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1 Introduction

1.1 ABC Proteins

1.1.1 General Background

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1.1.2 General Structure of ABC Proteins

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Figure 1.1 –Molecular architecture of ABC transporters

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Figure 1.2 –Phylogenetic relationships of Arabidopsis and rice proteins in ABC subfamily B

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1.1.5 Plant ABC Proteins

1.1.5.1 The TAP Proteins

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1.1.6 Bacterial ABC Proteins

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1.2 Stress Factors and Characterization of TAP1

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1.2.1 Metals as Stress Factors

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1.2.2 Non-Metal Stress Factors

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1.3 The Objectives of the Thesis

Studies of plant transporters can provide value to agriculture, by revealing the role of transporters in enabling plants to adapt to environmental stress conditions, which can impact plant production and crop yields. Additionally, it can provide insight into human transporters, and thereby contribute indirectly to developing methods to overcome chemical and drug resistance, and to gain novel insights into the cause of diseases.

The objectives of this study are:

1. To analyze the functional role of AtTAP1, primarily through the characterization of the mutant attap1 in response to different abiotic treatments, and by comparing expression patterns of AtTAP1 to AtTAP2.

2. To investigate on the evolution and conservation of AtTAP1 and AtNAP14.

3. To analyze the enzymatic function of the proteins AtTAP1, AtTAP2, and AtNAP14.

The results are presented in detail in this thesis, and the main results of AtTAP1 and AtTAP1 are presented in an article, submitted to Journal of Plant Research (Appendix 1).

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2 Materials and Methods

2.1 Materials

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2.1.3 Plant Material

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2.1.5 Vectors

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(40)

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(41)

