The Construction Of Expression Vector Of Proanthocyanidins Synthesized Key Gene Of ANR And Genetic Transformation Of Cryptanthus Bivittatus

Bromeliea is a perennial herbaceous monocots, it is the third largest tropical ornamental flowers only to orchids and anthurium. Cryptanthus bivittatus almost have no upright stems in the above ground. Dense clusters of leaves from the condensed stem, horizontal stretch a "rosette". Leaves are hard, wavy edge and have soft spines. The leaves are strip-shaped, there are two vertical stripe purple and dark red on base leaf and the back of leaf surface covered with white powder.In this research, we used C. bivittatus sterile leaf base as explants and established C. bivittatus regeneration and genetic transformation systems. First, the proanthocyanidins Synthesized key genes of ANR full length cDNA was cloned from anthurium spathe of ’Alabama’and inserted into a plant expression vector and constructed CaMV35S-GUS-ANR. Then, the ANR was transgened into C. bivittatus mediated with Agrobacterium EHA105. The transgenic plants were tested by PCR, RT-PCR and GUS staining methods and investigated by horticultural characters. The main conclusions of this research are as follows:(1) Established regeneration system of C. bivittatus:the culture medium of inducing buds was:MS+6-BA1.0mg/L+NAA0.2mg/L+30g/L sucrose+7.0mg/L agar powder, pH5.8; the culture medium for buds elongation was:MS+6-BA0.5mg/L+NAA0.01mg/L+30g/L sucrose+7.0mg/L agar powder, pH5.8; the rooting medium was:MS+NAA0.5mg/L+30g/L sucrose+7.0mg/L agar powder; pH5.8.(2) Established of the genetic transformation system of C. bivittatus:Selection:the median lethal concentration and lethal concentration of G418were50mg/L and60mg/L respectively. Selection medium was added with carbenicillin500mg/L. The best time of Agrobacterium infection C. bivittatus was20min, the transient expression was highest when OD600of the Agrobacterium suspension was0.8.(3) RNA was extracted from anthurium spathe in’Alabama’and reverse transcribed into single-stranded cDNA.The full-length cDNA of ANR was amplified by PCR technology.(4) Construction of ANR gene plant expression vectors:The ANR cDNA sequence was inserted into the multiple cloning site region of CaMV35S-GUS and constructed the plant expression vector of CaMV35S-GUS-ANR.(5) Molecular detection of transgenic plants:DNA and RNA were extracted from C. bivittatus. PCR and RT-PCR results showed that the positive transgenic plants conversion rate was67%, and gene stabal expressed after conversion.(6) Transgenic plants histochemical staining:the transgenic plants were detected by GUS staining, the results showed that transgenic plants leave appear blue plaques, intransgenic plants leave not appear blue plaques, this result proved that GUS reporter gene in plant expression vector has been integrated into the recipient cell genomic DNA.(7) Investigation of the transgenic plants indicated that the transgenic C. bivittatus leaves were changed, transformation from the wild-type purple into pure green, leaves shape have changed slightly.