CONSTRUCTION OF A PLANT TRANSFORMATION VECTOR … of a plant transformation...pussl khidmat maklumat...

CONSTRUCTION OF A PLANT TRANSFORMATION VECTOR CARRYING A MARKER GENE FOR EXPRESSION IN PLANTS

Ngien Leh N ah

Bachelor of Science with Honours (Resource Biotechnology)

2006

Pussl Khidmat Maklumat Akademik UNIVE~SlTI MALAYSIA SARAWAK

CONSTRUCTION OF A PLANT TRANSFORMATION VECTOR CA RRYING A MARKER GENE FOR EXPRESSION IN PLANTS

NGIEN LEH NAH

This project is submitted in partial fulfillment of the requirements for the degree of Bachelor of Science with Honours (Resource Biotechnology)

Faculty of Reso urce Science and Technology UNIVERSITI MALAYSIA SARAWAK

2006

ACKNOWLEDGEMENT

r want to give thanks to the Lord for His s trength and wisdom given to me throughout this

project I wou ld like to express my gratitude to my superviso r Dr Hairul Azman Ros lan for

his guidance ad vice and enco urage ment 1 would like to thank Mr Azis Ajim Mr Amin

Manggi and Ms Limjatai Patrick for their technical assis tance A spec ia l thanks to Ms

Yaso tha alp Sundaraj (MSc) Ms Patric ia Chong (MSc) Mr Ang Chung Huap (MSc) Mr

Tan Sia Hong (MSc ) Ms Wan Adnawani binti Meor Osman (MSc) and Ms Hashimatu l

Fatma binti Hashim (MSc ) for their invaluable sharings and helps Bes ides I wo uld like to

express my heartfelt th anks to the Sarawak Foundation for their financial support Last but not

least I would al so like to thank my family and mends for their m ora l SUppOlt and

encouragement upon completion of thi s project

Pusa Khidma Maklumt Akadcmik UNIVERSIl1 MALAYSIA SARAWAK

TABLE OF CONTENT

Page

ACKNOWLEDGEMENT

T ABLE OF CONTENT II

CHAPTER I INTRODUCTION

CHAPTER II LITERATURE REV IEW

LIST OF TABLES v

LIST OF FIGURES VI

ABSTRACTABSTRAK VII

2 1 pBl1 2 1and pUC l9 4

22 Alkaline lysis meth od in plasmid mi nipreparati on or 7

mlmprep

23 Plasmid conformation 8

24 Res tlic tion endonuc leases 9

25 The constnJ ction of binary vec to rs used in 10

Agrobaclerill m-medi ated plant trans fo1l11ation

CHAPTER HI MATERJALS AND METHODS

3 1 Mate li als 13

3 11 Plasmids 13

3 12 Bac te lial cell 13

32 Plasmid recovery of pEl l2 1 13

32 1 Preparation and maintenance of E culi stram 13

JM 109 cultures cany ing pBlI21

I

CHAPTER IV

32 2 Miniprep isolation o fp BIl2l 13

32 3 Restriction enzyme analysi s o f pBI1 2 1 IS

324 PCR fo r GUS geneofpB1l21 IS

33 Plasmid recovery of p UC 19 17

331 Preparation ofKCM competen t cells 17

332 Transfo nmation ofKCM competent cell s w ith 17

pUC 19 from stoc k co llection

33 3 Miniprep iso iationofp UC 19 18

33 4 Restri cti on analysis for pUC l9 18

3 4 Prime r design 18

35 Plas mid (pBI1 2 1) iso lati on by us ing QIAprep Spin 19

M iniprep kit

36 PCR for T -DNA and non-T-DNA 0 fpBl 2 1 19

3 7 Co nstruction and analysis of bina ry vector 20

3 7 1 Cloning of PCR produc t of T-DNA into p UC19 20

372 Confilmation tests to verify th e desired insert 20

3721 Restric tion anal ysis 21

37 2 2 PCR 21

38 Sequ enc ing of pBII 2 1 22

RESULTS AND DISCUSSIONS

4 I Restri ction analysis of pB 1121 and pUC 19 23

42 PCR forG USgeneo f pBl121 27

43 PC R for T-D NA and non-T-DNA o fpB1l21 28

III

CHAPTER V

REFERE CES

APPENDICES

Appendix A

Appendix B

Appendix C

44 ConstJUction and analysis of binary vector 30

44 1 Cloning of PCR product ofT-DNA into 30

pUC I9

45 Sequence analysis o f T -DNA 0 f pBJl 2 1 35

CONCLUSIONS AND RECOMMENDA TIONS 40

41

Lis t o f solu tions for alkaline lysis (Sam brook 1989) 45

Components o f 5X KC M used in transfonn ation with KCM

co mpetent ce ll s (JM 109)

Components of TSS (PH65) 46

Res tric tion sites of the pal1i al sequences of pB I1 2 1 47

IV

LIST OF TABLES

Table Page

31 The sequences o f primelS used in PCR to dete rmin e GUS gene insid e 16 iso lated plasmid

32 Compos ition of PCR reac ti on mixture fo r amplification of GUS gene 16

33 PCR parametelS fo r amplification orGUS gene 16

34 The primer sequences fo r ampli fyi ng regions of interest 19

3 5 Compos ition of PCR reac ti on mi xture for amplification 0 fT-DNA and non- 20 T-D A o fpBli 2 I

