Cloning And Functional Analysis Of Two New PPR Genes In Cotton

PPR ?Pentatricopeptide repeat? protein is a newly identified protein superfamily, which has been found in plant in recent years. The PPR widely exists in terrestrial plants, and it plays an important role in the process of plant growth and development. Most PPR proteins locate in chloroplasts and mitochondria, and through the combination with its transcript, the PPR prtoteins play the role of regulation in the mitochondria and chloroplast, including transcription, RNA splicing, RNA editing, translation, and so on. Research showed that lack of PPR gene in plants could cause delayed leaf etiolation, plant growth retardation, florescence delay, and seed abortion.This study used Gossypium hirsutum xuzhou 142 as experimental materials, where two PPR genes were cloned on the basis of ESTs from cotton using PCR techniques.The bioinformatics method was used to analyze the putative amino acid sequence, and real-time PCR were used to analyze the expression of target genes in different tissues and under different stress environment. We used mesophyll cell protoplast to analyze positioning of the two PPR proteins. To further study the function of the two genes in cotton, we construsted VIGS ?virus-induced gene silencing? vector, transformed into cotton, interfered the expression of GhPPR9 and GhPPR11, and then detected the physiological and biochemical indexes of genetically modified cotton.The results of our experiment are as follows:1. The full length ORF of GhPPR9 is 3072 bp, encoding 1023 amino acids, having no introns, the molecular weight is 114.2193kD, and the isoelectric point is 7.17. GhPPR9 belongs to the P subtribe of PPR gene families, and it has 25 PPR motifs. GhPPR9 has a close relationship with the PPR protien of cocoa ?XP₀07038120.1? and castor ?XP₀02511099.1?. The full length ORF of GhPPR11 is 1914 bp, encoding 637 amino acids, having no introns, the molecular weight is 71.5326kD,and the isoelectric point is 7.79. GhPPR11 belongs to the PLS subtribe of PPR gene families, and it has 13 PPR motifs. GhPPR11 has a close relationship with the PPR protien of cocoa ?XP₀07052071.1? and Clement pomelo ?XP₀06445271.1?.2. Prediction of subcellular localization shows that GhPPR9 may locate in mitochondria. The expression pattern of GhPPR9 can be found in root, stem, leaf, petal and in different development periods of fibers, and GhPPR9 has a relatively higher expression in petal and 5DPA fiber. The expression pattern of GhPPR9 is not affected by stress, which may be related with fiber development.3. GhPPRll may locates in nucleus and cytoplasm, and GhPPR11 has a low expression in different tissues. The expression of GhPPR11 increase under ABA, NaCl, drought and high temperature treatments, expecially at 6 h and 9 h after treatment, which suggests that GhPPR11 may be associated with cotton resistance tostress.4. To detect their phenotypic characteristics, the GhPPR9 and GhPPR11 are silenced using VIGS techniques. Compared to WT and the control, little distinction is detected in height, bolls'number is fewer, the chlorophyll content of GhPPR9 is slightly lower in experimental group.