A transient transformation system for gene characterization in upland cotton (Gossypium hirsutum).
A transient transformation system for gene characterization in upland cotton (Gossypium hirsutum).
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用于陆地棉（Gossypium hirsutum）基因表征的瞬时转化系统
DOI：
10.1186/s13007-018-0319-2
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发表时间：
2018
期刊：
影响因子：
5.1
通讯作者：
Miao Y
中科院分区：
文献类型：
--
作者：
Li H;Li K;Guo Y;Guo J;Miao K;Botella JR;Song CP;Miao Y
转基因棉花占世界棉花种植面积（2230万公顷）的64%。对棉花二倍体祖先种雷蒙棉和亚洲棉以及栽培陆地棉的基因组测序为作物改良提供了丰富的遗传信息。遗憾的是，由于现有的稳定转化方法效率低、耗时长和技术复杂，棉花基因功能鉴定落后于其他重要经济作物。本文提出了一种简单、快速、有效的G.可用于基因表征研究的多毛植物。我们建立了一个高地棉瞬时转化系统，用于基因鉴定。使用β-葡萄糖醛酸酶作为农杆菌介导的转化试验的报告基因，我们评估了多种转化参数，如农杆菌菌株、细菌密度、共培养时间、化学品和表面活性剂，这些都可能影响转化效率。在初始表征之后，选择农杆菌EHA 105菌株，并使用许多二元构建体进行基因表征研究。将7天龄的棉花幼苗与农杆菌共培养，并在植物感染后5天观察到瞬时基因表达。通过实时逆转录PCR（qRT-PCR）定量花椰菜花叶病毒（CaMV）35 S启动子控制下的两种不同转基因的转录物水平，其显示比在未感染的对照中观察到的水平增加3-10倍。研究了两个G. hirsutum基因以及其相应的蛋白质的亚细胞定位进行了研究，使用新的瞬时表达系统和我们的观察是一致的，与以前发表的结果使用拟南芥作为异源系统。农杆菌介导的瞬时转化方法是一种快速、简便的瞬时表达系统，能够在高地棉幼苗中实现高瞬时表达和转化效率。我们的方法可用于基因功能的研究，如启动子特性和蛋白质在棉花的亚细胞定位，避免了需要进行这样的研究，在异源系统，如拟南芥。本文的在线版本（10.1186/s13007-018-0319-2）包含补充材料，可供授权用户使用。
Genetically modified cotton accounts for 64% of the world’s cotton growing area (22.3 million hectares). The genome sequencing of the diploid cotton progenitors Gossypium raimondii and Gossypium arboreum as well as the cultivated Gossypium hirsutum has provided a wealth of genetic information that could be exploited for crop improvement. Unfortunately, gene functional characterization in cotton is lagging behind other economically important crops due to the low efficiency, lengthiness and technical complexity of the available stable transformation methods. We present here a simple, fast and efficient method for the transient transformation of G. hirsutum that can be used for gene characterization studies. We developed a transient transformation system for gene characterization in upland cotton. Using β-glucuronidase as a reporter for Agrobacterium-mediated transformation assays, we evaluated multiple transformation parameters such as Agrobacterium strain, bacterial density, length of co-cultivation, chemicals and surfactants, which can affect transformation efficiency. After the initial characterization, the Agrobacterium EHA105 strain was selected and a number of binary constructs used to perform gene characterization studies. 7-days-old cotton seedlings were co-cultivated with Agrobacterium and transient gene expression was observed 5 days after infection of the plants. Transcript levels of two different transgenes under the control of the cauliflower mosaic virus (CaMV) 35S promoter were quantified by real-time reverse transcription PCR (qRT-PCR) showing a 3–10 times increase over the levels observed in non-infected controls. The expression patterns driven by the promoters of two G. hirsutum genes as well as the subcellular localization of their corresponding proteins were studied using the new transient expression system and our observations were consistent with previously published results using Arabidopsis as a heterologous system. The Agrobacterium-mediated transient transformation method is a fast and easy transient expression system enabling high transient expression and transformation efficiency in upland cotton seedlings. Our method can be used for gene functional studies such as promoter characterization and protein subcellular localization in cotton, obviating the need to perform such studies in a heterologous system such as Arabidopsis. The online version of this article (10.1186/s13007-018-0319-2) contains supplementary material, which is available to authorized users.
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影响因子：
4.6
作者：
Chen X;Lu X;Shu N;Wang S;Wang J;Wang D;Guo L;Ye W
通讯作者：
Ye W
DOI：
10.1387/ijdb.130199bl
发表时间：
2013
期刊：
The International journal of developmental biology
影响因子：
--
作者：
通讯作者：
--
影响因子：
3.7
作者：
Pang J;Zhu Y;Li Q;Liu J;Tian Y;Liu Y;Wu J
通讯作者：
Wu J
影响因子：
1.2
作者：
Gao, Xiquan;Britt, Robert C.;He, Ping
通讯作者：
He, Ping
影响因子：
46.9
作者：
Li, Fuguang;Fan, Guangyi;Yu, Shuxun
通讯作者：
Yu, Shuxun