Cell-free cloning and biolistic inoculation of an infectious cDNA of potato virus Y

被引:48
作者
Fakhfakh, H
Vilaine, F
Makni, M
Robaglia, C
机构
[1] INRA,BIOL CELLULAIRE LAB,F-78026 VERSAILLES,FRANCE
[2] UNIV TUNIS,GENET LAB,TUNIS 1060,TUNISIA
关键词
D O I
10.1099/0022-1317-77-3-519
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Potato virus Y (PVY) full-length cDNA has been found to be refractory to cloning in Escherichia coli cells. A full-length 9.7 kb PW cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR) from the RNA of PVY (tuber necrotic strain, PVYNTN). Double-stranded DNA fragments were used as primers (ds megaprimers), to include signals for transcription in vivo (a cauliflower mosaic virus 35S RNA promoter and a nopaline synthase terminator) in the final PCR product. Biolistic bombardment with a helium particle gun was used to inoculate the amplified product to detached tobacco leaves. Inoculation of tobacco plants with ground inoculated leaves followed by northern blot, ELISA and immune-electron microscopy demonstrated that the DNA was highly infectious with up to 90% of bombarded leaves containing the virus. This methodology will allow the use of reverse genetics in the study of PVY-plant interactions and will also be useful for obtaining infectious cDNA from other viruses with large RNA genomes.
引用
收藏
页码:519 / 523
页数:5
相关论文
共 26 条
  • [1] PCR AMPLIFICATION OF UP TO 35-KB DNA WITH HIGH-FIDELITY AND HIGH-YIELD FROM LAMBDA-BACTERIOPHAGE TEMPLATES
    BARNES, WM
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (06) : 2216 - 2220
  • [2] INFECTIOUS TRANSCRIPTS AND CDNA CLONES OF RNA VIRUSES
    BOYER, JC
    HAENNI, AL
    [J]. VIROLOGY, 1994, 198 (02) : 415 - 426
  • [3] EFFECTIVE AMPLIFICATION OF LONG TARGETS FROM CLONED INSERTS AND HUMAN GENOMIC DNA
    CHENG, S
    FOCKLER, C
    BARNES, WM
    HIGUCHI, R
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (12) : 5695 - 5699
  • [4] DEBOKX JA, 1981, CMI AAB DESCRIPTIONS
  • [5] TAGGING OF PLANT POTYVIRUS REPLICATION AND MOVEMENT BY INSERTION OF BETA-GLUCURONIDASE INTO THE VIRAL POLYPROTEIN
    DOLJA, VV
    MCBRIDE, HJ
    CARRINGTON, JC
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (21) : 10208 - 10212
  • [6] INFECTIOUS INVITRO TRANSCRIPTS FROM CLONED CDNA OF A POTYVIRUS, TOBACCO VEIN MOTTLING VIRUS
    DOMIER, LL
    FRANKLIN, KM
    HUNT, AG
    RHOADS, RE
    SHAW, JG
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1989, 86 (10) : 3509 - 3513
  • [7] STABLE TRANSFORMATION OF MAIZE AFTER GENE-TRANSFER BY ELECTROPORATION
    FROMM, ME
    TAYLOR, LP
    WALBOT, V
    [J]. NATURE, 1986, 319 (6056) : 791 - 793
  • [8] INFECTIOUS INVITRO RNA TRANSCRIPTS DERIVED FROM CLONED CDNA OF THE CUCURBIT POTYVIRUS, ZUCCHINI YELLOW MOSAIC-VIRUS
    GALON, A
    ANTIGNUS, Y
    ROSNER, A
    RACCAH, B
    [J]. JOURNAL OF GENERAL VIROLOGY, 1991, 72 : 2639 - 2643
  • [9] INOCULATION OF PEPPERS WITH INFECTIOUS CLONES OF A NEW GEMINIVIRUS BY A BIOLISTIC PROCEDURE
    GARZONTIZNADO, JA
    TORRESPACHECO, I
    ASCENCIOIBANEZ, JT
    HERRERAESTRELLA, L
    RIVERABUSTAMANTE, RF
    [J]. PHYTOPATHOLOGY, 1993, 83 (05) : 514 - 521
  • [10] TRANSIENT PLANT GENE-EXPRESSION - A SIMPLE AND REPRODUCIBLE METHOD BASED ON FLOWING PARTICLE GUN
    GODON, C
    CABOCHE, M
    DANIELVEDELE, F
    [J]. BIOCHIMIE, 1993, 75 (07) : 591 - 595