| The Vitis quinquangularis which originated in Karst area of Guizhou has a strong resistance to drought. Aquaporin plays an important role in mediating plant moisture transport, maintaining moisture balance and dealing with all kinds of adversity stress. In order to study the structural features of aquaporin gene in wild grape and its role in in the process of drou ght resistance, in this paper, using V. quinquangularis ‘Huaxi-9’ as materials, V. vinifera ‘Red globe’ for comparison, the full length of PIP gene was obtained by RT-PCR and RACE technology. At the same time, the structure of PIPs, the expression of PIPs gene in tissue and under drought also were analysised, gene expression vectors were constructed, and the functions of PIPs gene were verified. This research will provide some information for researches 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(PIPs)gene,designated Vh PIPs, were cloned from tissue on wild grapevine(Huaxi-9)native to China(Vitis quinquangularis)by RT-PCR and RACE(Rapid amplication of c DNA ends). The full c DNA sequence of Vh PIPs were 1 087, 1 063, 1 089, 970, 867, 1 054, 1 111, 1 156, 860 bp,containing nine open reading frames(858, 861, 864, 861, 858, 852, 840, 864, 855 bp).They were named Vh PIP1;1, Vh PIP1;2, Vh PIP1;3, Vh PIP1;4, Vh PIP1;5, Vh PIP2;1, Vh PIP2;2, Vh PIP2;3, Vh PIP2;4. And the accession number of Gene Bank were KM026528, KP164979, KP164980, KP729192, KP765449, KP164981, KP164982, KP729193 and KP729194.2. Bioinformatics analysis demonstrated that Vh PIPs exhibited an internal symmetry showing two highly conserved NPA( Asn-Pro-Ala) motifs and a typical structure with six membrane-spanning domains and possessing the MIP family signal consensus sequence(SGGHINPAVT). And they all had the characteristic signal sequence of plasma membrane intrinsic protein(PIPs) of plant(GGGANXXXXGY and TGI/TNPARSL/FGAAI/VI/VF/YN). They encoded putative Vh PIPs protein with 286、286、287、286、285、283、279、287、284 amino acids. The relative molecular mass were between 29.6 k D and 30.7 k D. The Isoelectric point(p I) were between 7.7 and 8.9. The instability index were between 26.14 and 34.06, so these protein were instability, and they were all hydrophilic molecules(between 0.367 and 0.569), they were no signal peptide. Using DNAMAN bioinformatics software analyzed the homology of 35 PIPs amino acid sequence in Arabidopsis thaliana, V. heyneana, V. vinifera and V. berlandieri×V. rupestris. The result showed that the functional region of them highly-conserved and the homology up to 77.8%. The cluster analysis was carried out by using software, and 35 PIPs was grouped 2 clusters, the PIP1 s occupied one branch and PIP2 s occupied another.3. Quantitative real-time PCR results showed that nine PIPs expressed in roots,stems and leaves,and the expression level of Vh PIP1;1, Vh PIP1;2, Vh PIP1;4, Vh PIP2;1, Vh PIP2;2 and Vh PIP2;4 were the highest the roots of ‘Huaxi-9’, Vh PIP1;5 and Vh PIP2;3 had the highest expression level in leaves, and the expression level of Vh PIP1;3 had no significance in roots, stems and leaves. In ‘Red globe’, Vv PIP1;1, Vv PIP1;4 and Vv PIP2;3 had the highest expression level in roots, Vv PIP1;2 Vv PIP1;3, Vv PIP1;5and Vv PIP2;4 had the highest expression level in leaves, and the expression level of Vv PIP2;1 and Vv PIP2;2 had no significance in roots, stems and leaves.4. In the present study, under drought stress, the expression level of these nine PIPs changed with time extension. There were difference of expression level in different tissue of different grape varieties(plants).. There were four patterns of expression between isoforms:(1) no change(PIP1;2, PIP1;4, PIP1;5 and PIP2;3);(2) first rised and then dropped(Vh PIP1;1, Vh PIP2;1, Vh PIP2;2 and Vh PIP2;4 in the root of ‘Huaxi-9’; Vh PIP1;1, Vh PIP2;1 and Vh PIP2;2 in the stem of ‘Huaxi-9’; Vv PIP1;1, Vv PIP2;1 and Vv PIP2;2 in ‘Red globe’, Vv PIP2;4 in the leaf of ‘Red globe’);(3) decreased with water stress(Vh PIP1;3 in the root, stem, leave of ‘Huaxi-9’, Vh PIP2;4 in the stem and leaf of ‘Huaxi-9’; Vv PIP1;3 and Vv PIP2;4 in the stem of ‘Red globe’, Vv PIP1;3 in the leaf of ‘Red globe’.). So the expression level of PIP1;1, PIP1;3, PIP2;1, PIP2;2 and PIP2;4 had significant difference.5. The stomatal conductance(gs), transpiration rate(Tr), leaf water potential(Ψ), leaf relative water content(RWC) and root, stem and leaf of relative plasma membrane permeability(PMP) were significant differences in different drought resistance of grapes varieties(plants). Under drought stress, stomatal conductance, transpiration rate, leaf water potential and leaf relative water content gradually reduced along with the increase of stress time. The relative plasma membrane permeability of root, stem and leaf gradually increased, so cell membrane stability gradually decreased. There were positively correlated between the expression of PIPs gene and stomatal conductance, transpiration rate, leaf water potential and leaf relative water content. And there was negatively correlated between plasma membrane permeability and the expression of PIPs gene. The correlation analysis of various moisture physiological indexes and the PIP1;1, PIP1;3, PIP2;1, PIP2;2 and PIP2;4 were made. The result showed that there were the largest correlation between the moisture physiological indexes and Vh PIP2;1, Vh PIP2;2, and Vh PIP2;4 of ‘Huaxi-9’.6. The Vh PIP2;1 gene was inserted into multiple cloning sites of the vector p BI121 for obtaining the plant expression vector p BI121-35S-Vh PIP2;1. The vector was transferred into Arabidopsis genome by the combined methods of Agrobacterium-mediated and pollen tube pathway. The results showed that Vh PIP2;1 gene was successfully integrated into the Arabidopsis genome and expressed in Arabidopsis plants. The over-expression of Vh PIP2;1 genein T4 generation transgenic plants were treated, under drought stress, with PEG-6000. And comparing the germination and growth of seedings and the survival rate of transgenic Arabidopsis plants with wild-type Arabidopsis. The results show that over-expression Vh PIP2;1 gene plant were higher tolerance to drought stress than wild type plants. It was significantly higher survival rate than wild type plants, and also had a higher water potential. It would deduced that Vh PIP2;1 regulated the water potential of plant cell and influenced the plants tolerance to stress. |
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