Team:FAFU-CHINA/Proof

Proof

1.Introduction

    In our project, we transform theexpression vector of Cry and Cyt toxins. Based on the toxin groups,we construct six various toxins express devices as Figure1. To compare the toxicities, we also tested the toxicity of Bacillus thuringiensis strain LLP229.

2.Material, Condition and Method

    

The larvae of Ae. albopictus are feeding in the Key Laboratory of Biopesticideand Chemical Biology. The generation of Ae.albopictus is 11. The LLP229strain of Bacillus thuringiensis isprovided by Key Laboratory of Biopesticide and Chemical Biology. The concentration of LLP229is recorded in eachtable.

The CC530 and FA479 strains of Chlamydomonas reinhardtii and TAP mediumare bought from the Aquatic Species Research Institute in Wuhan, Hubei. The concentration of Chlamydomonas reinhardtii is recorded ineach table.

We contrasted various toxins groupexpression vectors and transform them into Chlamydomonas reinhardtii as following groups: Cry4Aa+2A+Cyt1,Cry4Aa+2A+Cyt2, Cry10Aa+2A+Cyt1, Cry10Aa+2A+Cyt2, Cry11Aa+2A+Cyt1,Cry11Aa+2A+Cyt2.

The toxicity analysis condition is in anenvironmentally controlled room (T=28±1 °C, RH=85%±5%, L: D=14:10).

Bioassay of variousconcentrations were assayed against 30 third-instar larvae in 20 ml of dechlorinatedwater. Each group was replicated 3 times. The level of mortality was recordedafter 48 hours. Then calculated the corrected mortality. The bioassay was thenrepeated such that a concentration range yielding 10–90% mortality was obtainedand the 50% lethal concentration (LC₅₀) was calculated.

Table 1.1 Toxicity of LLP29 to A. albopictus

Repeating group	Number of insects	Number of mortality	Corrected mortality (%)
1	30	30	100
2	30	30	100
3	30	30	100
Control	30	0	0

Note. Thenconcentration of LLP29 is 2.16 x 10⁹ (OD₇₅₀ of 1.0 = 8 x 10⁸ cells/ml,OD₇₅₀ =2.7A.)

 

Table  1.2 Toxicity of LLP29 to Culex quinquefasciatus

Repeating group	Number of insects	Number of mortality	Corrected mortality (%)
1	30	30	100
2	30	30	100
3	30	30	100
Control	30	0	0

Note. Thenconcentration of LLP29 is 2.16 x 10⁹ (OD₇₅₀ of 1.0 = 8 x 10⁸ cells/ml,OD₇₅₀ =2.7A)

Table 2.1 Toxicity of Chlamydomonas reinhardtii(Cry4Aa+2A+Cyt1) to A. albopictus

Repeating group	Number of insects	Number of mortality	Corrected mortality (%)
1	100	93	93%
2	100	89	89%
3	100	91	91%
Control 1 Control 2	100	0	0

           Note.1. The OD₇₅₀ of Chlamydomonas reinhardtii is 0.559

         2. The Control 1 group is Chlamydomonas reinhardtii (notransformation, OD=0.629)

         3. The Control 2 group is TAP medium

 

Table 2.2 Toxicity of Chlamydomonas reinhardtii(Cry4Aa+2A+Cyt2) to A. albopictus

Repeating group	Number of   insects	Number of mortality	Corrected       mortality (%)
1	100	89	89%
2	100	90	90%
3	100	88	88%
Control 1 Control 2	100	0	0

        Note1. The OD₇₅₀ of Chlamydomonas reinhardtii is 0.559

         2. The Control 1 group is Chlamydomonas reinhardtii (notransformation, OD=0.629)

         3. The Control 2 group is TAP medium

 

Table 2.3 Toxicity of Chlamydomonas reinhardtii(Cry10Aa+2A+Cyt1) to A. albopictus

Repeating group	Number of insects	Number of mortality	Corrected mortality (%)
1	100	90	90%
2	100	92	92%
3	100	91	91%
Control 1 Control 2	100	0	0

        Note1.The OD₇₅₀ of Chlamydomonas reinhardtii is 0.559

         2. The Control 1 group is Chlamydomonas reinhardtii (notransformation, OD=0.629)

         3. The Control 2 group is TAP medium

 

Table 2.4 Toxicity of Chlamydomonas reinhardtii(Cry10Aa+2A+Cyt2) to A. albopictus

Repeating group	Number of   insects	Number of mortality	Corrected mortality (%)
1	100	90	90%
2	100	90	90%
3	100	89	89%
Control 1 Control 2	100	0	0

        Note1. The OD₇₅₀ of Chlamydomonas reinhardtii is 0.559

         2. The Control 1 group is Chlamydomonas reinhardtii (notransformation, OD=0.629)

         3. The Control 2 group is TAP medium

Table 2.5 Toxicity of Chlamydomonas reinhardtii(Cry11Aa+2A+Cyt1) to A. albopictus

Repeating group	Number of   insects	Number of mortality	Corrected mortality (%)
1	100	90	90%
2	100	95	95%
3	100	91	91%
Control 1 Control 2	100	0	0

        Note1.The OD₇₅₀ of Chlamydomonas reinhardtii is 0.559

         2. The Control 1 group is Chlamydomonas reinhardtii (notransformation, OD=0.629)

         3. The Control 2 group is TAP medium

 

Table 2.5 Toxicity of Chlamydomonas reinhardtii(Cry11Aa+2A+Cyt2) toA. albopictus

Repeating group	Number of    insects	Number of mortality	Corrected mortality (%)
1	100	91	91%
2	100	90	90%
3	100	89	89%
Control 1 Control 2	100	0	0

        Note1. The OD₇₅₀ of Chlamydomonas reinhardtii is 0.559     

         2. The Control 1 group is Chlamydomonas reinhardtii (notransformation, OD=0.629)

         3. The Control 2 group is TAP medium

3.Reference

1.Zhang, L., Huang, E., Lin, J., Gelbič,I., Zhang, Q., Guan, Y., … Guan, X. (2010). A novel mosquitocidal strain LLP29isolated from the phylloplane of. Microbiological Research, 165(2), 133–141.doi: 10.1016/j.micres.2009.03.002