Abstract:

Objective: Nodulin 26-like intrinsic aquaporin proteins(NIPs) are transmembrane proteins essential for controlling the flow of water and other molecules, which play important roles in plant growth and stress tolerance. Temperature and water are important environmental factors for the growth and development of bamboo, the expression patterns of NIP family members of Moso bamboo(Phllostachys edulis) under temperature and drought stresses are of great significance to reveal their functions in response to stresses. Method: A genome-wide analysis of NIP genes and their promoter sequences in Moso bamboo was conducted comprehensively using bioinformatics software. The tissue expression patterns of PeNIPs were analyzed using the transcriptome data, and their expression patterns under temperature and drought stresses were validated by quantitative real-time PCR(qPCR). Two yeast expression vectors of PeNIPs were constructed to analyze their effects on drought and salt stress tolerance of yeast. Result: A total of 14 NIP genes were obtained from the genome of Moso bamboo (PeNIP1-1-PeNIP1-6, PeNIP2-1-PeNIP2-4 and PeNIP3-1-PeNIP3-4), of which the number of introns varied from 3 to 5. A variety of stress and hormone-related regulatory elements were found in the promoters of PeNIPs. The proteins encoded by PeNIPs had completely conserved domains of NIP family. The length of PeNIPs ranged from 235 aa to 297 aa with molecular weight of 24.03-31.84 kDa. The prediction of subcellular localization showed that all PeNIPs were localized on the plasma membrane. Collinearity analysis showed that a total of 27 pairs of fragments were found in 12 PeNIPs and 8 rice NIP genes. All the values of nonsynonymous and synonymous substitution ratios(K_a/K_s) were less than 1, indicating that these PeNIPs had undergone strong purification selection after replication in evolution. In the phylogenetic tree constructed with NIPs of P. edulis and other 5 species, all the NIPs were clustered into three groups(Ⅰ, Ⅱ, Ⅲ), which included 6, 4, and 4 members of PeNIPs, respectively. A total of 10 conserved motifs were found in PeNIPs, of which motif 1, 2 and 4 were common to all PeNIPs. Gene-specific expression analysis based on transcriptome data showed that there were some differences in the expression levels of different PeNIPs in 26 different tissues of Moso bamboo. For examples, PeNIP1-3, PeNIP1-4 and PeNIP1-5 of group I expressed in roots, but hardly expressed in bamboo shoots; four PeNIP2s of group Ⅱ as well as PeNIP3-1 and PeNIP3-2 of group Ⅲ had higher expression levels in roots and shoots. The results of qPCR demonstrated that with the extension of stress duration, 8 genes were up-regulated and 2 genes were down-regulated under 4℃ stresses, and 2 genes were up-regulated and 4 genes were down-regulated under 42℃ stresses. While under the drought stresses there were 3 genes up-regulated. The yeasts expressing PeNIP1-1 and PeNIP2-2 all grew better on the media supplemented with sorbitol or NaCl than the control. Conclusion: Fourteen PeNIPs belonging to NIP family were identified in Moso bamboo. There were some differences in the molecular characteristics and expression patterns of PeNIPs, suggesting that they might play different roles in the growth and development of Moso bamboo and in response to environmental stress. The tolerance ability of yeasts expressing PeNIP1-1 and PeNIP2-2 were improved to a certain degree, indicating that they might have important roles in the stress response of Moso bamboo.

Key words: Phyllostachys edulis, nodulin 26-like intrinsic aquaporin protein, molecular characteristics, gene expression