Screening Of Proteins Interacted With Splicing Factors Involved In Response To Heat Stress Using Yeast Two-Hybrid System

The splicing regulation of plant’s pre-mRNA plays an important role in the reaction mechanism which response to high temperature stress in Plants, our laboratory have gained the four arabidopsis mutants which related to high temperature stress by a forward genetic screening method for mutants in the previous experiments study. They are:hl1401, hl307, hl761 and hl111.Except for HL111, the other genes all may relate to splicing. In order to study the reaction mechanism of these splicing factor, we tried to use yeast-two-hybrid system to find the interacting proteins with the four genes by screening the arabidopsis cDNA expression library, and have successfully gained the information of these proteins.After a series of screening verification and eliminating of the false positive results respectively, we have found eighteen kinds of proteins in yeast interact with HL1401, and 13 of them are located in the nuclears, the others are mainly located in cytoplasm and the organs such as chloroplast.They mostly have different functions, some are helicases, some related to glycolysis, and some can combined with DNA and proteins and so on. Generally, the results agreed with our expectation that is because the subcellular localization of HL1401-GFP fusion protein was located in the nucleus in wild tobacco, most of these genes may interact with HL1401 in plants if our analysis from the localization result. However, as for the functional aspects, we still need further research and discussion.For HL307, we have found sixteen kinds of proteins, and eight of them are nucleoproteins, the other were mainly located in cytoplasm and the organs such as ribosome. Functionlly, there are also some proteins can combined with DNA, some are helicases and so on. To our joy, a nucleoprotein appears more than once in the screeing results also relate to stress reaction is a splicing factor, and we also found that HL307 is relate to splicing from the study of HL307, was also located in the nucleus. Therefore, the possibility of the interaction between the gene and HL307 is more higher. In addition, HL307-GFP fusion protein was also localized in the cytoplasm, so the other genes may interact with HL307 in plants if our analysis from the localization result.For HL761, we have found twenty-three kinds of proteins, and fifteen of them are nucleoproteins, the others were mainly located in cytoplasm and the organs such as chloroplast. Similarly, there are also some proteins can combined with DNA and proteins, some have the function of transcription factors and so on. Generally, the results also agreed with our expectation that is because HL761 was also localized in the nucleus. So these results can be reliable if our analysis from the localization result.For HL111, we have found eighteen kinds of proteins, there are only two nucleoproteins, and chloroplast proteins accounted for the largest proportion of them, the rest were mainly located in the cytoplasm and the organs such as mitochondria, and some individual proteins are unknown. Functionlly, there are also some proteins can combined with DNA and proteins, some proteins are hydrolase and so on. Of course, we can’t judge what from the above, unfortunately, the results didn’t agree with our expectation from the analysis on the localization which owing to the localization of HL111 was in the nucleus, and the above proteins are out of the nucleus except the individual proteins. But the later experiments show that HL111 also seems to be located in the chloroplasts, it explains our screening results can be reliable.In summary, the experiment try to find the interacting proteins with the target genes in yeast by screening library, then can prepare the ground for our further research about the mechanism of these genes which under heat stress through the verification and researching of the subsequent experiment in vivo and in vitro.