MODIFICATION OF SEED FATTY ACID COMPOSITION BY CRISPR/CAS9 TARGETING THE ...

116 Pages·2017·1.55 MB·English

MODIFICATION OF SEED FATTY ACID COMPOSITION BY CRISPR/CAS9

TARGETING THE FATTY ACID ELONGASE1 IN CAMELINA SATIVA

by

Mehmet Erkan Ozseyhan

A thesis submitted in partial fulfillment
of the requirements for the degree

of

Master of Science

in

Plant Science

MONTANA STATE UNIVERSITY
Bozeman, Montana

January 2018
©COPYRIGHT

by

Mehmet Erkan Ozseyhan

2018

All Rights Reserved
ii

DEDICATION
I would like to dedicate my thesis to my mother and father who always support
me to achieve my goals.
iii

TABLE OF CONTENTS

1. INTRODUCTION .......................................................................................................... 1

Fatty Acid Compositions in Plant Oils ............................................................................ 1
Understanding Fatty Acid Metabolism in Plants ............................................................ 5
Once Forgotten Precious: Camelina sativa ................................................................... 14
Engineering Fatty Acids in Camelina through Biotechnology ..................................... 19
CRISPR/Cas9-mediated Mutagenesis ........................................................................... 23

2. OBJECTIVES ............................................................................................................... 31

3. MATERIALS AND METHODS .................................................................................. 33

Plant Materials and Growth Conditions ........................................................................ 33
CRISPR/Cas9 Vector Construction .............................................................................. 34
Camelina Transformation .............................................................................................. 43
Screening and Confirmation of Transgenic Plants ........................................................ 45
Advancing Transformed Lines Towards Homozygosity .............................................. 46
Seed and Fatty Acid Analyses ....................................................................................... 48
Amplification of Individual FAE1 alleles ..................................................................... 49
Germination Test ........................................................................................................... 51

4. RESULTS ..................................................................................................................... 52

Section 1: Transgenic Line Creation, Confirmation & Analysis .................................. 52
Knocking out FAE1 alleles via CRISPR/Cas9 reduces
VLCFA content in Camelina seeds ....................................................................... 52
Reduction of VLCFA helps increase PUFA content ............................................ 66
Section 2: Fatty Acid Accumulation during Seed Development .................................. 69
Section 3: Mutation at the FAE1 Alleles of Cas9 Transgenic Plants ........................... 71
Section 4: Seed Weight, Plant Height, and Total Oil Content ...................................... 77
Section 5: Germination Test .......................................................................................... 79
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TABLE OF CONTENTS CONTINUED

5. DISCUSSION ............................................................................................................... 80

REFERENCES CITED ..................................................................................................... 86

APPENDICES .................................................................................................................. 99

APPENDIX A: Guide RNA Design Websites and Their Plant Databases ................. 100
v

LIST OF TABLES

Table Page

1. Transgenic Camelina Papers Published through 2012-2017 ............................ 20 

2. Examples of CRISPR/Cas9 Modification in Camelina sativa .......................... 29 

3. Golden Gate Cloning Protocol .......................................................................... 35 

4. Oligonucleotide Table ....................................................................................... 36 

5. Segregation of Red and Brown Seeds in the T2 Lines ...................................... 55 

6. Fatty Acid Composition of Brown Seeds of 14 T2 lines .................................. 56 

7. Fatty Acid Composition of Red Seeds of 14 T2 lines ...................................... 57 

8. Fatty Acid Composition of Brown Seeds from
………....Heterozygous T3 Lines ..................................................................................... 61 

9. Fatty Acid Composition of Red Seeds from
…………Heterozygous T3 Lines ..................................................................................... 62 

10. Fatty Acid Composition of Red Seeds from
…………Homozygous T3 Lines ...................................................................................... 63 

11. Fatty Acid Composition of Best T3 Lines ........................................................ 64 

12. Fatty Acid Composition of Best T4 Lines ........................................................ 64 

13. Fatty Acid Composition of Brown Seeds from
…………Homozygous T3 Lines ...................................................................................... 65 

