Hardware

Once we have constructed our overexpression vector using E. coli, how do we associate this vector with our plant? In other words, how can the constructed overexpression vector fulfill its gene editing role in the plant? After searching and exploring, our group finally found a tool that can help transform the overexpression vectors into plants-Agrobacterium tumefaciens.

Attachments: a picture of Agrobacterium petri dish, and a picture of shaking the bacteria when cutting seeds.

1. ####Transform the target plasmid into Agrobacterium

First we need to re-amplify a certain amount of the already constructed E. coli, and then extract the plasmid from it by plasmid extraction. Then thaw our Agrobacterium strain, add about 1μg of our extracted plasmid to the strain, place it on ice and ice bath for 5min, after ice bath, put it into liquid nitrogen for 5min, take it out of liquid nitrogen, put it into 37℃ water bath for 5min, take it out of liquid nitrogen, continue ice bath for 5min, after ice bath, add 300μl of liquid LB medium to it and incubate for 3min in a shaker at 28℃. Incubate in a shaker at 28℃ for 3-4 h. Finally, take about 100μl of the incubated product and evenly apply it to the Rif+Kana plate. 2-3d later, when Agrobacterium colonies grow on the plate, pick a single colony, add the Rif- and Kana-resistant LB medium, and shake it in a shaker at 28℃ for an amplification purpose, followed by adding glycerol, and keep it for spare use.

2. ####Agrobacterium infestation of plants

One day in advance, the preserved Agrobacterium solution was added to Kan-Rif culture medium and incubated to OD 0.6-0.8, and the organisms obtained by centrifugation were resuspended with the infestation solution to OD 0.2-0.3 and protected from light. After 1 day of seed germination, the hypocotyls of cucumber seedlings were carefully removed on an ultra-clean bench, the cotyledons were transected with a scalpel, the far hypocotyls were carefully removed, and 1/2 of the cotyledons close to the hypocotyls were taken and cut in half, which served as explants in the regeneration process of cucumber. Add the infiltration solution to infiltrate the explants in two times, each time for 90 s. Place the explants in the co-culture medium with the back side up. Three days later, the exoskeleton was inoculated vertically with the exoskeleton openings downward in the adventitious shoot induction medium, and placed on the biochemical culture frame for cultivation.

Conclusion: With the help of Agrobacterium, our overexpression vectors can be successfully transferred into our plant cells, which will enable them to perform gene editing on the plants themselves.

After a period of time under UV light to observe whether the fluorescent buds are produced, if so, it means that Agrobacterium infestation is successful, cut into the rooting medium to root, and then we move the young transgenic seedlings into our prepared substrate and let them grow. Due to the destruction of the gene, the general transgenic plant growth potential is particularly weak, the mortality rate is extremely high, and the requirements for the growth conditions are very harsh, we have designed a small artificial greenhouse after many attempts and expert consultation, you can artificially control the light, temperature, water and other environmental conditions, real-time monitoring of plant growth, to improve the survival rate of transgenic plants.

That is, to artificially create suitable environmental conditions for the growth of cucumber seedlings: the daytime temperature is maintained at 25-28℃, while the nighttime temperature is relatively low, roughly at 18-22℃; secondly, we use plant growth lamps to provide 12-16h of light per day, to ensure that the overexpression plants can receive sufficient natural light to promote their photosynthesis and growth; watering a small amount of watering several times, so that the substrate for the plant to grow In our artificial greenhouse, we also increase the ventilation to keep the air circulating and prevent the breeding of germs; in addition, when the growth of transgenic seedlings is weak, we will add a small amount of low concentration of liquid fertilizer, to avoid burning the roots, and at the same time, make the plant have enough nutrition to sustain their growth.

In our artificially constructed small greenhouse, all the appropriate environmental conditions required for the growth and development of transgenic cucumber plants were satisfied, which enabled them to grow robustly, thus creating a good foundation for the subsequent phenotypic observation and metabolite measurements.

The epidermal hairs play a huge role in cucumber production and are of great research value. First of all, the epidermal hairs of cucumber can resist pests and diseases, which makes the cucumber less likely to be attacked by pests and diseases in the process of growth. Moreover, some metabolites synthesized in cucumber epidermal hairs, such as flavonoids, are of high value. Take flavonoids for example, it has strong antioxidant and free radical scavenging ability, and has important biological activities such as regulating cardiovascular system, endocrine system, anti-cancer and anti-aging, which is of great significance to human health. And the fruit spines in this study belong to the epidermal hairs. Can we utilize cucumber fruit spines as a “plant factory” to produce flavonoids to get more flavonoids?

We interviewed the experts related to the development of cucumber epidermal hairs and discussed with the members of the group, and finally selected CsTBH, a spiny fruit development gene, and CsPAL, a key gene for regulating the rate-limiting of flavonoids, as the regulatory module for the production of flavonoids by cucumber spines, and proposed a transcriptional model for this purpose.

Firstly, in order to verify whether the two interact with each other, we carried out a yeast one-hybrid assay, in which the TBH gene was combined with the cis-acting regulatory element on the PAL promoter, and the result was that the yeast coloration was blue, which proved that the two could interact with each other.

Then, we constructed the overexpression vectors of CsTBH and CsPAL respectively, and introduced the two into the same cucumber plant through transformation, and then after cultivation, the overexpression plants were made to blossom and bear fruits, and then the phenotypes were observed.

After careful observation of the overexpression plants, we found that the fruits of the overexpression plants had a lot of black thorns compared with the wild type, which we guessed was caused by the increase of flavonoids. We guessed that this was the result of the increase in flavonoids. As a result, the amount of flavonoids in these spines was indeed significantly increased after professional measurement and identification. Thus, our hypothesis of utilizing cucumber fruit spines as a “plant factory” for flavonoid production to obtain more flavonoids was confirmed. This also provides a viable option for industrial utilization by humans.