Cloning And HpRNAi Expression Vector Constrction Of PMADS9 Gene,PMADS1 Gene And CHSJ Gene From Petunia Hybrida

RNA interference(RNAi) is the process of post—transcriptional gene silencing by which double-stranded RNA(dsRNA) directs sequence—specific degradation of homologous mRNA.Gene silencing trigered by RNAi have advantages of being high efficienct peculiar,simple, direct and so on.It has been applied in some aspects and will be used widely in the future。It reviews the mechanisms and the application of RNAi.Post transcriptional_silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals.In 2001,S.Varsha Wesley had demonstrated the potential for constructs encoding self-complementary 'hairpin'RNA(hpRNA) which can improve the efficiency of silence genes.Using hpRNA constructs containing sense/anti-sense arms can give more efficient silencing than both anti-sense and co-suppression.And the intron which these constructs contains had a consistently enhancing effect.Thus these features make hpRNAi more efficiently than anti-sense and co-suppression on plant gene silencing.MADS-box genes have been isolated from numerous eukaryotic organisms and encode transcription factors involved in growth and development.The MADS region linked specifically to the coalescent site of target gene and formed a homogenous or heterogenous dimer to regulate it, which can make different kinds of floral organs for many flowering plants.So they are important factors to control the development of floral organs and to make homeotic transformations.It is very useful to analyze the floral organ identity genes in order to understand the molecular model of plant floral organ development.AGL15(AGAMOUS-like 15) is a mermber of MADS-box family.It was found that overexpression of AGL15 affects floral organ senescence.The effects of AGL15 could be distinguished from the effects of the ethylene to senescence.PMADS9,a mermber of AGL15 subfamily,was isolated from petunia and the function was not conformed.This experiment obtained PMADS9,PMADS1 and CHSJ gene from Petunia(Petunia hybrida) young floral bud cDNA by PCR amplification,and constructed their hpRNAi expression vector respectively.We also introduced PMADS9 into petunia(Petunia hybrida) via Agrobacteriurn tumefaciens mediated method.The main results are as follows:1.Cloning of PMADS9 fragments and construction of the hpRNAi plant expression vectorsThe PMADS9 gene fragment was amplified by meas of PCR using petunia(Petunia hybrida) young floral bud cDNA as template and a pair of specific oligonucleotides at 5'- and 3'-end as primer.Comparison with previously published sequence showed 100%homologies in nucleotide sequence.The hpRNAi plant expression vectors pDH-1.2.Cloning of PMADS1 fragments and construction of the hpRNAi plant expression vectorsThe PMADS1 gene fragment was amplified by meas of PCR using petunia(Petunia hybrida) young floral bud cDNA as template and a pair of specific oligonucleotides at 5'- and 3'-end as primer.Comparison with previously published sequence showed 100%homologies in nucleotide sequence.The hpRNAi plant expression vectors pDH-2.3.Cloning of CHSJ fragments and construction of the hpRNAi plant expression vectorsThe CHSJ gene fragment was amplified by meas of PCR using petunia(Petunia hybrida )young floral bud cDNA as template and a pair of specific oligonucleotides at 5'- and 3'-end as primer. Comparison with previously published sequence showed 100%homologies in nucleotide sequence. The hpRNAi plant expression vectors pDH-3.