Mining Of Seedless Genes And Functional Study Of VvHB7 Gene In Grape

Grape(Vitis vinifera L.)is a global fruit,widely cultivated at home and abroad,and its annual output ranks at the top of the world fruit output.Rich in nutrients,lovely and exquisite grapes are favored by consumers.In particular,seedless varieties have become the mainstream of table grape production and consumption due to their characteristics such as meeting the current table manners and unique flavor,which is the main development trend and breeding direction of grape industry in the future.However,the formation mechanism and regulatory network of seedless grape are still not clear.Using RNA in situ hybridization and transgenic technology to find the key genes of seedless grapes and clarify their functions can provide new research ideas and methods for exploring the formation mechanism and regulatory mechanism of seedless grape,which plays an important role in creating new seedless germplasm resources.Based on the transcriptome analysis of different stages of seeded and seedless ovule development in our previous study,we screened and sequenced the seedless grape VvHB7 gene,and analyzed the bioinformatics,promoter sequence and subcellular localization of the VvHB7 gene.The expression characteristics of the gene tissue were determined by q RT-PCR.RNA in situ hybridization was used to explore the spatiotemporal expression patterns of genes,and transgenic technology was used to verify their functions.The main findings are as follows:1.Mining and cloning of seedless genes in grape.Analysis of expression levels of 26 candidate genes during ovule development using semi-quantitative PCR revealed that ten genes,including HB6,HB7,HB18,HB25,HB53,HB54,HB56,B3-RAV3,ERF60 and MADS21,were significantly higher in ovule development of seeded and seedless grape varieties.Four genes,HB17,HB38,B3-VAL1 and ERF16,were more expressed in ovules of seeded grapes.Vv HB6,VvHB7 and Vv HB18 were cloned from ovule of ’ Red Globe ’ and’Thompson Seedless’ respectively.The total length of DNA sequences of 2060 bp,588bp and636 bp were obtained,and compared with the sequence of grape genome website Pinot Noir.The analysis showed that the sequences of the three genes with different expression were conservative,and the similarity was above 90%.The results of amino acid sequence alignment showed that some amino acid sites of VvHB7 gene were different between ’ Red Globe ’ and’Thompson Seedless’.2.Promoter cloning and bioinformatics analysis of grape seedless associated gene VvHB7.VvHB7 gene is 588 bp in length,located on chromosome 2,contains 2 introns and 1 exon,encoding 192 amino acids.It belongs to HD-Zip subfamily class Ⅰ gene.Phylogenetic tree construction revealed that it has high homology with Arabidopsis ATHB12.Analysis of VvHB7 gene promoter shows that at around-145 bp,the Thompson Seedless promoter region is missing a sequence composed of 17 bases.Cis-acting element analysis shows that the type and number of regulatory elements in the Red Globe and Thompson Seedless promoter sequences are basically the same.There were many elements related to the synthesis of abscisic acid and gibberellin.Subcellular mapping experiments showed that the VvHB7 gene protein is located in the nucleus and may function in the nucleus.3.Spatio-temporal expression pattern analysis of grape seedless associated gene VvHB7.According to the cytological observation of ovule development in Thompson Seedless and Red Globe grape,the process of ovule abortion in seedless grape varieties was clarified,and the key period of ovule abortion in Thompson Seedless grape was determined at 30-40 d after flowering.The expression level of VvHB7 gene in the tissues,organs and ovule of Red Globe and Thompson Seedless grapes was detected by q RT-PCR.The results showed that the transcription level of VvHB7 gene in the fruits and ovule of seedless grapes was significantly higher than that in other organs.At flowering stage,10 d,20 d,25 d,30 d,35 d and 40 d,the expression level of VvHB7 in Thompson Seedless was significantly higher than that in Red Globe.RNA in situ hybridization analysis confirmed that the expression of VvHB7 in the Thompson Seedless was higher than that in the Red Globe ovule,and the positive signal was strongly expressed in the inner integument near the micropyle end at the critical stage of the Thompson Seedless ovule abortion.4.Study on the function of seedless grape gene VvHB7.The VvHB7 gene overexpression vector was constructed and hetero-transformed into tomato Micro-Tom.Through morphological observation,the overexpressed tomato lines showed dwarfing traits,increased branching,darker leaf color,and early fruit color transition.The pollen germination experiment showed that the pollen tube growth of the overexpression line was slower than that of the wild type under the same culture time.Statistical analysis of the data showed that the transgenic lines had smaller fruit volume and fewer seeds.The observation of tissue sections of tomato seeds showed that the endosperm cells of the seeds were more closely arranged and the cells became smaller.Quantitative real-time PCR analysis showed that the relative expression levels of genes involved in fruit ripening and seed development were significantly changed in the transgenic lines.