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Expression of ASGV coat protein (CP) in pET-32a(+) and pHIS expression
system

CP gene was amplified and cloned in pET-
32a(+) expression vector. Protein was induced
optimally in soluble fraction containing (1
mM) IPTG at 30°C (pET-32a)/28 °C (pHIS)
(Fig. 78).

The protein was purified and its identity was Fig. 78 ASGV CP as His Tag fusion protein. Lanes 1-4: 45
confirmed by Western blotting (Fig. 79). kDa His Tag fusion protein expressed after 1, 2, 3 and 4 h
of induction, respectively; Lane 5: Control with no IPTG.
Purified protein was used for rabbit Lane M: Prestained protein marker (Fermentas)

immunization. The IgG was extracted and

purified from the antiserum using Protein A antibody purification system.This IgG was used for

testing of field samples where the antisera developed was equally effective when compared to

commercial kits.

CSIR-IHBT Annual Report 2012-13 Fig. 79 The purified ASGV CP as (A) His Tag fusion protein in pET-32a (B) and pHIS parallel vectors (C) confirmation
by Western blotting

Detection and identification of ASGV
from nectarine, peach, wild cherry,
almond and apricot

Various stone fruits such as nectarine, peach, wild
cherry, almond and apricot were tested for ASGV
by ELISA and confirmed positive through RT-
PCR (Fig. 80).

Genetic transformation of apple rootstock Fig. 80 Amplifications with detection primers for
ASGV. M1kb ladder, 1: Nectarine, 2: Peach, 3: Wild
MM106 (Funded by Department of Biotechnology cherry, 4: Almond & 5: Apricot

Govt. of India)

In continuation to previous activity, transgenic lines of apple rootstock MM106 were tested
for RT-PCR expression of gus gene. Positive signals were obtained in the tested lines
(Fig. 81A). These were multiplied on BAP and NAAsupplemented MS medium (Fig. 81B) and rooted
prior to their transfer to soil for maintenance under contained polyhouse conditions (Fig. 81C).

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