Search

CN-121975852-A - Barnyard grass EcCYP A105 gene and application thereof

CN121975852ACN 121975852 ACN121975852 ACN 121975852ACN-121975852-A

Abstract

The invention discloses a barnyard grass EcCYP A105 gene and application thereof, belonging to the technical field of plant biology, wherein the nucleic acid sequence of the EcCYP A105 gene is shown as SEQ ID NO.1, the amino acid sequence is shown as SEQ ID NO.2, and the barnyard grass CYP81A105 gene is discovered for the first time, has the function of improving the drug resistance of rice to ACCase herbicides, and can be used as a resistance gene for cultivating herbicide-resistant rice germplasm.

Inventors

  • YANG QIAN
  • LV MIN
  • WEI TIAN
  • ZHU JINLEI

Assignees

  • 江苏里下河地区农业科学研究所

Dates

Publication Date
20260505
Application Date
20260319

Claims (8)

  1. 1. A barnyard grass EcCYP A105 gene for improving the drug resistance of rice is characterized in that the nucleic acid sequence of the EcCYP A105 gene is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2.
  2. 2. Use of the barnyard grass EcCYP a105 gene of claim 1 for increasing tolerance of rice to ACCase herbicides.
  3. 3. The method of claim 2, wherein the ACCase herbicides include pinoxaden, metamifop and fenoxaprop-p-ethyl.
  4. 4. A method for improving the tolerance of rice to ACCase herbicides is characterized by comprising the steps of transferring EcCYP A105 genes into pEGOEP S-H plasmid to construct a plant over-expression vector pEGOEP S-H-CYP81A105 containing EcCYP A105 genes; The over-expression vector pEGOEP S-H-CYP81A105 is transformed into agrobacterium EHA105, and the agrobacterium infects rice callus to obtain a transgenic plant with over-expression of EcCYP81A105 gene, so that the tolerance of the rice to ACCase herbicides is improved.
  5. 5. The method according to claim 4, wherein the nucleic acid sequence of the over-expression vector pEGOEP S-H-CYP81A105 is shown by SEQ ID NO. 3.
  6. 6. The method of claim 4, wherein the over-expression vector pEGOEP S-H-CYP81A105 is transformed into Agrobacterium EHA105, comprising, Placing competent Agrobacterium on ice, adding recombinant plasmid DNA and mixing well, standing on ice for 30min, quick-freezing in liquid nitrogen for 1min, water-bathing at 37deg.C for 5min, and ice-bathing for 5min; Adding an antimicrobial-free YEP liquid culture medium under the aseptic condition, carrying out shaking culture at 28 ℃ and 200r/min for 3-4h, centrifuging at 5000r/min for 2min, collecting thalli, re-suspending by using the antimicrobial-free YEP liquid culture medium, coating on a YEP solid culture medium containing rifampicin and kanamycin, and carrying out dark culture at 28 ℃ for 48h until single colonies appear.
  7. 7. The method of claim 4, wherein the infection of rice callus with Agrobacterium comprises, Selecting mature rice seeds, mechanically dehulling, selecting full sterile plaque-free high-quality seeds, sterilizing, and inoculating to a culture medium to induce callus; selecting a single colony of well-grown agrobacterium on a plate, inoculating the single colony into a YEP liquid culture medium containing rifampicin and kanamycin, and carrying out shake culture at 28 ℃ for 14-16h at 200 r/min; Adding into YEP liquid culture medium containing rifampicin and kanamycin, and performing 28 ℃ 200r/min expansion culture until OD600 = 0.6-0.8; Placing the cultured bacterial liquid into a centrifuge tube, centrifuging at 5000 r/min for 5min, and collecting bacterial bodies to prepare agrobacterium suspension; Picking out the grown callus, placing the callus in agrobacterium suspension for infection for 20min, and transferring the dried callus to a screening culture medium for first screening; Two weeks later, the initial callus with the resistant callus is transduced into a new culture medium, and the second screening is carried out; Selecting and screening vigorous growth resistant calli, transferring the calli into a culture dish filled with a differentiation medium, sealing the calli with a sealing film, and placing the calli into a constant temperature culture room () for waiting differentiation into seedlings; and after the seedlings grow to 1cm, transferring to a rooting culture medium to strengthen the seedlings, thereby obtaining the EcCYP A105 gene over-expressed transgenic plants.
  8. 8. The method according to claim 7, wherein the cultivation is carried out at a constant temperature, wherein the cultivation condition is a 12-hour photoperiod, the daytime cultivation temperature is 28℃and the nighttime cultivation temperature is 25 ℃.

