Molecular Cloning And Expression Analysis Of PIN Genes From Adiantum Reniforme VAR.sinense Y.X.LIN

Auxin plays critical roles in a variety of physiological and developmental processes of plants, such as apical dominance, development of inflorescence and phyllotaxis and embryogenesis. These processes depend on the establishment of the auxin concentration gradients. PIN genes encode auxin transport carrier protein, plays an important role in flower organs. To isolate the genes related the development of reproductive organs regulated by PIN superfamily, and carry out their expression analysis, the apagamous and sexual Adiantum reniforme var. sinense were selected as materials in this study. This study will not only be helpful to reveal the genes related the phenomenon of apogamy at the molecular level, but also the theoretical basis for the research of Adiantum reniforme var. sinense by egenetic engineering. Main results are as follows:1. Cloning and bio-informatics analysis of Ars PIN1 and Ars PIN2 genes were issued. Based on gene fragment information of transcriptome library in apogamy, PIN fragments were selected due to expression differences. According to conserved sequences of these fragments, primers were designed, and then TAIL-PCR technology was used to isolate total length of two PIN genes,named Ars PIN1 and Ars PIN2 respectively. The full length of c DNA of Ars PIN1 was 2440 bp, of which ORF was 2052 bp, encoding 683 amino acids; while Ars PIN2 was 2033 bp, of which ORF was 1923 bp, encoding 640 amino acids. The protein structure of two genes was analyzed: Ars PIN1 and Ars PIN2 might be unstable hydrophobic protein and located in cytoplasm membrane and endoplasmic reticulum. Alighment exhibits that Ars PIN1 and Ars PIN2 are highly homologous to other PINs. Both sides have a complete domain of transmembrane protein structure, and all are hydrophobic segment, the middle area are hydrophilic segments. Each hydrophobic segments are several fold across the membrane, the characteristic of structure is consistent with its function of across membrane transport.2. Specific expression of PIN genes were analyzed between gametophyte and sporophyte of Adiantum reniforme var. sinense by real-time quantitative technical. The results showed that: PIN expression shows the highest when sexual organs differentiate in the period of gametophyte. It suggests that PIN gene plays an important role in the development of sexual organ. PIN expression in the sporophyte was significantly lower than that in the gametophyte, We guess the down-regulation phenomenon of PIN gene expression from gametophyte to sporophyte is closely related to apogamy in ferns, and they may have some connection.3. Ars PIN1 and Ars PIN2 were digested and inserted into expression vector p CAMBIA2300 to construct the plant binary expression vector respectively. Genetic transformation by Agrobacterium-mediated method was used to transfer p CAMBIA2300-Ars PIN1 into Arabidopsis thaliana of wild type. A few PCR positive transgenic plants of Arabidopsis thaliana were obtained. The result showed that transgenic plants lagrely save the time of blossom and bearing compared with wild type.