Transgenic Technology Induction The Aurone Biosynthetic In Malus ’Snow Drift’

Flowering crabapples belong to the Rosaceae Malus, with high ornamental value. Theyare very popular and be attention because of their rich variation on leaves, flowers, fruit andtree. However, the colors of their petals only have white and different shades of red. It isdifficult to solve this problem by using traditional cross-breeding. AmAS1gene andAm4’CGT gene are key genes involved in the synthesis of aurone, which were cloned fromSnapdragon. This study plans to transfer the two genes to Malus ‘Snow drift’（a variety offlowering crabapple that has white petals） through Agrobacterium-mediated, in order toinduce the synthesis of aurone in M.‘Snow drift’, and obtain a new variety that has yellowpetals. So far, we have completed some work, like establishment and optimization of tissueculture propagation and regeneration system, cloned AmAS1gene and Am4’CGT gene,construction of expression vector, these achieves are necessary for transgenic. The mainfindings are as follows:（1） Obtained aseptic tissue culture plantlets from year-old stem segments, and then,studied the influence of different plant hormones to shoot proliferation and adventitious shootregeneration of Malus ‘Snow drift’ by two-factor completely randomized method, alsostudied the influence of different IBA concentration to rooting. The results show that there aretwo ways for shoot proliferation: adventitious buds from callus induction and adventitiousbuds from tissue-cultured axillary buds; the optimum medium for shoot proliferation is MS+6-BA2.5mg·L^-1+IBA0.3mg·L^-1, the proliferation value is9.11; in the medium of MS+TDZ0.2mg·L^-1+NAA0.1mg·L^-1, the regeneration efficiency was the highest, reached60.71%, the number of regenerated buds per leaf was5.03; in the medium of MS+TDZ1.0mg·L^-1+NAA0.5mg·L^-1, the number of regenerated buds per leaf was the highest，reached5.78，the regeneration was57.72%; the rooting efficiency could up to100%after added0.2mg·L^-1IBA in1/2MS medium.（2） Total RNA was extracted from the flower buds of snapdragon, and then, obtainedFirst-strand cDNA by reverse transcription PCR, the cDNA was used as template to obtainthe CDS sequence of the AmAS1gene and Am4’CGT gene by PCR amplification. The twogenes were connected to the pMD19-T Vector, constructed the cloning vectors AS1-T and4’CGT-T successfully. （3） Obtained GUS gene including the promoter and terminator from pBI121by doubledigested. Connected this fragments to the plant expression vector pCambia1302, constructedrecombinant vector GUS-pCambia1302successfully. Double digested the two reporter genesGUS and GFP sequentially, and replaced to AmAS1gene and Am4’CGT gene, constructedplant bivalent expression vector CGT-AS-pCambia1302successfully.（4） Explored transient expression of the two target genes in vivo petals of M.‘Snowdrift’ by using agrobacterium infiltration method.The results show that the target genetranscription have been completed,but the color of the petals was unchanged, there were paleyellow showed after preservated the petals in ultra-low-temperature. This phenomenon cannotexplaining until further analysis.