Genotype-independent Agrobacterium rhizogenes-mediated root transformation of chickpea: a rapid and efficient method for reverse genetics studies.
Genotype-independent Agrobacterium rhizogenes-mediated root transformation of chickpea: a rapid and efficient method for reverse genetics studies.
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DOI：
10.1186/s13007-018-0315-6
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发表时间：
2018
期刊：
影响因子：
5.1
通讯作者：
Chakraborty S
中科院分区：
文献类型：
--
作者：
Aggarwal PR;Nag P;Choudhary P;Chakraborty N;Chakraborty S
鹰嘴豆（鹰嘴豆）、重要的豆类作物是膳食蛋白质的主要来源之一。开发一种高效、可重复的转基因方法是加快农作物功能基因组学研究的当务之急。在这里，我们提出了一个优化的和详细的程序为发根农杆菌介导的鹰嘴豆根转化。在筛选培养基上，A.发根菌接种绿色荧光蛋白的表达进一步证实了转化的成功。我们证明，我们的方法充分转化鹰嘴豆根在早期发展阶段，具有高效率。此外，根转化被认为是基因型无关的，我们的协议的功效是最高的两个（Annigiri和JG-62）的七个测试鹰嘴豆基因型。接下来，我们通过表达参与原花青素生物合成的拟南芥透明种皮2（AtTT 2）基因，对鹰嘴豆毛状根的功能进行了分析。AtTT 2的过表达提高了毛状根中原花青素的水平，导致真菌病原菌尖孢镰刀菌的定殖减少。此外，转基因根的诱导不影响涉及真菌病原体感染根的功能研究。表达感兴趣的基因的转基因根将有助于使用反向遗传学研究进行下游功能表征。进行本研究中描述的根转化方案需要1天，并且表达转基因的根可以维持3-4周，为进一步的功能研究提供足够的时间。总体而言，目前的方法将大大有助于功能基因组学分析的候选基因在根际互作在这一抗旱，但经济上重要的豆科作物。本文的在线版本（10.1186/s13007-018-0315-6）包含补充材料，可供授权用户使用。
Chickpea (Cicer arietinum L.), an important legume crop is one of the major source of dietary protein. Developing an efficient and reproducible transformation method is imperative to expedite functional genomics studies in this crop. Here, we present an optimized and detailed procedure for Agrobacterium rhizogenes-mediated root transformation of chickpea. Transformation positive roots were obtained on selection medium after two weeks of A. rhizogenes inoculation. Expression of green fluorescent protein further confirmed the success of transformation. We demonstrate that our method adequately transforms chickpea roots at early developmental stage with high efficiency. In addition, root transformation was found to be genotype-independent and the efficacy of our protocol was highest in two (Annigiri and JG-62) of the seven tested chickpea genotypes. Next, we present the functional analysis of chickpea hairy roots by expressing Arabidopsis TRANSPARENT TESTA 2 (AtTT2) gene involved in proanthocyanidins biosynthesis. Overexpression of AtTT2 enhanced the level of proanthocyanidins in hairy roots that led to the decreased colonization of fungal pathogen, Fusarium oxysporum. Furthermore, the induction of transgenic roots does not affect functional studies involving infection of roots by fungal pathogen. Transgenic roots expressing genes of interest will be useful in downstream functional characterization using reverse genetics studies. It requires 1 day to perform the root transformation protocol described in this study and the roots expressing transgene can be maintained for 3–4 weeks, providing sufficient time for further functional studies. Overall, the current methodology will greatly facilitate the functional genomics analyses of candidate genes in root-rhizosphere interaction in this recalcitrant but economically important legume crop. The online version of this article (10.1186/s13007-018-0315-6) contains supplementary material, which is available to authorized users.
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影响因子：
14.8
作者：
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通讯作者：
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影响因子：
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影响因子：
14.8
作者：
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