中国农业科学 ›› 2015, Vol. 48 ›› Issue (22): 4417-4427.doi: 10.3864/j.issn.0578-1752.2015.22.003

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝光敏色素B基因的克隆及在拟南芥中异源转基因的功能验证

孙广华1,2,原换换1,2,樊晓聪1,2,顾海科3,宋梅芳2,3,肖阳4,孟凡华2,郭林2,杨青华1,詹克慧1,杨建平1,2   

  1. 1河南农业大学农学院,郑州 450002
    2中国农业科学院作物科学研究所,北京 100081
    3 北京市辐射中心,北京100875
    4 中国农业科学院研究生院,北京100081
  • 收稿日期:2015-06-22 出版日期:2015-11-16 发布日期:2015-11-16
  • 通讯作者: 詹克慧,Tel:15537182526;E-mail:kh486@163.com。杨建平,Tel/Fax:010-82105859;E-mail:yangjianping02@caas.cn
  • 作者简介:孙广华,Tel:010-82105851;E-mail:guanghuanuli@163.com。原换换,Tel:010-82105851;E-mail:yuanhuanhuan111@126.com。孙广华和原换换为同等贡献作者。
  • 基金资助:
    国家自然科学基金(31170267)、国家转基因生物新品种培育重大专项(2014ZX08010-002)、北京市自然科学基金(重点)(6151002)、中国农业科学院科技创新工程项目

Molecular Cloning and Arabidopsis Ectopic Expression of a Phytochrome B gene from Brassica oleracea

SUN Guang-hua1,2, YUAN Huan-huan1,2, FAN Xiao-cong1,2, GU Hai-ke3, SONG Mei-fang2,3, XIAO Yang4, MENG Fan-hua2, GUO Lin2, YANG Qing-hua1, ZHAN Ke-hui1, YANGJian-ping1,2   

  1. 1College of Agronomy, Henan Agricultural University, Zhengzhou 450002
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081
    3 Beijing Radiation Center, Beijing 100875
    4Graduate School, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2015-06-22 Online:2015-11-16 Published:2015-11-16

摘要: 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(BoPHYB),在拟南芥中验证异源基因BoPHYB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BoPHYB编码区cDNA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pJIM19-Myc-BoPHYB,通过农杆菌介导法转化拟南芥野生型Col-0和phyB-9突变体,并获得纯合转基因株系;比较BoPHYB与拟南芥光敏色素B(AtPHYB-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BoPHYB的编码区cDNA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 kD的蛋白质;与拟南芥和白菜的phyB结构域类似,BophyB包含1个GAF结构域、2个PAS结构域、1个HisKA结构域和1个HATPase_c结构域;氨基酸序列同源性分析发现,BophyB与拟南芥以及白菜的phyB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AtPHYB-GFP类似,在红光和白光下不但Myc-BoPHYB能互补phyB-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。

关键词: 甘蓝, 拟南芥, 光敏色素B, 去黄化反应, 避荫性

Abstract: 【Objective】Cabbage (Brassica oleracea L.) is one of the most widely grown vegetable crops in the world. Cabbage is grown in spring and fall, however varieties need to be made suitable for cultivation all the year round. Study on the response of cabbage phytochromes to light and temperature is an important basis for crop improvement with respect to extending growth periods. Phytochromes of interest here are the red/far-red reversible photoreceptors. Among them, phyB is an essential red light receptor that predominantly mediates seedling photomorphogenesis and shade-avoidance response. The objective of this study was to isolate the phytochrome B gene in cabbage (i.e., BoPHYB), to explore its functions on seedling de-etiolation and shade avoidance by ectopic expression in Arabidopsis, and evaluate its potential in cabbage breeding. 【Method】The cDNA sequence of BoPHYB was isolated from leaves of a self incompatible line 12C. Bioinformatic analyses were employed to predict its function domains and to build a phylognetic relationship tree among plant phyB homologs. The pJIM19-Myc-BoPHYB binary construct was electroporated into the Agrobacterium tumefaciens strain GV3101 and then introduced into Arabidopsis thaliana (both of the wild type and phyB-9 mutant) via a floral dip method. Transgenic plants were selected on germination plates containing 50 mg/mL kanamycin for Myc-BoPHYB. Homozygous T3 or T4 transgenic plants were used for testing the seedling hypocotyl elongation and shade avoidance responses. 【Result】The reading frame of BoPHYB possesses 3 507 bp and encodes 1 168 amino acid residues (128.9 kD) deduced from the DNA sequence. Similar to both phyB protein of Arabidopsis thaliana (i.e., AtphyB) and Brassica rapa (i.e., BrphyB), BophyB contains one GAF domain, two PAS domains, one HisKA domain, and one HATPase_c domain. Phylogenetic analysis indicated that BophyB belongs to the same branch as BrphyB and AtphyB, while showing low similarity to phyB proteins from monocotyledonous species, such as common wheat, corn and rice. Compared with the wild type Col-0, the transgenic lines overexpressing BophyB in A. thaliana exhibited much shorter hypocotyl elongation under red and white-light conditions. Their adult plants displayed dwarf stature with dark green leaves and short petioles under long-day and short-day conditions.【Conclusion】In this study, we cloned BoPHYB gene, and evaluated it with AtPHYB in crop improvement. Taking on similar structures and functions with AtphyB, BophyB promotes seedling de-etiolation under red and white-light conditions and inhibits shade avoidance responses in mature plants under long-day and short-day conditions.

Key words: Brassica oleracea L., Arabidopsis thaliana, phytochrome B, de-etiolation, shade avoidance