Koramutla Murali Krishna, Bhatt Deepa, Negi Manisha, Venkatachalam Perumal, Jain Pradeep K, Bhattacharya Ramcharan
National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute Campus New Delhi, India.
Department of Biotechnology, Periyar University Salem, India.
Front Plant Sci. 2016 Apr 18;7:457. doi: 10.3389/fpls.2016.00457. eCollection 2016.
Aphids, a hemipteran group of insects pose a serious threat to many of the major crop species including Brassica oilseeds. Transgenic strategies for developing aphid-resistant plant types necessitate phloem-bound expression of the insecticidal genes. A few known phloem-specific promoters, in spite of tissue-specific activity fail to confer high level gene-expression. Here, we identified seven orthologues of phloem-specific promoters in B. juncea (Indian mustard), and experimentally validated their strength of expression in phloem exudates. Significant cis-motifs, globally occurring in phloem-specific promoters showed variable distribution frequencies in these putative phloem-specific promoters of B. juncea. In RT-qPCR based gene-expression study promoter of Glutamine synthetase 3A (GS3A) showed multifold higher activity compared to others, across the different growth stages of B. juncea plants. A statistical method employing four softwares was devised for rapidly analysing stability of the promoter-activities across the plant developmental stages. Different statistical softwares ranked these B. juncea promoters differently in terms of their stability in promoter-activity. Nevertheless, the consensus in output empirically suggested consistency in promoter-activity of the six B. juncea phloem- specific promoters including GS3A. The study identified suitable endogenous promoters for high level and consistent gene-expression in B. juncea phloem exudate. The study also demonstrated a rapid method of assessing species-specific strength and stability in expression of the endogenous promoters.
蚜虫是半翅目昆虫，对包括油菜籽在内的许多主要作物品种构成严重威胁。开发抗蚜虫植物类型的转基因策略需要将杀虫基因在韧皮部中表达。一些已知的韧皮部特异性启动子，尽管具有组织特异性活性，但未能赋予高水平的基因表达。在这里，我们在芥菜（印度芥菜）中鉴定了七个韧皮部特异性启动子的直系同源物，并通过实验验证了它们在韧皮部渗出物中的表达强度。在韧皮部特异性启动子中普遍存在的重要顺式基序在芥菜的这些假定韧皮部特异性启动子中显示出不同的分布频率。在基于RT-qPCR的基因表达研究中，谷氨酰胺合成酶3A（GS3A）的启动子在芥菜植物的不同生长阶段显示出比其他启动子高几倍的活性。设计了一种使用四种软件的统计方法，用于快速分析启动子活性在植物发育阶段的稳定性。不同的统计软件根据这些芥菜启动子在启动子活性方面的稳定性对它们进行了不同的排名。然而，输出结果的一致性经验性地表明，包括GS3A在内的六个芥菜韧皮部特异性启动子在启动子活性方面具有一致性。该研究确定了适合在芥菜韧皮部渗出物中进行高水平和一致基因表达的内源性启动子。该研究还展示了一种评估内源性启动子物种特异性强度和表达稳定性的快速方法。
