| Most of seedless table grape varieties belong to the stenospermocarpy varieties, wherefertilization occurs normally but seeds fail to develop completely because some process likeabnormal PCD appears in ovule abortion cells embryo and/or endosperm.Vacuolar-processingenzyme (VPE) and metacaspases are both type of cysteine dependent protease, play anessential role in mediating and executing programmed cell death in plants. This study used thetypical stenospermocarpy seedless grape varieties: Thomspon and Younger and seed grapevariety: Pinot noir as the main experimental material, in order to analyze the function of βVPEand metacaspase family in different type of ovule development of grapes. The bioinformaticsanalysis showed that the structure, chromosomal location and the conserve motif of the geneβVPE and metacaspases family. Real-Time PCR and Western Blot were used to investigatethe expression pattern of βVPE and metacaspase family (only in transcription level) in thefield of tissue specificity and ovule development in different periods after flowering.Futhermore, GST-VvβVPE (cv.Pinot Noir) fusion protein were expressed by E.coli BL21,then were purified and used to prepare the polyclonal antiserum.The antiserum can be used inβVPE western blot detection in different grape varieties and transgenic plants. Last but notleast, the overexpression ananlysis of βVPE from cv.Pinot Noir and Younger in transgenicArabidopsis plants showed that grape βVPE played an important role in both the process ofthe germination and seedling development.This research obtained such experimental resultsas follows:1. The ovules were harvested from V.vinifera cv. Pinot Noir in8different developmentstages. RNA was isolated from these ovules. VvβVPE gene was obtained by RT-PCR, and themixture of cDNA was used as template. The result showed VvβVPE gene contained acomplete ORF (open reading frame) sequence (1485bp) encoding494amino acid (VvβVPE,Genebank accession number: KC136352). Chromosome positioning analysis showed thatVvβVPE was located in grape unknown chromosome and contained9exons and8introns.Protein sequence alignment analysis showed that the VvβVPE contained the typical VPEfamily H (177), C (219) catalytic dyad active structure and the R (112), R (389), S (395) substrate-binding pocket domain. Phylogenetic tree analysis showed that VvβVPE can bedivided into a subfamily with R.communis and A.thaliana. In addition, we cloned βVPE genefrom10seed and9seedless garap varieties then used their putative protein sequence tomultiple sequence alignment analysis, the result showed that βVPE protein sequencesimilarity from19seed and seedless grape viarities was99.13%.2. The full-length VvβVPE sequence was constructed into the prokaryotic expressionvector of pGEX-6P-1. An about81.3kDa fusion protein of inclusion body (GST-VvβVPE)was expressed in the E coli BL21through IPTG inducing. After optimizing the inducingcondition, two different methods were adopted to purify the protein, containing electrodialysis and dialysis renaturation by GdmCl treating, and then immunized the rabbit to obtainthe polyclonal antibody. Western blotting displayed a specific immunity-antigen signal,suggesting the obtained antiserum can be used for function analysis and detecting transgenicplants.3. Real-Time PCR was used to analyze the expression pattern of βVPE gene on thetranscriptional level. The results showed that all βVPEs from seed and seedless grape wereovule specific-expressed gene. In addition, there are two different expression patterns betweenseed and seedless grapes ovule development process. The βVPE expression quantity in theseed grapes was increased with the extension of ovule development. However, the βVPEexpression quantity in the seedless grapes was straightly at relatively low levels. Moreover,we also analyze the βVPE protein expression pattern on the translational level by western blotmethod. The results revealed that βVPE from seed grapes was existed as a55KD precursorVPE type（pβVPE）in the10day-20day of ovule development period; and then was existed asa40KD intermediate VPE type（iβVPE）in the25day-40day of ovule development period; andwas existed as a35KD mature VPE type（mβVPE）in the45day-70day of ovule developmentperiod in the end. Yet, βVPE from seedless grapes were always existed as the pβVPE type ineach stage of ovule developmental western blot detection. In addition, for further verifyingthe expression pattern of βVPE, we selected8varieties of seed grapes(Pinot Noir,8th Hutai,Carignane, Jiangxi-2, Tonghu-3, Zizhenxiang, Cabernet, Muscat Hamburg) and8varieties ofseedless grapes(Olmo Seedless, Perlette, Dalihong Seedless, Sultanina Rose, ThompsonSeedless, Younger, Flame Seedless, Long-fringe Thompson) ovules of30day and60day afterflowering to proceed expression analysis of βVPE on transcriptional and translational level.All the results verified the previous test consequences.4. Overexpression vectors of βVPE from cv.Pinot Noir and Younger were constructed,and then transformed them in Arabidopsis by agrobacterium-mediated method. Positivetransgenic plants and seeds were obtained for function analysis of grape βVPE gene. The results of SDS-PAGE phenotypic identification revealed that the protein phenotype oftransgenic seeds was similar with the wild type Arabidopsis, but quite different from the VPEquadruple mutant Arabidopsis. Enzyme activity test results showed that overexpression ofβVPE from cv.Pinot Noir and Younger to Arabidopsis could significantly improve the VPEenzyme activity in the transgenic seeds while the VPE activity almost could not be detected inVPE quadruple mutant Arabidopsis seeds. At last, the analysis results of seed germination testshowed that overexpression of βVPE from cv.Pinot Noir could significantly accelerated theArabidopsis seed germination rate.5. We identified the6Vitis vinifera metacaspases VvMC1-VvMC6, from the grapegenome, using BLASTN against the9known Arabidopsis metacaspases. We also obtainedfull-length cDNAs by RT-PCR. Each of the6grape metacaspases contains small (p10) and alarge (p20) conserved structural domains. Phylogenetic analysis of6grape and9Arabidopsismetacaspases showed that all metacaspases could be grouped into two classes: Type I andType II. Each phylogenetic branch shares a similar exon/intron structure. Furthermore, theputative promoters of the grape metacaspases contained cis-elements that are involved ingrape endosperm development. Moreover, the expression analysis of metacaspases usingreal-time quantitative PCR demonstrated that VvMC1, VvMC2were able to be detected in anytissue, and VvMC3. VvMC4, VvMC5, VvMC6exhibited tissue-specific expression. Lastly, incv. Thompson seedless grapes VvMC1, VvMC3, VvMC4were significantly up-regulated at the35DAF during ovule development, roughly same stage as endosperm abortion. We speculatethese genes might be associated with seedless grapes ovule abortive process. |
PDF Full Text Request