Cloning And Transformation Of Ubiquitin Gene In Winter Wheat And Its Expression

In some stage of the cell cycle, organisms must produce new proteins,which promote multiplication, differentiation and activation of the cells. At thesame time, it is also needed to degrade those invalid proteins that had finishedtheir functions already. In some cases, the protein death is as important as itssynthesization and post-translation,which plays a regulation role to theorganisms in physiological metabolism, growth and development. Strictly selective degradation of proteins is respond to spatio-temporalregulation accurately in cells. It will result in severe obstacle in cellularmultiplication or differentiation, and even death, if the time, speed or positionof this process becomes abnormal. Ubiquitin-dependent proteolytic pathway is the most important and highlyselective proteolytic pathway, which is called ubiquitin pathway or Ub/26Sproteasome pathway, in short. It is known that protein degradation involved in almost all aspects ofplant biology, including the cell-cycle, embryogenesis, photomorphogenesis,circadian rhythms, hormone signaling, homeosis, disease resistance and cellsenescene, etc. Some cDNA fragments were isolated by mRNA DDRT-PCR (mRNADifferential Display Revers Transcription Polymerase Chains Reaction) from 5冬小麦泛素基因的克隆与转化及表达特性研究wheat near-isogenic lines (NIL) in different drought resistance. NucleotideBlast showed that one of them was homologous to the second group of Ubgenes, which is called Ub-extension protein genes for whose C-terminusextension interacts with ribosomal subunits gene. Ubiquitin gene , Ub1 and Ub2, were cloned from wheat coleoptile. Onthis basis, Ub2 was transformed to tabacco. Northern blot analysis indicatedthat Ub involved in water stress of wheat. The main results were as follows: According to conserved amino acid sequences of Ub in other plants, wedesigned a pair of primers (UB1and UB2) and obtained a 204bp cDNAfragment from wheat coleoptile by RT-PCR. It shared high identity with Ubgenes in Genbank, which was registered in Genbank(AF413573). Using UB1 and B26 as pimers, a 450bp cDNA seguence was obtained byRT-PCR. It included the whole protein coding region (231bp) and 3ˊuntranslated region(219bp). The gene was named for Ub2 and registered inGenbank, the number was AF297059. The genomic DNA was isolated from wheat plants, then, transferred to aHybond nylon membrane filter. Using Ub2 cDNA as probe, Southern blotanalysis revealed that there were many copies of Ub in wheat genome. Wheat seedlings were treated by 20% and 30% PEG-6000 to inducewater stress in germinating stage, total RNA was isolated from coleoptiles,then the RNA was transferred to a Hybond nylon membrane filter. Using Ub2cDNA as probe, Northern blot analysis proved that the expression of Ub wasup-regulated by water stress, that is, in the conditions of untreated, low-gradewater stress and severe water stress, the expression of Ub increasedcorrespondingly. In the period of wheat budding, wheat seedlings was treated by 30%PEG-6000. Total RNA was isolated from coleoptile at different time, that isat 0h, 4h, 8h, 12h, 24h and 36h after treatment. The RNA was transferred to a 6山东农业大学硕士学位论文Hybond nylon membrane filter. Using Ub2 cDNA as probe, Northern blotanalysis revealed that the mRNA level was changing with the stress time goingon, the mRNA level of polyubiquitin was increasing before 8h, thendecreasing. But that of Ub-extension genes was increasing before 24h, thendecreasing. That is, the transcriptional expression of polyubiquitin genes wasnot coincident with Ub-extension genes. Sense and antisense Ub gene expression vectors under control of 35Spromoter were constructed and transformed into tobacco (Nicotiana tabacumL.). The results of detection by PCR showed that Ub gene had been integratedinto tobacco genome, which indicated that...