14. Expression Pattern of FAE1 Genes in Camelina sativa .................................... 69
vi

LIST OF FIGURES

Figure Page

1. Fatty Acid Profiles of Some Common Plant Oils ............................................... 4

2. Basic Fatty Acid Elongation and Desaturation Mechanism ............................... 7

3. Metabolic Pathway for Triacylglycerol Synthesis ............................................ 13

4. The CRISPR/Cas9 Mechanism in Bacterial Immune System .......................... 27

5. CRISPR/Cas9 Construct Design. ...................................................................... 40

6. The Map of pHEE401E Red2 Cas9 CsFAE1 Construct ................................... 42

7. Agrobacterium-mediated Camelina Transformation. ....................................... 44

8. Advancing CRISPR/Cas9 Lines Towards Homozygosity ................................ 47

9. Alignment of Partial Nucleotide Sequences of Three
…………Camelina sativa FAE1 Genes. .......................................................................... 50

10. Mutagenesis in the FAE1 Alleles in Camelina sativa ....................................... 54

11. Eicosenoic acid (20:1) Content in Single Seeds of
…………Wild Type and Cas9-modified Camelina ......................................................... 58

12. Chromatographs of Wild Type and Cas9-modified
…………Best T3 Lines .................................................................................................... 67

13. Changes in Fatty Acid Profile Between Wild Type and
…………Cas9-modified Camelina seeds ......................................................................... 68

14. Expression of FAE1 Genes in Various Parts of Camelina sativa ..................... 70

15. Cas9/sgRNA-induced Nucleotide Changes in T1 Plants .................................. 74

16. Cas9/sgRNA-induced Nucleotide Changes in T2Plants .................................. 75

17. Cas9/sgRNA-induced Nucleotide Changes in Best T3 Plants. ......................... 76
vii

LIST OF FIGURES CONTINUED

Figure Page

18. Comparison of Various Traits Between Wild Type and
…………Cas9-modified Camelina Seeds ........................................................................ 78 

19. Seed Germination Test Results ......................................................................... 79
viii

NOMENCLATURE

ACCase: acetyl-CoA carboxylase

ACP: acyl carrier protein

CoA: coenzyme A

CPT: CDP-choline:DAG cholinephosphotransferase

DAG: diacylglycerol

DGAT: acyl-CoA:DAG acyltransferase

DHAP: dihydroxyacetone phosphate

ER: endoplasmic reticulum

FAE1: fatty acid elongase1

FAS: fatty acid synthase

FatA: fatty acyl thioesterase A

FatB: fatty acyl thioesterase B

FAX1: fatty acid exporter1

G3P: sn-glycerol-3-phosphate

G3PDH: sn-glycerol-3-phosphate dehydrogenase

GlyceroPC: glycerophosphocholine

GPAT: glycerol-3-phosphate acyltransferase

GPCAT: glycerophosphocholine acyltransferase

KASII: β-ketoacyl-ACP synthase II

LACS: long-chain acyl-coenzyme A synthetase

LPA: lysophosphatidic acid
ix

NOMENCLATURE CONTINUED

LPAAT: lysophosphatidic acid acyltransferase

LPCATF: forward action of lysophosphatidylcholine acyltransferase

LPCATR: reverse action of lysophosphatidylcholine acyltransferase

LPCT: lysophosphatidylcholine transacylase

LysoPC: lysophosphatidylcholine

PA: phosphatidic acid

PAP: phosphatidic acid phosphatase

PC: phosphatidylcholine

PCho: phosphocholine

PDAT: phospholipid:diacylglycerol acyltransferase

PDCT: phosphatidylcholine:diacylglycerol cholinephosphotransferase

PLA : phospholipase A
2 2

PLC: phospholipase C

PLD: phospholipase D

PUFA: polyunsaturated fatty acid

SAD: stearoyl-ACP desaturase

TAG: triacylglycerol

VLCFA: very long chain fatty acid