Studies On The Creating Of Artificial Male Sterility Line In Plants With HSP70 Gene

HSP70, a kind of molecular chaperone, has the main functions of taking part in protein folding, protein degredation and reparation of DNA damadge and has important effects on the constructure and function of mitochondria .It has already been proved that there is a close correlation between HSP70 and the development of plants and animals.This paper deals with integrating sense and antisense cDNAs of HSP70 into tobacco DNA by constructing an expression vector of sense and antisense cDNAs of HSP70 and gene-transforming methods-genegun bombarding and Agrobacterium mediation . Provided expression of HSP70 gene is inhibited by sense and antisense cDNAs of HSP70 we can get male sterile plants so as to prove that antisense cDNA of HSP70 leads to male sterility1. Construction of male sterility expression vectorBy integration of artificial sense and antisense cDNAs of HSP70 into pUC18 and pUC19 respectively,we can obtain pSC and pAC .Tapertal specific expression promoter TA29 and terminator NOS are connected directionally with sense and antisense cDNAs of HSP70 extrected and purified from pSC and pAC.,then integrated into pUC18 and pUC19, by which we can build sense and antisense cDNA NOS(respectively named plasmid 650 and plasmid 651) of TA29-HSP70 .For the sake of better screening and examination of transformed gene,we cut plasmid 650 , plasmid 651 and 3301 (containing GUSgene bar screening marker gene)with HindIII and EcoR I enzymes,then connect purified fragments of 650and 651 with plasmid 3301 to construct the vector 3301+650 and 3301+651.Corroboration of whether sense and antisense cDNA-NOS is integrated into plasmid3301 can be made by plate screening and enzye-cutting analysis .2. Obtaining transgenic male sterile tobaccoIn order to prove that HSP70 antisense cDNA can lead to male sterility,with plasmid 3301+650 ,3301+651 we transformed 207 aspetic tobacco leaves by genegun bombarding and Agrobacterium mediation(109 by genegun bombarding, 98 by Agrobacterium).By cultivating them in blotting media containing basta 0.4 mg/1 ,weget 181 resistant leaves (98 by genegun bombarding, 88 by Agrobacterium mediating). When the resistant leaves budded, we select the teneral leaves for staining examination. 79.5% of 195 resistance germinations are tested positive .3. Molecular examination and biological observation of transformedplantsBy growing resistant germinations we got 30 transformed plants .PCR and spot blotting hybridization analysis indicate that HSP70 anti-sense cDNA had been integrated into tobacco genome .Through observation and measurement we found 12 completely sterile, 9hyper-sterile, 3 partially sterile and 6 fertile and significant differences between them in stamen, height, and fruit weight. The paraffin section analysis showed that there were no difference between transgenic sterile plants and control fertile plants in the development and structure of pollen sac. In order to analyze the sterility mechanism further, we handled the pollen mitochondria in normal temperature and heat shock response, then we extracted the mitochondrion proteins and make variance analysis . The results indicate that: in both normal temperature and heat shock , the contents of mitochondrion proteins of sterile plants and control plants are significantly different (P<0.01,n=10); the differences of contents of mitochondrion proteins of sterile plants in the normal temperature and heat shock response are not significant, which mean that the expression of HSP70 gene is inhibited and the function of mitochondrion is disturbed. Therefore,it is proved that HSP70 anti-sense cDNA inhibits the expression of HSP70, and leads to transgenic male sterility...