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CN-121975859-A - Efficient induction method for hairy roots of sugarcane based on WIND1-Ruby

CN121975859ACN 121975859 ACN121975859 ACN 121975859ACN-121975859-A

Abstract

The invention provides a high-efficiency induction method for sugarcane hairy roots based on WIND1-Ruby, belonging to the technical field of plant biology, the method comprises the steps of infecting the bud basal part of a sugarcane node stem segment with sprouting by utilizing co-expression agrobacterium rhizogenes containing WIND1 and Ruby reporter genes, and obtaining positive hairy roots of sugarcane through culture and induction. The Ruby reporter gene in the carrier used by the invention can lead the induced hairy root to present characteristic Ruby red, realize the visualization of transformation event, and can rapidly identify positive hairy root without complex dyeing or molecular detection, and further remarkably overcome the technical barriers of weak regeneration capability and low transformation efficiency of sugarcane bud basal part by introducing WIND1 transcription factor into the carrier, and the established sugarcane hairy root induction system has the advantages of simple operation, high transformation efficiency, visual screening, short period and the like.

Inventors

  • QUE YOUXIONG
  • SUN TINGTING
  • WANG PEILIN
  • WU QIBIN
  • ZHOU LILI
  • WANG DONGJIAO
  • ZHANG YUANYUAN
  • ZHAO WANYING
  • ZENG ZHEN
  • WANG WENZHI

Assignees

  • 中国热带农业科学院三亚研究院
  • 中国热带农业科学院热带生物技术研究所

Dates

Publication Date
20260505
Application Date
20260408

Claims (8)

  1. 1. A high-efficiency induction method for hairy roots of sugarcane based on WIND1-Ruby is characterized in that the method is characterized in that a co-expression Agrobacterium rhizogenes containing WIND1 and Ruby reporter genes is utilized to infect the bud basal part of a sugarcane node stem segment with sprouting, and positive hairy roots of sugarcane are obtained through culture and induction; Co-expression agrobacterium rhizogenes containing WIND1 and Ruby reporter genes are obtained by introducing a vector containing WIND1 and Ruby reporter genes into agrobacterium rhizogenes; The vector containing WIND1 and Ruby reporter genes is a modified plant binary expression vector with pCAMBIA1302 framework, wherein the T-DNA region only comprises a Ruby reporter gene expression cassette driven by a MAS promoter and a WIND1 transcription factor coding sequence driven by another CaMV 35S promoter; the sequence of the coding region of the Ruby gene is shown as SEQ ID NO. 1; The sequence of the WIND1 gene coding region is shown as SEQ ID NO. 2.
  2. 2. The method for efficiently inducing hairy roots of sugarcane based on WIND1-Ruby according to claim 1, wherein the method comprises the following specific steps: Activating and culturing the co-expression agrobacterium rhizogenes containing WIND1 and Ruby reporter genes to obtain an infection bacterial liquid; Selecting a sugarcane node stem section with sprouting; inoculating the infection bacteria liquid to the bud basal part of the sugarcane node stem segment; culturing the inoculated sugarcane node stem segment, and inducing the sugarcane node stem segment to generate hairy roots from an inoculated part.
  3. 3. The efficient induction method for sugarcane hairy roots based on WIND1-Ruby according to claim 2, wherein the OD 600 of the infectious microbe liquid is 0.6-0.8.
  4. 4. The efficient induction method for hairy roots of sugarcane based on WIND1-Ruby according to claim 2 or 3, wherein the specific preparation process of the infectious microbe liquid is as follows: Activating the strain, namely streaking the co-expression agrobacterium rhizogenes containing WIND1 and Ruby reporter genes on a solid plate, and culturing in an inverted mode; liquid culturing, namely picking corresponding single colony, inoculating the single colony into a liquid culture medium, and shaking culturing overnight; Expanding culture and collection, namely transferring overnight culture into a liquid culture medium, continuously culturing, centrifuging the obtained bacterial liquid, and discarding the supernatant; re-suspending, namely re-suspending bacterial precipitate by using an infection buffer solution, respectively adjusting OD 600 , standing and activating to obtain an infection bacterial solution.
  5. 5. The efficient induction method of sugarcane hairy roots based on WIND1-Ruby according to claim 4, wherein in the process of expanding culture and collection, overnight culture is continuously cultured in a liquid culture medium until the OD 600 value reaches 0.4-1.0.
  6. 6. A method for efficiently inducing hairy roots of sugarcane based on WIND1-Ruby according to any one of claims 1-3 and 5 is characterized in that the specific acquisition process of a vector containing WIND1 and Ruby reporter genes is as follows, a plasmid containing Ruby reporter genes is used as a template, the encoding region of Ruby gene is amplified, amplified products are subjected to NcoI and BstEII double digestion and then are inserted between a MAS promoter and a NOS terminator of a pCAMBIA1302 framework by a seamless cloning technology to construct a p1302-Ruby vector, then XhoI restriction endonuclease is used for carrying out single digestion on the p1302-Ruby vector to remove the original HygR screening marker genes, a plasmid containing Arabidopsis AtWIND encoding sequences is used as a template, the encoding region of ND1 gene is amplified, the linearized vector and the amplification products of WIND1 are subjected to seamless cloning connection, so that the ND1 gene replaces the original HygR marker, and the p1302-Ruby vector is obtained under the driving of double CaMV 35S promoters.
  7. 7. The method for efficient induction of hairy roots of sugarcane based on WIND1-Ruby according to any one of claims 1-3 and 5, wherein agrobacterium rhizogenes is strain K599 or other suitable agrobacterium rhizogenes strain.
  8. 8. The efficient induction method for hairy roots of sugarcane based on WIND1-Ruby according to any one of claims 1-3 and 5, wherein the culture conditions are that the temperature is 25-28 ℃, the relative humidity is 70-80%, and the photoperiod is 16h illumination/8 h darkness.

