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

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.

Structure of the nucleocapsid protein gene of rice yellow stunt rhabdovirus.

The complete nucleotide sequence of the N protein gene of rice yellow stunt rhabdovirus (RYSV) was determined by sequencing of cDNA clones derived from the viral genomic RNA. The 3' end of the N gene (messenger sense) was defined by sequence analysis of cDNA clones generated from the N protein mRNA by 3'RACE. The 5' end sequence of the gene was putatively assigned as 5'-AACAC-3'; this sequence is found in the presumed 3' leader/N gene junction region. The mRNA encoding the RYSV N protein is 1714 nt comprising a 15-nt untranslated 5' leader sequence followed by an open reading frame (ORF) of 1563 nt and a 136-nt untranslated 3' region. The calculated molecular mass of the N protein encoded by the ORF is 58,400 Da, which is larger in size than N proteins of other rhabdoviruses. Amino acid composition analysis shows that the RYSV N protein is rather basic with a predicted isoelectric point of 10.04; indeed, a large highly basic region could be found at the carboxy terminal portion of the protein. Amino acid sequence comparison between N proteins of RYSV and sonchus yellow net virus, both of which belong to the same genus Nucleorhabdovirus, revealed an overall 30% identity, with three relatively conserved blocks of 14-20 amino acid residues. Moreover, the hydropathy profiles of the two proteins are generally similar. The structural similarities between the N protein of RYSV and that of lettuce necrotic yellows virus, the type member of the genus Cytorhabdovirus, and those of animal rhabdoviruses, are less significant. Nucleotide sequence determination of 5' and 3' regions flanking the RYSV N gene identified a 14-nt common sequence that is very similar to the consensus gene junction sequences of other plant and animal rhabdoviruses.

Insect resistance of transgenic tobacco plants expressing delta-endotoxin gene of Bacillus thuringiensis.

The initiative B.thuringiensis delta-endotoxin (Bt toxin) gene clones TH12 and TH48 contain two different classes of homologous genes, the 5.3 kb class and 6.6 kb class, respectively. Bt toxin genes of both classes, modified at the 5'-end and truncated at the 3'-end, can still be expressed to produce the insecticidal, truncated toxin proteins in E. coli. The modified Bt toxin genes were inserted into the plant binary expression vector pBin 437 (a derivative plasmid of pBin 19) and were transferred into tobacco by Ti plasmid-mediated gene transfer system. Southern blot and DNA slot blot analysis indicate that the Bt toxin genes have been integrated into tobacco genome at a copy number of 1 to 5. Northern blot analysis of polyA+ RNAs from progeny of the transgenic plants revealed that Bt toxin genes of both 5.3 kb and 6.6 kb classes were expressed in transgenic plants, though the transcripts were degraded to RNAs of lower molecular weights. In insecticidal test, 5 plants from the progeny of 5.3 kb class gene-transformed SR1 tobacco plants and 3 plants from those of 6.6 kb class gene-transformed plants were found to be toxic to the testing larvea of H.assulta. In comparison with the control, mortality of the insects fed on transgenic plants reached 40-50% and the growth of the survived insects was remarkably inhibited. These results indicate that the modified Bt genes of the 5.3 kb and 6.6 kb classes were expressed in transgenic plants and could confer on the transgenic plants a new character of insect resistance.

Cloning and expression of delta-endotoxin gene of Bacillus thuringiensis in Escherichia coli.

Fragments (larger than 4 kb) of partially Sau 3AI-digested plasmid DNA from Bacillus thuringiensis subsp. kenyae 7404 and kurstaki HD-1 were cloned into the BamH I site of pBR322. Four transformants, containing the corresponding delta-endotoxin gene and producing proteins that reacted with antiserum against crystalline protein, were selected on the basis of results from in situ colony hybridization, radioimmunologic screening, and Western blot analysis. Three of those tested, i.e., the lysates of one transformant (TK89) carrying the delta-endotoxin gene of B.t. 7404 and two transformants (TH12 and TH48) carrying the delta-endotoxin gene of HD-1, were toxic for tobacco budworm (Heliothis assulta) and armyworm (Leucania separata Walker) caterpillars. This is the first report on cloning of the delta-endotoxin gene from B.t. subsp. kenyae, which is different in serotype from the well-studied subsp. kurstaki.

Study on the genome of cauliflower mosaic virus (Xinjiang isolate). Restriction mapping of virion and cloned viral DNA.

The genomic DNA of CaMV (Xinjiang isolate) was mapped on the virion DNA with a number of restriction endonucleases by double digestions and partial digestions of one-end 32P-labelled fragments. It contained the unique sites of BglI, SalI and XhoI. BamHI and HhaI each cut it at two sites, EcoRI at five sites (three of which were located), HpaII at seven sites, BglII at eight sites, and HaeIII at ten sites. The sites of all the enzymes used above, as well as HindIII which cut at ten sites were also mapped on the cloned CaMV DNA. Virion DNA and cloned viral DNA gave the same results in the number and location of cleavage sites of the restriction enzymes tested on both DNAs. Like most of the other isolates, CaMV-Xinjiang isolate has three single-stranded discontinuities. From the digestion patterns of SalI, XhoI, EcoRI and HhaI, it is concluded that the restriction map of CaMV-XJ genome is closely related to that of BKMV isolate from East Germany.

A comparative study of the cowpea and bean strains of southern bean mosaic virus.

Features of the primary structure and translation of the genomic RNAs of the cowpea and bean strains of southern bean mosaic virus have been investigated in order to assess the similarity of the two viruses. The sequence of 400 bases at their 3' termini have been determined. These include the 3' noncoding regions and extend well into the coat protein cistrons. The noncoding regions (136 bases for the cowpea strain RNA and 129 bases for the bean strain RNA) show no obvious sequence homology. However, extensive base as well as amino acid sequence homology exists in the coding region. RNAs from both strains have a small protein attached to their 5' terminus-the protein in the cowpea strain being the smaller of the two. In vitro studies show that there are similarities in the overall mode of translation of the genomes of the two viruses. Although corresponding proteins are synthesized they differ in size.