Cloning Of S-rnase Gene Promoter And Construction Of Female & Male Sterility Plant Expression Vector

There is an area of about 300 million hectares of natural and about 700 million hectares poplar in China.And there also has an wide distribution of Willow, Platanaceae Sycamore, Camphor etc. throughout the whole country.These trees result in a great amount of plant-caused pollution and severely affected to people's daily life and human health such as skin allergies, nasal congestion, runny nose, cough, or even trigger asthma, allergic rhinitis and so on. The fundamental solution to reduce plant-caused pollution is lies in culture new landscaping tree cultivars by genetic engineering using molecular breeding tools to nurture transgenic male or female sterility or male &female sterility technique. Male sterility can not effectively control plant-caused pollution.But female sterility or male&female sterility which cannot produce pollen, seeds or fruits can arrival this result.Pear S-RNase genes expressed in the pistil specifically,so its promoter is also pistil-specific expression. Isolation and Identification of pistil-specific expression S-RNase gene promoter and construction of female sterility gene expression vector or male&female sterility gene expression vector provides a useful molecular tools to foster female sterility or male&female sterility trees. The main results were as follows:1.This study isolated S12-,S13-,S21-RNase gene promoter with a length of 1448bp, 854bp and 1137bp successfully from'Jinhua','Maogong'(Pyrus pyrifolia) and'Yali' (Pyrus bretschneideri) genomic DNA by TAIL-PCR. GenBank accession number is HM047239,HM047240,HM047241 respectively.The core promoter regions and some upstream regulatory elements in the three fragments were analysed using the software of PLACE and PlantCARE and found that they all have the putative TATA box and CAAT box.And the promoters were affected by a variety of conditions regulation,such as light,abscisic acid,salicylic acid etc.According to the cis-regulatory elements of the S12-RNase promoter, full length and a series of 5'-deletion promoter fragments,and ligase into the Sal I and Xba I restriction enzyme sites of pBI101.2 vector and a series of 5'-deletion promoter fragments-pBI101.2-GUS fusions were constructed successfully and introduced into Arabidopsis thaliana plants (ecotype Columbia 0) by Agrobacterium mediated floral dip transformation method. A number of 4,48,17,19,10,19 transgenic lines were obtained by kanamycin and PCR detection.And there obtained 2,13,8,10,7,12 homozygous plants which can be used for further verify the promoter function.2. Broccoli (B. oleracea, var. italica) cultivar'Ryokurei'were used to isolate both the anther-specific gene promoter and candidate genes which has potential uses as cytotoxins BoCysPl for male sterility. Using PpS12pro-pBI101.2 as basic components, we constructed female-sterility plant expression vector PpS12pro-CysPl-pBI101.2 with CysPl gene into Xba I and Xma I restriction enzyme sites of pBI101.2 T-DNA sequence.And then constructed male&female-sterility plant expression vector BoA9pro-CysP1-PpS12pro-CysP1-pBI101.2 by insert the pre-connected BoA9pro-CysP1 gene reversely into Apa I and Cla I of pBI101.2.The female-sterility plant expression vector and male&female-sterility plant expression vector can used in genetically modification of female plants of dioecism and monoecious garden green plants in order to obtain new landscaping seedless&fruit varieties and stop the phenomenon of flying catkins effectively.