激发光源用于观察大豆寄生虫的研究

近日沈阳农业大学在线发表文献《Functional Identification of miR2119 Targeting ADHs in Modulating Soybean Resistance to Heterodera glycines》，文献中实验使用了LUYOR-3415用于大豆寄生虫的研究，使用LUYOR-3415RG观察GFP在大豆毛状根上的表达。

文献摘要如下:

Soybean cyst nematode (SCN, Heterodera glycines) is a sedentary endoparasite nematode that results in severe economic losses in soybean crops. miRNAs play crucial roles in plant responses to nematode. However, the role of miR2119 responding to SCN stress in soybean. Here, we demonstrated that the transcript levels of polycistronic precursors containing miR2119 and miR398a were significantly reduced in soybean upon nematode infection. Promoter of the miR2119-398a precursor analysis was conducted containing a GUS reporter gene. GUS activity assays demonstrated a decrease in miR2119-398a promoter during SCN infection. Overexpression of polycistronic precursor miR2119-398a (OE-premiR2119-398a) and miR2119 precursor (OE-premiR2119) rendered soybean more susceptible to SCN. Conversely, silencing miR2119 (STTM2119) increased soybean resistance against SCN. Furthermore, RNA-seq analysis revealed that miR2119 is involved in many defense signaling pathways. GUS reporter gene assays demonstrated that miR2119 targets GmADH1.1a and GmADH1.1b. Functional analysis indicated that ADHs act as a major role in responding to H. glycines by modulating reactive oxygen species (ROS) levels. Together, the findings reveal a novel mechanism by which the polycistronic precursor miR2119–398a coordinately regulates in response to H. glycines. Additionally, miR2119 becomes an essential element contributing to H. glycines by modulating ADH activity and ROS homeostasis in soybean.

Plasmid Construction and Agrobacterium rhizogenes Mediated Soybean Hairy Root Transformation

To analyze the GUS activity of miR2119-398a promoter, the approximately 2 kb promoter region of miR2119-398a was amplified from the Williams 82 template. Subsequently, the sequence was inserted in the recombinant vector pCAMBIA3301 with a GUS reporter gene. To characterize the functions of miR2119, miR2119-398a, and miR2119 target genes GmADHs, the soybean miR2119 precursor, miR2119-398a precursor, GmADH1.1a, and GmADH1.1b coding DNA sequences were amplified from the DNA template of Williams 82 and subsequently inserted into the pNINC2RNAi empty vector. The insertion was controlled by the CaMV 35S promoter, accompanied by an enhanced green fluorescent protein (EGFP) selectable marker. (40) Silencing miR2119 (STTM2119) was generated utilizing the short tandem target mimic (STTM) method based on Yan et al. (2012). (41) The STTM2119 sequence contained two copies of miR2119 complementary sequences that were linked by a 48 nt short spacer. The sequence was synthesized by GENEWIZ (Suzhou, China) and subsequently inserted into the binary vector mentioned above. The recombinant plasmid vectors were introduced into Agrobacterium rhizogenes K599 by thermal shock. Subsequently, the bacteria were injected into the hypocotyls of 4 day-old Williams 82 soybean seedlings, which were maintained in high humidity to facilitate the transformation of hairy roots. Hairy roots gradually developed from the infection site within 8–9 days. Positive transgenic hairy roots were screened with a hand-held lamp (Luyor, China) to visualize EGFP expression. The composite plants were then transplanted into new pots filled with vermiculite and continued to grow for about 15 days. (42)

文献地址：https://doi.org/10.1021/acs.jafc.4c05000

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