Clone And The Differential Expression Of Genes Related With Aborted Bud In Radish And Chinese Cabbage

Aborted bud of Chinese cabbage and radish is a disadvantageous phenomenon in breeding practise. It has important academic signification and practical importance to clarify the mechanism on molecular biology. In this paper, the mRNA differential display technology was adopted to research into the gene expression difference between aborted bud and normal bud of radish, and in silico cloning technology was adopted to clone full length of gene related; cDNA-AFLP technology was adopted to research into gene expression difference in different aborted phase of Chinese cabbage. The main research results were as follows:1. The effect of acidic phenol-guanidine thiocyanate-chloroform method and BIOZOL reagent on RNA extraction was compared with tender leaf and bud of Radish, and the main factors such as concentration of Taq enzyme, Mg²⁺, dNTPs, primer and cDNA in DDRT-PCR procedure were optimized, which obtain a proper, RNA extraction method and an optimal DDRT-PCR system in radish.2. The RNA of aborted buds and normal buds in radish male-sterile line were extreacted and analyzed by DDRT-PCR technology with 78 primer combinations combined 3 Anchor primers T₁₄A,T₁₄G,T₁₄C with 26 Random primers from B0301 to B0326. which indicated that: (1) 107 ESTs of differential expression gene were got in aborted buds, in which 13 ESTs are down regulating express and 94 ESTs are up regulating express in radish aborted bud; (2)By reclaiming, cloning and sequencing 89 valid ESTs were got and compared in NCBI with BLASTx order, it shows that there were 16 ESTs with BLASTx score greater than 80, 7 ESTs were related with energy, 5 ESTs were related with metabolism, 1 ESTs were related with human protein, and 3 ESTs are not known function; 15 ESTs which have not homology sequence in GenBank were got by BLASTn analysis, which are new discovered ESTs in radish, and have been loaded to GenBank, they are accessed number as follows: EST36-1:accession number FG124881 dbEST_Id:56786563; EST28-3:accession number FG124882 dbEST_Id:56786564; EST39-1:accession number FG124883 dbEST_Id:56786565; EST59-1:accession number FG124884 dbEST_Id:56786566; EST61-1:accession number FG124885 dbEST_Id:56786567; EST45-2:accession number FG124886 dbEST_Id:56786568; EST23-1:accession number FG124887 dbEST_Id:56786569; EST71-1:accession number FG124888 dbEST_Id:56786570; EST64-1:accession number FG124889 dbEST_Id:56786571; EST100-1:accession number FG124890 dbEST_Id:56786572; EST56-1:accession number FG124891 dbEST_Id:56786573; EST87-1:accession number FG124892 dbEST_Id:56786574; EST38-2:accession number FG124893 dbEST_Id:56786575; EST17-1:accession number FG124894 dbEST_Id:56786576; EST97-1:accession number FG124895 dbEST_Id:56786577;3. It was found that chloroplast, chloroplast maturation enzyme K (Mat K) and methylase have close relation with bud aborting in radish. By BLASTx analysis, we found that 7 related proteins were all from chloroplast and related with energy metabolism; 4 related proteins are related with metabolism, 3 of them are chloroplast maturation enzyme K and 1 is methylase therefore chloroplast have may some relations with radish aborted bud; especially the Mat K was amplified thrice in different primer conbination.4. BLASTn in GeneBank shows that 28 ESTs in aborted bud have homology with known function ESTs, 11 ESTs of them have relation with plant adversity intimidate, 7 ESTs of them have relation with senescence. It also found that genomic DNA in aborted bud represent regular DNA Ladder, while DNA in normal bud represent only single DNA strap. These show that radish aborted bud may be a cell programmed death as mechanism in PCD of plant resistance adversity intimidate and senescence.5. A part of RNA helicose gene and Photosystem I assembly protein Ycf4 gene were got. Two cDNAs which length are 941bp and 976bp respectively were obtained by RT-PCR depended on the sequence of EST14-2 and EST35-1 by in silico cloning, and they code 307 amino acids and 141 amino acids respectively. The amino acid sequence of cDNA from EST14-2 has 96% homology with RNA helicose gene of Arabidopsis thaliana, The amino acid sequence of cDNA from EST35-1 has 89%,96%,97% and 95% homology with Photosystem I assembly protein Ycf4 gene in Gossypium hirsutum, Draba nemorosa, Lobularia maritime and Arabidopsis thaliana.6. cDNA-AFLP was applied to analyzing gene express of aborted bud in Chinese cabbage with 256 primer combinations combined A4-A19 with T4-T19 and 192 differant display cDNAs which were between 100bp and 1 000bp were got.they were divided into four types according to expression of"aborted bud→normal bud":"strong→weak"type involve 56 ESTs (approximately accounting for 29.2%),"weak→strong"type involve 33 ESTs (approximately accounting for 17.2%),"present→absent"type involve 52 ESTs (approximately accounting for 27.1%),"absent→present"type involve 51 ESTs (approximately accounting for 26.5%), 88 cDNAs clearly differtial segments were reclaimed, cloned, sequenced and analyzed homology. 72 ESTs were gained, in which BLASTx score of 62 ESTs were larger than 80, they were divided into seven types:(1) 30 ESTs were related with energy and metabolism, approximately accounting for 48.4%,(2) 9 ESTs were related with membrane and transporting, approximately accounting for 14.5%,(3) 15 ESTs were related with transcription and translation,approximately accounting for 24.2%,(4) 3 ESTs were related with amino acid synthesis and processing,approximately accounting for 4.8%,(5) 1 EST were related with signal transduction,approximately accounting for 1.65%,(6) 1 EST were related with defense,approximately accounting for 1.65%,(7) 3 ESTs were related with unclear classified,approximately accounting for 4.8%。7. The EST54-1 was prolonged by In silico cloning and the RT-PCR was used to confirm the authenticity. One new gene of Chinese cabbage were obtained, which is malate dehydrogenase gene.