Cloning And Expression Regulation Of Aquaporin Genes From Cut Gladiolus Spikes

Gladiolus (Gladiolus hybridus) is one of the4most famous cut flowers in theworld. However, florets on cut gladiolus spikes are prone to lose water and wilt afterharvesting, which seriously damages the ornamental quality and commercial value.Aquaporins (AQPs), as members of membrane intrinsic proteins（MIPs）widelydistributing in plants, animals and microorganisms, are regarded as high-performancetransmembrane channels for water and some small molecules, e.g. glycerol. It iswell-known that plant AQPs are mainly in plasma membrane and tonoplast, and theyplay a key role in water metabolism. In this study, cut gladiolus (G. hybridus ‘Eerde’)flowers were used as the experimental materials, and3AQPs genes including1plasma membrane intrinsic protein (PIP) gene and two tonoplast intrinsic proteins(TIPs) genes were cloned for the first time from their petals of florets, and theirexpressions in the various tissues of cut gladiolus spikes, during the opening processof florets, and in the water-stressed detached florets were detected at thetranscriptional level. Additionally, the regulation of AQPs activity inhibitors on waterloss of detached petals was further investigated. The main results were as follows:1. One plasma membrane intrinsic protein（PIP） gene and2tonoplast intrinsicproteins（TIPs） genes were cloned by RT-PCR and RACE techniques, and werenamed as GhPIP1;1, GhTIP1;1and GhTIP2;1, respectively. GhPIP1;1was1130bplong with an867bp of opening reading frame（ORF）, encoding a protein of288amino acids and sharing high sequence homology at amino acid level (94%of identity)with Iris hollandica IhPIP1（BAF44223.1）. GhTIP1;1was1132bp long, encoding aprotein of278amino acids and showing83%identity with Zea mays ZmTIP1;1（NP001104896）. GhTIP2;1was1040bp long, encoding a protein of248aminoacids and showing81%identity with Zea mays ZmTIP2;1（ACG44020）.2. Three promoter sequences of GhPIP1;1, GhTIP1;1and GhTIP2;1wereobtained by Tail-PCR, they were1077bp,541bp and342bp long, respectively. Allof the3promoters contain some cis-acting elements responsive to ABA, GA, light and water-stress, and so on.3. Using the semi-quantitative RT-PCR and real-time quantitative PCR（q-PCR）,the expressions of above3AQPs genes in the various tissues of cut gladiolus spikes,during the opening process of florets and in water-stressed detached florets wereinvestigated. The results showed:（1）The AQPs genes were differently expressed inpetals, pistils, stamens, stems, leaves and bracts of cut gladiolus spikes. However,they all were highly expressed in petals;（2）During the flower bud stage to completeopening, the expressions of all the3AQPs genes were high,and thereafter obviouslydown-regulated（;3）The AQPs genes were down-regulated in water-stressed detachedflorets.4．The effects of different kinds and concentrations of AQPs activity inhibitorsincluding nano silver (NS), silver nitrate (AgNO3) and mercuric chloride (HgCl2) andrecovery agents β-mercaptoethanol (ME) on the water loss of detached petals offlorest on cut gladiolus spikes were investigated, the results showed（:1）Both25mg/LNS and50mg/L AgNO3solution could significantly delay water loss of petals;（2）100μmol/L HgCl2solution could significantly delay water loss of petals, however itcould be alleviated by the treatment with β-mercaptoethanol, a recovery agent forAQPs activity（;3）Compaired with100μmol/L HgCl2solution,25mg/L NS treatmentcould more effectively alleviate the water loss of detached petals.