The Establishing Of CDNA-AFLP System And The Cloning Of LFY Gene In Castanea Henryi Inflorescence

Chinquapin(Castanea henryi Rehd&Wils.) belongs to Fagaceae Castanea,withhigh nutritional value, which is the unique products and natural green food in northernFujian. It often leads to empty seededness because of poor flowering pollination andmalnutrition. The situation of the growth and development in chinquapininflorescence is an important prerequisite for flowering. If male inflorescence is toomuch, it makes chinquapin nutrition loss and shatter seriously, eventually leads tolow-yielding. In this study, chinquap inflorescences were taken as the material toestablish the technique system of cDNA-AFLP to find different gene relatedinflorescence differentiation, and clone chinquapin LFY gene. The research provide abasic theory in order to solve the differentiation and regulation of chinquapininflorescence, and reduce juvenile period of chinquapin. The main results were asfollows:1Established a suitable method of total RNA extraction in chinquapin inflorescenceThe study used two types of plant RNA Rapid Extraction kit, modified Trizolmethod and improved CTAB method to extract the chinquapin inflorescence totalRNA. Comparison of experimental results showed that: The improved CTAB methodwas most appropriate, which had high yield of obtained total RNA yield and relativelypure, complete. The total RNA could be used for subsequent reverse transcriptionreaction.2Established technique system of cDNA-AFLP in chinquapin inflorescenceThis study used EcoRI and MseI to double digest, selected eight EcoRI primersand eight MseI primers and were combined into64pairs of primers for selectiveamplification. It determined the final for the chinquapin inflorescence cDNA-AFLPsystem, of20μl.In this system,the template needed30ng, primer needed1μl each,10mM dNTPmix needed0.2μl,10×Ex-taq buffer required2μl and Ex-Taq(5U/μl)needed0.1μl. The research were collected20difference bands and recovered after thesecond amplification. Connecting with vector and sequencing. It obtained10sequences which were high homology through NCBI blastn or blastp.3The homology cloning of LFY gene in chinquapin inflorescenceIn this study, it was designed some up and down primers according to tho ORF of chestnut LFY gene sequences. And it was amplified and got the LFY gene fragmentof chinquapin inflorescence, the fragment lenth was1111bp. According to thisseguence to design3’RACE primers, then it got the LFY gene3’end of chinquapininflorescence. It shows99%homology with chestnut through NCBI blastn and blastp.Finaly, they were spliced together, and obtained1389bp sequence. The sequence hadbeen logged in GenBank.Accession No. JQ825167...