| Since salt cress sequencing is to be completed, the main task of the research work will be functional genomics study, while the most direct and efficient way of functional genome study is to construct large-scale mutant library by gene tagging. Among different methods of gene tagging, activation tagging can enable us obtain not only loss-of-function mutants, but more importantly can creat gain-of-function mutants, which has specific advantages of analysis on functional redundant genes and those that are required during multiple stages of the life cycle.In this experiment, activation tagging vector pSKI015 was introduced into salt cress by Agrobacterium tumefaciens-mediated flora dip transformation with an intention to construct an activation-tagged mutant library. Molecular analysis and cloning of the flanking genome sequences have been conducted in order to obtain Basta-resistant plants.In the process of the vernalization and transplanting of salt cress, it was found that seedings which completed vernalization earlier and had the better vegetative growth had higher rate of transplanting survival, and harvested more seeds after infection. The appropriate Infection interval is 5-6 days. TAIL-PCR method were used to obtain the flanking genome sequences of T-DNA insertion site for the Basta-resistant salt cress seedlings, and BLASTn analysis of those sequences have been done.Since transformation rate of salt cress is extremely low, not higher than 0.01%, only 380 Basta-resistant transplants were obtained by spraying 0.2% Basta.By TAIL-PCR, flanking genome sequences were amplified in 65 activation-tagged mutants and 23 flanking sequences, which were available to be sequenced, have been amplified. To each plant, we usually could obtain 1-2 special bands and the situation of 3 bands is very rare, all the length of which is shorter than 800bp. Besides, flanking sequences in only about 30% of the mutants could be successfully amplified with 3 special primers and 5 AD random primers. According to the PCR results, the successful rate of AD3 was the highest which was above 40% and the number of special bands was also lower. So primer AD3 can be selected preferentially to conduct TAIL-PCR. By pMD18-T sequencing, we have got 7 flanking sequences.In this experiment, only primary research was made on construction of salt cress activation-tagged mutant library and a library in large-scale is being constructed now. Though flanking sequences can be successfully amplified by TAIL-PCR, it is necessary to develop a more appropriate method of directly sequensing PCR products.FtsH is an ATP-dependent metalloprotease and chaperone which is involved in various biological functions and play important roles in organisms. In prokaryotes, functional studies have revealed an important role for bacterial FtsH in stress responses. Researches from higher plants show that plant FtsH homologs have closely relationship to light stress, cold acclimation, the hypersensitive reaction and so on.Under the stress of low temperature and strong light, ThftsH8-RNAi seedings showed the appearance of etiolated leaves. Inferred mutants with etiolated leaves are caused by ftsH8 gene silence. The mechanism is supposed that thylakoid FtsH8 protease is involved in the turnover of the D1 protein of the PSII reaction center in the context of its repair from photoinhibition and in the formation of thylakoid., we consider ThftsH8 is expressed under the induction of low temperature and strong light,and in this condition, ThftsH8-RNAi transformant is lack of ThFtsH8 because of gene silence,and show etiolated leaves, when temperature increase, ThftsH8 decrease expression level,correspondingly, ThftsH8-RNAi seedings etiolated leaves reduce.This is the preliminary study about ftsH8 gene function in salt cress, further studies will be launching in the future.
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