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客戶在《Plant Physiology and Biochemistry》發(fā)表的論文中采用我司NGS試劑盒純化PCR產(chǎn)物

Integrated analysis of ATAC-seq and transcriptomic reveals the ScDof3-ScproC molecular module regulating the cold acclimation capacity of potato

Xin Li a c d, Lin Chen b, Tiantian Liu a c d, Ye Chen a c d, Jin Wang a c d, Botao Song a c d
a National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, 430070, PR China
b Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (South China), Ministry of Agriculture and Rural Affairs, College of Horticulture, South China Agricultural University, Guangzhou, 510642, PR China
c Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan, 430070, PR China
d Potato Engineering and Technology Research Center of Hubei Province, Huazhong Agricultural University, Wuhan, 430070, PR China
Received 15 January 2024, Revised 27 March 2024, Accepted 28 March 2024, Available online 9 April 2024, Version of Record 11 April 2024.

Plant Physiology and Biochemistry
Volume 210, May 2024, 108576
https://doi.org/10.1016/j.plaphy.2024.108576

Highlights
? ATAC-seq and RNA-seq during the cold acclimation of wild potato were integrated.
? We obtained the responsive genes and potential pathways in cold acclimation.
? The ScDof3- ScproC module was shown to regulate freezing tolerance in potato.


Abstract
Low temperature severely affects the geographical distribution and production of potato, which may incur cold damage in early spring or winter. Cultivated potatoes, mainly derived from Solanum tuberosum, are sensitive to freezing stress, but wild species of potato such as S. commersonii exhibit both constitutive freezing tolerance and/or cold acclimation tolerance. Hence, such wild species could assist in cold hardiness breeding. Yet the key transcription factors and their downstream functional genes that confer freezing tolerance are far from clear, hindering the breeding process. Here, we used ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) alongside RNA-seq to investigate the variation in chromatin accessibility and patterns of gene expression in freezing-tolerant CMM5 (S. commersonii), before and after its cold treatment. Our results suggest that after exposure to cold, transcription factors including Dof3, ABF2, PIF4, and MYB4 were predicted to further control the genes active in the synthetic/metabolic pathways of plant hormones, namely abscisic acid, polyamine, and reductive glutathione (among others). This suggests these transcription factors could regulate freezing tolerance of CMM5 leaves. In particular, ScDof3 was proven to regulate the expression of ScproC (pyrroline-5-carboxylate reductase, P5CR) according to dual-LUC assays. Overexpressing ScDof3 in Nicotiana benthamiana leaves led to an increase in both the proline content and expression level of NbproC (homolog of ScproC). These results demonstrate the ScDof3-ScproC module regulates the proline content and thus promotes freezing tolerance in potato. Our research provides valuable genetic resources to further study the molecular mechanisms underpinning cold tolerance in potato.


低溫嚴重影響馬鈴薯的地理分布和生產(chǎn)，早春或冬季可能遭受寒害。栽培馬鈴薯主要來源于塊莖茄，對凍脅迫敏感，但野生馬鈴薯品種如S. commersonii，表現(xiàn)出組成型凍耐性和/或耐寒適應性。因此，這種野生物種可以幫助耐寒繁殖。然而，賦予冷凍耐受性的關鍵轉錄因子及其下游功能基因還遠未明確，阻礙了育種過程。在這里，我們使用 ATAC-seq（具有高通量測序的轉座酶可及染色質測定法）和 RNA-seq 來研究耐冷凍 CMM5 （S. commersonii） 在冷處理前后染色質可及性和基因表達模式的變化。我們的結果表明，在暴露于寒冷后，包括 Dof3、ABF2、PIF4 和 MYB4 在內(nèi)的轉錄因子有望進一步控制植物激素合成/代謝途徑中活躍的基因，即脫落酸、多胺和還原谷胱甘肽等。這表明這些轉錄因子可以調節(jié)CMM5葉片的耐凍性。特別是，根據(jù)雙 LUC 測定，ScDof3 被證明可以調節(jié) ScproC（吡咯啉-5-羧酸還原酶，P5CR）的表達。本氏煙草葉片中過表達ScDof3導致脯氨酸含量和NbproC（ScproC同源物）表達水平增加。這些結果表明，ScDof3-ScproC模塊調節(jié)了馬鈴薯的脯氨酸含量，從而促進了馬鈴薯的耐凍性。本研究為進一步研究馬鈴薯耐寒的分子機制提供了寶貴的遺傳資源。


2. Materials and methods
2.2. Construction of the ATAC-seq library
Leaf cells were washed with phosphate buffered saline (PBS) and centrifuged at 1000×g for 5 min at 4 °C. After removing the PBS, the washed cells were resuspended using a 200 μL of pre-cooled lysis buffer, gently blown on ice for 10 min, and centrifuged again at 500×g for 10 min. After removing the supernatant, 1 mL of washing buffer was added into the precipitate and mixed by inversion three times. Then, cell nuclei were collected after being centrifuged at 500×g for 10 min at 4 °C. The precipitation of nuclei was then resuspended in a 50-μL transposable reaction solution, gently blown, and mixed. The mixture was incubated in a PCR instrument at 37 °C for 30 min. DNA purification was performed using the DNA Clean Beads (NovoNGS, N240-01A, Suzhou, China). A primary library was obtained via amplification using P5 and P7 adaptor primers with 15 cycles. Finally, the library was purified and screened using 1.5 × NGS magnetic beads (PuriMag, K0004-100, Xiamen, China). After sequencing on the Illumina platform NovaSeq PE150 (paired end 150 bp), the sequence information of accessible chromatin could be obtained.