Construction Libraries And Isolation And Expression Analysis Of Peanut Resistance Related Genes

Penut (arachis hypogaea L.) is an important economic and oil crop for world as well as main vegetable oil and protein source for developing contries. So penut production plays great role in farmers'income and national oil security.Gynophore is the key organ for fruit formation of penut and is always paid much attention to by peanut community. During the growing storage, penut is easily infected by kinds of pathogen, especially aspergillus flavus, influencing penut production, processing and trade seriously. As obstacle of penut fruit, pericarp plays a key role in protecting kernel from invasion of germ or pests as well as stresses of environment. And the strong toughness testa also plays a great part in confronting aspergillus flavus invasion for its low permeability and breakage resistance. Other environment stress such as low temprature, drought and nutrition deficient can cause production and quality decline of penut. So cloning penut resistant genes will be usefull for investigating molecular mechnism of resistance favorable to penut resistance improvement.Construction and screening cDNA library is one of the important cloning gene methods, as to be a basic tool for finding out and study new gene at pressent. SMART technology, created by Clontech company, utilizes PowerScript TMRT reverse transcriptase and SfiⅠincision enzyme to construct full-length cDNA library quickly and simply. In this paper, four penut full-length cDNA libraries were suceessfully constructed with SMART technology, including a gynophore library, a gynophore library, a testa library and a treated library with different stresses using min-hua 6 as material. The titer of gynophore primary cDNA library was 1.3×10~6cfu, pericarp library was 1.3×10~7cfu, testa library was 1.23×10~6cfu, and press library was 8.86×10~6cfu. The recombination percentage of gynophore library was 10~0%, pericarp library was 95.5%, testa library was 93.3%, and press library was 97.6%. And the most inserted fragments were among 1.0-2.0 kb in length and the average length of inserted fragment was larger than 1.0kb.In this paper, 14 environment influenced genes were selected from minhua-6 penut 454 sequence results, and the expression of these genes under kinds of press was analyzed using semi-quantitative RT-PCR technology. The expression models of genes regulated by ABA, salicylic acid, ethephon or paclobutrazo were tested detailedly. Ads results, there were two genes'expression were induced by ABA, six by SA,two by ethphone and three by paclobutrazo. Moreover, the 14 genes'molecular functions and metabolic pathways were also annotationed by using blast2go technology.The constrction of libraries in this paper can provide platform for screening and cloning penut important genes and the analysis of the 14 genes can be helpful in studying penut resistance genes. Important genes'full sequence will be cloned next and further research of these genes will be conducted.