过表达桑树色氨酸脱羧酶基因(MnTDC)转基因烟草提高其耐盐性

Transgenic Over Expression of MnTDC Gene from Mulberry in Tobacco Enhances Its Salt Tolerance

褪黑素在植物生长、发育、生物与非生物胁迫中发挥重要作用。色氨酸脱羧酶(tryptophan decarboxylase,TDC)是褪黑素生物合成中第一个重要的酶。本研究克隆川桑TDC基因,并将其转入烟草中过表达,探讨MnTDC在盐胁迫下的作用机制。结果表明,成功构建过表达载体pLGNL-MnTDC,并经过农杆菌介导的愈伤组织转化获得3个独立转基因系。GUS染色、基因组PCR鉴定证明MnTDC成功转化到烟草株系中。qRT-PCR结果表明MnTDC在3个转基因烟草株系中的转录水平显著高于野生型(WT)。UPLC-MS/MS结果表明转基因株系显著提高了褪黑素的生物合成量。盐胁迫下,MnTDC过表达烟草与野生株系烟草相比,耐盐性显著增强。转基因烟草株系中的过氧化氢酶(CAT)、超氧化物歧化酶(SOD)及过氧化物酶(POD)活性均显著高于野生株系烟草,脯氨酸(Pro)等调节渗透压物质的含量和ERD10C等调节渗透压的基因表达量显著高于野生株系烟草,转基因株系中丙二醛(MDA)含量比野生株系烟草低。研究结果表明,MnTDC通过调节烟草细胞中褪黑素的含量进而影响了烟草植株整体的耐盐性。本研究初步揭示了MnTDC基因的抗逆功能,为桑树耐盐分子育种提供了重要候选基因和理论参考。

Melatonin plays an important role in plant growth,development,and biotic and abiotic stress.Tryptophan decarboxylase(TDC)is the first important enzyme in melatonin biosynthesis.In this study,we cloned the MnTDC from mulberry(Morus notabilis)and over expressed it in transgenic tobacco to explore the mechanism of MnTDC under salt stress.Results showed that the recombinant vector pLGNL-MnTDC was successfully constructed,and three independent transgenic lines were obtained by agrobacterium-mediated callus transformation.GUS staining and genomic PCR showed that MnTDC was successfully over expressed in the transgenic tobacco lines.qRT-PCR results showed that the transcriptional levels of MnTDC in three transgenic tobacco lines were significantly higher than those in wildtype(WT).UPLC-MS/MS results showed that the biosynthesis of melatonin in transgenic lines was significantly increased.MnTDC over-expressed tobacco showed signifi cantly enhanced salt tolerance compared with wild tobacco.The enzyme activity of catalase(CAT),superoxide dismutase(SOD)and peroxidase(POD)in transgenic lines were all significantly higher than those in wild lines of tobacco.The contents of substances regulating osmotic pressure such as proline(Pro)and the expressions of genes regulating osmotic pressure such as ERD10C were significantly higher than those in wild lines of tobacco.The content of malonaldehyde(MDA)in transgenic lines was lower than that in wild lines of tobacco.The results showed that MnTDC affected the salt tolerance of tobacco plants by regulating melatonin biosynthesis in tobacco.This study revealed the stress tolerance function of MnTDC gene,and provided important candidate gene and theoretical reference for molecular breeding of salt tolerance in mulberry.