Cloning Of The Genes Involved In Abortion Based On The ESTs Sequence In The Seedless Grape Cultivar Thomson Seedless

Seedless grapevines are conveniently edible and with good quality. They are widely used for the fresh market and raisnins. Seedless grapevines in cultivation are seed abortive. The fertilized ovules cannot develop the normal seed and only form seed trace when fruit mature. The abortion of ovules are highly invariable and not affected by external factors at various times and places. It is under the control of genes in seedless grapevines, which are valuable for research and utilization.In this study, a cDNA library was prepared from pre- and post abortive ovules of Thompson Seedless grapevines. EST and PCR techniques were used to clone the abortion-related genes and their up-stream regulatory sequences. Meanwhile, the activity of reactive oxygen species and reactive-oxygen-scavenging enzymes were analyzed in'Jingxiu'and seedless grapevine'Youngle'during ovules abortion/development, to reveal the trends of these enzymes and explore the relationship between the change of reactive oxygen species and seed abortion. The potential results are as follows:1. RNA extraction and constructing cDNA library. At 27d, 30d, 33 d, 36d, 39d, 42d, 45d and 48d post-anthesis, ovules were collected from Thompson Seedless and snap frozen in liquid nitrogen. Total RNA was isolated respectively from above samples with SDS/phenol method modified partially. The pool of total RNA at various times was used to construct the cDNA library. The titer of primary library was 5.87×105 pfu/ml. The effective library capacity is 5.55×105 pfu. The recombinant percent accounted for 96.60%. The size of insert fragments basically ranged from 0.5 kb to 2.0 kb with average of 0.9 kb. The titer of the amplified library was 2.315×109 pfu/ml.2. Sequencing and analysis of EST. 214 sequences with high quality were obtained. These sequences were searched in GenBank nr database for homologue using Blastn and Blastx programs and were further classified based on their functions. The results are as follows: 144 EST sequences accounting for 67.29% showed high homologue; 17 EST sequences accounting for 7.94% matched to the genes whose protein function were unclassified; 53 EST sequences accounting for 24.77% hit no any sequence in GenBank. 9 of the 53 represented novel ESTs. 60 full length sequences accounting for 28.03% of the library sequences were obtained. The 167 cDNA sequence have submitted to GenBank database, the GenBank accession numbers are as follows,EY254594,EY254595,EY254596,EY254922,EY254923,EY254924,EY254925,EY254926,EY254927,EY254928,EY254929,EY254930,EY254931,EY254932,EY254933,EY254934,EY254935,EY254936,EY254937,EY254938,EY254939,EY254940,EY254941,EY254942,EY254943,EY254944,EY254945,EY254946,EY254947,EY254948,EY254949,EY254950,EY254951,EY254952,EY254953,EY254954,EY254955,EY254956,EY254957,EY254958,EY254959,EY254960,EY254961,EY254962,EY254963,EY254964,EY254965,EY254966,EY254967,EY254968,EY254969,EY254970,EY254971,EY254972,EY254973,EY254974,EY254975,EY254976,EY254977,EY254978,EY254979,EY254980,EY254981,EY254982,EY254983,EY254984,EY254985,EY254986,EY254987,EY254988,EY254989,EY254990,EY254991,EY254992,EY254993,EY254994,EY254995,EY254996,EY254997,EY254998,EY254999,EY255000,EY255001,EY255002,EY255003,EY255004,EY255005,EY255006,EY255007,EY255008,EY255009,EY255010,EY255011,EY255012,EY255013,EY255014,EY255015,EY255016,EY255017,EY255018,EY255019,EY255020,EY255021,EY255022,EY255023,EY255024,EY255025,EY255026,EY255027,EY255028,EY255029,EY255030,EY255031,EY255032,EY255033,EY255034,EY255035,EY255036,EY255037,EY255038,EY255039,EY255040,EY255041,EY255042,EY255043,EY255044,EY255045,EY255046,EY255047,EY255048,EY255049,EY255050,EY255051,EY255052,EY255053,EY255054,EY255055,EY255056,EY255057,EY255058,EY255059,EY255060,EY255061,EY255062,EY255063,EY255064,EY255065,EY255066,EY255067,EY255068,EY255069,EY255070,EY255071,EY255072,EY255073,EY255074,EY255075,EY255076,EY255077,EY255078,EY255079,EY255080,EY255081,EY255082,EY255083,EY255084,EY255085.3. Cloning the genes related to metabolism of reactive oxygen species. VvMnSOD and VvAPX genes were cloned using PCR with universal and specific primers and liquor of the library as template. The full length of five genes related to metabolism of reactive oxygen species, including VvMT, VvTRX, VvDHAR were obtained from library random sequencing. These sequences were analyzed using bioinformatics methods. The cDNA of VvMT was 540bp with an ORF of 240bp, which