Cloning Of CP Gene Of Strawberry Vein Banding Virus And Development Of Genetic Transformation System Of Strawberry Cultivar

Strawberry(Fragaria ananassa Duch.)is a kind of perennial herb and susceptible to virus disease for the vegetative propagation. Strawberry vein banding virus (SVBV) is one of four kinds of strawberry latent virus. Many negative effects such as strain degeneration, yield and quality reductions may be caused in commercial strawberry cultivars infected with SVBV. No effective chemical agent to control strawbery virus disease, plant genetic engineering technology including high effective plant regeneration, plant genetic transformation, has been used in the studies of improvement of virus disease resistance in strawberry. Strawberry leaves SVBV-infected and seeding leaves in vitro of strawberry cultivars 'Toyonoka','Sachinoka'and 'Anna'were used as materials for strudying the detection of SVBV,cloning CP gene,high-frequency regeneration, genetic transformation systetem etc. The main results were as follows: 1. Total DNA, extracted from SVBV-infected strawberries leaves using CTAB method, was used as template to amplify by PCR. PCR amplification conditions and programs were optimized. About 600 bp fragment of SVBV was obtained based on optimum system. On the other hand, the total DNA was diluted to density gradient and amplified by PCR. The results showed that it can detect the virus in 2.5μg leaf tissue. PCR method is a reliable method of detecting SVBV. The PCR specific fragment was recycled, cloned into pMD18-T vector and sequenced. Compared with SVBV isolate that reported (GenBank accession number: Nc₀01725), it showed that the sequence of coat protein gene of this isolate is 89.2% and 96.3% identity at the sequence levels of nucleotide and deduced amino acid. GenBank accession number of the specific fragment sequence is AY862389. 2. The present primary strawberry cultivars 'Toyonoka','Sachinoka'and 'Anna'were used as experimental materials. To establish a reliable, rapid and effective in vitro regeneration system and to carry out the strawberry genetic transformation successfully, the factors affecting adventitious shoots regeneration were studied. The results indicated that Genotype was the main factor influencing regeneration system of strawberry in vitro, but the frequencies and amounts of shoot organogenesis were significantly affected by the hormone concentrations for a given genotype. Compared with 6-BA and TDZ, TDZ had a better effect on inducing shoot regeneration. The regeneration frequency of cultivar 'Sachinoka'reached the highest level of 86% in MS+TDZ8.0mg/L+IAA0.1mg/L and 42% in MS+6-BA1.0mg/L+IAA1.5 mg/L medium, while cultivar 'Toyonoka'obtained the highest level of 82% in MS+TDZ2.0mg/L+IAA0.1mg/L medium. and cultivar 'Anna'obtained the highest level of 7% in MS+TDZ2.0mg/L+IAA0.1mg/L medium. The corresponding numbers of shoots regeneration were 2.4,1.8,2.1 and 1.0 per leaf disc, respectively. To cultivar 'Toyonoka'and 'Anna', 6-BA can't induce shoot regeneration. While using the rooting medium of 1/2MS+IBA0.1mg/L,the rooting rate achieved nearly 100%. 3. Studying effects of different antibiotic on the regeneration of strawberry leaves in vitro and using transient expression of GUS gene, the optimal conditions for Agrobacterium-mediated transformation of shrawberry leaf discs was investigated. On medium containing 40 mg/L kanamycin, the shoot regeneration capacity of cultivar 'Sachinoka'was inhibited. The growth of Agrobactium was inhibited completely with 200 mg/L cefotaxine supplemented in the medium. As a result, 20 mg/L of kanamycin and 200 mg/L cefotaxine of were established as the concentration effective for selection of shoots and inibition of Agrobactium growth. Histochemical GUS assay showed that the transient expression rate was affected by pre-culture time, infected time, co-culture time and the concentration of adding acetosyringone. Pre-culture 0-2 d, infection 3 to 5 min,co-culture 72h and adding 25-100μmol/L acetosyringone contributed to the hoghest level of transient expression. The strawberry transgenic plants were developed with kanamycin resistance selection. Five transgenic lines were detected by GUS and PCR assays, and three of them show the positive band same as NPTâ…¡gene　and　CaMV35S.