Study On The Role Of HOX4 And Its Binding With Phosphatidic Acid In Cotton (Gossypium Hirsutum)

Cotton is one of the most important economic crops in the world. Cotton fiber is an important raw material for the textile industry, which is developed by the elongation of cotton ovule epidermal cells. Cotton fiber is a kind of single cell structure developed by the elongation and development of cotton ovule epidermal cells, which is similar to the developmental pattern of Arabidopsis trichome. Four homologous genes of AtGL2,GhHOX1-4,are cloned in cotton.GhHOX3 has been reported is a key factor involved in regulating cotton fiber elongation. Phosphatidic acid (PA) is a signaling molecule that is reported to regulate its activity by binding to a variety of target proteins and localization and function of nucleo protein. In this paper, the function of GhHOX4 and the interaction between PA and GhHOX4 protein were studied. The results were as follows:1. Effects of pH on the interaction of PA and GhHOX4 proteinPA is a pH Biosensor,the intracellular pH and PA protonation of the phosphorylated head affect the binding of PA to target protein. Four pH conditions were set up in vitro. Lipid-Protein Blotting experiment was used to study the interaction between PA and GhHOX4 protein under different pH conditions. The results showed that the binding ability of GhHOX4 and PA was stronger with the decrease of pH. When the plant is subjected to certain environmental stress, the change of pH in the cell will regulate the binding of PA to the target protein GhHOX4, thus affecting its biological function.2. Key domain analysis and truncation design of GhHOX4 proteinIn our previous study, we found that PA interacted with the GhHOX4 protein, so we cut the GhHOX4 protein in order to find the specific position of the PA and GhHOX4 proteins. Since GhHOX4 is a member of the HD-ZIP ? family, we cut the GhHOX4 protein into four segments according to the structure of the HD-ZIP ? family of transcription factors: the first segment is the HD domain of the amino acids 97 to 155.The second segment is the ZLZ domain of the amino acids 156 to 224, the third is the START domain of amino acids 297 to 522, and the fourth is the SAD domain of amino acids 523 to 749.3. The START domain is a key site for GhHOX4 binding to PAThe four truncated region was constructed on the prokaryotic expression vector pET28a or pMAL. And the soluble protein was obtained in Ecoli DE3 strain Rosetta.Lipid-Protein Blotting experiment was used to analyze which region of GhHOX4 was used to bind to PA. The results showed that PA could not bind to the START domain protein, but not with the other three regions, the protein HD, ZLZ and SAD domain,indicating that the PA binging motif resides in the START domain.4. The poly basic amino acid sequence KR-R-R is essential for the binding of GhHOX4 to PA.The four amino acid sites were mutated to alanine, named STARTK339A,STARTR340A, STARTK339A+R340A,STARTR340A and STARTR367A, respectively, in the START domain, and the poly basic amino acid sequence KR-RR, which could bind to PA, They were constructed on the pMAL protein expression vector and induced to express and purify in Ecoli DE3 strain to obtain soluble protein. Lipid-Protein Blotting experiments showed that the five proteins did not interact with PA,indicating that KR-R-R is critical for the binding of GhHOX4 protein to PA.5. Treatment of PA affects the initial differentiation and development of GhHOX4 transgenic Arabidopsis leaf trichome.The GhHOX4 transgenic Arabidopsis thaliana was treated with PA in vitro to study the effect of PA binding GhHOX4 on GhHOX4 function. The two weeks of GhHOX4 transgenic Arabidopsis thaliana and wild-type were sprayed with 20 ?M PA.The trichome on the fifth leaf of Arabidopsis thaliana was observed. The number of trichome was decreased and the proportion of the four branches of the trichome was also reduced.Previously, we used PA to treat GhHOX4 overexpressing fission yeast can make its length shorter. These results suggest that binding of PA to GhHOX4 may inhibit the transcriptional activity of GhHOX4 and play a negative role in the function of GhHOX4 in cotton fiber development.6. Preliminary study on GhHOX4 RNAi transgenic cottonThe constructed RNAi inhibitory expression vector was transformed into cotton to obtain GhHOX4 RNAi transgenic cotton plant. The positive transgenic cotton seedling was selected by gDNA level detection in field or greenhouse. The fibers were harvested at 6, 9, and 12 days after flowering and RNA was extracted and reverse transcribed into cDNA. The results of qRT-PCR showed that the expression of GhHOX4 gene was significantly decreased in the rapid elongation of cotton fiber. The results of vitro ovule culture showed that GhHOX4 gene could affect the elongation of fiber, and GhHOX4 RNAi transgenic cotton ovule was retarded and the fiber was short, which indicated that GhHOX4 could regulate cotton fiber elongation.