Cloning And Functional Identification Of TIPs Gene From Vitis Quinquangularis

Aquaporin plays an important role in mediating plant water transport,maintaining moisture balance and dealing with all kinds of adversity stress.The Vitis quinquangularis which originated in Karst area of Guizhou has a strong resistance to drought.In this paper,the full length of TIP genes were obtained by RT-PCR using V.quinquangularis ‘Huaxi-9' as test material,V.vinifera ‘Red globe' as the control material in order to study the structural features of aquaporin genes in wild grape and their roles in the process of drought resistance.At the same time,the structures and expressions of TIPs in tissues and under drought-rewatering conditions were also analysised,gene expression vectors were constructed,and the functions of TIPs were verified.This research canl provide some information for researchers on molecular mechanism of drought stress tolerance of plant and enlarge the gene for plant drought stress-tolerance genetic engineering.The main results were as follows.1.Nine plasma membrane intrinsic proteins(TIPs)gene,designated VhTIPs,were cloned from tissue on Chinese wild grapevine(Huaxi-9)by RT-PCR.They were named VhTIP1;1(KY379060),VhTIP1;2(KY379061),VhTIP1;3(KY391793),VhTIP1;4(KY391794),VhTIP2;1(KP276237),VhTIP2;2(KY379062),VhTIP3;1(KY922959),VhTIP4;1(KY379063),VhTIP5;1(KY922960),containing nine open reading frames(756,756,756,759,750,753,780,762,750 bp),respectively.2.The resulsts predicting by protein structure showed that VhTIPs exhibited two highly conserved NPA(Asn-Pro-Ala)motifs and a typical structure with six membrane-spanning domains and the MIP family signal consensus sequence(SGGHINPAVT)in internal symmetry.They encoded putative VhTIPs protein with 251,251,251,252,249,250,259,253,249 amino acids.The relative molecular masses were between 25.27 kD and 27.42 kD.The isoelectric points(pI)were between 4.96 and 7.11.The instability indexes were between 26.47 and 27.42,so these proteins were instability,and they were all hydrophilic molecules(between 0.657 and 0.923).The transmembrane regions of VhTIPs protein were predicted by TMHMM-2.0 software.Except for VhTIP1;4 had 7 transmembrane regions,the other 8 genes contained 6 transmembrane structures.Signal peptide was used to predict VhTIPs using SignalP 3.0 Server,and nine VhTIPs proteins have not signal peptide.Subcellular localization of these nine genes by Plant-mPLoc online software were all located on the vacuolar membrane.3.Quantitative real-time PCR results showed that the expression level of TIP1;4,TIP2;1,TIP2;2 and TIP4;1 were the highest in the roots of both ‘Huaxi-9' and ‘Red globe',they may be closely related to the regulation of root water transport and root pressure,TIP1;4 and TIP2;2 were only expressed in the root of these two grape plants,in the stems and leaves are almost undetectable,which indicated that the genes took part in the water transport in root.The expression levels of TIP1;4,TIP2;1,TIP2;2 and TIP4;1 in root of ‘Huaxi-9' were higher than that of in ‘Red globe',the expression of TIP1;2 was the highest in leaves,followed by stem and root.But that is relatively small,almost no expression in the ‘Red globe' root and leaf,which predicted that these genes may become the results of high-drought-resistance in'Huaxi-9'.TIP1;3 had the highest expression level in leaves of ‘Huaxi-9' and ‘Red globe',and could not be detected in roots.It may be mainly involved in leaf water transport.4.In the present study,under drought-rewatering stress,the expression level of these nine TIPs changed with time extension.There were differences of expression level in different tissue of two grape varieties(species).During drought process,the expression of VhTIP1;1 and VhTIP5;1 showed a tendency which rose first and then decreased in roos of ‘Huaxi-9',VhTIP1;2 and VhTIP2;1 reduced first and then increased in roots of ‘Huaxi-9'.VhTIP1;1,VhTIP2;1,VhTIP3;1,VhTIP4;1 and VhTIP5;1 could return to normal level after re-watering.VvTIP1;4 and VvTIP5;1 in roots of ‘Red globe' can be restored to normal level.VhTIP5;1 and VvTIP2;2 in the stem could return to normal level.VhTIP1;3,VhTIP1;4 and VvTIP2;1 in leaves could restored to normal level.5.The leaf relative water content(RWC),leaf water potential(?),net photosynthetic rate(Pn),stomatal conductance(gs),transpiration rate(Tr),hydraulic conductivity rate(Kh),leaf specific conductivity(LSC),specific conductivity(Ks)and relative plasma membrane permeability(PMP)in root,stem and leaf were significant differences between grapes varieties(species).Under drought stress,Pn,Gs,Tr,?l,RWC,Kh,LSC and Ks gradually reduced along with the stress time extension.The PMP of root,stem and leaf gradually increased,so cell membrane stability gradually decreased,but after the re-watering of these indicators can be restored to normal levels.There were positively correlated between TIPs expressions and physiological indicators.The correlation analysis showed that the expression of VhTIP1;1?VhTIP1;2?VhTIP2;2?VhTIP4;1 and VhTIP5;1 were positive correlations with various physiological indexes,VhTIP1;4 and VhTIP3;1 are negative correlations in ‘Huaxi-9',and TIP1;2 and TIP2;1 have the largest differences between ‘Huaxi-9' and ‘Red globe'.6.The VhTIP2;1 was inserted into multiple cloning sites of the vector pBI121 for obtaining the plant expression vector pBI121-35S-VhTIP2;1.The vector was transferred into Arabidopsis genome by Agrobacterium-mediated and pollen tube pathways.The results showed that VhTIP2;1 gene was successfully integrated into the Arabidopsis genome and expressed in Arabidopsis plants.And the overexpression of VhTIP2;1 gene was significantly higher than that of wild-type Arabidopsis thaliana,suggesting that VhTIP2;1 gene plays an important role in the process of drought resistance in Huaxi-9.