Cloning and Partial Characterization of Cotton Leaf Curl Burewala Virus From Khanewal

Fizza Akhter,Muhammad Tahir

doi:10.53992/njns.v3i1.24

Abstract

Begomoviruses are a serious threat to cotton production throughout the world. In Pakistan, enormous crop losses occur as a result of cotton leaf curl disease (CLCuD) caused by begomoviruses. Molecular characterization of begomoviruses has made possible the identification and analysis of begomoviruses prevalent in a host plant. Infected cotton leaf sample (C-59) was obtained from area around Khanewal during 2011. The total DNA was isolated from the infected sample by Cetyl trimethyl ammonium bromide (CTAB) method. An expected size band of approximately 1100bp, covering coat protein region of the virus, was amplified using universal primers. The amplified product was T/A cloned and sequenced to its entirety. DNA sequence showed 99% nucleotide sequence identity to each of Cotton leaf curl Burewala virus ((CLCuBuV; Accession No HF549Begomoviruses are a serious threat to cotton production throughout the world. In Pakistan, enormous crop losses occur as a result of cotton leaf curl disease (CLCuD) caused by begomoviruses. Molecular characterization of begomoviruses has made possible the identification and analysis of begomoviruses prevalent in a host plant. Infected cotton leaf sample (C-59) was obtained from area around Khanewal during 2011. The total DNA was isolated from the infected sample by Cetyl trimethyl ammonium bromide (CTAB) method. An expected size band of approximately 1100bp, covering coat protein region of the virus, was amplified using universal primers. The amplified product was T/A cloned and sequenced to its entirety. DNA sequence showed 99% nucleotide sequence identity to each of Cotton leaf curl Burewala virus ((CLCuBuV; Accession No HF549184)) and Cotton leaf curl Kokhran virus (CLCuKV; Accession No AJ002449)). Since CLCuBuV is a recombinant of CLCuKV and Cotton leaf curl Multan virus and the coat protein region of CLCuBuV was derived from CLCuKV that is most probable reason that the available sequence showed identity with CLCuBuV as well as CLCuKV. A complete characterization of full length virus will determine whether isolate C-59 is CLCuBuV or CLCuKV. Literature indicates that there is no existence of CLCuKV within the region and CLCuBuV is dominating within Indo-Pak184)) and Cotton leaf curl Kokhran virus (CLCuKV; Accession No AJ002449)). Since CLCuBuV is a recombinant of CLCuKV and Cotton leaf curl Multan virus and the coat protein region of CLCuBuV was derived from CLCuKV that is most probable reason that the available sequence showed identity with CLCuBuV as well as CLCuKV. A complete characterization of full length virus will determine whether isolate C-59 is CLCuBuV or CLCuKV. Literature indicates that there is no existence of CLCuKV within the region and CLCuBuV is dominating within Indo-Pak

Highlights

Begomoviruses have been divided into two groups based on genome organization
Coat protein gene (CP or V1) and the pre-coat protein (V2) gene are encoded on the virion sense strand whereas replication-associated protein gene (Rep or C1), transcriptional activator protein gene (TrAP or C3), replication enhancer protein gene (REn or C3) and the C4 gene are found on the complementary sense strand of DNA A
PCR amplification of the extracted DNA using universal primer designed for the partial characterization of begomovirus revealed a fragment of approximately 1.1kb on 1% agarose gel (Figure 3.1)

Summary

Introduction

Begomoviruses have been divided into two groups based on genome organization. The genome is either monopartite or bipartite. Monopartite genome constitutes only DNA A whereas bipartite genome is composed of DNA A and DNA B components. The genome replication takes place through double stranded DNA intermediates (Hanley-Bowdoin et al, 1999). Bipartite Begomoviruses have two genomic components designated as DNA A and DNA B (Lazarowitz, 1992). Coat protein gene (CP or V1) and the pre-coat protein (V2) gene are encoded on the virion sense strand whereas replication-associated protein gene (Rep or C1), transcriptional activator protein gene (TrAP or C3), replication enhancer protein gene (REn or C3) and the C4 gene are found on the complementary sense strand of DNA A. Movement protein (MP) and the nuclear shuttle protein (NSP) are coded on the DNA B component (Kallender et al, 1998)

Methods

Results

Conclusion