Karthik Sivabalan, Tuteja Narendra, Ganapathi Andy, Manickavasagam Markandan
1Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024 India.
2Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110 067 India.
3 Biotech. 2019 Jan;9(1):10. doi: 10.1007/s13205-018-1553-z. Epub 2019 Jan 2.
Protein p68 is a prototype constituent of DEAD-box protein family, which is involved in RNA metabolism, induced during abiotic stress conditions. In order to address the salinity stress faced by economically important soybean crop, we have transformed soybean cv. PUSA 9712 via direct organogenesis with marker free construct of gene by -mediated genetic transformation. The putative transgenic plants were screened by Polymerase chain reaction (PCR), Dot blot analysis and Southern blot hybridization. Reverse transcriptase-PCR (RT-PCR) and Quantitative real-time PCR (qRT-PCR) established that the gene expressed in three out of five southern positive (T) plants. The transformed (T) soybean plants survived irrigation upto 200 mM of NaCl whereas the non-transformed (NT) plants could not survive even 150 mM NaCl. The transgenic soybean (T) plants showed a higher accumulation of chlorophyll, proline, CAT, APX, SOD, RWC, DHAR and MDHAR than the NT plants under salinity stress conditions. The transformed (T) soybean plants also retained a higher net photosynthetic rate, stomatal conductance and CO assimilation as compared to NT plants. Further analysis revealed that (T) soybean plants accumulated higher K and lower Na levels than NT plants. Yield performance of transformed soybean plants was estimated in the transgenic green house under salinity stress conditions. The transformed (T) soybean plants expressing the gene were morphologically similar to non-transformed plants and produced 22-24 soybean pods/plant containing 8-9 g (dry weight) of seeds at 200 mM NaCl concentration. The present investigation evidenced the role of the gene against salinity, by enhancing the tolerance towards salinity stress in soybean plants.
蛋白质p68是DEAD-box蛋白家族的典型成员，参与RNA代谢，在非生物胁迫条件下被诱导产生。为了解决经济上重要的大豆作物面临的盐胁迫问题，我们通过直接器官发生法，利用基因介导的遗传转化，用无标记构建体对大豆品种PUSA 9712进行了转化。通过聚合酶链反应（PCR）、斑点印迹分析和Southern印迹杂交对推定的转基因植株进行了筛选。逆转录聚合酶链反应（RT-PCR）和定量实时聚合酶链反应（qRT-PCR）证实，该基因在五株Southern阳性（T）植株中的三株中表达。转化后的（T）大豆植株在高达200 mM的NaCl灌溉条件下存活，而非转化的（NT）植株甚至在150 mM NaCl条件下都无法存活。在盐胁迫条件下，转基因大豆（T）植株比NT植株积累了更高的叶绿素、脯氨酸、CAT、APX、SOD、RWC、DHAR和MDHAR。与NT植株相比，转化后的（T）大豆植株还保持了更高的净光合速率、气孔导度和CO同化率。进一步分析表明，（T）大豆植株比NT植株积累了更高的K含量和更低的Na含量。在盐胁迫条件下，在转基因温室中对转化后的大豆植株的产量性能进行了评估。表达该基因的转化（T）大豆植株在形态上与未转化植株相似，在200 mM NaCl浓度下，每株产生22-24个大豆荚，含有8-9 g（干重）种子。本研究通过增强大豆植株对盐胁迫的耐受性，证明了该基因对盐胁迫的作用。
