Construction Of Expression Vector And Functional Analysis Of The Triticum Aestivum L. TaNCED1 Genes

Wheat is one of the most important crops in the world. Since the earth is getting warmer, various extreme climates happened frequently, it is important to improve the adaptability of wheat to various stress environments to ensure high and stable yield. ABA plays important role in the adaptability of plants to various stresses. Genetic engineering can be used to change the expression of key genes about the ABA biological synthesis pathway to adjust the ABA level in plants to improve the tolerance of plants to various stresses.9-Cis-Epoxycarotenoid Dioxygenase (NCED), encoded by a complex gene family, which catalyze 9-cis-violaxanthin and/or 9'-cis-neoxanthin to produce xanthoxin is the key regulatory enzyme in the ABA biological synthesis pathway and plays important role in the regulation of ABA synthesis. Over-expression of NCED gene results in over-production of ABA in plants and then improving the tolerance of plants to various stresss, such as high-salt and drought.At the base of successful cloning of three TaNCED1 genes(TaNCED1-2, -3, -7), the main purpose of this study is to construct the expression vector of the three genes and transform them into Arabidopsis thaliana to analyze the function of NCED1 gene by different treatments of salt and ABA. The main results were obtained as below:1. Sequences of the three genes were obtained by PCR amplification using the cloning vectors of the three genes as template. Two restriction enzymes Kpn and XbaI were used to restrict the PCR product and expression vector pCAMBIA2300-35S. The ligation was transformed into E. coli DH5α, and the recombination plasmids were obtained by screening. PCR and restriction were used to make sure of the construction of the three expression vector of TaNCED1 and the constructed expression vectors were transferred into Agrobacterium GV3101 by freeze-thaw methods.2. Three TaNCED1 genes were transferred into Arabidopsis thaliana by floral dip method. 13, 24 and 45 transgenic plants were obtained, respectively ,and 79 positive transgenic plants were obtained by PCR screening with the transformation ratio of 96.3%. Twelve homozygous lines were randomly chosen through T2 and T3 generation screening for futher study. Every TaNCED1 gene has four homozygous lines named as S21 ~ S24, S31 ~ S34, S71 ~ S74, respectively.3. To analyze the function of TaNCED1, three transgenic lines S21, S31 and S71 and WT seeds were grown on growth medium(GM) containing various concentrations of ABA. On the medium containing no additional ABA, seeds germination of S71 and WT showed significant difference, and those of S21 and S31 were also affected, but not as significant as S71. On the medium containing 0.5μmol/L ABA, seed germination of S21, S71 and WT differed significantly, S31 also showed significant difference with WT, but not as significant as S21 and S71. The result indicated that overexpression of TaNCED1 improved the ABA concentration in transgenic line and prolonged seed dormancy which slow down the seed germination process, probably. There is difference among the three transgenic lines which showed the functional difference, probably.4. To evaluate the effect of TaNCED1 overexpression in transgenic plants on NaCl sensitivity, we germinated S21,S71 and WT seeds on GM containing five gradually increased concentations of NaCl (100, 120, 150, 180 and 200mmol/L). With the increasing of Nacl concentration, seed germination and seedling growth of S21 and S71 were seriously constrainted. At low Nacl concentration, transgenic plants and WT showed significant difference. While at high Nacl concentration, both WT and transgenic plants were all constrainted, but transgenic plants were constrainted much more than that of WT. S71 is constrainted apparently than that of S21, which indicated that TaNCED1-7 has more effect on ABA synthesis, probably.