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LUYOR-3415RG用于筛选拟南芥GFP

发布时间:2022-10-18   点击次数:117次

北京林业大学在《International Journal of Biological Macromolecules》发表了《Genome-wide identification and functional analysis of silicon transporter family genes in moso bamboo (Phyllostachys edulis)》,文献中使用了LUYOR-3415RG便携式荧光蛋白激发光源用于观察EGFP在拟南芥上的表达。

Silicon (Si) has crucial effects on plant development and stress resistance. Silicon transporters regulate Si absorption, transport, and distribution in plants. In this study, we identified and characterized the Si transporter gene family of moso bamboo (Phyllostachys edulis) and cloned seven putative Si transporter genes. Moso bamboo Si transporters contain conserved functional domains that mediate the accumulation of considerable amounts of Si. The analysis of gene duplication patterns and divergence times suggested that the expansion of the moso bamboo Si transporter family was mainly due to segmental duplications. The expression of moso bamboo Si transporter genes, which varied among organs, was significantly modulated by Si treatments. The subcellular localization analysis showed that Si transporters are plasma membrane proteins. The Si content increased in transgenic Arabidopsis overexpressing PeLsi1-1 or PeLsi1-2, which affected vegetative and reproductive growth. Our single-particle tracking analysis revealed the four diffusion modes of PeLsi1-1 on the plasma membrane. Moreover, the particle velocity, dwell time, and motion range of PeLsi1-1 decreased in response to Si treatments. The results of this study will further clarify the molecular mechanisms underlying Si absorption and accumulation in bamboo plants.

Genetic transformation and identification of transgenic Arabidopsis

Recombinant plasmids containing target genes were separately transferred into Agrobacterium tumefaciens strain GV3101 cells (Zomanbio, Beijing, China) via a freeze–thaw method for the subsequent transformation of Columbia-0 wild-type Arabidopsis with pBI121-35S::EGFP, PeLsi1-1, PeLsi1-2, and PeLsi2 according to the floral dip method [48]. Harvested T1 generation seeds were screened on half-strength Murashige & Skoog (MS) medium (Table S4) containing 50 mg/L kanamycin. The LUYOR-3415RG dual-wavelength fluorescent protein excitation light source (LUYOR, Shanghai, China) was used to screen for transgenic Arabidopsis lines with high EGFP expression levels (excitation and emission wavelengths were 440–460 and 500 nm, respectively). The kanamycin-resistant T1 seedlings were analyzed using the TransDirect® Plant Tissue PCR Kit (TransGen) and the EGFP-F/EGFP-R primers (Table S6). Twelve positive seedlings per transgenic Arabidopsis line were selected for the subsequent culture and examination. The T1 plants were irradiated using LUYOR-3415RG, after which three plants with high EGFP expression levels that also conformed to Mendelian inheritance were selected for each transgenic Arabidopsis line. AtActin2 (AT3G18780) was used as an internal control to identify T3 transgenic Arabidopsis plants via an RT-PCR analysis involving Si transporter gene-specific primers (Table S6).

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