客户在《Plant Physiology and Biochemistry》发表的论文中采用我司NGS试剂盒纯化PCR产物

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.


低温严重影响马铃薯的地理分布和生产，早春或冬季可能遭受寒害。栽培马铃薯主要来源于块茎茄，对冻胁迫敏感，但野生马铃薯品种如S. commersonii，表现出组成型冻耐性和/或耐寒适应性。因此，这种野生物种可以帮助耐寒繁殖。然而，赋予冷冻耐受性的关键转录因子及其下游功能基因还远未明确，阻碍了育种过程。在这里，我们使用 ATAC-seq（具有高通量测序的转座酶可及染色质测定法）和 RNA-seq 来研究耐冷冻 CMM5 （S. commersonii） 在冷处理前后染色质可及性和基因表达模式的变化。我们的结果表明，在暴露于寒冷后，包括 Dof3、ABF2、PIF4 和 MYB4 在内的转录因子有望进一步控制植物激素合成/代谢途径中活跃的基因，即脱落酸、多胺和还原谷胱甘肽等。这表明这些转录因子可以调节CMM5叶片的耐冻性。特别是，根据双 LUC 测定，ScDof3 被证明可以调节 ScproC（吡咯啉-5-羧酸还原酶，P5CR）的表达。本氏烟草叶片中过表达ScDof3导致脯氨酸含量和NbproC（ScproC同源物）表达水平增加。这些结果表明，ScDof3-ScproC模块调节了马铃薯的脯氨酸含量，从而促进了马铃薯的耐冻性。本研究为进一步研究马铃薯耐寒的分子机制提供了宝贵的遗传资源。


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.