Construction Of Screening System For Cd Stress-tolerance Related Genes In Sedum Alfredii Hance

Adverse environmental conditions, such as drought, low temperature, high soil salinityand heavy metals are among the most challenging factors for plant growth and survival.Adaptation to abiotic stress requires coordinate changes in metabolism, cell growth, divisionand differentiation, which depend on a large set of genes controlling complex regulatorymechanisms. Some genes such as stress relation genes and transcript factors whose expressionis upregulated by heavy metals, salt, cold or drought stress had been cloned by differentialRNA display. However, genetic approaches are better suited for identification of regulatorygenes than differential display techniques. In fact, the majority of genes responses to heavymetals, high salinity, drought and cold were cloned by forward genetic.A cadmium stress induced full-length cDNA Library of Sedum alfredii was constructedaccording to improved SMART cDNA library construction protocol. Total RNA were extractedfrom Sedum alfredii under cadmium stress, after synthesizing the first-strand cDNA usinglimited amount of total RNA, the double-strand cDNA was amplified with LD-PCR.full-length cDNA library was digested with Sfi I and then linked to pBI121GB, the product wastransferred into Agrobacterium tumefaciens EHA105, Clones were selected to sequencerandomly and bioinformatics analysis, the results showed that inserted fragments in the librarywere about1000bp. Then its were transferred into wide type Arabidopsis mediated by floraldip and more than1000positive transformed plantlets were obtained. Ferritin cDNA wascloned from line Sa8by PCR and expession analysis revealed that the target transcript wassignificantly increased. The results showed that S. alfredii genes had been cloned intoArabidopsis genome. Thus the gain-of-function mutagenesis system based on S. alfrediifull-length cDNA insertion was feasible.the present paper provides a novel method for identifying genes respond to Cd stress in S.alfredii, included cloning S. alfredii cDNA library into a vector suitable for overexpression inthe Arabidopsis, then transformated cDNA library into wild type Arabidopsis mediated by A. tumefaciens; screening tranformatants with antibiotic resistance and cloned Cd stress relatedcDNA from gain of functional mutants, then the function of the isolated cDNA was confirmedby physiological respond to Cd stress and genetics test. Therefore, FOX gene hunting systemwas viewed as an advanced and high throughput system with great potential in functionalgenomics research.