Identification Of Aquaporin Genes In Tea Plant And Study Of CsPIP1;5 Function

Aquaporins?AQPs?are a kind of membrane integrin which can transport water and other small molecular substrates efficiently and extensively on various biofilms in plants.AQPs mediate water transport on the membrane and determine the permeability of cell membrane,cell and plant tissue to water.Under the condition of adversity,the water channel protein of plants can affect gene expression by responding to the signal of external adversity stress,and change the switch,activity,transport and enrichment of channel molecules by using signal transduction pathway,so as to enhance the resistance of plants.Camellia sinensis is an important economic crop for leaf use in China.In the process of tea growth,it is often subjected to abiotic stresses such as high temperature,drought and low temperature,among which drought stress is the most common,which seriously affects the yield and quality of tea.There are few studies on the involvement of aquaporins in water stress in tea plants.In this study,"Fuding Dabai"tea varieties were used as materials to clone and identify the aquaporin gene in tea plants.Through natural drought and rehydration experiments,the expression mode of aquaporin gene response to drought stress in tea was preliminarily explored.The main results are as follows:1. Eight AQPs?aquaporins?genes were cloned and identified from tea leaves by RT-PCR.Amino acid sequence analysis showed that eight Cs AQPs contained SGGHINPAVT,a conserved signal sequence of MIP protein family,and two highly conserved NPA?Asn-Pro-Ala?units,all of which were stable proteins and hydrophobic proteins,and each Cs AQPs contained six transmembrane regions.Six Cs PIPs were mainly located in the plasma membrane and belonged to the PIP subfamily,while two Cs PIPs belonged to the TIP subfamily.2. The tissue-specific expression patterns of eight Cs AQPs genes in different tissues were analyzed.The results of real-time fluorescence quantitative PCR showed that Cs PIP2;4,Cs PIP 2;5,Cs PIP2;7,Cs TIP1;2 and Cs TIP1;4 had the highest expression in roots;Cs PIP1;5 and Cs PIP2;2 had the highest expression in leaves;Cs PIP2;8 had the highest expression in flowers,followed by leaves.3. The expression of eight Cs AQPs in tea plant under abiotic stress such as ABA,20%PEG,high salt,low temperature and waterlogging was studied.The results showed that seven aquaporin genes were down regulated,only Cs PIP2;8 was up regulated.The expression patterns of other stresses in our experiment can be divided into three categories:the first expression of transcription level is up-regulated,then down regulated,and then up and down;the second expression of transcription level is up regulated,then down regulated and up regulated;the third expression of transcription level is down regulated completely,but the degree of down regulated is different.4. Pot experiments of natural drought and rehydration were carried out to investigate the expression of eight aquaporin genes in roots and leaves.The results showed that after drought stress,the expression of 8 Cs AQPs genes in the roots of tea plant was up-regulated,and they were up-regulated to the highest level at 24 days after stopping irrigation,with a range of about 3.5-7.5 times,down regulated again at 29 days after stopping irrigation,and down regulated or kept stable after resuming irrigation.However,in the leaves,the expression patterns of aquaporin were different,Cs PIP2;2,Cs PIP2;5,Cs TIP1;2 and Cs PIP1;5 were slightly induced up-regulated,Cs PIP2;4 were inhibited,and the other three genes remained stable.5. Four high-score phosphorylation sites of Cs PIP1;5 were mutated and transferred to Saccharomyces cerevisiae for functional verification.The results showed that the growth potential of Cs PIP1;5^S207Atransgenic yeast was weaker than that of the control and other mutants under the same condition and dilution concentration,which further indicated that the phosphorylation of Cs PIP1;5 site 207 was indispensable for its essential function.In addition,Cs PIP1;5 was transferred into wild type Arabidopsis and homozygotes with high expression were screened.