1.1 Design

β-Carotene, as a precursor of astaxanthin, is widely found in higher plants. We plan to construct an expression system for astaxanthin in longan fruits, and the first step is to extract and detect the carotenoid content in longan tissues.

According to the carotenoid extraction methods consulted, commonly employed methods include supercritical fluid extraction, enzymatic catalysis, and ultrasonic or microwave assisted extraction method based on organic solvent. After analyzing and comparing these methods, we found that microwave-assisted technology can improve the extraction efficiency and yield of astaxanthin but may reduce the biological activity of the extract. After discussing with our supervisor, we decided to use a carotenoid detection kit. The extraction principle involves mixing the sample with an organic solvent to separate carotenoids from non-carotenoid components. Carotenoids have a characteristic absorption peak at 440nm. This method has good extraction efficiency and is relatively simple to operate.

1.2 Build

Using the Carotenoid Extraction Kit, Carotenoid Reagent 1 (product solution formula undisclosed): Liquid, 1000 mL × 2 bottles, stored at 4°C. Reagent 2 (product solution formula undisclosed): Liquid, 1000 mL × 1 bottle, stored at 4°C.Prior to use, mix Reagent 1 and Reagent 2 in a ratio of 2:1 to form the Extracting Solution, and store it sealed at 4°C to prevent evaporation.

1.3 Test

Add approximately 6 ml of the Extracting Solution to 0.2 g of tissue and allow it to soak for 20 minutes. Repeat the extraction process until the solution becomes colorless. Combine the extracts and adjust the total volume to 50 ml. Centrifuge the solution at 5000 rpm for 10 minutes, and then take the supernatant to measure its absorbance at 440 nm using a spectrophotometer. The final result can be obtained through a calculation formula.

V: total volume of extract, 50mL;

ε: Empirical absorbance coefficient of carotenoids, 2500;

d: cuvette aperture, 1cm;

W: Sample mass, g

1.4 Learning

The results showed that longan pulp, leaves, and other parts contain abundant carotenoids. Among them, the carotenoid content in mature leaves is higher than that in young leaves, while the content in longan pericarp is relatively low, and there is almost no carotenoid in the seed.

During the measurement process, we noticed that the extraction solution is volatile and toxic, so protective measures should be taken during operation. The measurement must be carried out quickly to prevent errors caused by volatilization.

2.1 Design

Based on the results of Iteration One, we preliminarily believe that our design has certain theoretical feasibility. Therefore, we further narrowed the target range of longan tissues, and continued to extract astaxanthin from tissues with high carotenoid content—flesh and leaves. Astaxanthin is a type of carotenoid, a liposoluble pigment that is insoluble in water but soluble in certain organic solvents such as acetone.

2.2 Build

Based on the reviewed literature, we have designed a method for extracting astaxanthin from longan fruits and leaves. DMSO was used as the extraction reagent. We will use instruments such as a balance, oven, 100 mesh sieve, glass tube, microplate reader, 96-well plate,50 ml centrifuge tubesand[2][3][4].

2.3 Test

The astaxanthin content is determined using the spectrophotometric method. Extract 10 mg of tissue powder with 5mLDMSO in a glass tube for 10 min, and then centrifuge the mixture at 4,000 rpm for 10 min to collect the supernatant.It is important to note that the extraction process should be performed away from light. Finally, the red supernatant is diluted to 25 mL with the extraction solvent, and the absorbance is measured at 530 nm using the Microplate reader.

2.4 Learning

The results of extracting astaxanthin from longan leaves and fruits were not ideal. At the apparent level, we found that the content of astaxanthin in both longan leaves and fruits is very low. This prompts us to conduct further exploration at the genetic level.

3.1 Design

After reviewing a substantial amount of relevant literature, we have tentatively identified the CrBKT gene and the HpBHY gene as having significant impacts on astaxanthin synthesis. Our plan is to screen the longan genome for endogenous genes that may have homologous relationships with these two genes.

3.2 Build

Based on previous research, locate the corresponding gene sequences (GenBank No. AY860820 and GenBank No. BD250390) and use the online analysis tool ExPASy Web (Artimo et al., 2012) (https://web.expasy.org/translate/)to translate them into amino acid sequences[1]. Utilize the TBtools analysis tool to conduct a preliminary BLAST comparison of the translated amino acid sequences with the longan genome. Compare the suspected homologous genes with the amino acid sequences of CrBKT and HpBHY genes to construct a phylogenetic tree in MEGA11.0 software using the maximum likelihood estimate method, with a Bootstrap value set to 1000 and specific parameters set to WAG+G. Construct the phylogenetic tree accordingly and use the online beautification tool iTOL (Interactive Tree Of Life) to enhance the visualization of the evolutionary tree.Then, according to the selected genes, TBtools software was used to draw the heat map of their expression in each tissue of longan.

