Screening Of Arabidopsis Activation Tagging Mutants

At present, the degree of soil salinization is seriously high in the world and has a rising trend. As an abiotic stress factor, the high salt in soil severely affects the growth of plants and greatly restricts the production and distribution of crops. Therefore, it is quite significant to improve the stress-resistance and stress-tolerance of plants especially crops. Many former researches showed that the stress responses of plants had a fairly complex process, and the responses of different plants to stress were specially different. The model plant Arabidopsis is a kind of annual herb plant and has the characteristic of small genome,short growth cycle,cleistogamy,simple genetic transformation,easy to plant and control,etc. At the same time, it is a typical kind of glycophyte. Although Arabidopsis was not salt-resistant, many researches showed that it had a lot of genes relating to salt-tolerance in its genome. In order to acquire more information about stress-resistant mechanism, Arabidopsis activation tagging mutant libraries were used as materials to screen salt-resistant and low K+-resistant mutants.Firstly, we screened three activation tagging mutant libraries by four kinds of MS medium that respectively contained 70 mM NaCl,200 mM NaCl,50μM K+/50 mM NaCl,50μM K+/150 mM NaCl. Subsequently, we used the short-root(sr)mutant as material to clone the mutant genes. The results were as follows:1. We have acquired a low-salt sensitive mutant lss. When transferring line1684 and col 0 into MS medium containing 70 mM NaCl, we observed that some seedings(lss) of 1684 showed the traits of low-salt sensibility, such as arrest of growth,curly and chlorotic cotyledon. The ratio of lss and normal plants was about 1:1. On the contrary, whole of the col 0 grew normally. Subsequently, we transferred T2 generation lss into the same medium. Some seedings(T2 lss) repeated low-salt sensitive phenotype. The ratio was about 1:1 similarly. The ratios of two generations were not line with Mendel's law of segregation. It should be identified by backcross.2. We have acquired two short-root mutants sr(short-root)and mr(middle-root). Accidentally, we observed that T1 line 1082 plants showed three kinds of phenotype when planted on normal MS medium 10 days latter: Roots of the first type(sr)were obviously shorter than wildtype(Ws); Roots of the second type(mr)were shorter than wildtype too, but longer than sr; Roots of the third type were similar to wildtype. The ratio of three types was about 1:1:1. After transferring sr,mr and Ws into soil about 50 days, we collected statistical data of sr,mr and Ws about some other phenotype such as plant height,length of root,number of leaves,number of pods and so on. By data analysis, we found that sr was the lowest one and Ws was the highest one.mr was between sr and Ws. Also, the number of leaves,pods of sr was the smallest; Roots of sr was the shortest. mr was almost always between sr and Ws, except that its number of base leaves was bigger than Ws. Subsequently, we planted T2 seeds of individual sr by the same method. 10 days latter, we found that whole of the T2 seedlings showed shorter root than Ws, without separation.3.We have used the short-root(sr) mutant as material to clone mutant genes.We first amplified the sequence of green fluorescent protein by PCR. There were objective bands at 300-400 bp, which has proved that both of the T-DNA sequences exsited in two mutants; Then we separated the flanking sequence near the T-DNA inserted site by TAIL-PCR. After the third time of amplification reaction, two mutants all had bands, about 1500 bp long; Through GeneClean, we regained the PCR fragments to connect them with pMD18-T vectors; Transforming the plasmids into Escherichia coli; Then screening the Ecoli by ampicillin and extracting the plasmids to identify by enzyme cutting. There was a band at about 1500 bp; But after sequencing, we found that the band was the sequence of vectors.At the same time,we have attempted to complete this work by plasmid rescue, but acquiring nothing. Perhaps the reason was that this method needed much higher-quality DNA.In view of the instability of TAIL-PCR and plasmid rescue, the gene clone work is still going on.The conditions for amplification reaction should be explored further.