Study on biological characteristics of some strains of Bacillus Thuringiensis producing Crystalline Protein to kill lepidopteran insects

VIETNAM INSTITUTE OF SCIENCE AND TECHNOLOGY

INSTITUTE OF ECOLOGY AND BIOLOGICAL RESOURCES

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NGUYEN THI HIEN

RESEARCH ON BIOLOGICAL CHARACTERISTICS OF SOME

Bacillus thuringiensis strain produces crystal protein to kill lepidopteran insects

MASTER'S THESIS IN BIOLOGY

Major: Microbiology

SCIENTIFIC INSTRUCTOR: ASSOCIATE PROFESSOR, DR. NGO DINH BINH

Hanoi, 2012

COMMITMENT

I hereby certify that this is my own scientific research work. The data and results in this thesis are true and have never been published in any other research work.

Thesis author

Nguyen Thi Hien

ACKNOWLEDGEMENTS

First of all, I would like to express my respect and deep gratitude to Associate Professor, Dr. Ngo Dinh Binh - Head of Microbial Genetics Department, Institute of Biotechnology, Vietnam Academy of Science and Technology, who has wholeheartedly guided and created favorable conditions to help me throughout the time of implementing and completing my graduation thesis.

I would like to sincerely thank the staff of the Microbial Genetics Department for their dedicated guidance and help in completing this graduation thesis.

I would also like to express my deep gratitude to my family and friends who have always been there to encourage, support, and create favorable conditions to help me study and complete my thesis.

