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CN-122012572-A - Application of tobacco NtWRKY gene, coding protein, vector and host cell

CN122012572ACN 122012572 ACN122012572 ACN 122012572ACN-122012572-A

Abstract

The invention discloses application of tobacco NtWRKY gene, coding protein, vector and host cell. NtWRKY6 was first found to promote the synthetic accumulation of tobacco polyphenols by NtPAL and NtWRKY 41. Genetic material with significantly increased levels of expression and polyphenol content of genes involved in polyphenol synthesis is obtained by transgenic technology through NtWRKY gene over-expression (over expression). Genetic material with a NtWRKY functional deletion resulting in a significant reduction in polyphenol content was obtained using gene editing techniques. Provides a new way for regulating and controlling beneficial metabolites of tobacco and obtaining new varieties.

Inventors

  • HUANG PINGJUN
  • LIU WANFENG
  • XU XIANGLI
  • WANG DONG
  • YANG XIAONIAN
  • WANG SHUAIBIN
  • GAO JUNPING
  • ZHANG XINYAO
  • PU WENXUAN
  • PENG YU
  • LI XIAOXU
  • HE XINXI

Assignees

  • 湖南中烟工业有限责任公司

Dates

Publication Date
20260512
Application Date
20241111

Claims (10)

  1. 1. The application of tobacco NtWRKY gene or protein in regulating plant polyphenol synthesis is characterized in that the NtWRKY gene sequence is shown as SEQ ID NO.1, and the NtWRKY protein sequence is shown as SEQ ID NO. 2.
  2. 2. The use of claim 1, wherein the plant comprises tobacco.
  3. 3. Use according to claim 1 or 2, characterized in that it promotes or inhibits the accumulation of tobacco polyphenols in particular.
  4. 4. The use according to claim 3, wherein the overexpression of the tobacco NtWRKY gene or protein results in an increase in polyphenol content and wherein the suppression of the expression of the tobacco NtWRKY gene or protein results in a decrease in polyphenol content.
  5. 5. An over-expression vector containing tobacco NtWRKY gene, a gene editing vector containing inhibiting tobacco NtWRKY gene, or the application of host cells of the vector in regulating and controlling plant polyphenol synthesis, wherein the NtWRKY gene sequence is shown as SEQ ID NO. 1.
  6. 6. The use of claim 5, wherein the plant comprises tobacco.
  7. 7. Use according to claim 5 or 6, characterized in that it promotes or inhibits the accumulation of tobacco polyphenols in particular.
  8. 8. The use according to claim 5 or 6 or 7, wherein the overexpression vector causes an increase in the polyphenol content.
  9. 9. The use according to claim 5 or 6 or 7, wherein said gene editing vector results in a reduced polyphenol content.
  10. 10. Use of the vector of claim 5, or of a host cell, in particular tobacco, for the preparation of a transgenic plant.

Description

Application of tobacco NtWRKY gene, coding protein, vector and host cell Technical Field The invention belongs to the field of genetic engineering, and in particular relates to application of tobacco NtWRKY gene, encoded protein vector and host cell in regulating polyphenol synthesis. Background Polyphenols in tobacco are important aroma precursors in tobacco, have important influences on tobacco color, cigarette aroma and smoke, and are one of important indexes for measuring tobacco quality. The polyphenols are positively correlated with the grade of tobacco products, the polyphenols content of high-grade tobacco leaves is higher, and the polyphenols content of low-grade tobacco leaves is low. Under reasonable mixing and aging conditions, the polyphenol compounds are degraded through a series of reactions to generate a series of degradation products, which can endow the tobacco products with elegant fragrance, increase the fragrance quantity of the tobacco products and play an important role in improving the quality of the tobacco products. The tobacco contains various phenolic substances, mainly chlorogenic acid, rutin, scopoletin and other polyphenol compounds, and has very small content of simple phenols. The contents of polyphenols in different types of tobacco are greatly different, the highest content in flue-cured tobacco can reach 7%, and the content in sun-cured tobacco is very low, which accounts for about 0.5%. The total amount of chlorogenic acid, scopoletin, rutin and polyphenol in flue-cured tobacco leaves in different aroma type tobacco areas is obviously different, the content of polyphenols is greatly different among flue-cured tobacco varieties of different genotypes, the total amount of polyphenols is not greatly changed in different years of the same genotype variety in the same tobacco area, the content of polyphenols in the same tobacco area is different for different genotype varieties, and genetic factors are the main reasons for causing the difference of the content of polyphenols in flue-cured tobacco leaves of different genotypes. The variety is taken as a genetic factor to directly influence the content of the polyphenols of the flue-cured tobacco, the differences of the content and the components of the polyphenols of the flue-cured tobacco varieties with different genotypes can be inherited to offspring, and the offspring polyphenols of the genotype varieties with high content of the polyphenols are also high. The different genotypes of different tobacco varieties have different polyphenol metabolism related enzyme activities and different gene expression amounts, so that the polyphenol levels are different. The larger the genotype difference between different varieties is, the larger the difference of the polyphenol content is. Therefore, the variety determines the metabolism and the content of the polyphenols in the tobacco leaves to a great extent. Polyphenols in plants are synthesized by the phenylpropane metabolic pathway (phenylpropanoid pathway) which starts from the aromatic amino acid produced by the shikimate pathway (SHIKIMATE PATHWAY), phenylalanine. Phenylalanine catalyzes the production of p-coumarate-CoA (p-Coumaroyl CoA) via phenylalanine ammonia lyase (PAL, PHENYLALANINE AMMONIA LYASE), cinnamic acid 4-hydroxylase (C4H, CINNAMATE 4-Hydroxylase) and 4-coumarate-CoA ligase (4 cl, p-Coumaroyl Coenzyme A Ligase) in sequence. P-coumarate-coa is a common precursor for the synthesis of various polyphenols, for example, chalcone synthase (CHS, chalcone Synthase) catalyzes the formation of naringin chalcone from one p-coumarate-coa and three malonyl-coenzymes A (manolyl CoA), which is the first step in the flavonoid biosynthetic pathway, followed by the production of flavonols, anthocyanins, procyanidins and other flavonoids. Chlorogenic acid (CGA) in plants is also synthesized via the phenylpropane metabolic pathway, which may have 3 synthetic pathways, with both pathway 1 and pathway 2 requiring p-coumaric acid coa as a substrate. In most Solanaceae plants CGA is synthesized by pathway 1, i.e. p-coumaric acid CoA is catalyzed by hydroxycinnamoyl CoA shikimate/quinic acid-hydroxycinnamoyl transferase (HCT, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase) to p-coumaryl shikimate, which is further catalyzed by p-coumarin3 '-hydratase (C3H, p-coumarate 3' -hydroxlase) and HCT to caffeoyl CoA. Caffeoyl-coa and quinic acid are condensed by hydroxycinnamoyl-coa quinic acid-hydroxycinnamoyl transferase (HQT, hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase) to produce CGA. Meanwhile, caffeoyl-CoA A O-methyltransferase (CCoAOMT, caffeoyl CoA O-METHYLTRANSFERASE) can also further catalyze caffeoyl-CoA to generate feruloyl-CoA, which is an important precursor for catalyzing synthesis of 6'-hydroxy feruloyl-CoA by feruloyl-CoA 6' -hydroxylase 1 (F6 'H1, feruloyl-CoA 6' -Hydroxylase 1). Scopoletin is produced by isomerisation and lactonization of 6' -