36 PCR thelmal cyc ling profi le forT-DNA amplification 20

37 PCR thermal cyc ling profil e for non-T-DNA amp lification 20

38 Sequenc es for M 13 fOIward and reverse primers 2 I

39 PCR therm al cyc ling profi le fo r MCS amplifi cation 22

v

LIST OF FIGURES

Figure Page

2 1 Restliction map of pB1121 5

22 Restriction map of a binary vector pBI 121 showing the T -DNA region and 5 non-T -DNA reg ion

23 Restriction map ofpUC l9 6

41 Agarose gel electrophoresis of resui ction enzyme digestions of p BI12 1 23

42 Agarose gel e lectrephoresis of restriction enzyme digestions of pB1121 24

4 3 Agarose gel electrophoresis of restric tion enzyme digestion ofpUC l9 26

44 Agarose ge l electrophores is of PCR products to dete rmine the GUS gene 27 insid e pB1121

45 Aga rose gel e lectropho resis of PCR product of T-DNA and non-T-DNA 29 amplifIcation

46 JMI09 growth observed on agar plate added with LB ampic illin X-ga l and 30 lPTG

47 Agarose gel e lectrophoresis ofPCR products ofMCS of pUCl9 33

48 Agarose gel e lectrophoresis o f li ga ti on product (5 fl) 34

49 BLAST result for T-DNA forward primer 35

4 10 BLAST resul t for pBII 2 1 (T-DNA reverse primer) 36

411 Panial sequences ofpBII2 1 (T-D NA) using fOlward primer ( 1002 bp) 37

412 Schematic representation of pBII2l (T-DNA) sequence forward primer 37

4 13 Pm1ial sequences of pBII21 (T -DNA) using reverse plimer (983 bp) 38

4 14 Schematic represen tation ofpBI121 (T-DNA) sequence reverse p limer 38

VI

Construction of a plant transformation vector carrying a marker gene for expression in plants

Ngien Lell Nah

Resource 8 iOlechn ology Faculty ofResoune Sc ience and Technology

Ulllverslty Malaysia Sarawak (UN IMAS)

ABSTRACT

T he constru ct ion o f plan t Iransfonnalion vecto r a b inary vecto r is importam espec ia ll y in pla nt trans fonna tion in volving Agrobaclenwn (umeaciens The resulta Ill binary veClor is trans fonned into AgJobaCferifll l1 lumeaclens as a carrier to be fun her transformed inla the plants In this s tud y th e transferred -DNA or T-DNA (- 55 kb) originated from bi nary vector pBl 12 l supplied by Ara bidop sis Bio log ic3 1 Resource Center (Co lumbus) was PCR-ampli fied in order to be subcl oned into pUCl9 vector The comp lete sequence of pB l1 2 1 (Accession number AF485783 ) was derived and primer designed for amplifica ti on of T-DNA and non-T-DNA regions T-DNA was successfully PCR-amp lified by using forward primer (5 - GCATATGTAGGTTTACCCGCCAATATATCCTGTCA - 3) and reverse primer (5 - GCATATG AGGCAGGATATATTGTGGTGTAAACA - 3 ) Several hia ls with different subc lo ning stra tegies were ca rried o ut to subelone the PCR fiagment (T-DNA) into pUC I9 The liga tion reac tion mi xture was evemua ll y trans fonlled inlo E coli strain JM 109 Less tha n 50 of white colo nies were obselved by using adaptors wi th BamHI site which we re added during liga tio n together w ith Bam HI-cut pUCJ9 However the gene clon ing was failed as no insert was detected both with res triction enzyme and PCR ana lyses The problem was main ly due to fuilure in li ga ti on reaction Th e work to form a helper plasmid fro m PCR-amplified non-T-DNA could not be carr ied out due to time constraint

Key words plant trans format ion vector binary vector pBI 121 Agrobacterium tumejaciens T-DNA PCR

ABSTRAK

Pembinlal1 veIlor lransjomw~i tumbll hal1 iailll vekior binan sangol pel1ll17g tel1rtamal1ya doam ral7~(ormsi [Ina man yang melibalk(ln Agrobacterium tume(ac iens Vektor binari yang beljaJJCI dilJ ina akan diflOl1Sjorm ke dahun Agrobacterilr11 wmeac iens yang bertindak sebagoi pembawa tlntllk drnmiddotons(orm ke daam furn bu17an Daam kajUln i1 transerred-DNA aa ll T-DNA (~55 kbp) beluw da rpada vektor bina pB1I21 yang dibekolkon oeh A(Ibl(lopsi Biologica l Resource Center Colllmbus lelah dw mpltfikasikan melalui Tinelok Ralas Beran (II Poill7erase (PCR) unuk disubklonkon ice doam vekor pUC 19 juuk(Jn penllh pBII21 (No mbor akses AF485783) dierbikan dan pencelS dtrekabenllk linilk amplijikasi bahagian T-DNA don bukan T-DNA Bahagian T-DNA btrjaya dlOmpliflkas ikan dengan mengflInakan pencelis ke hodopan (5middot GCATATGTAGGTITACCCGCCIATATATCCTGTCA 3 J dan pence ke belakang (5 - GCATATG AGGCAGGATATATTGTGGTGTAAACA - 3 J Beberapa perclIbaan dengan pendekatal1 s tra tegi pengsubklOl1al1 yang berbeza felall diambilll11tuk subklol1 frogmen peR (T-DNA) ke doam pUCI9 Akhirnya compural1 reakslligosi dillOns(ormkan ke dalom E coli strain J1v1l09 Kolol1lshykoloni p llllh (kllrang daripada 50) felah dlJ)erhallkol eli mana adaplors middot yang mempllnyai tempal pembatasan BmnHI felah ditam bahkon semasa proses igasl bersa ma-sama dCligan pUCJ9 yang telah dibatas oleh BamHI Walau bogaimanapul1 pengklonon gaged kerana fiada i-agm en sebesar 55 kbp dikesan dengal1 menggunakol1 enzim pembalosan dan unulisis PCR Masaah ini (hsebabkan lerulamonya oleh kegagalan dl dalam reuksf ligasi Ktlja pembmaan plasmid pembanlll dUlpada hast ampltjikasi PCR IIntuk bahagian bukan T-DNA tldak dapat dijalank(1I1 kerono keslIl1 wkan masa

Kala kUl1ci lIeklor nmuformasi tul11hllh(lI1 eklOr binari pRIl2 J I Agrobacteriwn lumeJoCle l1s T-DNA peR

Vll

CHAPTERl

INTRODUCTION

Plasmid is a small circ ul ar extrachromosomal double st randed DNA th at can replicate itself

and is not impol1ant in the survival of a bactelium (T0I1ora el af 2004) However linear

plasmids have been identified in both gram positive and gram negative bacteria (Towner and

Cockayne 1993 Ausubel et ai 1999) If a bacterial ce ll has plasmid it can survive under

certain environment such as the toxic env ironment For example Pseudomonas spp contain

plasmid which encode enzymes ca pable of degrading an enOlmous range of both natural and

synthet ic organic compounds such as to luene and camphor (Glover 1984) Therefore it is an

add itional advantage for the bactelia to survive in very diverse and challenging environments

Plasmid is an impor1ant biotechnological tool and serves as a cloning vector Molecular

biologists had genetically engineered naturally occuning plasmids which have these features

unique restriction sites small s ize replicates in host and selectable marker and or repor1er

genes (Haddock 2004) T he unique restriction sites are essen tial for introducing fo re ign DNA

Sam brook et of (1989) revealed illat the constru ction of small er plasmid vector will increase

the bacterial lranslormation efficiency The o lig in of replication (ori) in plasmid will ensure

autonomous rep lication within its host Selectable marker (eg anti biotic resistance gene)

ando r repol1er genes (eg t3-glucuronidase or GUS gene) will ease the identification of

recombinant bacterial cells

Accord ing to Scheppl er e 01 (2000) severa l transfonnation methods had been developed for

gene delivery in plant transfolmation process They are the Agrobaclerium (umejacien Ti

(Tumor-inducing) plasmid system chemical penneabilization of th e plasma membranes of

protoplas ts micro injection electroporation and biolis tics Biolis tics or microprojectile

bombardment is useful in trans fonning monocots (eg wheat lice and maize) while A

tllmejaciens Ti plasmid system is most success ful in transfOiming dicots (eg soybeans and

tomatoes) Howe ver recent studies have demonstrated the successfu l transfolmation o f

recalcitrant crops such as rice (Raineri et ai 1990 Afolabi et al 2005) Agroinfcction which

is essential in deli vering viral genomes to graminaceous hos ts were also repol1ed by Ellis

(1993)

Agrobacterium-mediated plant transfonnati on is the easiest system by which th e crown gallshy

producing soil bacterium A ume(aciens are modified with the remo va l of nonessential regions

(Scheppler e( aI 2000) Wu (1988) stated that the binary vecto r system will overcome the

problem ofthe big-sized (200 kb) Ti plasmid which cannot be used directly for cloning pUlpose

In the binalY vec tor system the A 11Il11e[aciens shou ld have two plasm ids onc plasmid canying

a marker gene within th e T -DNA region ano ther plasmid is the disarmed plasmid or helper

plasmid (no T-DNA region but has the virulence vir genes) Once the plant is infected by A

tumefaciens the vir genes of the helper plasmid will direct the incoJ1loration of the T-DNA into

the plant genome (Guerineau and Mullineaux 1993 Scheppler el ai 2000) Reeng ineering of

binary vector pBlJ2 [ by using peR approach to construct a smaller binary vector and a he lper

2

plasmid was allempted in this s tud y The knowledge of the co mplete sequence of the binary

vector pBII2l (C hen e l al 2003) was applied in thi s resea rch

Acco rding to Xiang and co-workers (1999) many useful and versa tile vecto rs have been

constructed in the past for the prod ucti on of transgenic plants but those vectors are fairly large

(more than 10 kb) and offe r fewer unique resllc tion sites for c loning gene of interest This

research ratio nale focuses on the reduct ion of plasmid vecto r size with more unique restriction

sites to accommodate larger fragme nt of foreign gene or gene of interest

The first aim of thi s resea rch is to veri fy the plasmid pBI12l and pUC l 9 from th e stock

co llection and to constlUct a s mall er binalY vec tor caJTying a marker gene and a helper plasmid

deri ved fro m pBII21 Subsequently series o f plant transfolTIl ation vecto rs wi ll be constlUcted

based on the newly-constlllc ted binary vecto r

3

CHAPTER II

LITERA TURE REVIEW

21 pBI121 and pUC19

In thi s research a mini binalY vector and a helpe r plasmid deli ved from pBI I21 (Figure 21)

will be construc ted by using Po lymerase Chain Reaction (PCR) approach T -DNA region

(Figure 2 2) will be amplified by PCR and subc lo ned into pUC 19 vec to r at multiple clo ning s ite

MCS (Figure 23) The mini binary vector will have essential e lements in plant tran s fonnation

process which are an Escherichia co li origin of repli cat ion (ori) and the T-DNA reg io n (RBshy

right borcie r express ion cassettes for NPTlJ-neomycin phosphotransfera se II as plant-selectahle

marker MCS-muItiple c lonin g s ite and the LB-Ieft border)

The non -T -DNA region (F igure 22) of pBI12 1 containing origi n of repli cation from plasmid

RK1 ori V NPTlJl 5 NPTIII CDS and RK2 fl fA wi ll al so be amplified and rec ircul arized to

fonm a helper plasmid to be trans fonmed into A IU11Iejac iens strai n LBA4404 RK1 oriV is the

vege tati ve and broad host-range plas mid origin of rep li cation whil e RK1 TlfA encodes lransshy

acting functions necessalY for repli ca ti on o f plasmid in its host ce ll s (Guelineau and

Mullineaux 1993 Ausube l el aI 1999)

4

l 94(

IT~

SI-il poundJ4It1

- 1

GaI U~ I 1amp111

)aofIJJ J 11U lill1 III 11 7 I

L l lI W I

l5j IV

1)1 U l fl1 ~

+ 141r - -~ r~~~

Ihl~ I ~ ~

Figure 2 1 Restriction map of pB1121 based on Brusslan (2000)

onNOS p(o~m ___ KlaC

~-

RKl

NOS prom

NPl1l COS

h -

NPTlII COS

pBI121

- 13kb

RK2 TriA

Telo 3

Figure 22 Restric tion map of a b inary vec tor pSfl21 showing the T-ON A region and non-TshyDNA regIOn (Adapted from Planl Molecular Genetics Lec ture 2 par1 3 o f 4 (n d ) Retrieved Jul y 8 2005 from hrtplwww umanitobacaafslp lant_sclencdCOURSESI39shy7681021023 hrllll )

5

Pusal Khidmat Maklumat Akademik UNIVE~SITI MALAYSIA S~RAWAK

E 1 1411

pUC19 pl asmids (Figure 2 3 ) will be used to cany the fo reign gene because they have a high

copy number (500-700) pUC 19 vecto rs express the amlno-tennin al fjagment of th e lacZ gene

product (6-galac tosidase) and show a-complementation in appropriate hosts (Sambrook el 01

1989) Po lyc loning site within the lacZ gene allows the insertion of fo reign DN A Disruption

of th e lacZ gene will ease the se lection of recombinant bacterial cells through his tochemical

staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011

Rgt f3m11fJ51 Hi94 p~ 6seYI 1110-------II

Cllli 1217

FIgure 23 Retrlction map of pU C19 The reslnCli on enzym e Sites within the mu illpl ( ~

clonIng SHe is not shown in the figure A dapted from Fenne nlas Description and Restric ll on Maps pUe i S pUCI9 (200 5) Retrieved Ju ly 13 2005 ITom hn p llwwwj-ennentasco mJtechin fonucie icac idshnappuc l SI9 htm

6

22 Alkaline lysis method in plasmid minipreparation or mini prep

Alkaline lysis miniprep takes ad vantage of th e fact that plasmid DNA is much s maller than

genomic DN A (Scheppler el 01 2000) Thus small er molecul es such as RNA and plasmid

will escape the lys is reaction once the genomic DNA and proteins are denatured and

prec ipitated with high-salt-concentration buffers

As explained by Bimbo im and Doly (1 97 9)50 mM glucose and 25 mM Tlis-HCI ac t as buffer

solution for pH control Lys is so lution (0 2 N NaOH and I sodium dodecyl sulphate SDS) is

then added 0 2 N NaOH is used to denature linear chromosomal DNA whil e 1 SDS a

detergent is added to break open the ce ll membranes and denatures the prote ins (Tumer el al

1997 Scheppler el ai 2000)

The preparation is then neutralized by adding So lution III (Turner el ClI 1997) Potass ium

acetate (KOAc) is used to cause the precipitati on of protein-SDS complexes and low molecular

weight RNA (Bimboim and Doly 1979) Following centrifugation after pheno l extraction

three ph ases can be observed name ly the lower phase containing hydrophobic cell components

a middle layer cont aining pro tein and an upp er phase w ith plasmid DNA (Walker and Rapley

1997)

Co ld absolute ethanol is used to precipitate and concentrate the DNA Finally the DNA is

pe lleted hy cenu fuga tion the pelle t is retained and washed w ith 70 e th ano l to remove

unwanted salts from the D NA The DNA pe lle t is air-dried to allow the evaporation of e thanol

7

Hence ethano l contamination which will interfere with subsequent analys is can be avoided

(ScheppJer et al 2000)

Fin ally the purified DNA is suspended in Tris-EDTA (TE) buffer As exp lained by Tumer et

al (1997) th e ethylene diamine te traace tic acid (EDT A) is added to chelate the ca tion Mg2+

which is the cofac tor of th e bacterial endogeno us nucleases the D ases Thus DNases are

inactiva ted and plasmid wi ll no t be degraded whilst the Tris -HCI will buffer th e so lution at

pH80

23 Plasmid conformation

ill 2003 Hayes reported th a t plasmid DNA is mostly maintained in a covalentl y closed circular

supercoi led form or FOlm 1 Plasmid conveI1s to an open circu lar fOlm or Form II when a ni ck

is introduced on one 0 f the strands of the doub le-stranded plasmid DNA Another fo rm is the

linearized o r FOlm III in which nic ks are introd uced at opposite posi ti ons on bo th DNA strands

Superco iled plasmid migrates the fastest on an agarase gel when subjected to electrophoresis

fo ll owed by linearized plasmid and the s lowest migrating fOlm is th e open circular plasmid

(Boffey 1986 Towner and Cockayne 1993 Martin 1996) Acco rding to Martin (1996) the

acc urate size o f ce lt ain plasmid can only be determined based on the migrat ion of its linear

fOlm compared to a set of linear mark ers

8

24 Restriction endonucleases

Res tric tio n endonucleases o r res l1ic tion enzymes (REs) are enzym es that can cleave o r d iges t

ce rtain specific sequences o fnu c leo tid es in DN A (Turner el aI 1997 Schepple r el aI 2000)

EcoRI was th e first isolated restriction enzyme in 1962 by Werner Arbe r fro m E coli

Naturally REs se v e as protec ti ve weapons in bacteri al cell s which will kill the in vaders

especially the bacterio phage o r fo re ign D A by digesting the unm eth ylat ed DN A According

to Williams and co-wo rkers ( 1993) the bacteri a l DNA is p rotected from its RE ac ti vity as its

own DNA is chemically modi fi ed (methylated)

REs have been used as an impo1ant too l in genetic engineeing Res tric tio n enzym es can be

di vided in to two types which are th e end onucleases and exonucleases Brown ( 1999) revealed

th at endon uc lcases diges t at th e middle of DN A mo lecul e while exonuc leases diges t either

from end of 5 to 3 or the end of 3 to 5 DNA molec ule REs c leavage th at produce sticky

ends (are not direct ly opposi te each o ther) in th e two strands o f a DN A are feat ured in mos t of

tbe REs (eg Bam HI o r HeoRI) In 1996 Dawson el al repo1ed that so me enzymes simply

cleave DNA with a double-s tranded brea k in the center of th e recognition sequence which

produces DNA fragments wit h temini called blunt ends (cg A lII) or Haelll ) This feature o f

e ither stic ky o r bl un t ends made it poss ible to c reate new recombinant DN A mo lec ules

According to Sa mbrook and co-workers ( 1989) REs have been classified into three gro ups

namely Type I II and 1Il Type [ and Type 1lI res trict ion end onucleases a re less useful in

mo lec ular c loning because the sequences otthe result ing fragments th ey cut are not prec ise ly

9

known (Brown 1999) Type 1I restric rion enzymes are routinely used in molecu lar cloning

which are mostly six-base cullers (Sambroo k el 01 1989)

25 The construction of binary vec to rs used in Ag robacterium-m ediated plant

tra nsfo rmation

The natural gene transfer system by using Agrobacteru11 has been used to produ ce transgenic

p lants in fl o ric ultural industry li ke Dendrobiwn orchid (Y u el 01 200 1) and for crops

imp rovement suc h as conon (UI-Haq 2004) c ano la (Basu el al 200 1) sorgh um (Zhao el al

2000) and Acacia hybrid (Pang el 01 2005) and fla x plants (Dong el 01 1993)

Sam broo k el I ( 1989) exp lai ned th at the trend for the future was to s treamline vectors to

reduce th e size to the mi nimum whi le increas ing the capacity to accept large fo reign DNA

The advantages o f constru ction o f small er plasmid vectors are to gain higher trans fo rmation

effic iency easier charac te ri zati on by restri c tion mapping and they can rep li ca te to higher copy

nu mbers

As describ ed by Bevan ( 1984) a bina ty vec to r called pB TN 19 (approximately 10 kbp) was

co nstruc ted by uti liz in g the Irans -ac ting functions o f the vir regi on in side tumo r-i nduc ing Ti

resident plasmid in A llimefaciens to trans fer T -D NA (Trans ferred -DNA) sequences in to the

nuclear ge nome of plants pBI 19 was relati vely small size which has large number of

restrictio n s ites and req ui res no reco mbinati ona l steps for inco rporation into the T i plasmid

10

An improved bin ary vector pBlN20 was designed to con tain a large multi-cloning site (MCS)

detived fimiddotom the superlinker found in pSL30 I and selectable kanamycin res istance gene nplfI

between the Ti border (Hennegan and Dann a (998) pBrN20 was derived from pBU21 and

has been proven to create lines of transgenic tobacco plant

As explained by Xiang e l al (1999) pBINI 9 binary vec tor was streamlined because of th e

avail abi lity of its complete sequence The nonessential sequences (ie Kille gene and i51

transposable element) were eliminated by usin g PCR-based approach The newly-constructed

mini binary vector pCB30l (35 kb) was tested fully functional in transtoD11ing Arabidop5is

plants As a res ult its seties (pCB30 I-based vecto rs) were also constructed fo r vari ous plant

transfo rmat ion tasks

According to Zhao and co-workers (2000) AgrobacleriLlm strain LBA4404 canying a supershy

binaty with a bar gene as a selectable marker for herbicid e resistance in the plant cells was

used to transfOllli so rghLUn It was the first successful repOli on the Agrobaclerium-mediated

transformation in producing stable transfonnants of sorghum plants

In 2003 Chen et al reponed on the reconst ruction o[the widely used expression vec tor pBI 121

by applying the knowledge of the complete sequ ence of pBI121 (accession number AF485783)

Chen and co-workers (2003) had cloned th e three di fferent plant genes into pB1l21 by

replacing the GUS coding re gion within T-DNA Then they PCR-amplifled the region and

subconed the PCR products into pGEM -T Easy vector

II

Tn recent s tud y conducted by Afolabi el al (2005) marker-free or clean gene transgenic lice

was prod uced by usin g multiple T-DNA approach This approach utili zes the novel

pGreenpSoup dual-binary vec tor system Both pGreen and pSoup vec tor would carry different

transgenes in its T -DNA and were co-transfonned into E coli and Agrobacerium

12

CHAPTER III

MATERIALS AND METHODS

31 Materials

311 Plasmids

pBl1 21 supplied by Arabidopsis Biological Resource Center Columbus and pUCI9 (MBI

Fennentas USA) were us ed in thi s study

312 Bacterial cell

The Escherichia coli strain JM 109 (Prom ega USA) were used as host ce ll [or propaga tion of

plasmid s

32 Plasmid recovery of pBIl21

321 Preparation and maintenance of E coli s train JMI09 cultures carrying pBIl21

E coli strain JMI09 f1-om stock co llecti on (C03 -388) was streaked on Luria-Beltani (LB) agar

with 20 ii i of kanamycin (50 ligiml) and LB agar witho ut kanamycin as negati ve control The

inoculated plates were then incubated at 37C for ovemight

322 Large scale isolation of pBIlll from overnight E coli strain JMI09 culture

Large scale preparations ofpBI 121 were canied out manually for reshiction analysis Half 0 f a

bac terial co lony was trans ferred into 100 ml o f Luria-B ertani (LB) broth containing 100 iiI of

13

kanamycin (50 jlgml) in a 250-ml conical flask The cultures were incubated overnight at

37degC wi th vigorou s shaking (200 rpm ) in the ionova r M 4000 incubator shaker 50 ml of th e

culture was trans fened each into two 50-ml Falcon tube and centrifu ged at 8000 rpm for 5

minutes at room temperature in the rotor centrifuge (KUBOTA 8800) The LB bro th was

removed by decanting and rccentlifuged at 8000 rpm for I minute Traces o f liquid media

were completely removed Iiom the tube

The bac te rial pellet was re suspended in I ml of ice-co ld Solution J (Appendi x A) by VOl1exing

briefly for 10 seconds and kept on ice I ml o ffreshly prepared Solution 11 (Appendix A) was

added and the mixture was mixed gently by invel1ing the tube terr times The tube was left at

room temperature and th e lysis reaction was allowed to occur fo l 5 minutes wi thout exceeding

5 minutes 2 ml of Solution [[[ (Appendix A) was added and mixed by inve ning the tube ten

times The mixture was centrifuged at 8000 rpm fo r 5 minutes to pe lle t the prec ipitate The

supern atant was transferred to a new Falcon tub e Two volume o f cold abso lute ethanol was

added to precipitate the DNA and the content was mi xed gentl y by in vel1ing the tube at leas t

ten times The DNA was pellcted by centrifuging at 8000 rpm for 10 minutes at room

temperature The supernatant was discarded and washed with I ml of 70 ethanol and

recenliifuged at 8000 rpm for I minute The s upernatant was discarded as much as poss ible

and air-dried The DNA pellet was resuspended in 35 jll ofTE buffe r Four rep lic ates o f largeshy

scale plasmid preparation were done and trarrs ferred into a 15 ml Eppendorftube by pipetting

14

323 Restriction enzyme analysis of pBII21

Several restriction enzymes were used for single and double digestions of pBI 121 such as

EcoR l HindJIl BamHI Ndel Eco811 Smal Noll and Xba l Bo th single diges tions and double

digestions ofp BI121 were perfolmed in 10 ILl reac tion with 4 ILl of pBI121 added A ll double

digestio ns were done by using Uni versa l Tangotrade Buffer (MB I Felmentas New Yo rk) The

react ion mixtures were inc ubated at 37degC overnight inside LABTECH water bath The

enzyme ac tivity was stopped by incubating at 6S oC for 20 minutes S ILl of undi gested and

digested samples were loaded with I ILl o f IX gel loading dye and electrophoresed at SOV for 2

ho urs in 08 agarose gel with ethidium bromide The agarose ge l was th en visuali zed und er

UV trans illuminator and photographed by us ing Po laro id 667

324 peR for GUS gene of pBII21

Besides the restIiction analysis perfo lmed on the iso lated pB 1I 2 1 to confirnl the desired

plas mid the DNA samples we re used as DNA template for amplification of GUS gene by using

th e fo rwa rd and reverse primers (Table 3 1) The compos itio n of PCR reaction mixtu re is

shown in Tab le 32 while paramete rs used in the PCR is shown in Table 33 The PCR was

ca rri ed out by using PCR th elmocyc1er (Biometra) Th e 3lmealing temperature 59C was

refelTed from previous PCR work done by Mohamed A li 2005 Posit ive control (pS RNpAGS

which con tains GUS gene) and negative control (without DNA samples) were inc luded

IS

Pussl Khidmat Maklumat Akademik UNIVE~SlTI MALAYSIA SARAWAK

CONSTRUCTION OF A PLANT TRANSFORMATION VECTOR CA RRYING A MARKER GENE FOR EXPRESSION IN PLANTS

NGIEN LEH NAH

This project is submitted in partial fulfillment of the requirements for the degree of Bachelor of Science with Honours (Resource Biotechnology)

Faculty of Reso urce Science and Technology UNIVERSITI MALAYSIA SARAWAK

2006

ACKNOWLEDGEMENT












TABLE OF CONTENT

Page

ACKNOWLEDGEMENT




LIST OF TABLES v

LIST OF FIGURES VI

ABSTRACTABSTRAK VII



mlmprep






3 1 Mate li als 13

3 11 Plasmids 13





I

CHAPTER IV












M iniprep kit






37 2 2 PCR 21






III

CHAPTER V

REFERE CES

APPENDICES

Appendix A

Appendix B

Appendix C



pUC I9



41






IV

LIST OF TABLES

Table Page










v

LIST OF FIGURES

Figure Page


















VI


Ngien Lell Nah



ABSTRACT



ABSTRAK



Vll

CHAPTERl

INTRODUCTION



























(1993)











2












3

CHAPTER II

LITERA TURE REVIEW

21 pBI121 and pUC19














4

l 94(

IT~

SI-il poundJ4It1

- 1

GaI U~ I 1amp111


L l lI W I

l5j IV

1)1 U l fl1 ~

+ 141r - -~ r~~~

Ihl~ I ~ ~



~-

RKl

NOS prom

NPl1l COS

h -

NPTlII COS

pBI121

- 13kb

RK2 TriA

Telo 3


5


E 1 1411






staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011


Cllli 1217



6
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS

ACKNOWLEDGEMENT












TABLE OF CONTENT

Page

ACKNOWLEDGEMENT




LIST OF TABLES v

LIST OF FIGURES VI

ABSTRACTABSTRAK VII



mlmprep






3 1 Mate li als 13

3 11 Plasmids 13





I

CHAPTER IV












M iniprep kit






37 2 2 PCR 21






III

CHAPTER V

REFERE CES

APPENDICES

Appendix A

Appendix B

Appendix C



pUC I9



41






IV

LIST OF TABLES

Table Page










v

LIST OF FIGURES

Figure Page


















VI


Ngien Lell Nah



ABSTRACT



ABSTRAK



Vll

CHAPTERl

INTRODUCTION



























(1993)











2












3

CHAPTER II

LITERA TURE REVIEW

21 pBI121 and pUC19














4

l 94(

IT~

SI-il poundJ4It1

- 1

GaI U~ I 1amp111


L l lI W I

l5j IV

1)1 U l fl1 ~

+ 141r - -~ r~~~

Ihl~ I ~ ~



~-

RKl

NOS prom

NPl1l COS

h -

NPTlII COS

pBI121

- 13kb

RK2 TriA

Telo 3


5


E 1 1411






staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011


Cllli 1217



6
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS


TABLE OF CONTENT

Page

ACKNOWLEDGEMENT




LIST OF TABLES v

LIST OF FIGURES VI

ABSTRACTABSTRAK VII



mlmprep






3 1 Mate li als 13

3 11 Plasmids 13





I

CHAPTER IV












M iniprep kit






37 2 2 PCR 21






III

CHAPTER V

REFERE CES

APPENDICES

Appendix A

Appendix B

Appendix C



pUC I9



41






IV

LIST OF TABLES

Table Page










v

LIST OF FIGURES

Figure Page


















VI


Ngien Lell Nah



ABSTRACT



ABSTRAK



Vll

CHAPTERl

INTRODUCTION



























(1993)











2












3

CHAPTER II

LITERA TURE REVIEW

21 pBI121 and pUC19














4

l 94(

IT~

SI-il poundJ4It1

- 1

GaI U~ I 1amp111


L l lI W I

l5j IV

1)1 U l fl1 ~

+ 141r - -~ r~~~

Ihl~ I ~ ~



~-

RKl

NOS prom

NPl1l COS

h -

NPTlII COS

pBI121

- 13kb

RK2 TriA

Telo 3


5


E 1 1411






staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011


Cllli 1217



6
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS

CHAPTER IV












M iniprep kit






37 2 2 PCR 21






III

CHAPTER V

REFERE CES

APPENDICES

Appendix A

Appendix B

Appendix C



pUC I9



41






IV

LIST OF TABLES

Table Page










v

LIST OF FIGURES

Figure Page


















VI


Ngien Lell Nah



ABSTRACT



ABSTRAK



Vll

CHAPTERl

INTRODUCTION



























(1993)











2












3

CHAPTER II

LITERA TURE REVIEW

21 pBI121 and pUC19














4

l 94(

IT~

SI-il poundJ4It1

- 1

GaI U~ I 1amp111


L l lI W I

l5j IV

1)1 U l fl1 ~

+ 141r - -~ r~~~

Ihl~ I ~ ~



~-

RKl

NOS prom

NPl1l COS

h -

NPTlII COS

pBI121

- 13kb

RK2 TriA

Telo 3


5


E 1 1411






staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011


Cllli 1217



6
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS

CHAPTER V

REFERE CES

APPENDICES

Appendix A

Appendix B

Appendix C



pUC I9



41






IV

LIST OF TABLES

Table Page










v

LIST OF FIGURES

Figure Page


















VI


Ngien Lell Nah



ABSTRACT



ABSTRAK



Vll

CHAPTERl

INTRODUCTION



























(1993)











2












3

CHAPTER II

LITERA TURE REVIEW

21 pBI121 and pUC19














4

l 94(

IT~

SI-il poundJ4It1

- 1

GaI U~ I 1amp111


L l lI W I

l5j IV

1)1 U l fl1 ~

+ 141r - -~ r~~~

Ihl~ I ~ ~



~-

RKl

NOS prom

NPl1l COS

h -

NPTlII COS

pBI121

- 13kb

RK2 TriA

Telo 3


5


E 1 1411






staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011


Cllli 1217



6
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS

LIST OF TABLES

Table Page










v

LIST OF FIGURES

Figure Page


















VI


Ngien Lell Nah



ABSTRACT



ABSTRAK



Vll

CHAPTERl

INTRODUCTION



























(1993)











