TransgenicNicotiana tabacum plants expressing a fungal copper transporter gene show enhanced acquisition of copper

Sudhir Singh, Premsagar Korripally,Ramachandran Vancheeswaran and Susan Eapen*

Supplementary information

*Corresponding author

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Supplementary Information S1

Cloning of tcu-1 in plant expression vector pCAMBIA1301

Wild type Neurospora crassa (FGSC#2489) was grown in the basal medium (copper deficient) for three days at 28°C in stationary mode. The mycelial mats were washed and total RNA isolated using TRI reagent (Sigma). First strand cDNA synthesis kit (Stratagene, La Jolla, CA, USA) was used for the preparation of cDNA using total RNA as template. The cDNA was used as template for amplification of tcu-1 gene open reading frame (765 bp) and cloned into the pTZ57R/T vector (Fermentas Life Sciences, Leon-Rot, Germany). Clone containing the insert (pTAtcu-1) was sequenced with universal primers for sequence identity. After sequence confirmation, NcoI-BamHI fragment containing tcu-1 was subcloned in between duplicated CaMV 35S promoter and the nos terminator in the vector pBI525. The 1.7-kb HindIII-EcoRI fragment containing 5’-35S-35S-tcu-1-nos-3’ was finally cloned into plant binary vector pCAMBIA1301 (CAMBIA, Brisbane, Australia). Besides tcu-1, the plasmid contained uidA as reporter gene and hptII as plant selectable marker (Supplementaryfig. S1).

Supplementary figure S1

Linear representation of T-DNA of pSS3, having tcu-1 under double 35S promoter, AMV as translational enhancer element, hptII as plant selectable marker and uidA as reporter gene (~ 7.3 kb)

Supplementary figure S2

a b c

Transient (a) and stable (b, c) expression of GUS in transgenic plants

Supplementary figure S3

PCR amplification of genomic DNA from independently developed putative transgenic lines using tcu-1 specific primers, 1-20= transgenic lines (C= control line, M= 100 bp ladder)

a

b

Supplementary figure S4:Zn and Cd accumulation incontrol and transgenic C-2-4 plants at different Zn and Cd concentrations, respectively in hydroponics medium. All the values are means of three replicates. Bars with different letters indicate significantly different values at p≤ 0.05 for Cd/Zn at a particular Cd/Zn concentration.

Supplementarytable S1. Primer pairs used in PCR amplification

Primer / Expected size of product / Sequence
CUTF / 785bp
(tcu-1) / 5’- CCA TGG CGC AAC ACG GCA CAA C-3’
CUTR / 5’- GTA CCT CAG CCA CAG CAA ACA GTG-3’
HYGF / 450bp
(hptII) / 5’-GAG GGC GAA GAA TCT CGT GC-3’
HYGR / 5’- CAC TGA CGG TGT CGT CCA TC-3’
GUSF / 1300bp
(uidA) / 5’-ATG GAT AAC AAT CCG AAC ATC AAA GA-3’
GUSR / 5’-TTA TTA GCC CTA GTT GGT TTG TAC A-3’

F= forward primer; R= reverse primer

Supplementary table S2. Inheritance of hygR in T1 generation of independent tobacco lines

Line / Resistant seedlings / Sensitive seedlings / χ2 test
(3:1)
Control / 0 / 125 / -
C-1-1 / 99 / 26 / 1.12
C-1-9 / 93 / 32 / 0.24
C-1-10 / 95 / 30 / 0.67
C-1-11 / 98 / 27 / 0.77
C-1-23 / 86 / 39 / 2.56
C-2-1 / 102 / 23 / 2.9
C-2-2 / 90 / 35 / 0.6
C-2-3 / 102 / 23 / 2.9
C-2-4 / 103 / 22 / 3.65
C-2-5 / 96 / 29 / 0.22

At p≤ 0.05 and n=1, χ2 table value is 3.84

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