Overview

   Our FAFU-china team aimed to establish a plant cell factory with high GABA production. To obtain a plant cell factory system for that, we constructed an overexpression GAD vector and a gene editing vector for knocking out CaM gene by using CRISPR/Cas9 gene editing technology, and our FAFU-china team also treated longan embryonic healing tissues with blue light, high temperature, drought stress and salt stress with the help of these series of means to increase the GABA content in longan.Laying a solid foundation for establishing a plant factory with high GABA production.
1. Modified genes (organisms)
   The content of GABA was increased by introducing exogenous recombinant genes into Escherichia coli and Agrobacterium tumefaciens for overexpression and knocking out individual genes.
2.Transform the environment
   In order to improve the content of GABA in longan callus, different treatment conditions were set up to regulate the physiological metabolism of longan cells, such as the effects of blue light treatment and high temperature treatment on the content of GABA in longan callus. The above describe that rationale behind the partial design of our system, as well as an overview of our experimental design.

Project advantages

   1.Professor Lai Zhongxiong in our laboratory established a longan embryogenic cell line which can preserve longan callus for a long time, providing a material basis for the experiment.
   2.Professor Lai Zhongxiong of our laboratory found that under the optimal conditions of somatic embryogenesis, the high frequency of somatic embryogenesis of longan embryogenic callus could be obtained by selecting the appropriate type of embryogenic callus, and selected the variety of Longan Red Seed.
   3.The laboratory has its own system in the study of longan.
  4.There are many ways to synthesize GABA, which provides a theoretical basis for the increase of GABA content.
  5.At present, cell factories focus on microbial fermentation, and there are few reports about plant cell factories. The experiment of this project is innovative.

Metabolic pathways

   There are two pathways of GABA synthesis and transformation in plants: one is the GABA shunt, in which glutamic acid is decarboxylated into GABA by glutamic acid decarboxylase (glutamic acid decarboxylase, GAD) (Fig. 1); The other is the conversion of polyamine degradation products to GABA, which is called polyamine degradation pathway (polyamine degradation pathway) (Fig. 2).
1. Glutamate-GABA pathway
2. GABA enrichment via the polyamine pathway

Specific implementation

Construction of overexpression vectors (not done because of the epidemic)
1. GAD gene cloning
   Carrying out PCR amplification by using F and R primers designed by DNAMan, carrying out agarose gel recovery on PCR products by using a GeneJET Gel Extraction Kit kit, The gel recovery product and plant vector expression pCAMBIA1301 + CaMV35S + NOS were digested with KpnI, PstI (GAD5) and KpnI, Sall (GAD2), and the gel recovery product was transferred to DH5a competence, coated on Karna resistant culture plates, and cultured overnight at 37 C. And that bacterial liquid is verify by PCR. After successful verification, the bacterial solution was sent to the company to feed back the sequence, and DNAMAN was used to compare the sequence to determine whether our target gene fragment was successfully connected to the vector. After success, the next experiment can be carried out.
2.Agrobacterium tumefaciens transformation
   One of the main goals of our project is to genetically engineer Agrobacterium tumefaciens to produce large amounts of GABA by overexpression of the GAD gene. In order to do this, we must first transfer the target gene fragment into Agrobacterium. The pCAMBIA1301 + CaMV35S + NOS vector we used is generally suitable for Agrobacterium-mediated plant transgenosis. From the map, the size of the vector was 12632 BP. A prokaryotic replication origin ori is used as an origin, and a replication origin containing pBR322 in turn from 5 'to 3', so that the vector can be replicated in prokaryotic cells such as Escherichia coli and Agrobacterium; Between the T border (L) and its corresponding T border (R) is a T-DNA region that can enter plant cells during Agrobacterium transfection, and in this T-DNA region, there is also a hygromycin resistance gene for screening positive plants or calli; Contains the 35S constitutive expression promoter; The GUS gene was expressed downstream of the promoter for histochemical staining. Therefore, the transformation of Agrobacterium tumefaciens with pCAMBIA1301 + CaMV35S + NOS shuttle vector is the core of our project.
   After the successful transformation of Agrobacterium tumefaciens, the longan callus materials in good condition were selected for infection experiment, and the samples were collected in a fixed number of days and stored in a refrigerator at -80 ℃ to determine the content of GABA in order to achieve the effect of high yield of GABA.
Overexpression vector I of GAD gene: GAD-2:1428bp KpnI SaII Overexpression vector II of GAD gene: GAD-5:1473bp KpnI PstI Gene editing
   FAFU-China successfully constructed a gene editing vector for knocking out CaM gene by using Genovo-w-3 (pGW3) vector through a series of steps of target primer annealing, BsaI enzyme cleavage of pGW3 plasmid, target ligation reaction, transformation, identification of positive clones, and transfer to Agrobacterium. Exogenous treatment
1. Exogenous GABA treatment
   One control, five GABA concentration treatments (2,4,6,8,10, unit µmol/L) were repeated for three times, and 2,4,6,8,10 µmol/L GABA was added to the culture medium respectively, and the proliferation and differentiation of longan callus somatic cells were observed under the microscope on 6/9/12 days respectively. And that polyamine content was determine. Index: Longan growth, endogenous hormone content (IAA, GA, ETH, etc.), endogenous GABA content
2.Exogenous salt stress treatment
   0 (CK), 50, 75, 100, 200 mmol/L NaCl solutions were added to the culture medium for three times, and the endogenous GABA content of longan embryogenic callus was determined after 6/9/12/20 days of culture. Different concentrations of NaCl were added to the M1 medium. Longan EC growing well on M1 medium for 20 days were inoculated on M1 medium containing four different NaCl concentrations.
Culture conditions: 25 ℃, dark culture.
3.Blue light treatment: (samples were taken at different times to establish a model of GABA response to different durations of light treatment)
Material: Longan callus (+ Ag) subcultured for 20 days was directly treated with blue light in culture flask
Number of treatments: 27 bottles of materials with relatively consistent growth
Blue light parameters: 32umol·m-2·S-2
Treatment time: 30min, 60min, 90min, 120min, 150min, 180min, 210min. Take out 3 bottles at each time point mix the samples on the filter paper, put them into the freezing tube, make corresponding marks, put them into liquid nitrogen, and store them in the refrigerator at -80 ℃.
The specific method was as follows: the materials treated for 20 days were transferred with black bags and placed in a blue light incubator, and the samples were mixed every 3 bottles when they were collected. The materials treated by blue light at different times were collected 0.1g per tube, and 6 tubes were collected respectively, and then put into liquid nitrogen immediately 4.High temperature stress treatment: (GABA responds to the high temperature treatment model of different time when sampling at different time)
Material: Longan callus (+ Ag) subcultured for 20 days was directly treated with high temperature stress in culture flask
Number of treatments: 30 bottles of materials with relatively consistent growth
Treatment temperature: 40 ℃
Treatment time: 30min, 60min, 90min, 120min,150min, 180min. Take out 3 bottles at each time point, mix the samples on the filter paper, put them into the freezing tube, make corresponding marks, put them into liquid nitrogen, and store them in the refrigerator at -80 ℃.
The specific method was as follows: the materials treated for 20 days were transferred with black bags and placed in the temperature treatment incubator, and the samples were mixed every 3 bottles when the samples were collected. The materials treated at high temperature for different times were collected 0.1g per tube, and 6 tubes were collected respectively, and then put into liquid nitrogen immediately