Hanoi, date…month…year 2012

Student

Nguyen Thi Hien

INDEX

INTRODUCTION 1

PROBLEM STATEMENT 1

CHAPTER 1. OVERVIEW 3

1.1. General information about Bacillus thuringiensis 3

1.1.1 . History of research and application of B. thuringiensis 3

1.1.2. Morphological characteristics of Bt 6

1.1.3. Biochemical characteristics 8

1.1.4. Classification characteristics 8

1.1.5. Classification of toxin genes of Bacillus thuringiensis 9 bacteria

1.1.6. Toxins of Bacillus thuringiensis 11

1.1.7. Structure of crystal toxin groups 13

1.1.8. Mechanism of action of crystalline proteins on insects 14

1.1.9. Research and application of Bt in Vietnam 15

1.1.10. Factors affecting the formation of spores and toxic crystals 16

1.1.11.Gene cry1C and subspecies Bacillus thuringiensis subsp. aizawai 18

1.2. General information about Lepidoptera 19

1.2.1. Lepidoptera 19

1.2.2. Test insects 19

CHAPTER 2. MATERIALS AND RESEARCH METHODS 24

2.1. Materials 24

2.1.1. Biological products 24

2.1.2. Chemicals and equipment 24

2.2. Research methods 26

2.2.1. Classification of Bt strains by serological reaction 26

2.2.2 Determination of Bt spore density 27

2.2.3 Test of insecticidal activity 27

2.2.4 Plasmid DNA extraction 28

2.2.5 Screening of Bt strains carrying cry1C gene by PCR 29

2.2.6 Cloning of cry1C gene segment 30

2.2.7 Determination of nucleotide sequence 32

CHAPTER 3. RESULTS AND DISCUSSION 33

3.1 Screening of Bacillus thuringiensis subsp. aizawai strains carrying cry1C gene

has activity against green leafhopper and silkworm 33

3.1.1 Classification of crystal shapes of studied Bt strains 33

3.1.2 Bt classification by serum 35

3.1.3 Insecticidal activity of Bta strains on green armyworm and 36-inch caterpillar

Spore concentration 36

Activity of Bta strains against green caterpillar and diamondback moth 37

3.2 Isolate and sequence the cry1C gene segment 39

3.2.1 Amplification of cry1C gene of Bta strains by PCR 39

3.2.2 Cloning of cry1C gene segment 40

3.2.3 Determination of the sequence of the cry1C gene segment 44

CHAPTER 4: CONCLUSION AND RECOMMENDATIONS 46

REFERENCES 47

LIST OF ABBREVIATIONS

STT

Acronym	Write in full
1	Amp	Ampicillin
2	Bp	Base pair
3	Bt	Bacillus thuringiensis
4	Bta	Bacillus thuringiensis subspecies aizawai
5	dH 2 O	Deionized water
6	DNA	Deoxyribonucleotide acid
7	E. coli	Escherichia coli
8	EDTA	Ethylene diamine tetra-acetic acid
9	OD	Optical density
10	PCR	Polymerase chain reaction
11	SDS	Sodium dodecyl sulfate
12	Sol	Solution
13	TE	Tris EDTA
14	X-gal	5- Bromo- 4 Chloro- 3 indolyl β- D- galactoside

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LIST OF TABLES

Page

Table 1.1. Classification of cry genes of Bt 10

Table 3.1. Crystal morphology diversity of Bt 34 strains

Table 3.2. Antibacterial activity of Bta strains against green leafhoppers after 3 days of testing.37 Table 3.3: Antibacterial activity of Bt strains against silkworms after 3 days of testing.38

LIST OF IMAGES

Figure 1.1. Spores and crystals of B. thuringiensis 7

Figure 1.2. Crystals of B. thuringiensis 8 bacteria

Figure 1.3. Three-dimensional structural model of the Cry1Ac 14 toxin protein

Figure 1.4. Insecticide mechanism of B. thuringiensis 15

Figure 1.5. Life cycle of silkworm 21

Figure 1.6. Cabbage damaged by pests 21

Figure 1.7. Life cycle of the green caterpillar 23

Figure 3.1. Shape of Bt colonies on MPA medium after 72 hours of incubation at 28ºC. 33

Figure 3.2. Spore and crystal shapes of Bt magnified 1000 times 34

Figure 3.3. Agglutination of TN 6.12 strain isolated with serotype under optical microscope at 400x magnification 36

Figure 3.4. Antibacterial activity of Bta strains studied against green leafhopper 37

Figure 3.5. Moth-killing activity of studied Bta strains … 38

Figure 3.6. Electrophoresis of PCR products of studied Bta strains 39

Figure 3.7. Green and white colonies appear on LBA medium after overnight culture 41

Figure 3.8 Electrophoresis of PCR colony products with primer M13 42

Figure 3.9. Electrophoresis of plasmid DNA cleavage products extracted from bacterial colonies on 1% agarose. 43

Figure 3.10. Electrophoresis of PCR products of cry1C gene from recombinant plasmid DNA….…44

INTRODUCTION

1. Problem statement

Vietnam has a tropical monsoon climate, with high humidity, which is a favorable condition for the development of agricultural and forestry pests. Lepidoptera is a large order in the insect class including butterflies and moths, on

180,000 species have been described and are present all over the world, they cause serious damage to the agricultural economy of the country.

To protect crop yields, farmers often use high concentrations of chemicals to spray immediately after a pest outbreak. On average, each hectare of crop must be sprayed with 5–7 kg of chemicals. However, the widespread use of insecticides has left residues in agricultural products, causing toxicity to users' health and environmental pollution. Instead of chemical measures, biological measures are encouraged. Currently, biological pesticides from Bacillus thuringiensis (Bt) account for more than 90% of the world's biological pesticide market share and are completely non-toxic to humans, animals and the environment [24].

The subspecies Bacillus thuringiensis subsp . aizawai (Bta) is the most studied of the 82 subspecies of Bacillus thuringiensis. Bacillus thuringiensis subsp . aizawai has the ability to synthesize crystalline proteins that are toxic to Lepidoptera insects, including silkworms ( Plutella xylostella ), green worms ( Helicoverpa armigera Hibber ) , armyworms ( Spodoptera litura ) and some Diptera insects. Cry1C is a toxic protein produced by Bta during spore formation, which has very strong activity against Lepidoptera insects . Therefore, in order to produce Bt pesticides specifically for Lepidoptera insects or transfer genes encoding crystalline proteins that are resistant to Lepidoptera insects into crops, we have conducted the following research: