核-质基因共调控的IGF-1在番茄果实质体中的表达特性研究

The potential to produce high levels of protein by plastid expression has recently increased interest in using plastid as biological factories. However, constitutive expression and overexpression of foreign proteins often lead to many constrains such as growth inhibition and toxicity to cells. In order to overcome this unfavorable pleiotropic effects, here we speculate a novel pattern of co-regulatory expression of nucleus-plastid genes which the foreign gene may specificly express in fruit plastid by the use of fruit-specific promoter and a transacivating fusion factor SIG1-32, targeting to plastid harboring the core RNA polymerase under the control of bacterial promoter groE. .Our prior experiments have successfully completed, including construction of nucleus expression vector with fruit-specific promoter E8 and SIG1-32 fusion facort and tomato plastid expression vector with groE::gfp cassette, and confirmation the co-regulatory function of E8 and SIG1-32 using GFP gene transient expression in tomato fruits. Furthermore, we are planning to construct the vectors which contain different fruit-specific promoters, PG and AGPL1, and transform them into tomato via agrobacterium-mediated method. After obtaining the mature fruits of regenerated transgenic plants, the suitable promoter will be screened out by analysis of GFP expression level using GFP gene transient expression system. Importantly, the IGF-1 plastid expression vector will be constructed and integrated into plastid genome using biolistic delivery system, based on the obtained nucleus transformed tomato plants with suitable promoter. By committing evaluation of IGF-1 expression level and the activity of IGF-1 protein, we may select the overexpression of IGF-1 gene plants and clarify the expression characteristics of IGF-1 gene in fruit plastid under the co-regulated effects of nucleus and plastid genes..This investigation would be of great potential significance in the production of foreign proteins in plant plastid and may help advance the field of protein production through effective transgene biotechnology. It may not only be able to offer a novel and controllable mode for transgene specific expression of foreign gene in plastid, but also create the material resources that would be used as oral vaccine to cure sugar diabetes and bioreactor to produce IGF-1 protein.

植物叶绿体中高量表达外源蛋白后，会导致植株生长缓慢或对细胞产生毒害，解决这一难题是目前质体表达研究的热点。本项目利用SIG1-32融合因子能与质体RNA聚合酶结合和识别原核启动子groE的特性，分别构建E8、PG和AGPL1果实特异性启动子表达载体，农杆菌法转化番茄，获得成熟果实后利用质体瞬时表达体系，对启动子进行筛选，探讨启动子对核-质基因表达元件调控机制；在此基础上，构建groE::IGF-1番茄质体表达载体，采用基因枪法对核转基因植株进行二次质体转化，将IGF-1导入番茄质体基因组，分析IGF-1在果实中的表达水平和蛋白活性、解析IGF-1高效表达对番茄植株生长发育的影响，最终阐明核-质基因共调控下IGF-1在果实质体中的表达特性，建立可控的质体转基因表达新模式。项目为利用质体特异表达外源蛋白提供技术和理论基础；也可为利用番茄口服治疗糖尿病和作为生物反应器生产IGF-1奠定材料基础。

摘要：植物叶绿体中高量表达外源蛋白后，会导致植株生长缓慢或对细胞产生毒害，解决这一难题是目前质体表达研究的热点。本项目首先利用番茄叶片作为外植体，采用正交优化设计，创新了番茄叶片高频再生体系；通过设计引物，分别从番茄中克隆了E8、2A11、PG、AGPL1启动子，从甜瓜中克隆了AOC、TP启动子，总共6个果实特异性启动子，并采用生物信息学软件对6个启动子进行了序列分析和比较；在此基础上，构建了5个包含不同启动子的番茄核表达载体，最后，采用正交设计法，对影响转化效率的浸染液等因素进行了优化，确定了最适宜的转化参数。经过农杆菌介导法，最终获得抗性植株41株，对获得的抗性植株进行PCR分子检测，共21株阳性植株，阳性率达51.21%。相关后续的蛋白表达工作正在进一步的开展和完善之中。项目的成果为下一步开展核质互作深入的研究奠定了坚实的材料基础。项目资助共发表论文7篇。申请专利6项。开展国内外交流共9人次，大会特邀报告１次。培养硕士生4名，其中2名已经取得硕士学位。新品种鉴定4个。项目投入经费50万元，支出41.08万元，各项支出基本与预算相符。剩余经费8.92万元，剩余经费计划用于本项目研究后续支出。