过表达和敲除NtabNAC087基因对烟草响应干旱胁迫的影响

Effects of Overexpression and Knockout of NtabNAC087 Gene on Response to Drought Stress in Tobacco(Nicotiana tabacum L.)

NAC基因家族作为植物特有的基因家族之一,其成员广泛参与烟草植株的生长发育调控和非生物胁迫应答。为了进一步鉴定早期在烟草中挖掘的拟南芥AtNAC072/RD26直系同源基因NtabNAC087响应干旱胁迫时的功能,本研究通过基因过表达和CRISPR/CAS9基因标记技术构建了对应普通烟草品种K326的遗传转化株系,对野生型K326和2种遗传转化烟草纯合株系幼苗分别进行干旱胁迫处理,观察叶片气孔结构并检测超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性、丙二醛(MDA)和脯氨酸(Pro)含量及基因表达量。结果发现,过表达株系芽期和苗期干旱胁迫下表现出更强的耐旱性,编辑株系则表现出干旱敏感性;过表达株系叶片与对照组相比气孔密度增加,孔径变小;SOD和CAT活性及Pro含量过表达株系较编辑株系相比显著提高,MDA含量过表达株系较编辑株系显著降低;NtabNAC087基因表达量均呈现先上升后下降趋势,且在处理6 h时达到最高。综上,NtabNAC087基因通过表达量或结构变化导致烟草叶片生理结构、抗氧化物酶活性和渗透调节物质含量发生显著变化。本研究结果为后续研究烟草干旱胁迫应答过程中的分子机制提供了理论支撑。

As one of the plant-specific gene families,NAC genes families are widely involved in growth and developmental regulation and abiotic stress response of plant.To characterize the function of tobacco gene NtabNAC087,a direct homolog of Arabidopsis AtNAC072/RD26 tapped earlier in response to drought stress.Genetically transformed strains from the common tobacco variety K326 were constructed by gene overexpression and CRISPR/CAS9 gene edit technology.Seedlings of wild-type K326 and two genetically transformed tobacco pure strains were treated with drought stress,the leaf stomatal structure was observed and the superoxide dismutase(SOD)and catalase(CAT)activities,malondialdehyde(MDA)and proline(Pro)contents and gene expression were detected.The results showed that the overexpressed lines showed stronger drought tolerance under drought stress at bud and seedling stages,while the edited lines showed drought sensitivity.Compared with the control group,the leaves of overexpressed strains had higher stomatal density and smaller pore size.The activity of SOD and CAT and the content of Pro in the overexpressed strain were significantly increased compared with the edited strain,while the content of MDA in the overexpressed strain was significantly decreased compared with the edited strain.The expression of NtabNAC087 gene showed an increasing trend and then decreased,and reached the highest level at 6 hours of treatment.The study indicated that the NtabNAC087 gene caused significant changes in the physiological structure,antioxidant enzyme activity and osmoregulatory substance content of tobacco leaves through altering gene expression or structure,which provided experimental data for subsequent studying on its molecular mechanism in the tobacco drought stress response process.