Isolation Of Differentially Expressed Gene Fragments Related To Opposite Character In Maize Using CDNA-AFLP Technique With Single Restriction Endonuclease

Maize with opposite character is both a good germ plasm resource for high yieldbreeding, and a precious variation for plant development research. In order to study themolecular and regulation mechanism deeply, in this study, based on constructing suitableNILs (near isogenetic line) of total RNA pools of opposite maize and mutual maize, singleendonuclease digestion cDNA-AFLP technology was applied to screen , isolation andbioinformatics analyze the gene differentially expressed fragments related to the oppositecharacter of maize. The results were as follow. 1. The quality and integrality of total RNA extracted with Trizol method were good, and were suitable to construct the total RNA pools of opposite maize and mutual maize equivalently. 2. The length of Double-strand cDNA retransfered by SMART^TM PCR cDNA Synthesis Kit from each pool was 100～1000bp. Its integrality was fine. 3. The length of digested fragment by a endonuclease —Sau3AⅠ,which can recognize a 4bp sequece,was100～1000bp. It could accord with the AFLP technology. 4. The ligation product was pre-amplified to get numbers of copies, after the endonuclease digestion fragment was linked to the artificial adapter. 5. Diluted pre-amplified product was systematically selective-amplified .A improved PCR method—TOUCH DOWN PCR program was adopted to get peculiar and high density copies. 6. Combined with urea denatured 6% PAGE and silver stain, 29 differentially expressed fragments had been obtained: 23 fragments enhanced expressed, 6 fragments restrained expressed in opposite maize. 7. 5 enhanced expressed fragments of opposite maize were cloned and sequenced, including S_TT-TT,S_TT-TC,S_TT-CC,S_TT-GC,S_CT-TG. The homology analysis showed that these 5 fragments had very high homologies with the maize mRNA that hade been separated . S_TT-GC had 80% homology with cytochrome b5 gene of N.tabacum . SCT-TG had over 90% homology with α-tublin gene of Z.mays , Eleusine indica, Hordeum vulgare, Oryza sativa and Setaria viridis. This study first applied the single endonuclease digestion cDNA-AFLP to analyzethe differentially expressed genes in maize, and made a series of improvement to thetechnology to find some differentially expressed fragments related to opposite character inmaize .Further research can be done with the obtained sequences to make probes ormolecular markers to locate ,clone the opposite gene ,and even study the gene expressioncharacters.