2












3

CHAPTER II

LITERA TURE REVIEW

21 pBI121 and pUC19














4

l 94(

IT~

SI-il poundJ4It1

- 1

GaI U~ I 1amp111


L l lI W I

l5j IV

1)1 U l fl1 ~

+ 141r - -~ r~~~

Ihl~ I ~ ~



~-

RKl

NOS prom

NPl1l COS

h -

NPTlII COS

pBI121

- 13kb

RK2 TriA

Telo 3


5


E 1 1411






staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011


Cllli 1217



6
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS

LIST OF FIGURES

Figure Page


















VI


Ngien Lell Nah



ABSTRACT



ABSTRAK



Vll

CHAPTERl

INTRODUCTION



























(1993)











2












3

CHAPTER II

LITERA TURE REVIEW

21 pBI121 and pUC19














4

l 94(

IT~

SI-il poundJ4It1

- 1

GaI U~ I 1amp111


L l lI W I

l5j IV

1)1 U l fl1 ~

+ 141r - -~ r~~~

Ihl~ I ~ ~



~-

RKl

NOS prom

NPl1l COS

h -

NPTlII COS

pBI121

- 13kb

RK2 TriA

Telo 3


5


E 1 1411






staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011


Cllli 1217



6
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS


Ngien Lell Nah



ABSTRACT



ABSTRAK



Vll

CHAPTERl

INTRODUCTION



























(1993)











2












3

CHAPTER II

LITERA TURE REVIEW

21 pBI121 and pUC19














4

l 94(

IT~

SI-il poundJ4It1

- 1

GaI U~ I 1amp111


L l lI W I

l5j IV

1)1 U l fl1 ~

+ 141r - -~ r~~~

Ihl~ I ~ ~



~-

RKl

NOS prom

NPl1l COS

h -

NPTlII COS

pBI121

- 13kb

RK2 TriA

Telo 3


5


E 1 1411






staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011


Cllli 1217



6
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS

CHAPTERl

INTRODUCTION



























(1993)











2












3

CHAPTER II

LITERA TURE REVIEW

21 pBI121 and pUC19














4

l 94(

IT~

SI-il poundJ4It1

- 1

GaI U~ I 1amp111


L l lI W I

l5j IV

1)1 U l fl1 ~

+ 141r - -~ r~~~

Ihl~ I ~ ~



~-

RKl

NOS prom

NPl1l COS

h -

NPTlII COS

pBI121

- 13kb

RK2 TriA

Telo 3


5


E 1 1411






staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011


Cllli 1217



6
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS










(1993)











2












3

CHAPTER II

LITERA TURE REVIEW

21 pBI121 and pUC19














4

l 94(

IT~

SI-il poundJ4It1

- 1

GaI U~ I 1amp111


L l lI W I

l5j IV

1)1 U l fl1 ~

+ 141r - -~ r~~~

Ihl~ I ~ ~



~-

RKl

NOS prom

NPl1l COS

h -

NPTlII COS

pBI121

- 13kb

RK2 TriA

Telo 3


5


E 1 1411






staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011


Cllli 1217



6
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS












3

CHAPTER II

LITERA TURE REVIEW

21 pBI121 and pUC19














4

l 94(

IT~

SI-il poundJ4It1

- 1

GaI U~ I 1amp111


L l lI W I

l5j IV

1)1 U l fl1 ~

+ 141r - -~ r~~~

Ihl~ I ~ ~



~-

RKl

NOS prom

NPl1l COS

h -

NPTlII COS

pBI121

- 13kb

RK2 TriA

Telo 3


5


E 1 1411






staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011


Cllli 1217



6
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS

CHAPTER II

LITERA TURE REVIEW

21 pBI121 and pUC19














4

l 94(

IT~

SI-il poundJ4It1

- 1

GaI U~ I 1amp111


L l lI W I

l5j IV

1)1 U l fl1 ~

+ 141r - -~ r~~~

Ihl~ I ~ ~



~-

RKl

NOS prom

NPl1l COS

h -

NPTlII COS

pBI121

- 13kb

RK2 TriA

Telo 3


5


E 1 1411






staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011


Cllli 1217



6
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS

l 94(

IT~

SI-il poundJ4It1

- 1

GaI U~ I 1amp111


L l lI W I

l5j IV

1)1 U l fl1 ~

+ 141r - -~ r~~~

Ihl~ I ~ ~



~-

RKl

NOS prom

NPl1l COS

h -

NPTlII COS

pBI121

- 13kb

RK2 TriA

Telo 3


5


E 1 1411






staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011


Cllli 1217



6
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS






staining

Ple l a (lsIN1 m

I IIltJcl 113

1 Ek1 235 Pdmt 219 A-~c[

Bqjl 2215

cat I t ~---

pUC1819 2686 bp

AIIIIPad 8011


Cllli 1217



6
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS
















1997)




7







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS







pH80











8





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS





















9




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS




tra nsfo rmation









nu mbers






10




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS




















II





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS





12

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS

CHAPTER III


31 Materials

311 Plasmids



312 Bacterial cell


plasmid s









13




















14



















IS




















14



















IS



















IS

CONSTRUCTION OF A PLANT TRANSFORMATION VECTOR … of a plant transformation...pussl khidmat maklumat...

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Transcript of CONSTRUCTION OF A PLANT TRANSFORMATION VECTOR … of a plant transformation...pussl khidmat maklumat...