| Grapes(Vitis vinifera)belong to Vitaceae Vitis.Breeding quality new varieties of seedless grape has been becoming the one of the main goals of grapevine breeding in the world,especially in developed countries.Male sterility is one of the important genetic factors of seedless.It could simplify the breeding procedure and improve the seedless rate that hybridized with male sterile varieties.’Zhongshanhong’(Male sterilty),’Wink seedling-3’ and ’Wink seedling-12’ were the selfing progenies of ’Wink’.The seeds were sowed in 2004;it was found that most inflorescences of ’Wink seedling-3’ and ’Wink seedling-12’ showed the abnormalities that were similar to the male sterility during the blooming and fructification in 2012.’Wink’(fertile),’Zhonshanhong’(male sterile),’Wink seedling-3’,’Wink seedling-12’ were examined.The pollen viability,pollen shape and their interior structure and the cytological features of microspore were observed,and then studied the characteristics of the gene regulation of pollen abortion.Analyzed the expressional pattern of genes related to the anther development using the real-time quantitative RT-PCR technique.VvAMS gene was cloned from grapes using the homology gene method,gene or protein structure characteristics were analyzed using the bioinformatics software.Related genes of male sterility VvMYB4 was transformed the tomato and then verified the function of its.The main results are as follows:1.The main features of two ’ Wink seedling’ grapes:Filaments curl and shorter than the style,little pollen in the anther,the pollen viability was about 15%.There were two kinds of pollen grain in anthers:normal prolate spheroidal pollen grains and approximately spheroidal pollen grains.The anther wall structure was not obvious,and the microspore mother cells were few and loose in the ’Wink seedling’ grapes.The tapetum of ’Wink seedling-3’ did not degrade completely in the binuclear stage,the nutrients of tapetum could not supply for the development of spores timely,which caused the abortion of microspores in the late developmental stage.The tapetum of ’Wink seedling-12’mostly disintegrated at the early mononuclear microspore,which caused the abnormal supply of nutrient and the abortion of microspores.2.Compared with the control,AG、A6、TDF1、AMS、ACOS5 and CYP71A of’Zhongshanhong’ were insufficient expression,MYB33/65 and MYB4 were overexpression,TPD1 was overexpression in the tetrad stage,DYT1 was insufficient expression in the meiosis stage and overexpression from the tetrad to the mature pollen stage,MS2 was overexpression in the meiosis stage and insufficient expression in the tetrad stage.AG\EXS\MYB4 and AMS of’Wink seedling-3’ were overexpression,SPL、A6、MYB33/65 and ACOS5 were insufficient expression,TPD1 and TDF1 expressed insufficiently in the meiosis stage,but TDF1 overexpressed in the mononuclear microspore stage,DYT1 expressed insufficiently in the meiosis stage,but overexpressed from the tetrad to mature pollen stage,MS2 and CYP71A overexpressed in the meiosis stage,but expressed insufficiently in the tetrad stage.AG.SPL、A6、MYB33/65、AMS、ACOS5 and CYP71A of ’Wink seedling-12’ expressions were not enough,EXS and MYB4 expressions were excess,TPD1 was overpression in the meiosis stage,TDF1 overexpressed in the binuclear stage,DYT1 expressed insufficiently in the meiosis stage,but overexpressed from the tetrad to mature pollen stage,MS2 was overexpression in the meiosis stage,but expression was not enough in the tetrad stage.The unusual expression of AG、EXS、SPL、TPD1 might affected the expression of downstream genes(A6、MYB33/65、TDF1)that related to the callose metabolism,and then changed the expression of genes that related to the tapetum degradation,caused the abnormal degradation of tapetum.Furthermore,the MS2、ACOS5 and CYP71A were influenced,the pollen wall developed abnormally.3.The full-length of VvAMS gene was 2241 bp and the open reading frame(ORF)was 1878bp.Gene sequence comprise 363bp 3’-untranslated region,3’-UTR contain the 25bp polyA structure,which encoded 625 amino acids.It has structure domain and function domain of the typical AMS gene.The phylogenetic tree analysis of the evolutionary relationship of AMS between different species indicated that VvAMS was closed to Theobroma cacao.The protein molecular weight is 69.57 kD,isoelectric point is 4.94.Compared with other sequences of AMS protein in NCBI,the sequence similarity between the deduced amino acid sequences of VvAMS and other species were more than 90%.The encoding protein has not the transmembrane region and signal peptide.It is a hydrophilic protein,α-helix is the major component and P-extended strand,random coil and β-turn distributed in the protein sequence.4.The sequences similarity of VvMYB4 from ’Zhongshanhong’,’Wink seedling-3’ and’Wink seedling-12’ was 99.80%.The similarity of their encoding proteins was 99.77%.VvMYB4 had very high similarity among the grape varieties.VvMYB4 was transferred to’Micro Tom’ tomato by the agrobacterium mediated transformation.The transgenic strains were obtained after the resistant selection of hygromycin.7 resistant strains were positive through the GUS staining.After PCR amplification,5 GUS positive strains were screened.After RT-PCR amplification,4 PCR positive strains were screened.It showed that the VvMYB4 gene was successfully transferred into tomato.Compared with the non-trangenic plant,the transgenic strains anthers did not connect closely with the stylus.There were few pollen grains in the transgenic tomatos,and the pollen viability and germination were more lower than the non-transgenic tomato.Expression levels of some genes related to the phenylpropanoid biosynthetic pathway were detected using qRT-PCR technology.The results showed that MYB4 gene overexpressed in transgenic tomato.It restrained the relative expression of PAL、C4H、C3H、COMT、DEX、FLP、CHS、CHI、F3H and FLS genes in the transgenic tomato.The insufficient expression of genes were suggeated that influenced the synthesis of the sporopollenin and flavonoids,and then affected the formation of the pollen wall. |
PDF Full Text Request