Isolation and expression analysis of partial sequences of heavy metal transporters from Brassica juncea by coupling high throughput cloning with a molecular fingerprinting technique
Isolation and expression analysis of partial sequences of heavy metal transporters from Brassica juncea by coupling high throughput cloning with a molecular fingerprinting technique
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DOI：
10.1007/s00425-011-1376-1
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发表时间：
2011-07-01
期刊：
影响因子：
4.3
通讯作者：
Seal, Anindita
中科院分区：
文献类型：
--
作者：
Das, Soumita;Sen, Monali;Seal, Anindita
重金属转运蛋白在植物体内重金属的积累和转运过程中起着重要的调控作用。这些基因是重要的候选基因，在金属耐性和积累植物中研究，以它们在环境净化中的潜在用途。我们耦合简并引物为基础的RT-PCR方法与分子指纹技术的基础上扩增的rDNA限制性分析（ARPINCE），以确定新的EST对应的重金属转运蛋白从金属积累芥菜。我们利用该技术以高通量方式克隆天然抗性相关巨噬细胞蛋白（NRAMP）和黄条样蛋白（YSL）的几个家族成员，以区分来自异源多倍体B的密切相关的同种型和/或等位基因变体。Juncea。获得了23个芥菜NRAMP和27个YSL的部分克隆，它们与已知的拟南芥和蓝芥NRAMP和YSL基因具有相似性。克隆的转运蛋白在结构域中表现出拟南芥特异性的变化，这可能具有重要的功能后果。半定量RT-PCR为基础的表达分析所选的成员表明，即使是密切相关的异构体/等位基因变体的BjNRAMP和BjYSL有不同的组织特异性和金属依赖性的表达，这可能是必不可少的适应性健身和重金属耐受性。与此一致，发现BjYSL 6.1和BjYSL 5.8在B的镉处理的芽和铅处理的根中特异性地显示出升高的表达。juncea分别。
Heavy metal transporters play a key role in regulating metal accumulation and transport in plants. These are important candidate genes to study in metal tolerant and accumulator plants for their potential use in environmental clean up. We coupled a degenerate primer-based RT-PCR approach with a molecular fingerprinting technique based on amplified rDNA restriction analysis (ARDRA) to identify novel ESTs corresponding to heavy metal transporters from metal accumulator Brassica juncea. We utilized this technique to clone several family members of natural resistance-associated macrophage proteins (NRAMP) and yellow stripe-like proteins (YSL) in a high throughput manner to distinguish between closely related isoforms and/or allelic variants from the allopolyploid B. juncea. Partial clones of 23 Brassica juncea NRAMPs and 27 YSLs were obtained with similarity to known Arabidopsis thaliana and Noccaea (Thlaspi) caerulescens NRAMP and YSL genes. The cloned transporters showed Brassica-specific changes in domains, which can have important functional consequences. Semi-quantitative RT-PCR-based expression analysis of chosen members indicated that even closely related isoforms/allelic variants of BjNRAMP and BjYSL have distinct tissue-specific and metal-dependent expressions which might be essential for adaptive fitness and heavy metal tolerance. Consistent to this, BjYSL6.1 and BjYSL5.8 were found to show elevated expressions specifically in cadmium-treated shoots and lead-treated roots of B. juncea, respectively.