2.1.6 Primers

Table 2.1 –Primers used in this study

$OOSULPHUVZHUHRUGHUHGIURP6LJPD$OGULFK Primer name Sequence^a

J/ ¶$7**&7&$*&$$*7$&7&**&7*&$&¶

J5 ¶7$$*$&**&$7&*7777*7&7&77¶

J.SQ,/ ¶$$7**7$&&$7**&7&$*&$$*7$&7&*¶

J.SQ,5 ¶$$7**7$&&7$$*$&**&$7&*7777¶

7$33URPRWHU/ ¶$7&&$7**$**77$$&*$77***&$*$$*¶

7$33URPRWHU5 ¶$7**&*&*&&77$7*77&**7**$&7$$$*¶

6SURPRWHU/ &$&$$7&&&$&7$7&&77&*&$$*$&&¶

<)35 ¶$&$&*&7*$$&77*7**&&¶

1<5 ¶7$*&**&&*&77$&$7*$7$7$*$&*77*7**&¶

126 *$7$$7&$7&*&$$*$&&**&$$&$**$¶

/% ¶*&&7777&$*$$$7**$7$$$7$*&&77*&77&&¶

/% ¶7$*&$7&7*$$777&$7$$&&$$7&7&*$7$&$&¶

6$,/BB%/ ¶$$**$$$$$*&77*$$*&*7&¶

6$,/BB%5 ¶7*777&&&7&$77&*777&$&¶

7SURPRWHU ¶7$$7$&*$&7&$&7$7$***¶

7WHUPLQDWRU ¶*&7$*77$77*&7&$*&**7*¶

7$3/ ¶*$&77**&7&$*$&7*7&$*&$¶

7$35 ¶&&$&77&$&&$**$7*&$&**$¶

7$3D/ ¶&$7777*777$&77*777$&$*&¶

7$31KH,/ ¶$7*&7$*&$7**&7&$*&$$*7$¶

7$3%DP+,5 ¶$7**$7&&7&$7$$*$&**&$7&*7777*7&¶

6$,/BB&/ ¶$**$7&*77***77$7&77&*¶

6$,/BB&5 ¶*7$7&$*777&$*&7*&&$**¶

$FWLQ)RUZDUG ¶7*&&$$7&7$&*$***777&¶

$FWLQ5HYHUVH ¶*$$&&$&&*$7&&$*$&$&7¶

S-(7)RUZDUG ¶&*$&7&$&7$7$***$*$*&**&¶

S-(75HYHUVH ¶$$*$$&$7&*$7777&&$7**&$*¶

7$31KH,/ ¶7$*&7$*&$7***&$$&$$*$$$¶

7$3%DP+,5 ¶7$**$7&&7&$&$$**7**7$$&¶

7$3,QVLGH/ ¶*&*&77$7$**$*77**777&$¶

7$3,QVLGH5 ¶&$&&*&77**7&&7$&7$*7*&¶

1$31KH,/ ¶$7*&7$*&$7**&$*7*7&*$&*7777&*¶

1$36DF,5 ¶$7*$*&7&&7$$$*7**7**$&*77&7*&¶

1$3,QVLGH/ ¶$7&&7*$&77*&77&&&$&7*¶

1$3.SQ,/ ¶$7**7$&&$7**&$*7*7&*$&*7777&*¶

1$3.SQ,5 ¶$7**7$&&$$*7**7**$&*77&7*&$$&¶

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(42)

2.2 Methods

2.2.1 Bioinformatics Methods

Alignments. 3URWHLQ VHTXHQFHV ZHUH REWDLQHG IURPncbi.nlm.nih.gov, DQGDOLJQHGZLWK%LR(GLWVRIWZDUHYHUVLRQ+DOO

Phylogenetic tree. $ SK\ORJHQHWLF WUHH ZDV EXLOW IURP DOLJQPHQWV WR LQGLFDWH HYROXWLRQDU\ UHODWLRQVKLSV XVLQJ WKH %LR(GLW SURJUDPPH 1HLJKERU-RLQLQJ83*0$PHWKRGYHUVLRQDDSSOLFDWLRQ

Identities and similarities. 3HUFHQWDJH LGHQWLW\ DQG VLPLODULW\ RI SURWHLQV ZHUH GHULYHG XVLQJ WRRO SURJUDPPH EMBOSS Needle KWWSZZZHELDFXN7RROVSVDHPERVVBQHHGOH

Chloroplast transit peptides. 3UHGLFWLRQRIFKORURSODVWWUDQVLWSHSWLGHV F73ZDVH[HFXWHGXVLQJ&KORUR33UHGLFWLQJ6HUYHU(PDQXHOVVRQ 1LHOVHQDQGYRQ+HLMQH

2.2.2 Plant methods

2.2.2.1 Plant growth and stress treatments

Arabidopsis. 6WHULOL]HG VHHGV IURP Arabidopsis thaliana HFRW\SH

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(43)

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2.2.2.2 Sterilizing Seeds

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(44)

2.2.2.3 Plant Transformation

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<)3IOXRUHVFHQFHIURPFKORURSK\OOXVLQJD1LNRQ$5FRQIRFDOODVHU VFDQQLQJPLFURVFRSH

2.2.2.4 Biolistic Transformation of Tobacco

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(45)

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2.2.2.5 General Staining Method/GUS Staining

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2.2.3 Bacterial Methods

2.2.3.1 Chemically Competent DH5αCells

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(46)

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2.2.3.2 Transformation of Chemically Competent E. coli Cells

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2.2.3.3 Chemically Competent ABI Cells

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(47)

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2.2.3.4 Transformation of Chemically Competent ABI Cells

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2.2.4 Vectors Constructed

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PromoterTAP1--pPCR-Script Cp^R. 7KH XSVWUHDP QXFOHRWLGH SURPRWHU UHJLRQ RI WKHAtTAP1 JHQH ZDV DPSOLILHG IURP '1$ XVLQJ 3:2SRO\PHUDVHDQGWKHSULPHUV7$33URPRWHU/7$33URPRWHU5 7DEOHDQGFORQHGLQWRWKHYHFWRUS3&56FULSW&S⁵7KHYHFWRUZDV WUDQVIRUPHG LQWR '+Į DQG EDFWHULD FRORQLHV VHOHFWHG RQ /%$JDU PHGLDFRQWDLQLQJFKORUDPSKHQLFRODQG;JDO

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(48)

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2.2.4.1 Sequencing

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