Identification Of AQP Gene Family And Analysis Of The Abiotic Stress Resistance Function In Jerusalem Artichoke

Aquaporins（AQPs）is the members of major intrinsic protein（MIP）widely existing in animals and plants.In plants,AQPs are involved in many physiological processes,such as root water absorption and transport,regulation of stomatal closure,transmembrane transport of small molecules including the H₂O₂and CO₂.Previous studies have shown that the expression of the AQPs has specificity.Salt treatment can induce or inhibit the expression of a lot of genes encoding the AQPs,which may play an important role in plant response to salt stress.As a perennial herb,Jerusalem artichoke（Helianthus tuberosus）is belonged to Compositae family,which has a strong ecological adaptability.However,the understanding of the function of genes such as Jerusalem artichoke HtAQP is still very limited.Therefore,this study uses Jerusalem artichoke transcriptome data,screening,identification and analysis of the HtAQPs by bioinformatics.And the expression pattern of the HtAQP genes in Jerusalem artichoke was analyzed by q RT-PCR.On this basis,three stress response genes,the HtPIP1-5,HtPIP1-6 and HtTIP2-2 were selected for cloning,and their structural characteristics,subcellular localization,transfer water activity,and function in response mechanism to abiotic stress were analyzed.The purpose is to lay a theoretical foundation for the study of the AQPs in Jerusalem artichoke.The main research results as follows:（1）There are a total of 59 HtAQP genes were screened and identified from the Jerusalem artichoke transcriptome database.According to phylogenetic trees analysis,59 Jerusalem artichoke HtAQPs belonged to five subfamilies:PIPs（18 subtypes）,TIPs（18 subtypes）,NIPs（15 subtypes）,SIPs（5 subtypes）and XIPs（3 subtypes）.The analysis results of physicochemical properties,conserved domain,characteristic site and subcellular localization prediction of HtAQPs showed that the structural characteristics and subcellular localization of the HtAQPs had diversity and subfamily specificity.（2）The genes expression level of the HtAQP in leaves,stems,roots and tubers of Jerusalem artichoke was analyzed,the results showed that the expression of the HtAQPs could be detected in the four tissues,and about half of HtPIPs and HtTIPs had high transcription levels in the four tissues.In addition,some genes,such as HtPIP2-6,HtTIP1-5,HtTIP1-6,HtTIP2-2 and HtTIP2-6,are highly expressed in stems and tubers,while HtTIP2-1 is specifically expressed in roots and leaves.（3）The genes expression of the HtAQP is affected by abiotic stress.Under salt stress,the expression level of most HtAQPs showed an overall down-regulated,and the up-regulated genes mostly belong to HtPIPs or HtTIPs subtypes.In the initial stage of drought stress simulated by PEG6000,the number of up-regulated and down-regulated HtAQPs was roughly the same.But with the extension of stress time,the number of down-regulated HtAQPs was increased gradually.In the leaves of Jerusalem artichoke,there are six PIP subtypes including HtPIP1-5,HtPIP1-6,HtPIP2-2,HtPIP2-3,HtPIP2-6 and HtPIP2-7,and seven TIP subtypes including HtTIP1-1,HtTIP1-2,HtTIP1-5,HtTIP1-6,HtTIP2-2,HtTIP2-5,HtTIP2-6 and HtTIP2-4 was up-regulated expression under salt（200 m M Na Cl）and drought（15%PEG6000）stress.These results suggest that HtAQPs may play an important role in plant responses to water stress.（4）Jerusalem artichoke aquaporin HtPIP1-5 and HtPIP1-6 belong to plasma membrane intrinsic proteins,and they have high homology with the sunflower HtPIP1-4 and HtPIP1-3.The subcellular localization of the HtPIP1-5 and HtPIP1-6 was verified by using the protoplast transient expression system.The results showed that the both were located in the plasma membrane and endoplasmic reticulum of Arabidopsis protoplasts.This has been confirmed that the HtPIP1-5 and HtPIP1-6 are channels for transmembrane transport of water molecules.Moreover,ectopic expression of the HtPIP1-5 and HtPIP1-6 could increase tolerance of the Saccharomyces cerevisiae to salt stress.（5）Ectopic expression of the HtPIP1-5 and HtPIP1-6 could promote the vegetative growth of Arabidopsis.The primary root length,cotyledon and rosette leaf area of Arabidopsis plants overexpressing the HtPIP1s are significant better than those of the wild type plants,and the stomatal conductance,intercellular CO₂concentration and transpiration rate were also higher.In addition,overexpression also increased the expression level of the At AP、At FT、At LFY and At SOC1 in Arabidopsis rosette leaves,inhibit the expression of the At FLC,and promoted early bolting and flowering of the transgenic Arabidopsis.（6）Ectopic expression of the HtPIP1-5 and HtPIP1-6 could enhance the tolerance of Arabidopsis to high salt and drought stress.Physiological data showed that the transgenic Arabidopsis plants with HtPIP1s had higher germination rate and longer primary roots than the wild type plants under drought stress,and the RWC of leaves was also higher than the wild type,while the ion leakage rate（IL）,the proline,H₂O₂and MDA contents were lower.Under high salt stress,the stomatal conductance,transpiration rate,Ca²⁺content,photosynthetic pigment content and chlorophyll fluorescence parameters（such as Fv/Fm,NPQ and Rfd）of transgenic Arabidopsis were also significantly higher than wild type.（7）The ORF of Jerusalem artichoke HtTIP2-2 gene is 747 bp,its expression level is relatively high in tongue flower,stem and tuber.The HtTIP2-2 expressing in the leaf and root tissue is induced by salt and drought stress.The HtTIP2-2 belonging to the tonoplast membrane intrinsic protein subfamily.The sequence similarity between HtTIP2-2 and sunflower HtTIP2-1 is as high as 98.79 percent.The HtTIP2-2 has water transfer activity,and it’s mainly located in the plasma membrane and PM surrounding vesicles.Ectopic expression of the HtTIP2-2 could increase the tolerance of Saccharomyces cerevisiae to salt stress.（8）Ectopic expression of the HtTIP2-2 could promote the root hair growth of Arabidopsis and endow with stronger stress tolerance.Physiological data showed that the expression of the HtTIP2-2 could inhibit stomatal closure induced by ABA.Under salt stress,the transgenic Arabidopsis with a HtTIP2-2 gene had higher stomatal conductance and transpiration rate than the wild type,and the K⁺content,Ca²⁺content,chlorophyll content and K⁺/Na⁺in leaves were also significantly higher.Moreover,the plants with HtTIP2-2 gene had higher RWC under drought stress,while the H₂O₂content,MDA content and IL in leaves were significantly lower than wild type plants.This study provides the family information about Jerusalem artichoke AQPs,and preliminarily reveals the role of HtPIP1-5,HtPIP1-6 and HtTIP2-2 in plant stress resistance,which would be helpful for further studies on the function and regulation mechanism of HtAQPs.