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【目的】甘蓝(Brassica oleracea L.)是世界上最广泛种植的蔬菜作物之一,其种植已由春、秋两季种植转变为四季栽培,因此,研究甘蓝对光温的反应能力是品种改良的重要基础。光敏色素是植物最重要的一类光受体,主要响应远红光和红光的变化,其中,光敏色素B是红光的最主要受体,与光形态建成、避荫性以及生物产量等性状密切相关。克隆甘蓝的光敏色素B基因(Bo PHYB),在拟南芥中验证异源基因Bo PHYB在光形态建成和避荫性反应中的作用,丰富植物光敏色素基因功能研究,评价光敏色素在甘蓝品种改良中应用的潜在价值。【方法】利用RT-PCR的方法克隆、测序验证得到甘蓝自交不亲和系12C的Bo PHYB编码区c DNA序列;利用生物信息学软件分析其编码的氨基酸序列及结构域,并与其他植物的光敏色素B进行序列比对,分析它们之间的同源关系;构建其35S启动子驱动的植物表达载体p JIM19-Myc-Bo PHYB,通过农杆菌介导法转化拟南芥野生型Col-0和phy B-9突变体,并获得纯合转基因株系;比较Bo PHYB与拟南芥光敏色素B(At PHYB-GFP)的转基因株系在不同光质下的下胚轴伸长和成株期的表型,验证其在光形态建成和避荫性反应中的作用。【结果】从甘蓝中克隆了Bo PHYB的编码区c DNA序列,该基因编码区含有3 507个核苷酸,编码具有1 168个氨基酸残基、分子量为128.9 k D的蛋白质;与拟南芥和白菜的phy B结构域类似,Bophy B包含1个GAF结构域、2个PAS结构域、1个His KA结构域和1个HATPase_c结构域;氨基酸序列同源性分析发现,Bophy B与拟南芥以及白菜的phy B同源性最高,与小麦、玉米、水稻等单子叶植物的同源性较低;与At PHYB-GFP类似,在红光和白光下不但Myc-Bo PHYB能互补phy B-9极端黄化的表型,而且转基因株系均表现为下胚轴加剧缩短;长日照和短日照下成株期转基因导致植株矮化、叶片深绿和叶柄缩短。【结论】克隆了甘蓝光敏色素B基因,甘蓝光敏色素B与拟南芥和白菜的光敏色素B具有相似的结构和功能,在红光和白光下促进幼苗的去黄化反应,在成株期显著抑制长日照和短日照下植株的避荫性反应。
【Objective】 Cabbage (Brassica oleracea L.) is one of the most widely grown vegetable crops in the world. Its cultivation has been changed from planting in spring and autumn to planting in four seasons. Therefore, The important foundation. Phytochrome is one of the most important photoreceptors of plants, and mainly responds to far red and red light changes. Among them, Phytochrome B is the most important receptor of red light, which is related to light morphogenesis, shade avoidance and biological yield The traits are closely related. In the Arabidopsis thaliana, Bo PHYB was used to verify the role of the heterologous gene Bo PHYB in light morphogenesis and shade-avoidance response. The phytoalexin gene function was enriched in Brassica napus. The potential value of applying improvements. 【Method】 The cDNA sequence of Bo PHYB coding region of 12C of selfing incompatible lines of cabbage was cloned and sequenced by RT-PCR. The amino acid sequence and its coding region of Bo PHYB were analyzed by bioinformatics software and compared with other plants Of the phytohemagglutinin B sequence alignment analysis of their homology; construct its 35S promoter-driven plant expression vector pJIM19-Myc-Bo PHYB by Agrobacterium-mediated transformation of Arabidopsis wild type Col- 0 and phy B-9 mutants were obtained, and homozygous transgenic lines were obtained. Hypocotyl elongation at different light qualities of transgenic lines of Bo PHYB and Arabidopsis thaliana B (At PHYB-GFP) Strain phenotype, to verify its role in the formation of light and shade avoidance response. 【Result】 The c DNA sequence of Bo PHYB coding region was cloned from cabbage. The coding region contains 3 507 nucleotides and encodes a protein with 1 168 amino acid residues and a molecular weight of 128.9 kD. Similar to the phy B domain of cabbage, Bophy B contains one GAF domain, two PAS domains, one His KA domain and one HATPase_c domain. The amino acid sequence homology analysis showed that Bophy B and qunnan Mustard and Chinese cabbage have the highest phy B homology and low homology with monocotyledonous plants such as wheat, corn and rice. Similar to At PHYB-GFP, not only Myc-Bo PHYB can complement phy B in red and white light -9 extreme yellow phenotype, and the transgenic lines showed hypocotyls intensified shortening; long-day and short-day transgenic plantlets led to plant dwarfing, dark green leaves and petiole shortening. 【Conclusion】 The B gene of Brassica napus was cloned. Brassinolone B has similar structure and function to the phytochrome B of Arabidopsis thaliana and Chinese cabbage. It promotes the de-etiolation of seedlings in red and white, Significant inhibition of long-day and short-day under the shade of plants response.