Construction Of Marker-free Bivalent Expression Vector For Potato Antivirus Transformation

Potato is a sort of important crop in the north of China.Degeneration caused by virus infection is the problem of primary importance in potato production.Potato Leafroll Virus(PLRV) and Potato Spindle Tuber Viroid (PSTVd) are viruses which cause cultivars degeneratation and yield reduction of potato,and spread extensively.Previous researches indicated that the spreading of PLRV by aphis redounded to the propagation of PSTVd. Therefor,it is especially important for potato cultivars to resist PLRV and PSTVd together.It is an effective and economic method to cultivate virus-resistant potato cultivars by plant genetic engineering.Nowadays,RNA interference structure of ssRNA virus gene could confer transgenic plants with higher virus resistance.One of the most effective method to resist viroid is to cleave virus nucleic acid with ribozyme.In this research,with two pairs of specific primers based on PLRV genomic conservative sequences lying on the both sides of Intergenic Sequence(IS) reported in GenBank,two cDNA fragments(369bp) containing the IS(IS-ClaI-BstpI and IS-Kpn-BglII,with different restriction enzyme sites) were amplified by reverse transcription polymerase chain reaction(RT-PCR) using the total RNA of PLRV Inner Mongolia isolate as a template.The fragments were cloned into pBS-T vector and checked by PCR, restriction enzyme degestion and nucleotide sequencing.The cloned IS sequence was compared with the homologous squences of thirteen PLRV isolates in GenBank.The results showed that it had high homology with those isolates.The highest homology reached 100%of nucleic acid squence with the average of 97.90%.The cloned IS was also conservative in the sequences of PLRV complete genome.There was a "UUAUAUU" squence highly conservative in the IS for all the analysed isolates.It indicated that intergenic sequence was important for plant virus to reproduce.Meanwhile,di-component hammerhead fibozyme targeting two sites on negative strand of PSTVd was designed,synthesized and cloned according to its action manner.The results of PCR,restriction enzyme digestion and nucleotide sequence analysis indicated the ribozyme was accurately synthesized.Marker-free bivalent expression vector p3301-DR-Isir with inverted repeat sequence of PLRV IS and ribozyme specificly cleaving PSTVd RNA(-) was constructed through five steps recombination of intermediate transformation vector pSKN-DR,pSKN-35S-DR,p3301-DR and pKAN-IS. In the course of vector construction,every recombinant plasmid was detected by PCR and restriction enzyme analysis.The results of nucleotide sequencing of expression vector p3301-DR-Isir proved that it was successfully constructed.Marker-free bivalent expression vectors p3301-DR-Isir was transferred into Agrobacteriurn LBA4404 through freezing and melting method.Then potato cultivars of Northeast White and Jin No.7 were transformed with p3301-DR-Isir mediated by Agrobacterium turnefaciens.In this test the effects of explants,the Agrobacteriurn density,the durations of pre-culture and co-culture on transformation frequency were investigated and the optimum transformation condition was established:the explants were pre-cultured for 2 days,OD600 of Agrobacterium was 0.3-0.5,and the durations of infection and co-culture were 10 minutes and 4 days, respectively.Both stem segments and leaf discs could be used as explants.In this research marker-free bivalent expression vector p3301-DR-Isir with RNAi structure of PLRV IS and di-component ribozyme specificly cleaving PSTVd RNA(-)was constructed successfully,which could be used as gene source to transform potato cultivars for high resistances to PLRV and PSTVd.