Cloning And Expression Vector Construction Of Seed Coat Special Promoter From Gossypium Arboretum L

Cotton, an important crop, plays a decisive role in the field of agriculture, industry and nationaldefense construction. Improving the fiber quality is one of main breeding objectives in cotton breeding.Genetic engineering is a more effective method than the traditional protocol, which has the defection oflong period and slow effect. Promoter, the determiner of the positon of transcriptional start point and thefrequency with which the gene is transcribed, has the theory significance and the practical significancein the mechanism of differentiation and development and improvement of cotton fiber as an importantmolecular tools. The CaMV35S promoter, a typical representative of the constitutive promoter, waswidely used in the improvement of the genetic engineering of crops on account of its sustained, stableand efficient expression characteristics. However, further study and practical application has revealed itsdeficiencies and potential security issues. With the development of genetic engineering these years,promoters being tightly regulated were desperately needed. Seed-coat specific promoters are being usedto drive gene expression in the epidermal cells efficiently and specifically. It′s expected to reveal themechanism of cotton fiber differentiation and development in the molecular level and provide usefultools for fiber improvement by further research and utilization of ovule-promoters. The Arabidopsisseed coat microarray data, gene expression profiles data and cotton genomics data were exploited toobtain the outer-seed-coat-specific promoter from Gossypium arboreum L by homology cloning. Themain results are as follows:1. Arabidopsis seed coat microarray data was analysed for genes expressed in the wild type seedcoat,7genes showed a seed-coat specific expression pattern, with the code name: AT1G17260,AT1G56100, AT1G61720, AT2G30810, AT3G49270, AT5G35550and AT5G47350.2.3seed-coat specific candidate genes, GaPPT1, GaPPT2and GaPPT3were gained by amino acidsequences alignment of protein-encoding genes between Arabidopsis seed-coat specific candidate genesand Asian cotton genome-wide data.3.6expression vectors were successfully constructed with the GUS seclected as a reported gene,including the pOCA28-GaPPT1, pOCA28-GaPPT2, and pOCA28-GaPPT3recombinant vector withthe cotton seed-coat specific promoter; The pOCA28-AT5G47350recombinant vector with theseed-coat specific promoter from Arabidopsis; The positive control CaMV35S promoter recombinantvector and the negative control recombinant vector pOCA28-GUS.4. The method to recover DNA fragments from agarose gel electrophoresis may not be very applica-ble for the construction process of expression vector. It′s feasible to get Restricted DNA product afterethanol disposition and combined with proper vector directly.