l-/d-limonene synthase

Agarose gel electrophoresis of l-/d- limonene synthase gene

In Fig.1 limonene synthase gene is inserted into the backbone pSB1C3. Then the plasmids are digested by Pst I and EcoR I. The length of the l-LS gene, d-LS gene and backbone are 1735bp, 1904bp and 2624bp,respectively. The target genes are marked with arrows, and the figure shows that limonene synthase gene was transferred into the E.coli BL21 cells successfully. 1.2 SDS-PAGE analysis of l-/d- limonene synthase

SDS-PAGE analysis of l-/d- limonene synthase

In Fig 2，we want to verify whether the limonene synthase and GPPS gene are expressed or not. The molecular weight of limonene and GPP synthase is 89 kDa (with GST on pGEX-4T-1 plasmid) and 49 kDa respectively. In Fig.2, the target proteins are marked with arrows, and the figure shows that synthases are expressed in the cell successfully.

Verification of attraction of limonene towards C. elegans

In order to know whether limonene attracts C. elegans, filter paper is dropped of 5 \(\mu\)L5% limonene (DMSO aq) and put on one side. The control is put on the other side with only 5 \(\mu\)L DMSO. To eliminate the effect of DMSO, two other control groups are made.

a. The distribution of the C. elegans on the plates of control group Control group is used to eliminate the effect of DMSO, and some other experimental factors.

b. The distribution of the C. elegans on the plates of experimental group

This group shows significantly biased movement by compareing l-limonene with DMSO.

We find that nematodes of experimental group show significantly biased movement when we count the number of nematodes (Fig.1). However, nematodes of control group shows no significant difference about the distribution of nematodes on the plate.

C stands for the control group in which the filter paper is dropped with 5 \(\mu\)L DMSO; T stands for the experimental group in which the filter paper is dropped with 5 \(\mu\)L 5% l-limonene.

According to the data we get, we did simple analysis which means we used the ratio of the number of nematodes distributed on two sides and drew a histogram(Fig.2). In the histogram, nematode distribution of experimental group shows significant difference that nematodes prefer limonene.

During our verification of attraction of limonene, we analyzed 20 samples. In order to verify if there is any difference between test group and the control group at statistic level, we used paired t test to verify.

The methods are shown as the following:

The confidence of the following is 0.05, α=0.05. Firstly, we used the normality test. We selected the single sample K-S test to verify whether it had normality.

1. Judging standard of the single sample K-S test
   If P > 0.05, it has normality, otherwise it doesn’t have normality.
2. We used SPSS to derive the data and output the results

Analysis

The p value of the control group is 0.598, while that of the test group is 0.366. Both of these two groups meet normality.

After testing the normality, we did a significance test——paired t test to verify if there was any difference between these two groups.

1. Set up a hypothesis

$$H_0: \mu1 = \mu2$$

$$H1: \mu1 < \mu2$$

Among them, \(\mu_1\) is the mean of the control group, \(\mu_2\) is the mean of the test group.

2. The judging standard of the paired t test.

If p < 0.05, we reject H₀ and accept H₁ and the limonene can attract the nematodes. And if p > = 0.05, we accept H₀ and the limonene has no effect on the nematodes.

3. Output the results