Studies The Mechanism Of Male Sterile In New Gapevine Selection’zhong Shan Hong’

Plant male-sterile is an important tool to utilize plant heterosis, having enormous value in seed production. The mechanism of male-sterile involves many aspects, and so much research has been done in the whole world.’Zhongshan Hong’is a male sterile grape line which is selected from the self cross progeny of the cultivar’Wink’which belongs to the species of Vitis vinifera.L by Nanjing Agricultural University. In this study, we employed ELIS A to evaluate the changes of phytohormone contents in anther abortion of male sterile new grapevine selection’Zhongshan Hong’. VvMS2and VvMYB4were cloned from ’Zhongshan Hong’ and ’wink’, expressions of these genes were detected by real-time quantitative RT-PCR (qRT-PCR), and their functions were analyzed through overexpressing in transgenic tobacco plants. We preliminarily analysised its function. The main results are as follows:1. We employed ELISA(enzyme-linked immunosorbent assay) to evaluate the contents of IAA(indole acetic acid), GAs(gibberellic acid), CTK(cytokinin)and ABA(abscisic acid) at different developmental stages of anther, including sporogenous stage, microspore mother cell stage, meiosis stage, tetrads stage, mononuclear microspore stage, binuclear stage and mature stage. The results showed that the contents of IAA and GAs in the floral buds were not significant differences between’Zhongshan Hong’ and’Wink’ before meiosis, but they were much lower in’Zhongshan Hong’than in’Wink’ at the stages of tetrad and uninucleate. The content of ABA began to decrease at the stage of mononuclear microspore in’Wink’, while at binuclear stage in’Zhongshan Hong’. The ratios of IAA/ABA, GAs/ABA were remarkably higher in’Zhongshan Hong’than’Wink’before binuclear stage. The results suggested that the deficiency of IAA, GAs and CTK, the delayed reduction of ABA and the imbalance of the ratios of the IAA/ABA, GAs/ABA contributed to the male sterility of grape.2. Specific primers were designed based on the grape sequence (CBI29968) in NCBI which is highest homology with Arabidopsis male sterility gene MS, and the cDNA and genomic DNA of VvMS2were cloned by PCR. VvMS2Gene structure characterizations were analyzed using the bioinformatics software. Quantitative real-time PCR (qRT-PCR) was performed to determine the expression pattern of VvMS2in different tissues of’wink’ and in different development stage in bud between’Zhongshan Hong’and’wink’. The full length ORF cDNA sequence of VvMS2with1755bp was cloned. Bioinformatics analysis showed that this gene encodes584amino acids, containing NAD-binding-4structure domains and sterile structure domains. This gene contains9exons and8introns. The protein molecular weight is64.74kD, isoelectric point is8.84.Comparing with other sequences of MS2in GenBank, the deduced amino acid sequences of VvMS2shared40%-71%in homology. VvMS2only specially expressed in floral buds and at the stages of tetrad to dinucleate. The expression level in button at uninucleate stages in’wink’ is remarkable higher than that in’Zhongshan Hong’. The Conclusion is that the abnormal gene expression is related with male sterility in’Zhongshan Hong’.3. The full length cDNA sequence of VvMYB4with978bp was cloned. Bioinformatics analysis showed that this gene encodes325amino acids, containing Myb-like DNA-binding domain. This gene contains3exons and2introns. The protein molecular weight is35.81KDa, isoelectric point is6.83. Comparing with other sequences of Myb4in GenBank, the deduced amino acid sequences of VvMYB4shared58%～85%in homology. VvMYB4only specially expressed in floral buds and at the stages of uninucleate. The expression level in button at uninucleate stages in’wink’is remarkable lower than that in ’Zhongshan Hong’.4. Transformation of pMD19-T vector was carried out in this paper; then, VvMYB4gene was inserted into this vector. Fusion plant expression vector was constructed successfully through digested and inserted into the plant expression vector. Transgenic VvMYB4tobacco plants were obtained through the method of Agrobacterium-mediated. Compared with wild-type strains, transgenic tobacco lines showed thinner anther, less pollen, shorter filaments and lighter color petals. Pollen germination experiments show that transgenic tobacco pollen germination rate is very low or no germination. Scanning electron microscopy shows that the transgenic tobacco line1’s pollen shapes were changed and the transgenic tobacco line2’s pollens were shriveled, no germination ditch. Bagged before flowering, most of the transgenic plants cannot produce offspring. All of those indicate that the VvMYB4gene can induce the male fertility.