Cloning of papaya Ringspot virus coat protein gene and transformation of ‘solo’ papaya through microprojectile bombardment

Issue Date

12-2011

Abstract

A unique coat protein gene of the Philippine Papaya Ringspot Virus (PRSV) strain was isolated from the yellow 'Solo' papaya using standard molecular cloning techniques. The coat protein gene was characterized using restriction enzyme digestion, gene sequencing and sequence alignment. The polymerase chain reaction product representing the coat protein gene is about 900 base pairs fragment as revealed by the restriction enzymes Bam HI and Xba l. Multiple sequence alignment (MSA) using the Aligr X-MSA program of Vector NTi showed that the coat protein gene is highly homologous to the coat protein gene sequence of the Papaya Ringspot Virus of Thailand (97.92%), Japan 1 (92.06%), Australia (91.07%) and China (91.07%). The coat protein gene was inserted into the binary vector p2k7 and multiplied in Escherichia coli. The induction of somatic embryos was promoted significantly by culturing 90-120 day old papaya zygotic embryos wherein one of the cotyledonary leaves was removed. Using microprojectile bombardment, transient expression studies using the plasmid pBI 121 conferring gus expression in papaya somatic embryos showed that a pressure of the helium blast of 1,200 kPa promoted high number of gus expressing cells. On the other hand, a distance of 12.5 cm of the target somatic embryos away from the filter unit containing the plasmid DNA promoted high number of gus expressing cells. Stable regeneration of 359 putative transgenic papaya plantlets 4-6 mo after transformation. Morphologically, the transformation using a locally optimized protocol for microprojectile bombardment resulted to the putative transgenic plants, are not phenotypically different from the untransformed yellow 'Solo' papaya. The putative transgenic To plants are susceptible to PRSV like the untransformed ' Solo' control. Likewise, the 11 T, transgenic lines derived from the To transgenic plants are also PRSV-susceptible. However, percent infection varied from 17-93%, indicating a large variability in the susceptibility of the different lines, and resistance could be unstable in these lines.

Source or Periodical Title

Philippine Journal of Crop Science

ISSN

0115-463x

Volume

36

Issue

3

Page

45-56

Document Type

Article

College

College of Agriculture and Food Science (CAFS)

Physical Description

illustrations ; chart ; tables ; references

Language

English

Subject

gus expression kanamycin, microprojectile bombardment, papaya, stable transformation, transient expression, transgenic plants

En – AGROVOC descriptors

CARICA PAPAYA; GENE CLONING; COAT PROTEINS; PLANT VIRUSES; SOMATIC EMBRYOGENESIS; SEQUENCE ALIGNMENT; PCR

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