| Phosphorus, an essential mineral element for plants, is involved in plant biosynthesis, signal transduction, energy transferring, and other important metabolic processes.In some cases, for various reasons, soil may have very low phosphate content, and phosphate absorption may also be difficult, thus limiting the plant production of the soil. During the long term of evolution, plants have evolved a complex set of methods to adapt to phosphate deficiency, including morphological and biochemical mechanisms, such as changing root system architecture, secreting large amounts of organic acids, symbiosis with fungi, and alterative metabolic pathways. Meanwhile, the rapid development in the field of molecular biology elicits several key regulatory factors which play important roles in this adaptation, including transcription factors, phosphate transporter proteins, non-coding RNA, and phosphate-induced genes. However, most of these studies focus on model plants, and there is a lack of research on soybeans, with only a single report on GmPT. Therefore, there is important theoretical and practical significance in investigating soybeans.In the present study, two transcription factors, namely AtWRKY6 and AtWRKY75, which are related to tolerance to low phosphate stress and have relatively high homologies, were cloned from soybeans, and respectively named as GmWRKY75 and GmWRKY6. The bioinformatics analysis and function study results showed the following:1. The sequences of AtWRKY75 and AtWRKY6 in Arabidopsis were used as templates to search the sequences of AtWRKY75 and AtWRKY6 in soybeans in the Phytozome and PGDD database. The respective ORFs of GmWRKY75 and GmWRKY6 were 588 bp and 1674 bp, and were located on chromosomes 16 and 15 in the soybeans, with homologies of 86% and 80% with Arabidopsis. The Bioinformatics analysis showed that the GmWRKY75 and AtWRKY75 in the soybeans had the same domain and similar gene structure with those in Arabidopsis. Multiple sequence alignment and phylogenetic analysis showed that GmWRKY75 and GmWRKY6 had a common conserved domain with Arabidopsis WRKY. GmWRKY75 and AtWRKY75 were in the same class, Ⅱc in the subfamily Ⅱ of WRKY, while Gm WRKY6 and AtWRKY6 were in the same class, Ⅱb.2. Under the stress of low P, N, K, and lack of Fe, the soybeans, Kefeng 1, were cultured in hydroponic media. The RT-PCR analysis showed that the levels of GmWRKY75 and GmWRKY6 were the same in the four treatment conditions, characterized by a slight decrease followed by a significantly increase. However, the time points corresponding to the maximum expression level were different, i.e.48 h for low P,36 h for low K,24 h for low N, but rise slowly after 6 h for lack of Fe.3. The fusion expression vectors pJIT166-GFP-GmWRKY75 and pJIT166-GFP-GmWRKY6 were constructed, and transfected into the onion epidermal cells by means of a gene gun. Then their subcellular localizations were detected, and the results showed that both GmWRKY75 and GmWRKY6 were located within the nucleus.4. The plant overexpression vectors pCAMBIA3301-GmWAT75 and pCAMBIA3301-GmWRKY6 were constructed and transfected into Arabidopsis using the Floral dip method,Obtain T3 transgenic plants, phosphorus culture found, from the seedling stage to the flowering and maturity stage, the T3 generation of transgenic Arabidopsis exhibited better growth than the wild-type. In addition, the efficient phosphorus test showed that the T3 generation of transgenic Arabidopsis had a more significant rising trend of phosphate content than that of the wild-type.5. Agrobacterium-mediated cotyledon node transformation was used to transfect GmWRKY75 into soybean plants, Further optimization of the transformation system GmWRKY75, providing a foundation for the Phosphorus-resistant transgenic soybean breeding. |
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