Preliminary Cloning Of The Promoter Region And Functional Analysis Of Plasma Membrane Intrinsic Protein Gene CcPIP1in Hickory

Hickory(Carya cathayensis Sarg) is the important oil woody plants plants. The hickory fruit isthe famous dried fruit and woody oil plant and is endemic to China, with high economic value.Grafting is an important means to solve the gardening and seed cultivation of hickory. Water is animportant component of the plant cell, the plant aquaporin is rapid water transport channel ofintercellular and intracellular, as a subfamily of plant aquaporin, the plasma membrane intrinsicprotein PIPs located in the plasma membrane are typical water selective channel proteins.GenomeWalker Universal technology was applied to clone the promoter of plasma membraneintrinsic protein of hickory (CcPIP1). Real time RT-PCR technology was applied to preliminary studyits expression and controlling in hickory grafting seedling. The main results are summarized asfollowing:1. The total RNA with high quality, integrality and no-impurity extracted with modified CTABmethod was suitable to gene clone and expression analysis. The cDNA of hickory grafting seedlingreversely transfered by using SMARTTMPCR cDNA Synthesis Kit were integrate, and touch therequirement of experiment.2Using digested DNA as template, we designed the special primers WK1, WK2, WK3,according to the cloned sequence and obtained CcPIP1promoter by Genome Walker Universaltechnology. The total length of obtained CcPIP1promoter is876bp. According to PlantCARE andPLACE online software, the sequence has the general characteristics of eukaryotic promotersequences, two CAAT box, seven TATA box and one G-box. Also the other types of cis-actingregulatory elements were found.3Using quantitative RT-PCR technology,we analysis the hickory CcPIP1gene expression ofthe different treatments (control,50μM ABA,100mM NaCl,250mM mannitol, and5mM DTT)during the different grafting days (0d,1d,3d,5d,7d,14d). The results show that: under control,CcPIP1gene expression in the scion decreased sharply3days after grafting and increased in thesubsequent4days, and then decreased. While CcPIP1gene expression in the rootstock decreasedsharply1day after grafting and increased in the subsequent6days. And the expression level in7daysafter grafting researched the highest, and then decreased. Under100mM NaCl treatments or50μMABA treatments, the model of CcPIP1gene expression was the same with the control, but the geneexpression level was lower than that in the control during the different days after grafting respectivelyunder100mM NaCl treatments. While the gene expression level was higher than that in the controlduring the different days after grafting respectively under50μM ABA treatments. Under DTTtreatment, the CcPIP1gene expression both in the rootstock and scion decreased at first day aftergrafting, then increased till14d after grafting. CcPIP1gene expression decreased sharply1day after grafting In the subsequent13days after grafting, CcPIP1gene expression increased gradually andresearch the highest14days after grafting. But under mannitol treatment, there is no regular pattern ofthe CcPIP1gene expression.4The relative conductivity in the hickory during the different grafting days (0d,1d,3d,5d,7d,14d) under different treatments (Control,50μM ABA and5mM DTT) was measured. The resultsshow that: Under control, the relative conductivity decreased gradually during the grafting. Therelative conductivity was51.7%before grafting in the scion while the reative conductivity decreasedto39.2%14days after grafting. But in the rootstock, the range of relative conductivity was from33%to72%. Under50μM ABA treatment, the relative conductivity was51.7%and45.4%in the scion androotstock respectively before grafting while that decreased to33.6%and26.9%14days after graftingin the scion and rootstock respectively. Under DTT treatment, the relative conductivity in the scionand rootstock decreased14days after grafting.