Studies On Strawberry Regeneration In Vitro And Construction Of PlB Gene-Targetting SiRNA Expression Vector

Strawberry is one of the most important commercial fruit crops in the world. Because of the genetic limitations associated with high heterozygosity and polyploidy,it is laborious,costly,and time-consuming to improve strawberry quality and yield by the conventional methods of breeding,which usually results in a low frequency.Recent advances and applications in genetic engineering technology provide a new path for strawberry breeding and selection by way of incorporating foreign genes into plant genome for desired agronomic traits.However,an efficient tissue culture system is thought to be crucial to the success of plant genetic engineering,since the efficiency of Agrobacterium-mediated transformation is considered to be dependent on two primary factors,one being the regeneration ability of the infected tissue,and the other,the infection efficiency of Agrobacterium.In this research,we have studied the effects of a series of cultural factors(genotypes, basic media,hormone concentrations,dark incubation and AgNO₃ concentrations) on the inducing adventitious shoot of strawberry,respectively.Therefore,we have obtained ideal efficient regeneration system through above of studies.Pectate lyase gene exhibite a close correlation with softening of ripe strawberry fruit,the usage of siRNA,an intermediate in the silencing pathway,to target the pectate lyase gene would be a valuable tool to study its function.Oligonucleotides expressed siRNA were designed and synthesized according to the target protein gene of plB and subcloned to the binary vector pBI121 after annealling.The major results can be summarized as below:(1) The strawberry regeneration system has been established.'Hongjia' showed higher adventitous shoot regeneration compared with 'Toyonoka'. Through the analysis of regeneration rate,shoots per leaf and the quality of adventitious shoots,the optimal initiation medium for shoot regeneration of each cultivar was respectively as follows:For 'Hongjia',shoot regenerated best on MS supplemented with 3.0 mg/L 6-BA and 0.1 mg/L 2,4-D with regeneration rate of 56.4%.For'Toyonoka',48.2%regeneration rate was gained on the MS medium containing 2.0mg/L TDZ plus 0.1 mg/L NAA.CTK played an important role in the shoot regeneration from the leaves of strawberry.Compared to 6-BA,TDZ was more effective in inducing shoot regeneration from leaves of strawberry cv.Toyonoka.To compare the effects of three basic media,MS,MS+B₅ and B₅,on the shoot regeneration from the leaves of strawberry,MS with high salt and N concentrations was more benefitial to the shoot regeneration than the others for both of genotype,and adventitious shoots regenerated from MS exhibited higher quality.For 'Toyonoka',Two weeks of dark treatment increased the shoot regeneration frequency from 48.2%to 52.2%, however,the shoot regeneration rate decreased with the extension of dark periods.The effects of dark treatment on adventitious shoot regeneration of strawberry cv.'Honjia' was contrary to 'Toyonoka'.On the best combination of plant growth regulators for shoot regeneration,the addition of AgNO₃ varied from 2.0 mg/L to 8.0 mg/L into the medium could not help to stimulated shoot regeneration,and with the higher concentration of AgNO₃,more obvious inhibit results were observed.(2) A siRNA expression vector has been constructed.For screen of BLAST,19 bases oligonucleotides which is GCCTACAGGAAATGCCATG(767-785) were ascertain from useing the known genic sequence of p/B's mRNA(AF339024) of GenBank.After the hairpin DNA of siRNA annealing,the results of agar-gel electrophoresis analysis showed that there was an obvious stripe whose molecular weight was similar to the expected.Plant binary vector pBI121 was digested by Sac I and BamH I,the results were identified by agar-gel electrophoresis where two strip can be seen.The annealling products was recombined with pBI121 vector,which was transformed into DH5a.After screen,the positive clone(pBI121-PL) was gotten.Recombinant pBI121-PL vector was further identified by digestion with Hindâ…¢and BamHI,Hindâ…¢and SacI,respectively.The result demonstrated that 70 bp had been inserted the expected site.