amiRNA技术沉默C-3氧化酶编码基因StCPD对马铃薯抗旱性的影响

CPD(constitutive photomorphogenesis and dwarf)基因编码C-3氧化酶,为油菜素内酯(brassinosteroid,BR)生物合成途径中的限速酶,在植物响应逆境胁迫过程中具重要调控作用。本研究利用人工micro RNA(artificial micro RNA,ami RNA)技术,构建马铃薯CPD基因(StCPD)的干扰表达载体p CPB121-ami Rcpd,通过根癌农杆菌介导法将其转入马铃薯栽培品种"紫花白",获得转基因植株(Ci1~Ci5),其中Ci1和Ci3的StCPD基因干扰程度分别为78%和90%。基因组织表达特异性分析表明,StCPD在马铃薯试管苗叶片中表达量最高,是茎和根中表达量的3.05倍和1.65倍。转基因植株株高、茎粗、根长、鲜重及薯的大小和鲜重等指标均较非转基因(NT)植株显著下降,表明StCPD基因干扰表达后,植株的长势明显受到抑制。模拟干旱胁迫处理下,转基因植株叶片中丙二醛(malondialdehyde,MDA)含量显著高于NT植株,而脯氨酸含量显著低于NT植株。转基因和NT马铃薯中,StCPD基因的表达量、MDA和脯氨酸含量均显著高于对照;且随着胁迫处理时间延长,基因表达量呈持续增强趋势,MDA和脯氨酸含量随之增加。结果表明,StCPD基因干扰表达能明显降低马铃薯对干旱胁迫的抵抗能力,为进一步研究BR对马铃薯生长发育和对干旱胁迫的响应奠定了基础。

Effect of Silencing C-3 Oxidase Encoded Gene StCPD on Potato Drought Resistance by amiRNA Technology

The constitutive photomorphogenesis and dwarf (CPD) gene encodes C-3 oxidase as a key rate-limiting enzyme in the brassinosteroids (BRs) biosynthesis pathway, which plays a vital role in response to abiotic stress in plant. In this research, the potato CPD gene (StCPD) interference expression vector pCPB121-amiRcpd was constructed using the plant expression vector pRS-300 and amiRcpd skeleton precursor of Arabidopsis miR319a, and transferred into the potato genome mediated by Agrobactium tumefacienses L., so that transgenic plants (Ci1-Ci5) were obtained. The analysis of real time fluorescence quantitative polymerase chain reaction (qRT-PCR) showed that the interference degree of StCPD gene expression reached 78% and 90% in the transgenic lines Ci1 and Ci3 respectively. StCPD gene expressed in the roots, stems and leaves of the transgenic and non-transgenic (NT) plants, and expression level in the leaves was 3.05 and 1.65 fold higher than that in stems and roots. The malondialdehyde (MDA) content in transgenic plant leaves was significantly higher, whereas the proline content was significantly lower than those in NT under drought stress. The expression level of StCPD gene, MDA and proline contents in transgenic and NT plants under drought stress were significantly higher than those of control, and increased at different sampling times. These results indicated that StCPD gene interference expression could depress the effects of drought stress on potato. These results lay a foundation for further research on BRs regulation in potato development, which will help to reveal the molecular mechanisms of BRs regulation in potato.