Isolation of a genomic DNA for Jatropha curcas ribosome inactivating protein and its tobacco transformation

Genomic DNA for Jatropha curcas ribosome inactivating protein （JRIP） was cloned from total DNA of its leaves by polymerase chain reaction （PCR）. The no intron character was confirmed. The plant expression vector pBI121-JRIP was constructed by inserting the JRIP gene into pBI121 plasmid. The recombinant Agrobacterium EHA105 strain harboring pBI121-JRIP was constructed by conducting pBI121-JRIP to strain EHA 105. PCR and Southern blotting were carried out, and the results proved that the JRIP gene was integrated into tobacco genome. It might provide a new material for disease resistance tobacco species breeding.

Construction of RNAi Vector Which Resist Cucumber Mosaic Virus and Transformation of Tobacco

[Objective] Aimed to construct RNAi vector resistant to cucumber mosaic virus and transferred this vector into tobacco. [Method] RT-PCR method was used to amplify cucumber mosaic virus NS04 and process RNA2 gene sequen of tomato isolates. The analysis results of phylogenetic tree demonstrated that the sequence in RNA2 encoded CMV-2a had 98.0% and 96.5% homology with nucleotide and amino acid of DQ412731 isolate of Zhejiang,China. The replicase fragment in CMV RAN2 gene was taken as target sequence to construct pBi35SCR2 eukaryotic expression vector,then the expression vector was identified. Through agrobacterium-mediated method,the expression vector was transferred into tabacco and PCR method was used to check the transfer. The PCR results demonstrated that the experiment had successfully construct eukaryotic expression vector of pBi35SCR2 and the expression vector was successfully transferred into tabacco. [Conclusion] The obtained transgenic tobacco could be used as challenge test material in following experiment and provided foundation for studying processing tomato resist cucumber mosaic virus.

Genetic Engineering of Tobacco with Double Resistance to Both Virus and Insect

Expression vector pE14 with double resistance to virus and insect was constructed by inserting CW-cp gene and Bt-toxin gene one by into T-DNA of the same binary vector pE3. Tobacco was then transformed with Agrbacterium tumefaciens (At)GV311-SE carrying PE14. Nopaline assay and PCR amplification confirmed that both CW-cp gene and Bt-toxin gene had been introduced into tobacco genome by T-DNA of PE3. Test attack with virus and demonstrated, in some of the transgenic plants, double resistance to both infection by CMV and damage by Manduca sexta.

Isolation and molecular characterization of the FLOWERING LOCUS C gene promoter sequence in radish(Raphanus sativus L.)

Both bolting and flowering times influence taproot and seed production in radish. FLOWERING LOCUS C （FLC） plays a key role in plant flowering by functioning as a repressor. Two genomic DNA sequences, a 3 046-bp from an early- and a 2 959-bp from a late-bolting radish line were isolated and named as RsFLC1 and RsFLC2, respectively, for they share approximately 87.03% sequence identity to the FLC cDNA sequences. The genomic DNA sequences, 1 466-bp and 1 744-bp, flanking the 5＇-regions of RsFLC1 and RsFLC2, respectively, were characterized. Since both of them harbor the basic promoter elements, the TATA box and CAAT box, they were designated as PRsFLC1 and PRsFLC2. The transcription start site （TSS） was identified at 424 and 336 bp upstream of the start codon in PRsFLC1 and PRsFLC2, respectively, cis-regulatory elements including CGTCA （MeJA-responsive） and ABRE （abscisic acid-responsive） motifs were found in both promoters, while some cis-regulatory elements including TCAelement and GARE-motif were present only in PRsFLCI. These sequence differences lead to the diversity of promoter core elements, which could partially result in the difference of bolting and flowering time in radish line NauDY13 （early-bolting） and Naulu 127 （late-bolting）. Furthermore, to investigate the activity of these promoters, a series of 5＇-deletion fragment-GUS fusions were constructed and transformed into tobacco. GUS activity was detected in PRsFLCI-（1 to 4）-GUS-PSlaG-3 and PRsFLC2-（1 to 4）-GUS-PS1aG-3 transgenic tobacco leaf discs, and this activity progressively decreased from PRsFLC-1-GUS-PSlaG-3 to PRsFLC-5-GUS-PS1aG-3. Deletion analysis indicated that the cis-regulatory elements located at -395 bp to ＋1 bp may be critical for specifying RsFLC gene transcription.