Identification And Expression Analysis Of VP Gene Family In Cotton

Cotton,one of the most important fiber crops,is a pioneer crop in the saline-alkali fields.The research on the mechanism of salt tolerance is increasingly important to improve the salt tolerance of cotton.H+-translocating Inorganic Pyrophosphatase(H+-PPase),an proton pump in vacuolar membrane of the plant cells,is presented as pyrophosphate(PPi)substrate,and usually plays an important role in secondary active transport process,such as the regulation of expansion of cells,H+ electrochemical gradient,inorganic ion,organic acids and sugars.The enzyme activity increases to facilitate the expression of VP under the stress of cold stress,drought and high salt stress,which can enhance the resistance to the stress by regulating the inner balance in cells.This study systematicly analysed the VP gene family memebers in cotton for the first time.Gossypium raimondii L.,Gossypium arboreum L.(Shixiya 1)and Gossypium Hirsutum L.(Zhong 9807?ZhongS9612?Zhong9409?GK50?Y2067 and GZ)were chosen,from which VP gene family members were cloned.According the expression differences of all members under the stress of 0.4% Na Cl and seed germination ability variation of transgenic seeds,we analysed the function of VP gene.The main research results are as follows:1.VP gene family members were comparatively systematicly analysed in cotton genome A and D.Based on cotton genome database of(A genome and D genome),13 VP family genes,including 6 in genome A and 7 in genome D,were identified with the bioinformatics method,using the sequence GhVP(Gene Bank No.: ADN96173.1)as the query sequence and their gene sequences and chromosome location and gene structure information were obtained.The result showed that the VP family was relatively conservative in plant evolution.2.The evolution of VP gene family was analysed.From the analysis of the phylogeny tree,VP genes had a high sequence similarity and a relatively close relationship within the same species.Genetic structure analysis showed that the number of exons in most VP genes is 7-8,and only a few VP genes had a large quantity variance,ranging from 1-14.From the analysis of conservative structure domain,VP gene members of the VP family had a multiple relatively conservative structure domain,such as N-myristoylation site,Casein kinase II phosphorylation site,protein kinase C phosphorylation site and so on,which can provide an important basis for the further study of the function of VP gene family in cotton.3.The expression differences of VP gene family were analysed.The qRT-PCR showed that a different expression level of gene members in the VP family existed in different tissues,e.g.,highest in leaves and lowest in fibers,suggesting that expression of the VP genes is tissue-specific.Moreover,with the salt stress intensity increases,the changes of expression level of the VP genes were different.4.Gene GhVP was cloned.In this study,pBI121-GhVP::GFP fluorescence expression vector was constructed and was transformed into onion epidermal cells with the gene gun technology.The findings indicated that gene GhVP was located on cytomembrane.Fluorescence expression vector pBI121-GhVP::GFP was constructed and transformed into cotton pollen.The results showed that the green fluorescence of the pollen was enhanced,indicating gene pBI121-GhVP::GFP could express in cotton,which could provide the evidence for the study of GhVP in cotton.5.The expression difference of GhVP and GhNHX was performed.Fluorescence expression vector pBI121-GhVP::GFP,over-expression vector pBI121-GhVP::NHX and RNAi vector pBI121-PH::VP were constructed successfully,and transformed into cotton with gene gun living conversion technology.The results of germination test of transgenic seeds indicated VP was associated with salt resistance,and salt resistance of cotton seeds can be improved through the transformation of VP.The study analysed the VP gene family members and the expression difference of VP in cotton and tested the germination ability of transgenic seeds,laying the foundation for the research of molecular mechanism of salt tolerance in cotton.