VHA Gene Family Cloning And Gene Expression In Eriobotrya Japonica(Thumb.)Lindl.

The plant vacuolar H+-ATPases(V-ATPases)are transporter proteins with complex functions on the plasma membrane,which is responsible for acidification of intracellular compartments,proton transport across the plasma membrane,and providing membrane-transport energy by hydrolysis ATP,thereby affecting the secondary metabolites synthesis and transportation in vacuolar,such as accumulation and degradation of organic acids.In this study,we used five different tissues of’Jiefangzhong’(high-acid cultivar)loquat,including fruit,stem,callus,leave and flower,and various developmental stages of loquat fruit as experimental materials taking use of RACE,RT-PCR,green fluorescent protein fusion and tranformation technology,we got nine members of EjVHA gene family:EjVHA-1,EjVHA-2,EjVHA-3,EjVHA-4,EjVHA-5,Ej VHA-6,EjVHA-7,EjVHA-8 and EjVHA-9.And EjVHA-6,EjVHA-7 and EjVHA-9 regulated organic acid synthesis in loquat fruit were revealed,while EjVHA-4,EjVHA-5 and EjVHA-8 degraded the concentration of organic acids.We also identifed the EjVHA-7 and EjVHA-8 which are belong to chloroplast VHA,and the transgenic tobacco plants of EjVHA-7 promoted plant growth at low temperature stress.The principal results were as follows:1 Cloning and bioinformatics analysis of VHA family genes from loquat fruitThe full-length cDNA of VHA genes were cloned from loquat fruit(’Jiefangzhong’)by RACE method according to the RNAseq database of loquat V-ATPase gene fragment.We have obtained nine VHA gene sequences EjVHA-1(KJ544915),EjVHA-2(KP201499),EjVHA-3(KJ195449),EjVHA-4(KJ195450),EjVHA-5(KJ195452),EjVHA-6(KF766121),EjVHA-7(KJ195451),EjVHA-8(KJ195447)and EjVHA-9(KJ195448),and the all full-length cDNAs were:2 362 bp,2 133b p,1 333 bp,888 bp,797 bp,1 178 bp,1 347 bp,624 bp,615 bp respectively.The nucleotide and protein sequencing homology analysis by BLAST of NCBI disclosed that the cloned fragments are V-ATPase subunits and are highly identical to the published sequences.We cloned seven gDNA sequences of VHA gene family.The full-length of genomic EjVHA-3 is 3 047 bp,containing 9 introns and 10 exons.EjVHA-4 and EjVHA-5 full-length gDNA are 2 513 bp and 1 400 bp respectively,both of them are composed of 2 introns and 3 exons with the same structure.EjVHA-6 genome DNA is 780 bp without intron.The gDNA of EjVHA-7 is 2 017 bp comprising 5 introns and 6 exons;EjVHA-8 and EjVHA-9 gDNA are independently 1 141 bp and 1 155 bp,driven by 2 introns and 3 exons,the main distinction between EjVHA-8 and EjVHA-9 gDNA is the length of two introns.The bioinformatics analysis showed that all these VHA proteins did not have a typical signal peptide,indicated that they were not secretory protein.However,EjVHA proteins were filled with abundant specific and non-specific domains,including specific domains of ATP-synt-ab,V-A-ATPase-A,ATP-synt-ab-Nand non-specific domains of ATP-synt-ab,V-ATPase-C and COG5127.The transmembrane structure of EjVHA proteins were also ample,apart from EjVHA-1,EjVHA-4 and EjVHA-5,EjVHA proteins did not have a transmembrane domain.In EjVHA protein,phosphorylation sites mainly involved serine,threonine and tyrosine,which was dominated by serine,phosphorylation of threonine and tyrosine acted as a supplement.Threonine sites were not existed in EjVHA,while EjVHA-9 and EjVHA-8 had no tyrosine sites.The results of subcellular localization prediction unveiled that loquat VHA family gene members were not only existed in the chloroplast and cytoplasm,but also distributed in the vacuole,chloroplast thylakoids,nucleus and mitochondria.The phylogenetic tree illustrated that EjVHA protein had the closest relationship with Malus domestica followed by Citrus sinensis.Notwithstanding,it is far away from Arabidopsis thaliana.2 Analysis of EjVHA expression in different tissues from loquatRT-PCR was used to analysis the expression of EjVHA gene among five different tissues of loquat,including fruit,stem,leave,callus and flower.The results showed that