3.3 Test

After performing a preliminary BLAST comparison between the amino acid sequences predicted from related genes and the third-generation genome sequence of longan, nine gene members were suspected to have homologous relationships. However, after further analysis using DNAMAN software, the results indicated that the identity between the exogenous gene and the endogenous gene in longan was only 35.85%.

3.4 Learning

Therefore, we preliminarily conclude that there are no relevant homologous genes in longan, clarifying at the genetic level the reason for the lack of astaxanthin content in longan. This also suggests that the feasibility of increasing astaxanthin content by overexpressing genes related to astaxanthin synthesis in longan is low.

4.1 Design

Based on the results of Iteration Three, we designed a plan to transfer the exogenous genes, CrBKT from Chlamydomonas reinhardtii and HpBHY from Haematococcus pluvialis, into callus tissue. The objective is to explore the impact of these exogenous genes on astaxanthin synthesis in longan, thereby validating the feasibility of subsequent high-astaxanthin expression vectors in longan.

4.2 Build

We used pCAMBIA1301+CaMV35S+NOS vector modified by pCAMBIA1301 vector to facilitate the regulation of initiation and termination of transcription.By designing reverse primers, we performed inverse PCR to linearize the vector. Using SnapGene software, we designed primers for the target genes and employed seamless cloning technology to ligate the CrBKT gene extracted from Chlamydomonas reinhardtii and the HpBHY gene from Haematococcus pluvialis into the linearized pCAMBIA1301+CaMV35S+NOS vector. Next, the recombinant gene was transformed into DH5α competent E.coli cells, resuscitated, and then coated in LB solid medium containing kanamycin for screening to obtain recombiners.Finally, we verified the successful construction of the recombinants through bacterial liquid PCR and sequencing.

4.3 Test

(1)Screening

Screening Strategy: Kanamycin is used to cultivate the Escherichia coli after transformation, and a screening method is employed to select recombinant colonies.

Confirmation Method: The large fragment recovered after performing inverse PCR on the vector pCAMBIA1301-CrBKT-HpBHY is ligated with the recovered gene fragment, and the ligation product is transformed into E.coli. By selecting a culture medium, the clones are tested for positivity. Further colony PCR reveals that a single band of 3432bp is produced. This indicates that the CrBKT gene and the HpBHY gene have been successfully cloned into the vector.

(2)Transformation, Antibiotic Preparation, and Agrobacterium Activation

Prepare 50 mg/ml of kanamycin sulfate and 25 mg/mL of rifampicin. Add these antibiotics to LB liquid medium in a volume ratio of 1:1000, respectively, and mix well.

Take the Agrobacterium competent cells and add 0.01-1 μg of plasmid DNA per 100 μL of competent cells, mixing thoroughly. Allow the mixture to stand on ice for 5 minutes, then sequentially place it in liquid nitrogen for 5 minutes, followed by a 5-minute water bath at 37°C, and finally another 5-minute ice bath. Subsequently, under a 28°C environment, add 700 μL of LB liquid medium without antibiotics and shake the culture for 150 minutes. Centrifuge to collect the bacteria and retain some supernatant to resuspend the bacteria. Spread the resuspended bacteria onto an LB plate containing kanamycin. Invert the plate and incubate it in a 28°C incubator for 48 hours. The monoclonal colonies were selected and transferred to LB liquid medium containing kanamycin for extended culture at 28°C and 200 rpm for 24 hours.

(3)Infection Process

Infect the well-grown embryogenic callus (EC) of longan with Agrobacterium suspension at an OD600 value of 0.6-0.8 for 30 minutes. Inoculate the infected callus in small clusters and spread them evenly onto the culture medium for cocultivation. After cocultivation on MS medium for 3 days, perform GUS staining for identification. Subsequently, extract RNA separately to identify positive transgenic cell lines, which will be utilized for subsequent experimental research.

(4)Inoculation

Remove the mixture of Agrobacterium suspension and longan callus from the shaker. After disinfecting or sterilizing all the experimental equipment, proceed to work in Biosafety Cabinet.Pour the bacterial solution and longan callus mixture from the centrifuge tube into the filter paper funnel and let it sit for a few moments to allow the bacterial solution to fully filter out.Inoculate the longan callus, which has been drained of excess bacterial suspension, onto MS solid medium. After sealing the plates, incubate them at 25°C in the dark for 3 days to promote the growth of the callus.

4.4 Learning

Based on this, we have introduced exogenous genes into the embryogenic callus of longan through a series of procedures including gene cloning, Agrobacterium-mediated transformation, infection of the embryogenic callus, and inoculation and cultivation. Preliminary and cultivation were also conducted, successfully verifying the technical feasibility of establishing a high-expression system for astaxanthin in longan.