fermentation liquor, and collecting supernatant, and carrying out enrichment extraction on the diisopropylpeptide in the supernatant by using macroporous adsorption resin or an organic solvent to obtain a crude product; S5, taking the crude product, performing primary separation by 200-300 mesh silica gel column chromatography and ODS (oxide dispersion medium) compression chromatography, combining with TLC (thin layer chromatography), and performing HPLC (high performance liquid chromatography) purification and NMR (nuclear magnetic resonance) analysis to obtain the cyclic (valine-valine) dipeptide, namely the diisopropylpeptide 。 Further, the nucleotide sequence of the knockout primer of S1 is shown as SEQ ID NO.1 and SEQ ID NO.2, and the common penicillium (Penicillium commune) is preserved in China general microbiological culture Collection center with the preservation date of 2025, 11 months and 27 days and the preservation number of CGMCC NO.42330 in the China national academy of sciences of China general microbiological culture Collection center with the Xila NO. 3 of the national academy of sciences of China, north Star in the Korean area of Beijing, S1. Further, the condition of the fermentation culture in the step S3 is that the control temperature in the early stage of fermentation is 33-35 ℃, the control temperature in the middle and later stages of fermentation is 28-30 ℃, and the total fermentation time is 72 hours. Further, in the fermentation culture of S3, the OD 600 of the recombinant engineering bacterium BA-VV01 is 2.8, and the inoculation volume of the recombinant engineering bacterium BA-VV01 is 10% of the volume of the fermentation medium. The application of the diisopropylpeptide prepared by the preparation method of the diisopropylpeptide in agriculture. Further, the application in agriculture is an application in improving crop quality. Further, the improvement of the crop quality comprises at least one of increasing the vitamin content, the soluble sugar content, the single fruit weight, the commodity rate and the storage endurance of the crop, and further comprises at least one of reducing the nitrate content, the malformed fruit rate and the post-harvest rotten fruit rate. Further, the