[Cloning of ACC oxidase gene and inhibition of endogenous gene expression with RNAi in cauliflower].
A fragment of 1202 bp of the candidate ACO gene was amplified from the cauliflower (brassica oleracea Var. botrytis) genome using the degenerated primers which were designed according to the consensus sequence of ACO amino acids among various plant species. The result of BLAST showed the sequence presented a very high match with the ACO genes from other plants; the homologue was from 83% to 99%. Three exons and two introns were identified in this sequence. The spliced length of mRNA was 756 nt and encoded 252 amino acids. The putative new gene was denominated BoACO, and submitted to GenBank (AY676466). Using the sequence, we constructed an RNA interference (RNAi) transformation vector through the way of BP cloning. The transformation into cauliflower was performed. Five regenerated plants with kanamycin resistance were obtained. And the transgene integrated into cauliflower genome was proved with PCR and Southern blotting. The expression of this ACO gene is down-regulated based on the Northern blotting in the transgenic plants. The activity of ACO enzyme was depressed significantly.
Chen
Yin-Hua
YH
National Key Laboratory of Crop Genetic Improvement, Wuhan 430070, China. lonewalls@hotmail.com
Zhang
Jun-Hong
JH
Ouyang
Bo
B
Li
Han-Xia
HX
Ye
Zhi-Biao
ZB
D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001937:Brassica / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D034622:RNA Interference; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
D018744:DNA, Plant; D010940:Plant Proteins; D000594:Amino Acid Oxidoreductases; C059460:1-aminocyclopropane-1-carboxylic acid oxidase