Clone And Sequence Analysis Of Plant Fruit-Specific E8 Promoter And Antisence Polygalacturonase Gene

Peach〔Prunuspersica (L.) Batsch.〕is tipical respiratory climacteric fruits and attains the maturity appears to breathe the high peak soon during the post harvest period. Most of fruit ripe in hot and moist season.That leads to softening and rotting of fruit flesh. Therefore,the effective way to solve the difficult problem is to breed varieties that can endure storage transportation. This is the main problem that restricts production of peach. Low temperature storage is the main method to prolong peach storage,but it easliy brings low temperature injury.So it is not the better way. Thought air control storage can reduce fruit respiration and ethylene synthetical rate to prolong storage and transportation time .But the cost is much and the effcet is not very good . Practice proved that it is hard to adjust the market serve time by imporve storage method. It has hope to solve the problem by tranditional breeding,but peach is perennial plant so breeding time is very long and germ resource has limitation.So it hard to get breakthrough in short time . To close the gene translation which lead fruit flesh soft by antisense gene technology may delay the time and extent of fruit flesh getting soft .It also can improve storage effect and increase the economic value of peach.Similar research alredy applied to improve germ plasm on tomato, muskmelon,cherry and so on.Polygalacturonases,a kind of cell wall hydrze enzyme,is highly expressed in the organization particularity , synthesized during fruit ripe process and plays a key role in fruit softening. Fruit-specific gene E8 is promoter of answer ETH. It control the E8 gene special expression during fruit ripe process.This study described promoter cloning of fruit-specific gene E8 and antisense gene PG. Plants were collected explants of tomato'Maofen 802'and peach flesh. It laid down a solid foundation and provided beneficial information for a prolonged shelf life using genetic engineering technology. The results were as fallow.The DNA was extracted from cotyledons of tomato'maofen802'by high salt and low PH,step by step acentric ,SDS,CTAB and improved CTAB method.The improved CTAB method is the best method in the DNA extract.We use this DNA as stencil and amplified by PCR ,the product of which was reclaimed and subcloned into pMD18-T Simple Vector.After identification by PCR and restriction enzymes,the recombinant pMD18-E8 vectors were subjected to sequence analysis.As indicated by homology analysis,the resultant tomato fruit-specific E81.1 promoter was highly conservation, with GenBank submission,proved to share 99.1% homology.Therefore,tomato fruit-specific E8 promoter has been successfully cloned.The RNA was separated from peach by Trizol, improved Trizol 1, improved Trizol 2 method.The improved Trizol 2 method is the best method in the RNA extract and one fragment about1188bp was cloned by RT-PCR.But the RNA was separated from peach leaf and young peach and peach shuck which haven't one fragment about 1188bp was cloned by RT-PCR .It is said ,the PG gene is short in these tissue. This fragment was connected with pGEM-T Vector System by TA cloning to get recombinant plasmid pGEM–PG.The fragment was PG gene by sequence analysis, with GenBank submission ,proved to share 98.57% homology.Therefore antisense gene PG has been successfully cloned.