1.1 Experimental preparation

(1) Experimental design

Carotenoids are fat-soluble pigments that are insoluble in water, but soluble in organic solvents such as acetone, petroleum ether, and chloroform. Commonly used extraction methods include organic solvent method, supercritical fluid extraction method, enzyme-catalyzed reaction method, and organic solvent-based ultrasonic or microwave-assisted extraction method. In this experiment, carotenoid content kit will be used to extract and quantitatively detect and carotenoids in plant tissues [1].

The principle of detection of carotenoids is based on their insoluble properties in water and soluble in organic solvents. After crude extraction by organic solvents, the absorbance (A) of the solution can be calculated using the Lambert-Beer law, which is proportional to the solute concentration (C) and the thickness of the liquid layer (L), expressed as A = αCL, where α is the absorbance coefficient. In this kit, carotenoids show a maximum absorption wavelength at 440. Based on the empirical formula, the content of total and carotenoids can be calculated. This kit is mainly used in the field of scientific research, especially for the extraction and quantitative detection of and carotenoids in plant tissues, and is not suitable for clinical diagnosis or other purposes.

(2) Preparation of experimental materials

Kit: The carotenoid content kit was purchased from Horui Corporation

Instruments: balance, oven, 100 mesh sieve, beaker, funnel, gauze, glass test tube, visible spectrophotometer,microplate reader, trace quartz cuvettes, 96-well plate.

(3) Reagent composition and preparation

1．Reagent 1: liquid (product solution formula not announced), 1000 mL × 2 bottles, store at 4°C.

2．Reagent 2: liquid (product solution formula not published), 1000 mL × 1 bottle, store at 4°C.

3．Extracting Solution: Mix reagent 1 and reagent 2 in proportion (2:1) before use, and seal and store at 4 °C to prevent volatilization.

1.2 Experimental operation

(1) Sample handling

1. The longan fruit, peel, and pit (powder) are dried and crushed at 60 °C, and sieved through a 100 mesh sieve.

2. Weigh about 0.2 g of the sample and place it in a glass test tube.

3. Add 6 mL of the extract solution to the tube and extract for 20 minutes.

4. Filter the extract into a 50 mL centrifuge tubesand repeat the extraction until the residue is colorless.

5. Combine the filtrate and set the volume to 50 mL to be measured.

(2) Absorbance measurement

1. Add the extract solution to the trace quartz cuvettes.

2. After zeroing with the extraction solution, the absorbance was determined by a microplate reader.

3. Absorbance recorded at 440 nm (A) respectively.

4. Calculate the absorbance difference:

[NOTE]:Blank tube only needs to be measured once

1.3 Data analysis

The formula for determination with a trace quartz cuvette is as follows:

V: total volume of extract, 50mL;

ε: Empirical absorbance coefficient of carotenoids, 2500;

d: cuvette aperture, 1cm;

W: Sample mass, g

1.4 Precautions

1. The extract solution is volatile and toxic, so take protective measures during operation.

2. The measurement must be rapid to prevent errors caused by volatilization.

2.1 Experimental preparation

(1) Experimental design

Astaxanthin is a kind of carotenoid, which is a fat-soluble pigment, insoluble in water, but soluble in organic solvents such as acetone, petroleum ether, and chloroform. In this experiment, organic solvent extraction method was used to extract and quantitatively detect astaxanthin-containing materials such as longan leaves.

The detection principle of astaxanthin is based on its insoluble properties in water and soluble in organic solvents. After crude extraction by DMSO, the absorbance of the solution ($A_{530}$) can be calculated using a calculation formula[2][3][4].

(2) Preparation of experimental materials

Instruments: balance, oven, 100 mesh sieve, glass tube, microplate reader, 96-well plate,50 mL centrifuge tubesand.

(3) Reagent composition and preparation

DMSO, 500 mL × 1 bottle, and keep in constant temperature sealed away from light.

2.2 Experimental operation

(1) Sample handling

1. Dry and crush the longan young leaves, longan old leaves and longan flesh at 60°C and sieve through a 100-mesh sieve.

2. Weigh about10 mg of the sample and place it in a glass tube.

3. Add 5mL of DMSO to the tube and extract for 10 minutes.

4. Centrifuge the mixture at 4,000 rpm for 5min and collect the supernatant.

5. Filter the extract into a 50 mL centrifuge tubesand, and repeat the extraction until the residue is colorless.

6. Combine the filtrate and set the volume to 25 mL to be measured.

(2) Absorbance measurement

1. Add the extract solution to 96-well plate.

2. After zeroing with DMSO, use the microplate method.

3. Absorbance ($ A_{530} $) recorded at 530 nm respectively.

2.3 Data analysis

The formula is as follows:

Where $C_{A}$ is the astaxanthin concentration (mg/L),

$A_{530}$ is the absor bance value at 530nm,

$m (g)$ is the weight of the Longan tissue powder[4].

2.4 Precautions

1. The extract solution is volatile and toxic, so take protective measures during operation.

2. The measurement must be rapid to prevent errors caused by volatilization.

3. Away from light during extraction.

3.1 Overview

HpBHY and CrBKT were used as target genes to construct the pCAMBIA1301-CrBKT-HpBHY expression vector. We designed CrBKT-HpBHY seamless cloning primers according to the published sequences through SnapGene software, amplified the target gene by PCR, designed pCAMBIA1301+CaMV35S +NOS reverse primers according to the published vector sequences. The vector was linearized by reverse PCR and then obtained pCAMBIA1301-CrBKT-HpBHY after linking, transformation and screening.

