棉纤维次生壁增厚相关基因的cDNA克隆与分析

Cloning and Analysis of cDNA Related to the Genes of Secondary Wall Thickening of Cotton (Gossypium hirsutum L.) Fiber

以陆地棉品种“徐州 14 2”植株开花后 14、18、2 4、30d的棉纤维和胚珠离体培养 12d、并分别在添加 0 (对照 )和10 0 μmol·L- 1 ABA的培养基中继代培养 5h的棉纤维为材料 ,用mRNA差异显示技术分析其基因表达差异。回收到 2 0条差异条带。利用反向Northern杂交鉴别和分析阳性差异条带 ,结果表明 :PG39 4在次生壁开始增厚以后表达 ,又被ABA诱导表达 ,为质的差异 ;PC39 1为ABA增强表达 ,PG10 2、PG2 0 3和PC2 0 4为ABA减弱表达 ,这 4个差异条带为量的差异。BLASTX分析表明 ,这 5个EST与拟南芥的蛋白激酶、普通烟草的细胞色素P4 5 0单加氧酶、乙二醛酶Ⅰ、异黄酮还原酶同系物和拟南芥的异黄酮还原酶的相似性分别为 5 1%、92 %、85 %、4 4 %和 6 5 %。进而推测这 5个EST与棉纤维次生壁增厚相关。

As experimental material cotton ( Gossypium hirsutum L. cv Xuzhou142) fibers and ovules were collected from field-grown plants on 14, 18, 24, 30 days post-anthesis (DPA), respectively, and cultured for 12 days and sub-cultured on the medium contained 0 (control) or 100 μmol·L -1 ABA for five hours. DDRT-PCR (mRNA differential display of reverse transcriptional-PCR) was used to analyze the difference of the gene expression of the fibers described as above. Twenty differential cDNA fragments were recovered. The method of reverse Northern hybridization was adopted to identify and analyze positively differential fragments. It was shown that PG39-4 expressed after the onset of secondary wall thickening, and the expression of PG39-4 was induced by ABA. It was the qualitatively differential fragment. The expression of PC39-1 was enhanced by ABA, but the expression of PG10-2,PG20-3 and PC20-4 was supressed by ABA. They were quantitatively differential fragments. Based on BLASTX analysis, the five ESTs were identified, and their similarity with protein kinase in Arabidopsis thaliana , cytochrome P450 monooxygenase in Nicotiana tabacum, glyoxalase Ⅰ, phenylconmaran benzylic ether reductase homolog and Isoflavone reductase in Arabidopsis thaliana was 51%, 92%, 85%, 44% and 65%, respectively. The five ESTs were further supposed to be related to the onset of secondary wall thickening of cotton fiber.