过表达EuCuZnSOD提高巨尾桉的耐寒性研究

摘要

Copper/zinc superoxide dismutases are important antioxidant enzymes to guard cells against superoxide toxicity. The over?expression of CuZnSOD can improve the resistance and recovery ability of plants to abiotic stresses such as salt,drought,cold and high temperature stress.The plant genome encodes three types of CuZnSOD,the CuZn?SOD1 in cytoplasm,the CuZnSOD2 in chloroplast and the CuZnSOD3 in peroxisome or extracellular. The cytosolic Eu?CuZnSOD1 gene was cloned from Eucalyptus grandis×E.ophylla(GenBank Accession Number:JX138573)and the bi?ological function was verified by prokaryotic expression system.The fusion vector of sense EuCuZnSOD1 gene derived by CaMV35S promoter was constructed and transformed to Eucalyptus grandis ×E. ophylla by Agrobacterium. Using Kana?mycin selection and PCR analysis,ten transgenic eucalyptus lines were obtained. Compared with the control plants the tissue culture transgenic eucalyptus No. 40 and No. 41 could be well tolerant in -13.1℃ for 5 h and recovery without obvious damage.There was no distinct increase in SOD enzyme activity in transgenic plant paralleled with wild plant un?der room temperature and 4℃ for 48 h. However, the real?time PCR analysis showed that the expression of EuCuZn?SOD1 was raised to about nine times higher than wild line under room temperature.After 4℃ for 36 h,the expression of EuCuZnSOD1 in No.40 and No.41 line was respectively increased about 16 and 36 times higher than the wild plant and then down to the normal level at 4 ℃ for 48 h. The data indicated that the increased expression of EuCuZnSOD1 im?proves the cold tolerance of Eucalyptus. The klason lignin content of transgenic Eucalyptus was reduced especially the No. 41 Eucalyptus was decreased to 56% of the control plant except for the No. 40 plant was no significantly different from the control plant. In conclusion,over?expression of the cytosolic EuCuZnSOD1 in Eucalyptus grandis ×E. ophylla could enhance the cold?tolerance and improve the recovery ablity.%铜锌超氧化物歧化酶(CuZnSOD)是一种清除超氧阴离子自由基的金属酶,与多种抗逆性相关.该研究利用CaMV35S启动子融合巨尾桉EuCuZnSOD1基因通过根癌农杆菌介导的叶盘转化法导入巨尾桉中,经卡那霉素初步筛选和PCR鉴定,得到10株转基因阳性植株.结果表明:通过-13.1℃暗处理5 h的抗寒性实验,发现40和41号植株在低温下无叶片萎蔫现象,组培室培养100 d后全株存活无白化叶片,说明这两株转基因巨尾桉的抗低温和冻害后恢复能力较强.在4 ℃低温胁迫下跟踪监测转基因温室苗的SOD酶活性发现,与对照相比转基因植株的SOD酶比活力无规律性且变化不大,但是相同条件下实时定量PCR检测结果表明抗寒性较强的40和41号植株在常温下EuCuZnSOD1的表达量较对照增加了约9倍,4 ℃处理36 h后EuCuZnSOD1的表达量相比对照植株分别增加了16倍和36倍,说明EuCuZnSOD1在低温胁迫下的表达量增加提高了巨尾桉的耐寒性.分析转基因组培苗全株的硫酸木质素含量,表明EuCuZnSOD1高表达不影响甚至降低了巨尾桉的木质素含量,40号植株的木质素含量与对照相同,而41号整株植株的木质素含量相比对照降低了56%,其他检测转基因植株的木质素含量均有不同程度的降低.该研究结果表明巨尾桉中大量表达EuCuZnSOD1基因可以提高巨尾桉的抗寒能力以及寒害后的恢复能力,不影响甚至可能是负调控了转基因巨尾桉的木质素生物合成.