Tariq Muhammad, Khan Anwar, Tabassum Bushra, Toufiq Nida, Bhatti Muhammad Umar, Riaz Saman, Nasir Idrees Ahmad, Husnain Tayyab
Department of Genetics, Hazara University , Mansehra, Khyber Pakhtunkhwa , Pakistan.
Turk J Biol. 2018 Feb 15;42(1):45-53. doi: 10.3906/biy-1709-17. eCollection 2018.
We evaluated transgenic lines of sugarcane modified with the barley chitinase class-II gene to create resistance against the red rot causative agent Colletotrichum falcatum Went. Local sugarcane cultivar SP93 was transformed with a 690-bp coding sequence of the chitinase-II gene under the influence of a polyubiquitin promoter. Transgenic sugarcane lines (T 0) overexpressing the chitinase gene were obtained through a particle bombardment method with 13.3% transformation efficiency. Four transgenic sugarcane lines, SCT-03, SCT-05, SCT-15, and SCT-20, were tested for resistance against red rot by in vitro antifungal assays. Crude protein extracts from transgenic sugarcane plants SCT-03, SCT-05, SCT-15, and SCT-20 inhibited the mycelial growth of C. falcatum by 49%, 40%, 56%, and 52%, respectively, in a quantitative in vitro assay. Our findings revealed that two transgenic lines, SCT-15 and SCT-20, exhibited the highest endochitinase activity of 0.72 and 0.58 U/mL, respectively. Furthermore, transgenic lines SCT-15 and SCT-20 exhibited strong resistance against inoculated C. falcatum in an in vitro bioassay, as they remained healthy and green in comparison with the control sugarcane plants, which turned yellow and eventually died 3 weeks after infection. The mRNA expression of the transgene in the C. falcatum-inoculated transgenic sugarcane lines increased gradually compared to the control plant. The mRNA expression was the highest at 72 h in both transgenic lines and remained almost stable in the subsequent hours.
我们评估了用大麦II类几丁质酶基因修饰的甘蔗转基因株系，以培育对红腐病病原体镰刀状炭疽菌（Colletotrichum falcatum Went）的抗性。当地甘蔗品种SP93在多聚泛素启动子的作用下，用一段690 bp的几丁质酶II基因编码序列进行转化。通过粒子轰击法获得了过表达几丁质酶基因的转基因甘蔗株系（T0），转化效率为13.3%。通过体外抗真菌试验，对4个转基因甘蔗株系SCT-03、SCT-05、SCT-15和SCT-20进行了红腐病抗性测试。在定量体外试验中，转基因甘蔗植株SCT-03、SCT-05、SCT-15和SCT-20的粗蛋白提取物分别抑制了镰刀状炭疽菌菌丝生长49%、40%、56%和52%。我们的研究结果表明，两个转基因株系SCT-15和SCT-20分别表现出最高的内切几丁质酶活性，为0.72和0.58 U/mL。此外，在体外生物测定中，转基因株系SCT-15和SCT-20对接种的镰刀状炭疽菌表现出强抗性，与对照甘蔗植株相比，它们保持健康和绿色，对照甘蔗植株在感染后3周变黄并最终死亡。与对照植株相比，接种镰刀状炭疽菌的转基因甘蔗株系中转基因的mRNA表达逐渐增加。在两个转基因株系中，mRNA表达在72 h时最高，在随后的时间里几乎保持稳定。
