| Banana is an annual and herbaceous plant, it likes high temperature and humidity. The leaves become thinner, the fruit becomes shorter and grows delay when lacking of water, it will be resulting in reduction of output. It is necessary to breed for stress tolerance. Therefore, looking for the stress resistance genes from the bananas and researching for the mechanism of stress tolerance is important. For this purpose, we obtained the cDNA fragment of the Aquaporin gene family from the suppression subtractive hybridization library(SSH) in the early stage of the banana’s ripening. On the basis of cloning the Aquaporin gene from the banana fruit by RACE technology, we used the model plant Arabidopsis thaliana to further explore aspects of the gene in some of the features and stress tolerance mechanism. We hope to solve the problem in the stress tolerance by manipulating the expression of these genes.AQPs locate in the specific cell membrane area in the plant. The water, small molecules such as glycerol and some ions can penetrate the membrane. MaAQP1 (Musa acuminata aquaporin)contains 861 bp open reading frame and six putative transmembrane domains with two highly conserved motifs of asparagine-proline-alanine (NPA) and aromatic/arginine (ar/R) region. In the vital biomembrane, it exits in the tetramer form. Many experiments show that the plant can response to some kinds of the abiotic stress by regulating the AQPs activity.When analysing the MaAQP1 expression in different organs of banana, the results demonstrates that MaAQPl is expressed in all organs and the expression level is higher in banana root, suggesting it may play important role in these tissues. When treated by a series of abiotic stress, including salt, drought, waterlogging, low temperature, ethephon, harm, we found that the expression of MaAQP1 was sharply induced when treated by drought and salt stress except other stress, indicating that MaAQP1 gene mainly participate drought and salt stress responsive pathway.During the process of banana fruit postharvest ripening and induced by exogenous ethylene, the mRNA level of MaAQPl gene decreased gradually.On the opposite, the expression level of MaAQPl increased gradually when treated with 1-MCP. It shows that MaAQP1 responses to ethylene signals in the fruit ripening process by regulating the transport of water so that negative regulating on ethylene biosynthesis.To understand the MaAQP1’s function in stress and its molecular mechanism, we inserted the coding region of MaAQP1 to the pCAMBIA1304 plant expression construction driven by 35S promoter. We used the model plant Arabidopsis thaliana which the genetic background is clear to study the stress response mechanism of MaAQP1.The plant expression vector pCAMBIAI304-MaAQP1 was transformed into Arabidopsis thaliana. As a result,35 genetic transformant T1 plants with hygromycin-resistance have been obtained. The result of PCR test showed that 29 genetic transformant T1 plants were positive. Then we obtain the Homozygous lines in T3 plants from the T2 plants with 3:1 segregation ratio. Selecting 5 lines for Southern blot analysis, the result verified that the MaAQP1 was integrated into the genomes of Arabidopsis thaliana with both single-copy and multi-copy integration of the target gene. These five transgenic plants were named L16, L13, L6, L8, Ll-5. RT-PCR analysis showed that MaAQP1 expresses in all organs of transgenic Arabidopsis thaliana and the expression level is higher in root. This result was consistent with the expression lever of MaAQP1 in the banana.Analysising the transgenic lines and the wild-type, we find that the root of the transgenic lines is longer than the wild-type.And its root hair is much more when growing to 3 days. LI6 and L13 represent two independent lines which show different copy integration of the target gene, for researching the resistance stress mechanism of MaAQPl. When treating with drought and Nacl, L16 and L13 exhibited stronger stress tolerance than the wild-type during the stage of seeds germination. And we found that the larger amount of transgenic lines survived than wild type when treating adult plants of all the lines with 350mM NaC1 and drought. Therefore, the change of the root in the transgenic lines may promote the water transporting and regulation, suggesting that it is associated with increasing the resistance stress.To deeply explore whether MaAQPl confered stress tolerance in Arabidopsis depending on ABA signal pathway or not, we analysed the change of ABA/stress responsive gene expression under drought and NaCl stress. In the ABA signal pathway, some genes both response to ABA and stress, and the others are independent on ABA but only response to stress. The result showed that the expression change of the gene which both response to ABA and stress in the transgenic lines is the same with the wild-type under drought and NaCl stress. The expression of these genes was induced lower in the transgenic lines than in the wild-type, which revealed that MaAQPl could improve the ability against the stress.Therefore, the stress response genes in plants will be induced strongly only under the heavy external stress. And the expression mode of the other response genes which were independent on ABA were different in the transgenic lines and the wild-type. We speculated that MaAQPl-mediated stress resistance mechanisms in transgenic plants may be dependent on ABA signal transduction pathway.For the regulation of plant roots growth by MaAQPl, we studied the root growth mechanism in the transgenic lines. Root growth is regulated by a variety of factors, one of the major factor is the influence of the water and nutrients on the growth of roots. Another is the plant hormone. AQPs mainly transport the water and other small molecules in plants. So we would like to know that MaAQP1 whether involved in regulatory pathways of the plant hormone for promoting the plant growth. Then we selected some plant hormones including auxin, cytokinin, ethylene, brassinosteroid genes to study. We found that the expression of these genes was higher in the transgenic lines than in the wild-type. The expression of the auxin, cytokinin and brassinosteroid genes was higher than ethylene genes. And the expression ratio of these three hormone genes and ethylene-like genes was higher in the transgenic lines than in the wild-type. It showed that the genes which promoting the plant growth playing a major role in the transgenic lines. And the growth of plant roots was not only regulated by a single hormone, but may be the network constituted by a series of hormone. It is connected with the ratio between the hormone. |
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