[Cloning of AHA gene from Amaranthus hypochondriacus and it's aphid inhibitory effect in transgenic tabacco plants].

ABSTRACT

Using total DNA isolated from Amaranthus hypochondriacus as template, Amaranthus hypochondriacus agglutinin AHA gene was amplified by PCR and cloned. Sequence analysis results showed that this gene is consisted of 2453 base pairs including one 1538 bp intron and two exons of 212 bp and 703 bp respectively. After inverse PCR amplification, coding region of AHA gene was obtained. AHA gene with it's intron (AHAg) and withou intron (AHAc) were inserted downstream of 35S promotor in the binary vector pBin438 resulting in the construction of two plant expression vectors pBAHAg and pBAHAc repectively. Leave explants of Nicotinana tabacum var. SR1 were transformed with A. tumefaciens LBA4404 harbouring the above expression vectors. Results from PCR and Southern blot analysis showed that AHA genes were inserted into the genome of transformed tobacco plants. Immunodot blot analysis indicated that AHA was expressed in transgenic plants. The results from insect bioassay with peach aphid (Mizus persicae) showed that the transgenic plants of pBAHAg and pBAHAc were aphid resistant, evidenced by a 57%-48% reduction in insect population density, some plants were more than 85%. The aphid resistance of transgenic plants transformed with AHAg gene as judged by aphid inhibition rate was higher than that of plants transormed with AHAc gene indicating that the intron in AHAg may be favorable for expression of AHA in transgenic plants.