Team:Nanjing/Basic Part
Basic Part One:
This gene is found in red chicken-feet cotton, and it belongs to Glyco-hydro-19's subfamily. This gene is composed of 975 bases and codes for the synthesis of class I chitinase which is made up of 324 amino acids. When the cotton is physically attacked by aphids, the gene Ghchi6 gets overexpressed that the plant starts to secrete chitinase. The overexpression of Ghchi6 can improve the aphid-resistance in cotton, and we plan to transfer this favored gene to tobacco to improve its aphid-resistance. Through modifications we have transferred the gene to Agrobacterium and then to the tobacco through infection. Normally the expression of chitinase in plants is very low under common conditions, but in cottons the expression is triggered by external factors like mechanical damage.
Aphid is one of the most notorious economic pests in the world. They feed on phloem sap of the host plant and deprive the plant’s nutrients. They have a great impact on the plant’s yield. It is hard to control this kind of pest due to their small size and strong fertility. In addition, aphids control now rely on chemicals like pesticides that may allow the development of resistance. As a result, the aphids are harder to be controlled. Thus, developing a new way to control the aphid and promote yield is a general goal. In a paper named Functional study of cotton Chitinase gene Ghchi6 on the resistance to aphid which was done by Liang Luo and Prof. Chuanren Li, researchers transferred the gene Ghchi6 to a wild type of Arabidopsis to make a transgenic type. The Line 1, 2, and 4 have different expressions of the gene Ghchi6 in their leaves, and Line 4 has the highest expression, following by Line 1 and 2. Line 2 is a sample that contains a small expression of the gene, which may work similar to a wild type.
The aphids feed through their sucking mouthparts. The following graphs illustrate the expression levels of Ghchi6 gene in red-chicken feet cotton’s different parts (leaves, roots, stems) 24 hours after the attack from the aphids’ needling. As we can see, the expression of Ghchi6 occurs that all three parts are suppressed by the attack from aphids. This implies that the aphids have some specific techniques to control the expression of Ghich6 in plants to reduce the chitinase secrete by the plants. On the contrary, the pure mechanical stimulus like tearing part of the leave will trigger the overexpression of the gene, meaning that the protective mechanism has already started to secrete chitinase as the plant feels the threat from external environment. Although the aphid may use its saliva to reduce the expression of Ghchi6, it is still obvious that this gene can enhance the aphid-resistance.
In the analysis of the Arabidopsis ‘s expression of chitinase, the figure clearly shows that all the transgenic types, Line 1, 2 and 4, are showing relatively higher chitinase activity, especially the Line 4 one which has a substantial improvement, implying that the overexpression of Ghchi6 is promoting the chitinase activity. Therefore, the overexpression of Ghchi6 can enhance the plant’s aphid-resistance.
Moreover, in that research, the use of green peach aphids, or the Aphis gossypii, to perform the experiment of testing the aphid-resistance. They place the green peach aphids with the wild type and transgenic types in a choice test. The test lasted for 72 hours and the researchers record the number of green peach aphids on the line ones (transgenic types Line 1, 2 and 4) and wild types every 12 hours. The result is significant.
As the figure tells, for each of the Line comparing with their wild type counterpart, the aphids which choose them to feed on are much fewer than those on the wild types. For Line 4 the difference is most obvious – almost 5 times larger on wild types than on the Line 4, and the difference increases as the experiment time increase. The aphid-resistance reduces with the expression of gene Ghchi6. The Line 2 contains minimum amount of expression so the aphid-resistance is similar to that of the wild type. In another no choice test, the green peach aphids, along with those transgenic and wild type, produces similar results as the choice test, demonstrating that the aphids prefer the wild type and ignore the ones with Ghchi6 genes. This suggests that the aphid resistance is improved through the transfer of gene.
The mechanism of aphid-resistance of Ghchi6 is not only limited to the expression of gene and chitinase. It is also about soluble sugar within the plant. As we all know that the aphids eat the plant’s phloem liquid which contain much sugar in it. The study down by Liang Luo also tells that the soluble sugar content within the plants will also be affected by the existence of the gene Ghchi6.
The soluble sugar content in wild type is significantly higher that those within the transgenic types, meaning the latter ones are not as attractive to the first one to aphids from the prospective of nutrition content. It’s more efficient for the aphids to feed on the wild type. This mechanism also implies another possibility of the reasons for the aphid-resistance of the transgenic plants.
Another unexpected finding is about the content of callose of the plants. Callose often exists in higher plants, having the function of enhancing the cell wall and prohibiting the damage from insects including aphids. As the figure shows, the expression of callose in the transgenic types are higher than the wild type. This can also contribute to the special aphid-resistance of Ghchi6.
In general, through previous study we can know that the gene Ghchi6 is a new method to improve plant’s aphid resistance by trigger the secretion of chitinase in plants and use this enzyme to harm aphids. Although the study shows that the aphids has developed some techniques to repress the expression of Ghchi6 through some unknown chemicals in their saliva to reduce the level, the choice and no choice test still figure out the in the same condition the aphids prefer to feed on those ones without this gene than the edited ones.
Basic Part Two:
We adapted the part to be able to assemble our gene of interest (BBa_K3253003) with the cloning vector (BBa_K3253005) to test whether our gene of interest has already been successfully transferred to the Agrobacterium and our target plant. This florescent gene was expected to have its greatest excitement wavelength at 510nm, which is an indicator of the result of our transfer. Due the delay of the gene posting from the IGEM official for a month. The official promised that the gene would arrive at 10th August, but the actual Ghchi6 sequence arrived at China at 7th September. This is the main reason that we haven't finished our experiment on time. Please take this into consideration. We have shown our progress in our demonstration.