Fermenting HpRNA By RNase Ⅲ Deficient Strain To Resist Tobacco Mosaic Virus

This study was based on RNAi technology,first we regarded TMV coat protein gene sequence as a template to construct an expression vector containing IR (inverted repeat),then transformed the vector into RNaseⅢdeficient strain DY330 to acquire the engineered bacteria which expresses hpRNA,at last we collected hpRNA through cultivating engineered bacteria and sprayed tobacco leaves with hpRNA to resist tobacco mosaic virus. The main contents were as follows:1 Construction of engineered bacteria DY-hp which expresses hpRNAAccording to TMV coat protein gene sequence to design primers, through extracting total RNA of tobacco, reverse transcription and PCR(Polymerase Chain Reaction) to obtain the cDNA of coat protein,and the alignment result was 98.33% in accordance.Regarding cDNA as the template to obtain two sequences by polymerase chain reaction, then linked them to T vector to get the cloning vector containing IR,and the alignment result was 99.73% in accordance. By linking the IR to expression vector pET-21a,the vector was transformed into RNaseⅢdeficient strain DY330 to acquire the engineered bacteria DY-hp which expresses hpRNA at last.3 Optimization of fermenting hpRNA by DY-hp .By measuring the growth curve of DY-hp, we found hpRNA’s yield was highest four hours after IPTG induction.The induced concentration of IPTG and the induced concentration of bacteria were optimized,the results were that when induced concentration of IPTG was 0.4mM,and OD600 of bacteria was 0.4,the average highest yield of hpRNA was 23μg per milliliter bacterial cultures.4 Spraying tobacco leaves with hpRNAA total of six experimental treatments:spraying hpRNA three days before inoculating TMV; spraying hpRNA three days after inoculating TMV;?spraying extracts from DY-control which contains the blank vector pet-21a;only inoculating TMV;only spraying hpRNA ; blank control. The experiment was repeated three times, the resistance rate of hpRNA’s preventing TMV was 65%,and the resistance rate of hpRNA’s curing TMV was 40%.