| Ramie (Boehmeria nivea (L.) Gaud) is an important perennial fiber crops. The yield and quality of ramie fiber is significantly inhibited by the reproduction growth, especially a lot of flowering and fruiting on the third crop. Ramie is hermaphrodite plant. So, cross-pollinated and the potential of heterosis utilization is great in theory. However, due to the high heterozygosity of ramie varieties, the separation of hybrid seed F1 is actually serious and isolated female flower and emasculation process is time-consuming. The ananthous or unisexual ramie varieties is good for exploitation. Previous studies found that the flower development and sex expression of ramie are closely related to the ethylene. Therefore, cloneing and characterizing the critical ethylene biosynthetic enzyme genes, analysizing spatio-temporal expression of these genes can contribute to further understanding the molecular mechanisms of sex determination and improving ramie varieties.In this paper, full length cDNA sequence of two ACO genes and one ACS gene in GBN-08, GBN-09 were cloned. The bioinformatics of these genes were analysized. And spatio-temporal expression of these genes were analysized. Overexpression vector of BnACO2 gene was constructed and transgenosis Arabidopsis plants was obtained. The main results are as following:1. The full length sequences of BnACO1 gene, BnAC02 gene, BnACS 2 gene are 1476 bp,1377 bp and 1680 bp, respectively. The ORFs of three genes are 981 bp,957 bp and 1 503 bp, and which encoded 326 amino acids,318 amino acids and 500 amino acids, respectively. The similarity comparison revealed that the homology of BnACO1 gene nucleotide sequence is over 79% with the ACO genes of Ficus carica and other species, and the similarity of the amino acid sequences with that of these species was over 78%. The similarity comparison revealed that the homology of BnACO2 gene nucleotide sequence was over 82% with the Morus alba and other species ACO gene, and the similarity of the amino acid sequences with that of these species is over 84%. The similarity comparison revealed that the homology of BnACS2 gene nucleotide sequence was over 70% with the Rosa rugosa and other species ACS gene, and the similarity of the amino acid sequences with that of these species was over 65%. Conservative structure domain analysis showed that BnACO1 and BnACO2 gene belonged to 2OG-FeⅡ_Oxy superfamily and BnACS2 gene belonged to aspartate aminotransferase family(AAT_Ⅰ superfamily) with pyridoxal phosphate (PLP) as a coenzyme.2. The results of semi-quantitative RT-PCR showed that the BnACO1 expressed in root, stem, shoot tip and blade, female flowers and male flowers, with the higher expression level in male flowers, while the lowest expression level in root and stem. The results of real-time PCR showed that the BnACO1 was induced by ABA, dehydration and NaCl. It may play a role in gender differences of ramie and have distinctive expression pattern in the regulation of development and stress responses. And BnACO1 BnACO2, BnACS2 expressed in the early development of female flower, especially in the female flower bud length significantly differentially expressed at 2.0cm.3. The BnACO2 gene could express the target protein under 37℃ and 1.0 mmol/L IPTG induction. The prokaryotic expression vector pQE-BnACO2 recombined protein was about 36.15 kDa in size, which were consistent with the predicted protein size. The overexpression vectors of pRJ101-BnACO2 was constructed and agrobacterium-mediated transformation of Arabidopsis obtained preliminary positive transgenic plants and transgenic Arabidopsis seeds. |
PDF Full Text Request