Genome-wide Analysis Of AQPs Family In Phyllostachys Edulis And Functional Analyses Of PeTIP4;1-1 And PePIP2;7

Water balance is essential for the normal life activities of plant.Aquaporins(AQPs)can regulate the flow of water in cells and participate in many physiological processes in plant,such as water uptake,nutrient uptake,and tissue expansion.AQPs are required to maintain cell turgor pressure for the plant cell division and cell elongation.The stable turgor pressure can provide the impetus for cell division and enlargement,which induces the expansion of cell wall and promotes the growth of plant.Bamboo is one of important forest plants,which can complete high growth in a short period of time.It has been confirmed that the fast growth is determined by both cell division and elongation,and the root pressure determines the growth height of bamboo.Therefore,the systematic study of the relationship between AQPs gene function and water balance in bamboo has important scientific significance for comprehensively analyzing the fast-growing mechanism of bamboo.In this study,the genome-wide identification and characterization analysis of aquaporin gene family in moso bamboo(Phyllostachys edulis)were conducted.Based on the comprehensive analyses of the aquaporin gene family members in moso bamboo,the gene expression patterns in different tissues and response to different stresses,and the changes of root pressure during fast growth of moso bamboo were studied.The functional analysis of PeTIP4;1-1 and PePIP2;7 were further conducted.Main results are as follows:1.Genome-wide identification of aquaporin gene family in moso bamboo.Bioinformatics methods were used to identify the aquaporin genes genome-widely in moso bamboo.A total of 26 and 63 PeAQPs were identified in the first and second versions of moso bamboo genome respectively,using the AQPs of Zea mays,Oryza sativa as query sequences and deleting the sequences with incomplete conserved domain.The gene structure and scaffold location,the basic physical and chemical characteristics,the subcellular location,the conserved domains,and the predictded function of the proteins encoded by the aquaporin gene family members in moso bamboo were analyzed respectively.It was clarified that 63 PeAQPs belonged to four distinct subfamilies of PIPs(22 members),TIPs(20 members),NIPs(17 members)and SIPs(4 members)respectively,while XIPs was not presence in moso bamboo.2.Gene expression profile analysis of PeAQPs.The transcriptome data and qPCR technique were used to analyze the expression profiles of PeAQPs.The results showed that there were significant differences in the expression profiles of PeAQPs in different tissues,and in roots and shoots at different stages of moso bmbooo.For examples,PePIP2;3 and PePIP2;4 were mainly expressed in root,and PePIP2;7 was specific expressed in leaf.Nine PIPs,four TIPs and only PeNIP2;3 were up-regulated continuely with the elongation of roots.Five PIPs,eight TIPs and only PeNIP2;1 were up-regulated in the shoot tips with the growth of shoots.The expression of three and nine PeAQPs in root and leaf respectively were significantly induced by drought stress,and that of eight and two genes were significantly up-regulated in response to NaCl stress.The difference in the expression of PeAQPs indicated that there were certain differences in their functions.3.Correlation analysis of root pressure and PeAQPs gene expression in bamboo.The measurement of root pressure during the bamboo shoot period indicated that the shoot had a strong root pressure,which was up to 69.9 kPa in the 6 m shoots.The root pressure of bamboo shoots showed a circadian rhythm,which was negative pressure during the daytime and positive pressure at night,causing by the water flowed in and out the bamboo shoots.A strong root pressure could transport the water from the base to the top of bamboo shoot.Meanwhile,some PeAQPs with high expression levels were found near the transition points of the changing root pressure,such as PePIP1;4,PeTIP4;1 and PeNIP3;3.Besides,the expression levels of PeTIP4;2 and PePIP2;8 showed positively correlated with the root pressure.Therefore,it is speculated that these PeAQPs could be involved ing regulating the balance of water through their expression,which had an important role in the formation of bamboo root pressure in the shoot period.4.Functional analysis of PeTIP4;1-1 gene.The gene PeTIP4;1 highly expressed in the turning point of root pressure was isolated,which genomic sequence was 1,777 bp containing three exons separated by two introns.The ORF of PeTIP4;1-1 was 756 bp encoding a peptide of 251 amino acids.PeTIP4;1-1 contains six transmembrane helices and two conserved ‘NPA' motifs.PeTIP4;1-1 was predicted to be mainly located on the tonoplast.PeTIP4;1-1 was constitutively expressed with the highest level in culms,which had an increasing expression trend with the bamboo shoot increasing.Besides,PeTIP4;1-1 was significantly up-regulated in both bamboo roots and leaves under drought and salinity stresses.Many stress responsive elements were found in the promoter region of PeTIP4;1-1(1497 bp),and the promoter activity was further analyzed by transient expression.PeTIP4;1-1 confers stress tolerance might be associated with various pathways regulating drought and salinity responses in transgenic plants.These results indicated that PeTIP4;1-1 might be one of the key genes involved in response to drought and salinity stresses,which was a candidate gene for stress tolerance breeding in other crops through genetic engineering.5.Functional analysis of PePIP2;7 gene.PePIP2;7,another aquaporin homologous gene,was isolated from moso bamboo,whose genomic sequence was 1265 bp containing four exons separated by three introns.The ORF of PePIP2;7 was 864 bp encoding a peptide of 287 amino acids.PePIP2;7 contains six transmembrane helices and two conserved ‘NPA' motifs.PePIP2;7 was mainly located in plasma membrane.The expression of PePIP2;7 exhibited to rise first and then descend in response to drought and NaCl stresses.Over-expression of PePIP2;7 in Arabidopsis thaliana can improve the tolerance ability in response to low temperature with higher Y(II)and NPQ values.Thus,PePIP2;7 could response stresses by regulating water balance in leaves.This study not only laid a foundation to better understand the function of AQPs in moso bamboo,but also provided a new reference for fully analysis of the rapid growth mechanism of bamboo in terms of water transport with AQPs,which was beneficial to the molecular breeding of bamboo in the future.