大豆叶片特异表达基因GmRCAβ和GmCRB1启动子的分离及其在耐除草剂转基因大豆研发中的应用  

Isolation of the promoters of leaf speciffc expression genes GmRCAβand GmCRB1 and their application in the development of herbicidetolerant transgenic soybean

作　　者：胡晓莺 朱佳美 寿惠霞 HU Xiaoying;ZHU Jiamei;SHOU Huixia(Institute of Modern Seed Industry,College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,China;Seed Engineering and Industrialization Group,Hainan Institute of Zhejiang University,Sanya,Hainan 572000,China;College of Life Sciences,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江大学农业与生物技术学院现代化种业研究所,杭州310058 [2]浙江大学海南研究院种子工程与产业化团队,海南三亚572000 [3]浙江大学生命科学学院,杭州310058

出　　处：《植物生理学报》2025年第2期179-190,共12页Plant Physiology Journal

基　　金：转基因生物新品种培育科技重大专项(2020ZX08013-01B)。

摘　　要：转基因大豆的目标蛋白在食用种子中存在较高的表达,这一现象使得部分公众对其食用安全性产生顾虑。为了实现目的基因在叶片的定点表达,本研究利用大豆数据库中不同组织的转录组数据,筛选了大豆叶片特异表达的基因。本研究从中选取了叶片特异表达的大豆二磷酸核酮糖羧化酶编码基因GmRCAβ和大豆叶绿体RNA结合蛋白编码基因Gm CRB1。为了进一步验证其表达模式,本研究将上述基因的启动子连接β-葡萄糖苷酶基因(GUS)创制了稳定的遗传转化材料。GUS染色结果表明,2个载体培育的转基因大豆叶片均具有较高的GUS活性,而种子中GUS染色不可见,该结果进一步证实了这两个启动子的叶片表达特异性。用这两个基因的启动子驱动草甘膦耐性基因g10-epsps的表达,创制转基因大豆材料。酶联免疫吸附反应表明,转化体G10-EPSPS蛋白在种子中的积累量显著低于以Ca MV35S启动子创制的耐草甘膦转基因大豆ZUTS-33。大田除草剂耐受性测试表明,田间喷施0、200和400 m L·667 m^(–2)草甘膦时,转基因大豆与非转基因对照相比具有显著的耐草甘膦能力,而转化体RCA-1、RCA-2及CRB-1种子中G10-EPSPS的蛋白含量低于检测线,较ZUTS-33转基因大豆显著下降。综上,本研究成功创制了叶片特异表达的耐除草剂转基因大豆材料,为进一步提升耐除草剂转基因大豆的安全性和种质资源提供了重要保障。The presence of target protein of genetically modiffed soybean(Glycine max)in the edible seeds has caused food safety concerns in public.In order to achieve site-speciffc expression of the target gene in leaves,the transcriptome data from soybean database were used to screen the genes that are speciffcally expressed in leaves.Two leaf-speciffc expression genes,the ribulose carboxylase gene GmRCAβand the soybean chloroplast RNA-binding protein coding gene GmCRB1 were selected.To further verify the expression pattern,the promoters of GmRCAβand GmCRB1 were linked to theβ-glucosidase gene(GUS)to generate stable genetic transformation events.GUS staining of the corresponding transgenic soybean showed that the blue staining of GUS expression was high in leave,but not visible in seeds.The promoters of GmRCAβand GmCRB1 were then used to drive the glyphosate tolerance gene g10-epsps to generate transgenic soybean.Enzyme-linked immunosorbent reaction(ELISA)showed that the accumulation of G10-EPSPS protein in seeds was signiffcantly lower than that of glyphosate-tolerant transgenic soybean ZUTS-33 created with CaMV35S promoter.Field herbicide tolerance test showed that when 0,200 and 400 mL·667 m^(–2) Roundup®was sprayed with in the ffeld,the transgenic soybean had signiffcant tolerance compared with the non-transgenic control.Meanwhile the protein contents of G10-EPSPS in the seeds of transgenic events of RCA-1,RCA-2 and CRB-1 were signiffcantly reduced compared to ZUTS-33 transgenic soybean.In conclusion,this study successfully generated herbicide-tolerant transgenic soybeans with the target-protein speciffcally highly expressed in leaves but almost none in seeds,which provides an important approach for further improving the safety and germplasm resources of herbicide-tolerant transgenic soybean.

关 键 词：大豆 叶片特异型启动子 耐草甘膦 转基因 

分 类 号：R73[医药卫生—肿瘤]