Cloning Of Fruit Ripening Related Genes From Pear (Pyrus Pyrifolia Nakai) And Their Construction Of Rnai Vectors

Sand pear(Pyrus pyrifolia Nakai) originated in central,southern and western of China, and it is suitable for cultivation in the warm and wet area with some advantages such as heat-resisting,combats drought,the fire blight-resistant and so on.But its fruit storage ability is poor,which brings a problem in pear industrialization:how to improve the storage ability of pear fruit.The present research is mainly concentrated on elongating the fruit shelf life according to the physiological characteristic of the sand pear fruit by improving the storage conditions for fruit and using some ethylene inhibitors,but these methods can not solve the problem ultimately.In this paper,the different kinds of culture medium,the proportion and kinds of hormones in regeneration of 'Wakahikari' pear leaf in vivo were studied.The ripeness related genes were cloned from fruit and their RNAi expression vectors were constructed.The expression characteristics of interference genes were studied through Agrobacterium mediated gene transformation of pear,tomato and tobacco,which make it possible to obtain a new pear germ plasm with good storage ability in the future.1,In order to obtain high regeneration frequency and provide conditions for gene transformation of pear(Pyrus pyrifolia 'Wakahikari'),the effects of different hormonal combinations of varieties and concentrations on regeneration of pear leaves in vitro were studied.The results showed that 'Wakahikari' leaves had 63.2%regeneration frequency in NN₆₉ medium with TDZ 1.0mg/L,IBA 0.15mg/L,AgNO₃ 0.1mg/L,cultured for 30 days in light after 21 days in dark.2,A pair of specific primer were designed and synthesized according to the AC03 sequence (GenBank with accession number AB042107) from pear(Pyrus pyrifolia Nakai).The upstream regulatory sequence was amplified from genomic DNA of pear(Pyrus pyrifolia 'Wakahikari') leaves by Genomewalker,the DNA fragment was subcloned into pMD19-T vector.A 715bp fragment was obtained and sequenced.A core promoter element frame of TATA-Box at 89bp upstream of initial site was detected by landing the PLACE database and predicting cis-acting element of plant DNA online.In addition,substructure analysis showed that there were some specific elements related to regulating gene expression,thus it is likely an inducible promoter of ACC oxidase gene in pear fruit.3,The total RNA was isolated from mature fruit of pear(Pyrus Pyrifolia 'Wakahikari').A pair of specific primer was designed by software of Primer premier 5 according to the mRNA conserved region sequence of ACC oxidase gene.A 401 bp fragment was obtained by RT-PCR.The cDNA sequence was 99%homologous to ACC oxidase gene(AC03) of Pyrus pyrifolia by sequencing.It showed that it was ACC oxidase gene of sand pear.Two pairs of specific primers with different sites of restricted enzyme were designed according to the sequence.And antisense and sense ACC oxidase gene were amplified,then ligated with the YYT gene,which was related to carotenoid synthesis in Subcocci as interval sequence.The fusion gene was digested by two restricted enzymes and cloned into plant expression vector to construct the recombinant plasmid pYF028,which could express dsRNA ofACC oxidase gene.4,To study the expression characteristics of ACO interference gene in plant,ACO interference gene was transfered into tomato by Agrobacterium mediated system.Kmr buds were screened from 520 explants cultured on selective medium with MS+ZT 1mg/L+ IAA 0.5mg/L+Km 50mg/L+Cef 250mg/L+sucrose 30g/L+agar 7g/L,and gained 34 Kmr plants after two subcultures.Six plants with roots were obtained on MS+IAA 0.5mg/L+Km 50mg/L+Cef 250mg/L+sucrose 30g/L+agar 7g/L for 30d.By GUS detection,PCR and RT-PCR analysis,it indicated that the ACO interference gene was transferred and integrated into the genome of tomato.The result showed that the release quantity of ethylene in transgentic tomato was significantly higher than the untransgentic control after detecting the release of ethylene.5,The total RNA was isolated from mature fruit of sand pear(Pyrus pyrifolia 'Wakahikari').A specific fragment with predicted size was amplified from cDNA of Pyrus pyrifolia by RT-PCR with degenerate oligonuelectide primers designed from the conserved domain of other plant lipoxygenases.The cDNA fragment was 827bp.Amino acid sequence revealed that the cDNA fragment had conserved domain of lipoxygenase and was 77.5%homologus with potato through blasting and clustering analysis.It is likely that the cDNA fragment was lipoxygenase gene of Pyrus pyrifolia named as LOX1.The cDNA sequence of this clone was deposited in GenBank with accession number EF215448.The specific primers with restricted enemzy site were designed from an open read frame(ORF) of the cDNA sequences according to the principles of construction of its corresponding RNAi vector.Two fragments were amplified by PCR,one is postive orientation,the other is reverse orientation,ligated with YYT interval region.The fusion fragment was introduced into plasmid of pYF7713,it was successful that the recombinant plasmid pYL028 was constructed,which could express dsRNA of pear LOX gene.6,To study the expression characteristic of interference gene in plant,we transfered LOX1 interference gene into tobacco through Agrobacterium mediated system.Km^r buds from 113 explants cultured on selective medium with MS+6-BA 2mg/L+NAA 0.1mg/L+Km 50mg/L+Cb 250mg/L+sucrose 30g/L+agar 7g/L with pH 5.8 wre screened out,and 16 Km^r plants were obtained after two subcultures.Five plants with roots were obtained on I/2MS+IBA 0.5mg/L+Km 50mg/L+Cef 250mg/L+sucrose 30g/L+agar 7g/L with pH 5.8 for 30d.By GUS detection and PCR and RT-PCR analysis,it showed that the LOX1 interference gene was transferred and integrated into the genome of tobacco.It was found that 37.8%of LOX activity was inhibited in transgenic plant compared with control under low temperature stress,but the activities of SOD,CAT and POD were enhanced and the content of MDA was significantly lower than that of the control.7,A specific fragment of the predidicted size was amplified from eDNA of mature fruit in Pyrus pyrifolia by RT-PCR with degenerate oligonuclectide primers designed according to the conserved domain of other plant lipoxygenases.Two 827bp fragments coding 275 amino acids were obtained and sequenced.The amino acid sequences revealed that the cDNA fragments had lipoxygenase conserved domain by BLAST online.We deduced that they were another two fresh members from lipoxygenase gene family in Pyrus pyrifolia Nakai.We named them LOX2 and LOX3.The cDNA sequences were deposited in GenBank with accession number EF215449 and EF215450.The results showed that LOX2 gene expressing in pear(Pyrus pyrifolia Nakai) fruit was related to seed development in fruit,the LOX1 and LOX3 expression in fruit was induced by stress.