摘要
本研究将从极度抗旱、耐盐植物柽柳(Tamarix androssowii)中克隆得到的泛素交联酶基因(ubiquitin conjugating enzymes E2s),成功构建到植物表达载体pROKⅡ上,利用农杆菌介导法将泛素交联酶基因导入模式植物烟草基因组中,通过对转基因烟草的盐、旱胁迫试验,对泛素交联酶基因进行功能验证。
对烟草进行遗传转化,共获得68株卡那霉素抗性芽,从中选出长势最优的15个株系,进行PCR检测,结果均为阳性;从15个株系中随机对6个株系进行PCR-Southem杂交,各转基因株系均出现杂交谱带,证明泛素交联酶基因己整合到烟草基因组中;进一步对6个株系进行RT-PCR和实时荧光定量RT-PCR检测,结果表明外源基因能够表达,但各株系外源基因的表达水平不尽相同,表达量最高的为TL3和TL1,TL10和TL11次之、最低的为TL5和TL7。
分别对6个转基因株系进行了NaCl胁迫和干旱胁迫试验,分析逆境胁迫条件下转基因烟草与非转基因对照烟草的丙二醛(MDA)含量、相对电导率、超氧化物歧化酶(SOD)活性、叶绿素含量、相对生长量、生根率等指标的变化,结果表明,不同浓度NaCl胁迫处理后,各株系的相对电导率、MDA含量都逐渐增大,在NaCl浓度为440mmol.L~(-1)时,各转基因株系相对电导率平均值低于对照烟草的27.1%,MDA平均含量低于对照的22.6%;NaCl胁迫下,各转基因株系SOD平均活性最低的TL5也高于对照烟草的47.5%,叶绿素平均含量最高的TL10高于对照烟草的64.4%,最低TL3也高于对照烟草的29.7%:当NaCl浓度220mmol.L~(-1)时对照烟草生根率只有30%,而转基因烟草生根率保持在50～70%,综上说明,盐胁迫条件下,转基因烟草受害程度明显小于对照。
干旱胁迫处理10d时,转基因烟草相对电导率平均值低于对照烟草的19.6%;对照烟草MDA含量为7.7μmol.g~(-1),而转基因烟草MDA含量大多在4.9～6.3μmol.g(-1)左右,低于对照烟草;此时转基因烟草的叶绿素平均含量为1.01mg.g~(-1),高于对照的64.5%,而SOD平均活性高于对照的32.3%;对转基因株系干旱胁迫10d时相对生长量进行调查,各转基因株系相对生长量在59%～83%范围之间,而对照株系的相对生长量为29.7%,说明泛素交联酶基因的导入提高了烟草的抗旱能力。
综合以上结果,泛素交联酶基因的组成型表达能够不同程度提高转基因烟草逆境胁迫下细胞膜稳定性、SOD活性、叶绿素含量及相对生长量,说明泛素交联酶基因具有提高植物抗旱、耐盐性的功能。
The gene encoding a ubiquitin conjugating enzymes (E2s) was obtained from the Tamarix androssowii cDNA library. The plant genomic expression vector, pROK II, with the E2s gene was constructed and transferred into Nicotiana tabacum L via Agrobacterium-mediated transformation procedure. Moreover, the function of the exogenous E2s gene was examined under salt and drought stress.Sixty-eight resistant lines were obtained through kanamycin selection, and 15 well grown lines which subjected to PCR assay all showed positive. In PCR-Southern hybridization, we checked 6 transgenic plants selected from the 15 plants randomly. The result showed that the foreign target gene had been integrated into tobacoo genome. Real time RT-PCR method was employed to study the expression of the E2s gene, indicated that it was expressed but the expression rates varied. Among the six lines, TL3 and TL1 had the highest expression rate, TL10 and TL11 lines are lower, and TL5 and TL7 had the lowest expression rate.Contents of malondialdehyde (MDA) , activity of superoxide dismutase (SOD) . rate of conductivity, growth rate, content of chlorophyll and radication rate of the 6 lines were determined and analysised under NaCl and drought stress. When the transgenic lines were treated with 440mM NaCl. the average relative conductivity and MDA content are respectively 27.1% and 22.6% lower than the control. The SOD activity of TL5 was lowest in all the transgenic lines, which is still 47.5% higher than the control. The TL10 and TL3 have the highest and the lowest chlorophyll content among the six lines, they are 64.4% and 29.7% higher than the control. The radication rate of transgenic lines was about 50-70%, whereas the control line was only 30% in 220mM NaCl stress condition. The above results indicated that the transgenic lines suffered minor damage under NaCl stress.In the tenth day of drought stress, the average relative conductivity is 19.6% lower than control line, the MDA content of transgenic and control lines is 4.9~6.3μmolg~-1 and 7.7μmol g~-1 respectively. The average chlorophyll and SOD content is 64.5% and 32.3% higher than the control, the average SOD content of transgenic lines is 1.01mg/g. In this time, relative growth rate was ranged from 59% to 83% but the control line is about 29.7%. These results demonstrated that the introduced exogenous E2s gene enhanced the drought stress tolerance of tobacco.All the experiments results indicated that the constitutively expressed E2s gene from T. androssowii can improve the stability of cell membrane, increase the SOD activity and
chlorophyll content, and enhance the relative growth rate under adverse environment. That was Els gene could ehance the tolerance of salt and drought stress of plant.
引文
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