Description

Barnyard grass EcCYP A105 gene and application thereof Technical Field The invention belongs to the technical field of plant biology, and particularly relates to a barnyard grass EcCYP A105 gene and application thereof. Background Barnyard grass (Echiinochloa crus-galli (L.) Beauv) is one of the most widely distributed and seriously damaged malignant weeds in rice production areas in China, and the weed community mainly containing barnyard grass seriously threatens the quality and yield of rice in China. Research shows that barnyard grass weeds in the field not only can lead to yield reduction of rice, but also can lead to reduction of rice processing and taste quality. At present, in large-area production of paddy rice in China, chemical herbicide is mainly adopted for controlling weed harm by vast paddy rice planters, but due to excessive dependence and long-term use of a single chemical barnyard grass remover in local areas, the occurrence and rapid development of resistant barnyard grass population are caused, and great difficulty and challenges are brought to paddy barnyard grass prevention and control. Acetyl-CoA carboxylase (ACCase) inhibitor herbicides are main agents for preventing and killing gramineous weeds in paddy fields in China, and the action targets are single, so that drug resistance is easy to generate and the trend of continuous expansion is presented. The development of resistant weeds is to be suppressed in a green and efficient manner, and the resistance genes and functions thereof must be mined. At present, functional studies on the participation of barnyard grass CYP81A105 in weed drug resistance are not reported. Disclosure of Invention This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. The present invention has been made in view of the above and/or problems occurring in the prior art. Therefore, the invention aims to overcome the defects in the prior art and provide the barnyard grass EcCYP A105 gene for improving the drug resistance of rice. In order to solve the technical problems, the invention provides the technical scheme that the barnyard grass EcCYP A105 gene for improving the drug resistance of rice is provided, the nucleic acid sequence of the EcCYP A105 gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2. It is still another object of the present invention to overcome the deficiencies of the prior art and to provide an application of barnyard grass EcCYP A105 gene in improving rice tolerance to ACCase herbicides. As a preferred scheme of the application of the invention, the ACCase herbicide comprises pinoxaden, metamifop and fenoxaprop-p-ethyl. Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for improving the tolerance of rice to ACCase herbicides, comprising transferring EcCYP A105 gene into pEGOEP S-H plasmid to construct plant over-expression vector pEGOEP S-H-CYP81A105 containing EcCYP A105 gene; The over-expression vector pEGOEP S-H-CYP81A105 is transformed into agrobacterium EHA105, and the agrobacterium infects rice callus to obtain a transgenic plant with over-expression of EcCYP81A105 gene, so that the tolerance of the rice to ACCase herbicides is improved. As a preferred embodiment of the method of the present invention, the nucleic acid sequence of the over-expression vector pEGOEP S-H-CYP81A105 is shown in SEQ ID NO. 3. As a preferred embodiment of the method of the present invention, wherein the over-expression vector pEGOEP S-H-CYP81A105 is transformed into Agrobacterium EHA105, comprising, Placing competent Agrobacterium on ice, adding recombinant plasmid DNA and mixing well, standing on ice for 30min, quick-freezing in liquid nitrogen for 1min, water-bathing at 37deg.C for 5min, and ice-bathing for 5min; Adding an antimicrobial-free YEP liquid culture medium under the aseptic condition, carrying out shaking culture at 28 ℃ and 200r/min for 3-4h, centrifuging at 5000r/min for 2min, collecting thalli, re-suspending by using the antimicrobial-free YEP liquid culture medium, coating on a YEP solid culture medium containing rifampicin and kanamycin, and carrying out dark culture at 28 ℃ for 48h until single colonies appear. As a preferred embodiment of the method of the present invention, wherein said Agrobacterium infects rice callus, comprising, Selecting mature rice seeds, mechanically dehulling, selecting full sterile plaque-free high-quality seeds, sterilizing, and inoculating to a culture medium to induce callus; selecting a single colony of well-grown agrobacterium on a plate, inoculating the single colony into a YEP liquid culture medium containing rifampicin and kanamycin, and carrying out shake culture at 28 ℃ for 14-16h at 200 r/min; Adding into YEP liquid culture medium containing rifampicin and kanamycin, and performing 28 ℃ 200r/min expansion culture until OD600 = 0.6-0.8