coded a protein with 14 Cys, accounting for 17.72% of total amino acid residues.The cDNA sequence have submitted to GenBank database, the GenBank accession numbers is EU280163. The protein sequence of VvMT contained C-x-C, C-x-y-C and C-C motifs and highly conserved cysteine rich region, which are the main features of MT genes. VvMT belonged to Type2 and the 15th member of MT family. The DNA sequence of VvMT was 998bp, including 3 exons and 2 introns. Its GenBank accession numbers is EU280165. Still, the 783bp up-stream regulatory sequence of VvMT was cloned using enzyme digest, adaptor ligation and nested PCR. The core promoter region spanned from 685bp to 708bp and contained 12 regulatory elements of TATA-box,A-box,CAAT-box,G-Box,ARE,Box-4,CCGTCC-box,GAG-motif,HSE,Skn-1_motif,TC-rich repeats,repeatscircadian. Skn-1_motif was the conserved sequence specifically expressed in endosperms.The cDNA of VvTRX was 561bp with an ORF of 381bp coding a protein of 126 amino acid residues. Its GenBank accession numbers is EU280164. Thioredoxin spanned from 13 to 117 of the protein and the active site WCXP[C/S] was highly conserved. VvTrx belonged to the 3rd subfamily of thioredoxin h. The DNA sequence of VvTrx was 989bp, containing 3 exons and 2 introns. Its GenBank accession numbers is EU280166.The cDNA of VvMnSOD, the member of MnSOD revealed by Cluster analysis was 896bp with an ORF of 687bp coding a protein of 228 amino acid residues. DvWEHAYY, the Mn and Fe binding domain of SOD spanned from 189 to 196 amino acid residues. Its GenBank accession numbers is EU280161.The cDNA of VvAPX was 1044bp with an ORF of 762bp coding a protein of 253 amino acid residues. The peroxidase domain spanned from 19 to 226bp, ascorbate_peroxidase from 4 to 249bp and a Peroxidases active site signature, APLMLRLAWHSA from 33 to 44. VvAPX was one cytoplasmic ascorbate peroxidase. Its GenBank accession numbers is EU280159.The cDNA of VvDHAR was 990bp with an ORF of 639bp coding a protein of 212 amino acid residues. VvDHAR belonged to 1st family of DHAR and probably located in cytoplasm. Its GenBank accession numbers is EU280162.4. Cloning the CysP and CPI gene related to protein destination. The EST of VVTOV384 had full 3' poly (A) tail and not complete 5'end. VvCysP was cloned based on this EST using 5'RACE. The cDNA of VvCysP which was a cysteine proteinases of papain, was 1409bp with an ORF of 1134bp, coding a protein of 377 amino acid residues. This protein was a precursor and the mature one probably contained 233 amino acid residues and was an extracellular protein. QGsCGSCWSfST, spanned from 163 to 174 was the cysteine active site of cysteine protease in eukaryotes, LDHGVLLVGYG, spanned from 310 to 320 was the histidine active site, YWIiKNSWgenWGenGFYkI, spanned from 334 to 353 was the asparagine active site. Its GenBank accession numbers is EU280160.The cDNA of VvCPI was 872bp with an ORF of 759bp coding a protein of 252 amino acid residues. CY domain of CYSTATIN spanned from 5 to 125, the active site, EQVVAGKIYyLTLE from 81 to 94 and the signal peptide from 1 to 22. This protein was a precursor and the mature one was an extracellular protein.5. The change of the concentrations of reactive oxygen species and the activities of the antioxidant enzymes in the ovule of grape. The concentrations of and H2O2 and the activities of the antioxidant enzymes in the ovule of seedless grape'Youngle'and seeded grape'Jingxiu'were studied in the different periods after bloom. The results indicated that the concentrations of rose all the time, and the increasing rate in the ovule of seedless cultivar'Youngle'was greater than that in the ovule of seeded cultivar'Jingxiu'during middle and later periods of development; H2O2 concentration increased initially and decreased before it increased again. The subsequent increase in the seedless cultivar'Youngle'was significantly greater than seeded cultivar'Jingxiu'. The activities of SOD, CAT and AsA-POD increased initially and dropped drastically after ovule abortion in the seedless cultivar'Youngle'. But it maintained high level in seeded cultivar'Jingxiu'. The activity of POD in the ovule of the seedless cultivar'Youngle'displayed a similar trend to the other enzymes, but the activity in seeded cultivar'Jingxiu'increased constantly, but the increasing speed was lower in later stages. The changes of reactive oxygen content and the activity of antioxidant enzymes in ovules have some relevance to ovule abortion in the seedless cultivar'Youngle'.