Cloning And Expression Analyses Of Half-size ABCG Gene Family Members In Grape

Pseudoparthenocarpy seedless grapes’ fertilized embryos undergo various degrees of ovule abortion because of a process like programmed cell death(PCD). Former researches have demonstrated that VvABCG20 is related to ovule abortion in seedless grapes. We identified Vitis vinifera half-size ABCG genes from the PN40024 grape genome and bioinformatic analyses were performed. Full-length complementary DNAs(cDNAs) of them were cloned from the ‘Vitis vinifera cv. Pinot Noir’. The expression profiles of half-size ABCG genes were analyzed via quantitative real-time PCR(qRT-PCR) and semi-quantitative RT-PCR(sqRT-PCR) technologies to identify the relationships between half-size ABCG gene subfamily members and embryo development. We also constructed interference vector of AtABCG20, a homolog of VvABCG20 gene in Arabidopsis thaliana, to look for the function of VvABCG20 in embryo development. The main results were as followings:1. The half-size ABCG gene family members from the Arabidopsis thaliana were utilized as queries to search for grapevine half-size ABCG gene subfamily members and 30 sequences were obtained after screening. The results of bioinformatic analyses revealed that 30 half-size ABCG gene subfamily members were widely distributed in chromosomes of Vitis vinifera and have 7 kinds of exon/intron structures. The numbers of the transmembrane domain ranged from 4 to 7. Multiple sequence alignment was performed for 30 grapevine and 28 Arabidopsis thaliana half-size ABCG protein sequences. It was found that each of the half-size ABCG gene family members share a a typical structure of ABC protein which contained Walker A and Walker B motifs, an ABC signature which located between Walker A and Walker B motifs, the H loop, the D loop and the Q loop, with the exception of VvABCG3 and AtABCG3. The results of a phylogenetic analysis revealed that the 58 half-size ABCG gene subfamily members formed 5 large branches, and each branch contained ABCG proteins from both grapevine and Arabidopsis thaliana. The exon/intron structures of members in one phylogenetic branch were similar. The result of synteny analyses of grape and Arabidopsis thaliana suggested that 8 paired homologs of half-size ABCG genes were located in syntenic genomic regions, indicating these syntenic orthologs may share a common ancestor and have similar functions. Thus we could know the functions of grape half-size ABCG genes through their Arabidopsis thaliana homologs.2. The full-length cDNAs of 20 identified half-size ABCG gene family members were isolated from ‘V. vinifera cv. Pinot Noir’ using RT-PCR approaches. The results of sequences analyses suggested that the length of cDNA ranged from 1827 bp(VvABCG10.2) to 2508 bp(VvABCG11.7). At the meantime, the ORFs ranged from 1794 bp(VvABCG8) to 2406 bp(VvABCG1), and the length of the encoded polypeptides ranged from 598 to 801 aa, with predicted molecular masses ranging from 66.93 to 90.33 kDa.3. The expression level of 30 genes in stem, root, leaf, tendril, alabastrum, flowers, pericarp tissues and the mixed cDNAs of ovules at 8 developmental stages of ‘V. vinifera cv. Pinot Noir’ were measured via semi-quantitive RT-PCR and found that they exhibited distinct expression patterns in the examined tissues. VvABCG1, VvABCG11.4 and VvABCG11.5 were specifically expressed in the root whereas the VvABCG11.7 was expressed in all tissues except root. VvABCG20 was specifically expressed in ovules whereas VvABCG7 was expressed in all tissues except ovules. VvABCG6, VvABCG15.3 and VvABCG22.2 were expressed in alabastrum and flowers and VvABCG15.2 was expressed in all tissues except stem. VvABCG15.1 and VvABCG26 were expressed in all tissues whereas the expression levels were different.4. qRT-PCR expression analysis of the 13 ovule-expressed VvABCG genes during the development of ovule between seeded(‘V. vinifera cv. Pinot Noir’) and seedless(‘V. vinifera cv. Thompson Seedless’) variety revealed that they have distinct expression profiles. VvABCG20 was differentially expressed in later ovule developmental stages between seeded and seedless grapevines. The expression level of VvABCG6, VvABCG11.1, VvABCG15.4 and VvABCG11.8 in ‘V. vinifera cv. Thompson Seedless’ were higher than ‘V. vinifera cv. Pinot Noir’. These five genes may associate with ovule abortion in pseudoparthenocarpy grapes.5. The interference vector of AtABCG20, a homolog of VvABCG20 gene in Arabidopsis thaliana, was constructed and then transformed into Agrobacterium tumefaciens GV3101 by electropration.