GDS-80手持基因枪在橡胶树遗传转化上应用的参数优化与配件改进  

Parameter Optimization and Accessory Improvement of GDS-80 Handheld Gene Gun for Rubber Tree Genetic Transformation

作　　者：范璟璋 华玉伟[2] 范月婷 辛士超[2] 戴雪梅[2] 黄天带[2] 黄华孙[2] 李季[2] FAN Jingzhang;HUA Yuwei;FAN Yueting;XIN Shichao;DAI Xuemei;HUANG Tiandai;HUANG Huasun;LI Ji(College of Tropical Crops,Hainan University,Haikou,Hainan 570228,China;Rubber Research Institute,Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Biology and Genetic Resources of Rubber Tree,Ministry of Agriculture&Rural Affairs,Haikou,Hainan 571101,China;Xixian Senior Middle School Affiliated to Central China Normal University,Xixian,Henan 464300,China)

机构地区：[1]海南大学热带作物学院,海南海口570228 [2]农业农村部橡胶树生物学与遗传资源利用重点实验室/中国热带农业科学院橡胶研究所,海南海口571101 [3]华中师范大学附属息县高级中学,河南息县464300

出　　处：《热带作物学报》2021年第9期2458-2467,共10页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金项目(No.31770717,31872708);中国热带农业科学院基本科研业务费专项资金(No.1630022019013,No.1630022018001)。

摘　　要：GDS-80手持基因枪是美国Wealtec公司2009年推出的基因传递系统,具有操作简便、高效的特点。已报道的橡胶树基因枪转化研究都是使用台式基因枪。本文首先利用考马斯亮蓝轰击滤纸去除明显不适合的参数,接着以绿色荧光蛋白(GFP)基因作为报告基因,轰击巴西橡胶树体细胞胚以及愈伤组织,用荧光显微镜观察绿色荧光,探究适合橡胶树的轰击参数。而且比较了Image J软件和肉眼统计荧光斑点数及GDS-80手持基因枪与PDS-1000/He台式基因枪的差异。结果表明:对于橡胶树体细胞胚和愈伤组织,仅依靠原厂配件难以获得较好的转化效果。本文设计了直径3 cm(与轰击范围相同),高度2.5 cm,孔径60目的过滤网。同时改进装试验材料的培养皿,用刀片在培养皿的中心割出一个直径3 cm的圆圈,轰击胚状体时,将上述设计的过滤筛网正向卡在圆圈上,胚状体放入过滤网中,轰击时将培养皿用试管架架高,压力就从筛网及底部的空洞分解,微弹完全轰击到试验材料。轰击胚状体最优参数为:轰击压力为60 psi,针状调节阀为3圈,目标间隔盘为6 cm。轰击愈伤时将材料放到普通培养皿中心的轰击范围内,反向盖上过滤筛网。最优轰击参数为:轰击压力为50 psi,针状调节阀为4圈,目标间隔盘为6 cm。本文采用Image J软件进行荧光斑点计数,准确率与肉眼相当,但较肉眼省时。GDS-80手持基因枪与PDS-1000/He台式基因枪转化效率相当,但GDS-80手持基因枪每打一枪比PDS-1000/He台式基因枪快12 min。研究结果为橡胶树遗传转化提供了高效的基因枪转化体系,为转基因研究提供了一个高效的统计荧光数的软件。GDS-80 handheld gene gun is a gene delivery system launched by Wealtec Company in 2009,which is easy to operate and efficient.All the published researches on the transformation of Hevea brasiliensis by gene gun are based on desktop gene gun.In this paper,Coomassie Brilliant Blue was firstly used to bombard filter paper to screen out obviously not suitable parameters,and then the green fluorescent protein(GFP)gene was used as the reporter gene to bombard the somatic embryos and callus of H.brasiliensis,finally the green fluorescence was observed by fluorescence microscope to explore the suitable bombardment parameters for H.brasiliensis.The difference in fluorescent spot numbers calculated by Image J software and naked-eye and the difference between GDS-80 handheld gene gun and PDS-1000/He desktop gene gun was compared.The results showed that it was difficult to obtain good transformation efficiency by using original accessories for somatic embryos and callus of H.brasiliensis.In this paper,a filter screen with a diameter of 3cm(same as the bombardment range),the height of 2.5 cm and pore diameter of 60 mesh was designed.At the same time,a petri dish containing the experimental materials was improved.A circle with a diameter of 3cm was cut out in the center of the petri dish by a blade.The filter screen designed above was clamped on the circle with forwarding direction,and the embryos were put into the filter screen.When bombarding,the petri dish was put on the test tube rack,and the pressure would decompose from the mesh and the cavity at the bottom,and the micro bullet would completely bombard the embryos.The optimal parameters for embryos were as follows:bombardment pressure 60 psi,needle-shaped regulating valve 3 circles,and target spacing plate 6 cm.When the callus was bombarded,the material was placed in the center of a traditional petri dish and covered by the designed filter screen reversely.The optimal bombardment parameters for callus were as follows:bombardment pressure 50 psi,needle-shaped regulating v

关 键 词：巴西橡胶树 GDS-80 基因枪 GFP基因 Image J 

分 类 号：S794.1[农业科学—林木遗传育种]