Cloning And Analysis Of VPE Family And The Differentially Expressed Gene In Ovule Abortion Of Seedless Grape

For study the formation mechanism of the seedless grape in the future,the molecularmarker technology was used to scan the grape ovule abortion relate gene. in this study,theSRAP-cDNA technology was used to scan the genes which have different expression patternsduring ovule development between the V. vinifera cv. Youngle and V. vinifera cv. Pinot Noir.Analysis the different expression EST in two grape,then,cloned them after sequenceanalysis,9genes were selected to analysis their expression profile in different tissues andexpression in the ovule development of V. vinifera cv. Youngle and V. vinifera cv. Pinot Noir.finally,YG7931and YG1891were choosed to do more test. Their sequence were cloned formV. vinifera cv. Thompson Seedless,and the qRT-PCR was used to analysis their expression inthe ovule development of V. vinifera cv. Thompson Seedless and V. vinifera cv. Pinot Noir. theresults suggested the VvWRKY20and VvABCG2were the grape ovule abortion relate gene.The VPE family was also studed in the genomic and transript level. The main results are asfollows:1,the SRAP-cDNA technology was used to amplified and the differentially expressedfragments between the V. vinifera cv. Youngle and V. vinifera cv. Pinot Noir were analysised.Then25EST sequences was cloned,preliminary analysis this genes whether was the grapeovule abortion relate gene.2,9genes from the25EST were selected to analysis their expression profile in differenttissues of V. vinifera cv. Youngle.And found the YG1891was only expressed in flowers, budsand ovules,the YG2951was not expressed in the root, peel and pulp.other genes wasexpressied in all tissues.3,The qRT-PCR was used to analysis the expression of the9selected genes in the ovuledevelopment of V. vinifera cv. Youngl. and V. vinifera cv. Pinot Noir. finally, and foundYG1891,YG38122,YG5571E,YG7931may be the grape ovule abortion relate gene.4,The VvWRKY20was cloned from V. vinifera cv. Thompson Seedless, according to theYG7931sequence information. the qRT-PCR was used to analysis the expression ofVvWRKY20in the ovule development of V. vinifera cv. Thompson Seedless and V. vinifera cv.Pinot Noir,found the expression in Thompson Seedless were significant difference,but in the Pinot Noir were relatively stable,and the expression level is smaller than that of ThompsonSeedless.this results suggested the VvWRKY20may be the grape ovule abortion relate gene.5,The VvABCG2was cloned from V. vinifera cv. Thompson Seedless, according to theYG1891sequence information. Sequence analysis found that a78bp sequence fromThompson Seedless was missed compared with Pinot Noir', and the missing sequence washappen to translation a transmembrane domain. The qRT-PCR was used to analysis theexpression of VvABCG2in the ovule development of V. vinifera cv. Thompson Seedless and V.vinifera cv. Pinot Noir, the expression pattern of VvABCG2in two grape was different, andthe expression level is higher in seedless than in the seeded. These results suggested theVvABCG2may be the grape ovule abortion relate gene.6,In the research of vacuolar processing enzyme(VPE),βVPE,γVPE,δVPE werecloned from V. vinifera cv. Pinot Noir. γVPE,δVPE were cloned from V. vinifera cv.Thompson Seedless. βVPE were cloned from V. vinifera cv. Youngle and found7alternativeSplicing. In the grape δVPE, one of the three crucial amino acids Ser was chang as Ala.Tissues-specific expression analysis found that βVPE was expressed in roots, flowers, budsand ovules. The γVPE was expressed in various tissues. The δVPE was only expressed inflowers, buds and ovules. The expression of βVPE were differently in the late ovuledevelopment of seeded and seedless grape. The genomic sequence of βVPE form V. viniferacv. Youngle,V. vinifera cv. Thompson Seedless and V. vinifera cv. Pinot Noir have a highindentity as98.76%. The promoter of βVPE has a little differently between V. vinifera cv.Thompson Seedless and V. vinifera cv. Pinot Noir, the number of Skn-1_motif was differently,which is a cis-acting regulatory element required for endosperm expression, especially, thesequences near the Skn-1_motif group were missing in V. vinifera cv. Thompson Seedless.these results suggest the βVPE was the the seedless grape ovule abortion related gene.