Functional Identification And Mechanism Analysis Of Pear V-ATPase Gene PbVHA-B1

Pear（Pyrus）is one of the most economically valuable fruits in China with a wide range of distribution.It has won the favor of consumers with its unique flavor and excellent quality.Fruit flavor is the most important quality in fruit standard evaluation in addition with appearance.The organic acid together with sugar,soluble solids and other substances codetermine the fruit flavor.According to the content and composition of organic acid,the fruit can be divided into three different acid types:malic acid type,citric acid type and tartaric acid type,most of pear fruit belong to malic acid type and a few of them partein to citric acid type.Compared with citric acid,malic acid is more palatable and acceptable,so the level of malic acid greatly affects the flavor of pear fruits.Due to customer preference and the wide range of cultivation,the cultivation of pear trees also face with many challenges of abiotic stress,among which salt stress has a great impact on the cultivation of pear.Therefore,mining the genetic factors related to abiotic stress tolerance and searching for functional genes related to organic acid accumulation is an important task to promote the development of pear industry.Many important physiological processes in plant life depend on the regulation of plant vacuolar H+-ATPase.V-ATPase make use of the energy released by ATP hydrolysis to establish a transmembrane electrochemical gradient（ΔΨ）,which provides a strong driving force for the transport of ions in cells and the transmembrane transport of metabolites.As a B subunit involved in the assembly of V-ATPase,corresponding studies in different species have shown that it can respond to both salt stress and organic acid accumulation.However,the research of V-ATPase gene in pears is still rare.In this study,33 V-ATPase genes were screened from pears and clustered with V-ATPase genes from apples,PbVHA-B1 gene was selected as the research object ultimately.We analyze the expression levels of PbVHA-B1 gene under different stress conditions,and the expression patterns in different varieties of pear fruits.Then we studying the function of PbVHA-B1 gene in improving plant salt tolerance by identifying the functions of transgenic lines and wild-type Arabidopsis thaliana,functional verification of transgenic and wild-type Arabidopsis thaliana,virus-induced gene silencing pear fruits and untransformed pear fruits to investigate the response of PbVHA-B1 gene to malate accumulation.The main research results shown as follows:1.In this study,33 V-ATPase genes were identified in pear by BLAST and were divided into many subfamilies with the V-ATPase genes in apple by phylogenetic analyses.The expression pattern show that the expression of PbVHA-B1 is the most sensitive one under salt stress and the expression level of PbVHA-B1 increased under the salt,dehydration,ABA and low temperature stresses.Overexpression the PbVHA-B1 could improve the salt tolerance of arabidopsis transgenic lines.The transgenic lines had higher germination rate,the root grow longer,leaf senescence delayed and had higher Chlorophyll Content.The lower malondialdehyde MDA accumulation and conductivity level indicated that the membrane of transgenic lines was in good condition.Meanwhile,the transgenic lines could regulate the activity of peroxidase（POD）,catalase（CAT）and superoxide dismutase（SOD）to relieve the accumulation of hydrogen peroxide（H2O2）and superoxide anion（·O2-）.The overexpression of PbVHA-B1 could help transgenic lines to absorb K+ and eliminate Na+ to matain the ion balance and cellular homeostasis.These results indicated that Pb VHA-B1 plays an important role in improving salt tolerance of plants.2.The results show that organic acids of different components are contained in the fruits of 8 pear varieties of 3 different cultivars,’Dananguoli’ ’Nanguoli’ ’Dangshansuli’ ’Cuiguan’’Yali’ ’Jingbaili’ ’Huangguan’ ’Yuluxiang’,of which malic acid is the main organic acid.During the dynamic development of ’Dangshansuli’,PbVHA-B1 gene had highest levels of expression in the early stage of fruit development and then decreased.Interestingly,the expressin pattern of Pb VHA-B1 was consistent with the content of malic acid in the fruit of’Huangguan’ ’Yali’ and ’Yuluxiang’.The content of total acid and malic acid in the Arabidopsis transgenic lines overexpressed PbVHA-B1 was higher than the wild lines.Meanwhile,down-regulation of PbVHA-B1 in the fruit of ’Huangguan’ by virus-induced gene silencing（VIGS）resulted the lower content of total acid and malic acid.These results indicated that the expression of PbVHA-B1 is positively related with the accumulation of malic acid in plants.