Cloning And Functional Analysis Of CHS And Its Promoter In Lilium

Lily flower is one of the five world famous cut flowers, and it is widely favored by people for the characters of the big flower, varied flower color, graceful posture, and strong fragrance. But like other fresh cut flowers, lily will decay rapidly while blooming. How to prolong the longevity of lily flower has become one of the main subjects.On the basic research of senescent mechanism in Lily cut flower, building RNAi expression vectors, further transferring them to the cells of Lily, and controlling the senescence of Lily by manipulating the expression of ethylene, the propose of prolonging the longevity of lily flower can be attained.1. With the material of oriental hybrid'sorbonne', extracted and purified the total RNA , then the ds-cDNA was synthesed by reverse transcriptase. The primers was designed by homologous sequence of the CHS, by the RT-PCR method,a fragment about 900bp was cloned and sequenced.The sequencing result showed that the fragment was 873bp,homology analysis showed that the sequence was over 91% homology to the reported sequence.The result showed that we got the right CHS cDNA sequence,The sequence was submitted to the Genbank,and the accession number is DQ471950.2. By the PCR method,we got a fragment about 1300bp,sequencing and homology analysis result showed that the sequence was 1307 bp. Comparison the CHS cDNA and DNA sequence showed that the DNA have two extons interrupted by a 82bp intron. The Genbank number is DQ471951.3. By using the total DNA of the oriental hybrid'sorboone'as template, and by using the chromosome walking technology with linker, together with the feature of nested-PCR, touchdown-PCR, hot-start PCR and two stages PCR, 896bp fragment was cloned, which is the 5'-upstream regulatory sequence and was called'PCHS'.It was demonstrated by plant CARE analysis that 5'-upstream sequence from ATG had the feature of promoter: TATA-box ,G-BOX and CCAAT-box involved in the transcriptional start and the translation respectively, and other regulatory sequence related to flower-specific expression. it was speculated that the regulatory sequence would have function specifically in the flower development.