Heterogeneous expression of Peu-miR473a gene confers drought tolerance in Arabidopsis thaliana.
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摘要: 为了探究胡杨miR473a基因的功能,本文克隆了miR473a的前体Pre-Peu-miR473a,并利用农杆菌花序侵染法将其遗传转化入拟南芥。通过普通PCR和β-葡萄糖苷酸酶(GUS)组织化学染色检测获得CaMV35S: miR473a过表达植株,然后对转基因和野生型植株在甘露醇模拟高渗环境与土壤自然干旱条件下的生长状况及各项生理指标进行评价。结果表明,胡杨miR473a前体长度为100 bp,与毛果杨前体序列相似度为100%,可以形成完美的二级茎环结构。相比于野生型,过表达胡杨miR473a的拟南芥在200 mmol/L甘露醇胁迫条件下的萌发率、根长和生长状况都优于野生型;在土壤干旱处理条件下,转基因拟南芥的株高、相对含水量、脯氨酸含量以及光系统Ⅱ(PSⅡ)最大光合效率均显著高于野生型10%以上(P0.05)。半定量PCR检测显示:Peu-miR473a参与胡杨受干旱胁迫的正调控,杨树中预测的靶基因Potri.012G093900、Potri.007G100200、Potri.009G165300、Potri.004G204400受干旱胁迫的负调控;拟南芥中预测的靶基因AT1G24530、AT5G45000、AT5G46070及AT3G52950在转基因株系中表达量下降。初步预测它们有可能被miR473a靶向调控。本研究表明,miR473a基因在干旱胁迫条件下通过调控植株的抗脱水能力和渗透调节能力发挥一定抗旱作用。Abstract: In order to study the function of miR473a in Populus euphratica, we cloned the precursor of miR473a, Pre-Peu-miR473a, and used floral dip method to transform miR473a into Arabidopsis thaliana. Using PCR and histochemical staining of β-glucuronidase (GUS) detection methods, we obtained the CaMV35S:miR473a transgenic plants. Then we validated the growth status and physiological indexes of transgenic plants and wild-type plants under hypertonic environment simulated by D-mannitol and natural soil drought conditions. The results indicated that the miR473a precursor has a length of 100 base pair, sharing 100% homology with miR473a homologue in Populus trichocarpa, and forming perfect secondary stem loop structure. Compared with wild-type A. thaliana, the transgenic plants exhibited higher germination rate, longer root length,and better growth conditions under osmotic stress (200 mmol/L D-mannitol); In soil drought condition, the plant height, relative water content (RWC) and maximum photosynthetic efficiency of transgenic plants were all significantly higher, 10%, than that of the wild type A. thaliana (P0.05). The semi-quantitative PCR results showed that Peu-miR473a gene is involved in up-regulation in P. euphratica under drought stress. The putative target genes in Populus Potri. 012G093900, Potri.007G100200, Potri.009G165300 and Potri.004G204400 are down-regulated by drought stress. Expressions of the potential target genes in A. thaliana AT1G24530, AT5G45000, AT5G46070 and AT3G52950 were all decreased in transgenic plants, indicating that they may be target-regulated by miR473a gene. Our study suggests that miR473a gene plays a role in plant drought tolerance by regulating the plants’ ability to resist dehydration and drought under stress.
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Keywords:
- miR473a /
- Populus euphratica /
- overexpression /
- Arabidopsis thaliana /
- drought resistance
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