the expression of EjVHA-1 was highest in stem,while the expression in the callus is the lowest;a high level of EjVHA-2 expression was occured in all tissues,and it was most expressed in stem;all of EjVHA-3,EjVHA-4 and EjVHA-5 were stem-specific;EjVHA-6 was flower-specific;EjVHA-7 was highly expressed in stems and flowers,and the expression in other tissues is generally low;whereas both of EjVHA-8 and EjVHA-9 had the similar expression pattern in various tissues,for one thing,they were all expressed at a high level in the stem and flower,for another,they had the highest expression level in stem and lowest expression in fruit.So the major difference between them was the expression levels,namely,the levels of EjVHA-8 gene in different tissues were higher than that in EjVHA-9.3 Determination of organic acids content and EjVHA gene expression analysis during fruit developmentA simple and accurate high-performance ion-exchange chromatography(HPIC)was used to determine the organic acids content in loquat fruit during fruit development period,the results suggested that the contents were increased in the early stage and then the contents of organic acid accumulation were peaked at 123 days after full bloom(DAFB),organic acid concentration rapidly drops when it started expand and became mature.RT-PCR detected the expression of EjVHA genes during loquat fruit growth,and it was found that the expression pattern of EjVHA gene family members is not the same.According to the correlation between the content of organic acids during its growth and the expression of EjVHA genes,they are mainly divided into three types,one is the genes which involve the process of synthesis of organic acid involving EjVHA-6,EjVHA-7 and EjVHA-9.The second one is related to organic acid degradation,including EjVHA-4,EjVHA-5 and EjVHA-8;as for EjVHA-1,EjVHA-2 and EjVHA-3,they are classified into the last type,which has no evident connection with organic acid.4 Study on EjVHA-7 and EjVHA-8 gene sublocation and EjVHA-7 transformation of Nicotiana tabacum L.The EjVHA-7-GFP and EjVHA-8-GFP subcellular location vector were constructed,and the recombinant plasmid of 35S::EjVHA7-GFP and 35S::EjVHA8-GFP were transformed into the leaves of tobacco,then observed under fluorescence confocal microscopy,the results revealed that EjVHA-7 and EjVHA-8 have a strong green fluorescence signal in the chloroplast of tobacco,in conclusion,the EjVHA-7 and EjVHA-8 are located in the chloroplast.The pCAMBIA1301-35S:EjVHA7 over-expression vector was constructed,and transferred into tobacco.Transgenic tobacco plants consisting of the target gene were screened by PCR detection.And the results of qRT-PCR determination indicated that the expression level of EjVHA-7 in transgenic tobacco is highest,but the expression of non-transgenic tobacco plants and pCAMBIA1301 empty vector transgenic tobacco plants are low.The seeds of EjVHA-7 transgenic tobacco(T1 generation)were placed in culture medium at 25℃ containing hygromycin unearthed that non-transgenic seeds are unable to grow normally,most of transgenic tobaccos are able to grow normally as well and germination rate is at the high level.T1 generation of EjVHA-7 transgenic tobacco,pCAMBIA 1301 transgenic tobacco and non-transgenic plants were seeded in MS culture medium and plug tray with nutrient soil under 25℃,disclosed that T1 of EjVHA-7 transgenic tobaccos grow generally better than those of pCAMBIA 1301 transgenic tobacco and non-transgenic plants in MS culture medium.The seed germination rate of transgenic and non-transgenic tobacco at condition of 15℃ are slower than that at 25℃.Moreover,the seed germination of transgenic tobacco is two days earlier,come with a growth faster than non-transgenic tobacco at 15℃.Above all,the over-expression of EjVHA-7 transgenic tobacco has the advantage of growth and phenotype is more obviouse at low temperature,which suggested that EjVHA-7 could response to low temperature and may play an important moderating role in the process of plant stress resistance.