To explore the structure and evolution characteristics of cotton NLP transcription factors in the whole genome, and further understand their expressions patterns, so as to lay a foundation for the further function research and utilization of NLP genes.

The NLP transcription factor family members in the whole genomes of four cotton species, Gossypium arboreum (G. arboreum, Ga), Gossypium raimondii (G. raimondii, Gr), Gossypium barbadense (G. barbadense, Gb) and Gossypium hirsutum (G. hirsutum, GH), were identified using two strategies, BLASTP and HMM search. Further bioinformatics analysis was carried out on the confirmed cotton NLP family members. The molecular weights, theoretical isoelectric points and other physical and chemical properties were predicted using online software Expasy; the MEGA 7 software was used to build the phylogenetic tree; protein conservative motifs were analyzed through MEME website; online software GSDS 2.0 was used to analyze gene structures; TBtools was used to view the chromosome localizations; McscanX was used to analyze the replication genes of cotton NLP family members; the PlantCARE website was used to predict the cis-acting elements in the promoters of cotton NLP family genes. The heat maps of cotton NLP genes expression levels of different tissues and under abiotic stresses were drawn through TBtools to analyze the tissue expression characteristics and abiotic stresses response characteristics. The expressions of GHNLPs in cotton under nitrogen starvation and nitrogen resupply treatments were analyzed by RT-qPCR.

A total of 11, 11, 21 and 22 NLP members were screened from the four cotton protein databases of G. arboreum, G. raimondii, G. barbadense and G. hirsutum, respectively. These NLP family genes encoded 693-996 amino acids. The relative molecular masses ranged from 76.92-110.02 kDa and the theoretical isoelectric points were 5.13-7.77. The subcellular localization prediction results showed that almost all the NLP members located in the nucleus. Promoter analysis found a large number of cis-acting elements related to phytohormone and stress response. Phylogenetic analysis showed cotton NLPs were divided into three groups, I, II and III. Gene replication analysis showed that fragment replication was the main force for NLP members expansion in cotton. All the Ka/Ks values were less than 1, indicating that evolution of NLP family in cotton mainly underwent purification selection. The results of expression analysis also confirmed that GHNLPs responded to nitrogen starvation and nitrogen resupply.

From the whole genome of G. arboreum, G. raimondii, G. barbadense, and G. hirsutum, 11, 11, 21 and 22 NLP transcription factor members were identified respectively. They had high conservatism and some degree of differences. The expression levels of GHNLPs changed significantly during nitrogen starvation and nitrogen resupply processes, which may play a role in the response of cotton to nitrate.