3.2 Construction of target gene expression vector

The pCAMBIA1301-CrBKT-HpBHY expression vector was constructed using HpBHY from Haematococcus pluvialis and CrBKT from Chlamydomonas reinhardtii as the target genes.

1. Design HpBHY-CrBKT seamless cloning primers according to published sequences;

2. PCR amplification of the target gene according to the designed primers, and gel extraction of the target gene band.

3. The reverse primer of pCAMBIA1301+CaMV35S+NOS was designed according to the published vector sequence, and the 1301 was linearized at no load and gel extraction of the linearized carrier band.

4. Ligation via T4 DNA ligase overnight at 16°C; DH5α competent was transformed and plated into Kana-containing LB solid medium, monoclonal was selected and colony PCR was performed, and finally the pCAMBIA1301-CrBKT-HpBHY recombinant vector was obtained.

3.3 Preparation of antibiotics

50mg/mL kanamycin sulfate was prepared by mixing kanamycin sulfate 0.5 g with 100mL double distilled water. Rif 0.25 g and 100 mL Dimethyl sulfoxide were used to prepare 25 mg Rifampicin antibiotics. 50mg/mL kanamycin sulfate and 25mg/mL rifampicin antibiotics were added into LB liquid medium at the volume ratio of 1:1000 and mixed.

3.4 Transformation method

1. Agrobacterium receptor state stored at -80℃ needs to be slightly melted at room temperature or in the hand, and inserted into the ice when in the mixed state of ice and water.

2. Add 0.01-1 μg of plasmid DNA per 100 μL of competent (To improve transformation efficiency, we performed a pre-experiment to determine the plasmid amount before first use), gently dial the bottom of the tube and mix well, and let it stand on ice for 5 minutes, liquid nitrogen for 5 minutes, a 37°C water bath for 5 minutes, and finally an ice bath for 5 minutes.

3. 700 μL of antibiotic-free LB liquid medium was added to the mixture and cultured at 28℃ for 2-3 hours.

4. Agrobacterium were collected at 6000 rpm centrifugation for 1 min, retaining about 100 μL of supernatant and lightly beating the resuspended block, and then coated on LB plates containing the corresponding antibiotics, culture was inverted in a 28 ℃ incubator for 2-3 days;

5. The monoclonal colonies were selected and transferred to liquid medium for extended culture.

6. Agrobacterium solution was added into the LB medium containing antibiotics, the sealing film is sealed, and the mixed bacterium liquid is oscillated. The bacteria was shaked in shaking bed vibration culture at 180 rpm under 28℃ for 2d.

Agrobacterium, as an aerobic bacterium, needs a lot of oxygen to grow. Successful liquid shakes require sufficient dissolved oxygen in the medium:

(1) Use triangular bottles;

(2) Keep the cross-section of the culture medium large and the thickness small;

(3) Add antibiotics as little as possible, and use low concentrations when they must be added.

3.5 Activation of Agrobacterium tumefaciens

3.5.1 Adjust the OD value of Agrobacterium

(1) Preservation of bacterial solution

500 μL of activated Agrobacterium was transferred into the cryopreservation tube, 60% glycerol was added into the same volume, and then the liquid nitrogen was quickly frozen and stored in the ultra-low temperature refrigerator at -80 ℃.

(2) Centrifuge

The remaining bacterial solution was balanced and placed in a centrifuge at 28 ℃ and 5000 rpm for 10 minutes.

(3) Preparation of culture medium

At the volume ratio of 1:10000, 50 mmol/L acetosyringone and 10 mmol/L MgCl2 were added into MS liquid medium in turn, and shaken to mix.

(4) Suspension precipitation

After centrifugation, the supernatant was discarded. The MS Medium and the mixed solution of acetosyringone and MgCl2 were added into the sediment, beat well, re-suspension of the block.

(5) Cuvette treatment

Prepare 5 clean trace quartz cuvettes, rinse twice with distilled water and once with re-suspension to ensure no impurities in trace quartz cuvettes.

(6) Determination of absorbance

In the super-clean working table, the suspension was added to the trace quartz cuvettes, another one was taken out and rinsed, and MS liquid medium was added as the control. UV photometer was used to determine absorbance at 600 nm and the OD value was adjusted to 0.6-0.8.

(7) Culture treatment

The modified OD value of Agrobacterium was cultured in a dark shaker at 28℃ and 180 rpm for 1 hour in order to prepare for subsequent experiments.

3.5.2 Infection

Agrobacterium tumefaciens with OD600 of 0.6-0.8 was used to infect EC of embryogenic calli of longan with good growth condition for 30 min. After infection, longan EC was filtered and sucked up the residual bacterial liquid, and inoculated in the medium in a small round way for co-culture. After co-culture on MS Medium for 3 days, GUS staining was performed, identification, RNA was extracted separately for identification of positive transgenic cell lines, and positive transgenic cell lines were added to the liquid medium shaker for later use.

3.5.3 Inoculate

(1) Preparation

Fold the filter paper into a funnel shape and place it on the flask. Flasks and parafilm are sterilized with 75% alcohol and then placed in a sealing film for UV sterilization to ensure a sterile environment.

(2) Separation

Remove the Agrobacterium liquid centrifuge tube containing the longan callus from the shaker. 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.

(3) Drain

When there is not much bacteria solution left in the funnel, use a medicine spoon to carefully remove the longan callus, place it on a clean filter paper sterilized by the flame of an alcohol lamp, and drain it to make the bacteria on the material fully absorbed by the filter paper.

(4) Inoculation

The longan calli of the filtten solution were inoculated on MS solid medium, sealed and placed in a dark culture at 25°C for 3 days to promote the growth of callus.