In this section, we present and analyze the results obtained from the engineering part. Through detection, we determined the carotenoid and astaxanthin contents in wild-type Dimocarpus longan, and based on this, inferred the feasibility of constructing a high-astaxanthin-expressing system in longan. Following this, based on the chemical synthesis pathway of astaxanthin, we successfully constructed the pCAMBIA1301-CrBKT-HpBHY vector as the foundation.

Agrobacterium-mediated transformation technology was used to introduce it into longan callus, hoping to culture the longan plants with high astaxanthin expression and explore its application potential in Longan production.

Since astaxanthin and its precursors belong to the carotenoid family, we use the carotenoid content in longan as a direct indicator to assess the potential for astaxanthin production in various tissues of longan. To this end, we sampled five tissues from wild-type longan, including old leaves, young leaves, pericarp, seed, and pulp, with three biological replicates and technical replicates for each tissue. Carotenoids were extracted and the results are as follows.

1.1 Result Analysis

The highest carotenoid content was found in older leaves, consistent with their role in photosynthesis[4]. Given that carotenoids such as astaxanthin are synthesized to protect plants from oxidative stress, the high levels in mature leaves suggest that they are crucial for photoprotection and chlorophyll stabilization[3]. This tissue may be a potential focus for further astaxanthin production[1].

Although carotenoid content in young leaves is lower than in mature leaves, they still show a significant amount of carotenoids, which may indicate active biosynthesis during early developmental stages. The growth process from young to mature leaves provides valuable insights into the regulatory pathways of carotenoid biosynthesis, which is important when considering astaxanthin production.

The moderate carotenoid content in the fruit peel indicates its role in protecting the fruit from environmental factors such as UV radiation and pests. Carotenoids in the peel may contribute to the plant's defense mechanisms[2]. Compared to leaf tissues, the peel may be less ideal for astaxanthin production but could still serve as a secondary source[3].

The carotenoid content in the seeds is negligible, making them unlikely to be a viable raw material for astaxanthin extraction.

The carotenoid content detected in the fruit pulp is minimal, indicating that extracting astaxanthin directly from the fruit may not be highly satisfactory. However, given the edible value of the fruit, increasing its astaxanthin content still holds potential and significant scientific importance.

This preliminary data suggests that focusing on leaves may produce the greatest amounts of astaxanthin. Further research could explore optimizing astaxanthin extraction methods or enhancing the specific biosynthetic pathways of astaxanthin in Dimocarpus longan tissues.

Unlike the potential for increasing astaxanthin content indicated by the tissues responding to carotenoid detection, this analysis directly measures the percentage of astaxanthin relative to the dry weight of wild-type Dimocarpus longan, providing a direct representation of the astaxanthin content in wild-type Dimocarpus longan to explore its scientific value.

The extraction results indicate that the astaxanthin content in wild-type Dimocarpus longan is extremely low. Due to this minimal amount, the experimental results are significantly affected by the precision of the experimental equipment, resulting in a low reliability of the specific numerical values. However, it can still be inferred that wild-type Dimocarpus longan plants may contain either very low concentrations of astaxanthin or none at all, suggesting that there is no value in directly extracting astaxanthin from wild-type Dimocarpus longan.

Nevertheless, the carotenoid extraction experiments from wild-type Dimocarpus longan indicate that the carotenoid content is considerable. Based on Dimocarpus longan, there is theoretical feasibility and technical direction for constructing a high-expression astaxanthin system through the extraction of carotenoids and utilizing synthetic biology.

We used SnapGene software to design primers according to the sequence of pCAMBIA1301-CrBKT-HpBHY, and confirmed whether the vector pCAMBIA1301-CrBKT-HpBHY was successfully constructed by colony PCR.

We transformed the plasmid into DH5α-receptive E.coli. PCR identification was performed to confirm the construction of vector pCAMBIA1301-CrBKT-HpBHY. The results are as follows:

3.1 Result Analysis

Colony PCR was performed on the obtained E.coli according to the primers designed by us. After PCR amplification and electrophoresis, if the vector was successfully constructed, the corresponding fragment CrBKT-HpBHY was amplified by PCR, and the size of the PCR product should be 3432 bp. If the construction is not successful, there is no PCR band.

The electrophoresis results showed that the band is located between 3000 bp and 4000 bp. This confirms that we have successfully constructed the vector pCAMBIA1301-CrBKT-HpBHY and successfully transformed the gene pCAMBIA1301-CrBKT-HpBHY into the DH5α-competent E.coli

3.2 Infection

At present, we are using Agrobacterium to infect longan callus.

Our future plans are as follows:

1.Observation of Callus Growth

We will regularly monitor the growth of the Dimocarpus longan embryogenic callus tissue on the MS solid medium to ensure that it remains in good condition. If abnormal growth or contamination is observed, appropriate measures, such as changing the medium or adjusting growth conditions, will be taken promptly.

2.Molecular Verification

Once the callus growth is stable, we will perform PCR analysis for verification at the gene level.

3.Gene Expression Verification in Transgenic Plants

Finally, we will conduct further molecular analysis, such as Western blot, to confirm the expression level of the target gene. If successful, we will evaluate the gene's functional performance to understand its specific effects on Dimocarpus longan.