Description

Efficient induction method for hairy roots of sugarcane based on WIND1-Ruby Technical Field The invention belongs to the technical field of plant biology, and relates to a high-efficiency induction method for hairy roots of sugarcane based on WIND 1-Ruby. Background Sugarcane (Saccharum officinarum) is a global important sugar crop and energy crop, has large and complex genome and difficult genetic transformation, and severely restricts the research of sugarcane functional genomics and the molecular breeding process. The hairy root culture system is a large amount of proliferated adventitious roots generated after agrobacterium rhizogenes (Agrobacterium rhizogenes) infects plants, has the characteristics of rapid growth, hormone autonomy, genetic stability and strong secondary metabolite synthesis capability, and is an ideal material for researching gene functions, root system biology, plant-microorganism interaction and producing secondary metabolites. At present, hairy root induction systems such as cotton, soybean, etc. have been successfully established in a variety of plants. However, there is no report on the hairy root induction system of the important crop of sugarcane. The reason for this is that the whole plant tissue of sugarcane (including stem segments, leaves, bud bases, etc.) has the common problems of poor regeneration capability, insensitivity to agrobacterium infection, difficulty in stable transformation of induced adventitious roots, etc., especially when the bud bases are used as explants, the cells are in a relatively static state, and root primordium differentiation is difficult to start, so that the existing method has extremely low or unstable transformation efficiency. In the existing method, explants such as leaves and stem segments are used for co-culture, and the problems of complex operation, unstable transformation efficiency, time and labor consumption in positive identification and the like exist. Furthermore, traditional hairy root positive identification often relies on molecular means such as PCR, GUS staining, etc., which are not only time consuming but also destructive. In recent years, the advent of visual reporters such as Ruby has provided new possibilities for real-time, non-destructive monitoring of transgenic events. The Ruby gene can enable the transformed tissue to accumulate betalain and display vivid red, thereby greatly facilitating the screening of positive materials. However, the team of the present invention found in earlier studies that treatment of sugarcane shoot bases with Agrobacterium rhizogenes alone carrying Ruby reporter genes was still not ideal for hairy root induction efficiency, indicating that there was an inherent stuffy regeneration of sugarcane shoot bases. Therefore, it is necessary to establish a sugarcane hairy root induction method which is easy to operate, efficient and capable of visually screening, and the method has important significance for promoting basic research and biotechnology application of sugarcane. Disclosure of Invention Aiming at the problems, the invention provides a high-efficiency induction method for hairy roots of sugarcane based on WIND 1-Ruby. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: A high-efficiency induction method of hairy roots of sugarcane based on WIND1-Ruby comprises the steps of infecting a bud basal part with a germinated sugarcane node stem segment by utilizing co-expression agrobacterium rhizogenes containing WIND1 and Ruby reporter genes, and culturing and inducing to obtain positive hairy roots of sugarcane; Co-expression agrobacterium rhizogenes containing WIND1 and Ruby reporter genes are obtained by introducing a vector containing WIND1 and Ruby reporter genes into agrobacterium rhizogenes; The vector containing WIND1 and Ruby reporter genes is a modified plant binary expression vector with pCAMBIA1302 framework, wherein the T-DNA region only comprises a Ruby reporter gene expression cassette driven by a MAS promoter and a WIND1 transcription factor coding sequence driven by another CaMV 35S promoter; the sequence of the coding region of the Ruby gene is shown as SEQ ID NO. 1; The sequence of the WIND1 gene coding region is shown as SEQ ID NO. 2. Further, the method comprises the following specific steps: Activating and culturing the co-expression agrobacterium rhizogenes containing WIND1 and Ruby reporter genes to obtain an infection bacterial liquid; Selecting a sugarcane node stem section with sprouting; inoculating the infection bacteria liquid to the bud basal part of the sugarcane node stem segment; culturing the inoculated sugarcane node stem segment, and inducing the sugarcane node stem segment to generate hairy roots from an inoculated part. Further, the OD 600 of the infectious microbe liquid is 0.6-0.8. Further, the specific preparation process of the infectious microbe liquid comprises the following steps: Activating the str