Construction And Identification Of Tansgenic Wheat Resistant To Wheat Yellow Mosaic Virus

Wheat (Triticum aestivum L.) calli induced from immature embryos were transformed with plasmid containing mutant Escherichia coli RNase III gene (rnc, in pAMM2025) through Agrobacterium. Six transgenic wheat lines (T0 and T1 progeny) are PCR positive.Wheat yellow mosaic virus (WYMV) coat protein gene, WYMV-72kD protein gene, WYMV-NIb protein gene, human 2-5A-RNase L gene were delivered into Wheat calli induced from immature embryos by others in our lab from 1999 to 2002. Seeds from these transgenic lines confirmed by PCR were sowed in fields heavily contaminated with WYMV to carry on field trail and molecular detection.As for transgenic wheat with WYMV CP, PCR and PCR-Southern results show that the transgene is integrated stably in T5 generation. Western blot result proves the expression of it in transgenic wheat. One line of these CP-transformed wheat show exhibited good resistance to WYMV in field trial, and all backcross lines have good resistance and are PCR positive. In the detection of transgenic wheat with WYMV 72kD, PCR and PCR-Southern results showed that the transgene is integrated in T5 generation. Marker-free transgenic lines are obtained by PCR selection. Two lines of these transgenic wheat with 72kD have good resistance to WYMV in field trial. As for transgenic wheat with WYMV NIb, the foreign gene can be detected in T2 generation by PCR, and one line show good resistance. In the experiment of transgenic wheat with 2-5 A-RNase L, Southern blot analysis proved the existence of 2-5A in transgenic wheat genome (T2 generation). Ten transgenic line exhibited good resistance to WYMV in field trial.Furthermore, two expression vectors-pET2-5A5' and pKGRL, which contained the 5'-terminus of 2-5A gene and RNase L gene respectively, were constructed and two fusion proteins (N-terminus of 2-5A protein and RNase L protein are fused with His tag and GST respectively) were successfully expressed in Escherichia coli by induction with IPTG In addition, the specific antiserum to N-terminus of 2-5 A protein was obtained from the immuned rabbits.