Transformation Of Tobacco With MCT Gene And The Functional Analysis Of LsNHXs Genes From Limonium Sinense

Chapter 1: Transformation of tobacco with MCT geneMethyl bromide is one kind of highly effective fumigant used widely in the whole world, but it plays the destructive effect to the ozone layer and the environment. So methyl bromide was appealed to be eliminated by the International Organization as soon as possible. The development of alternative technologies for methyl bromide is imminent.The methyl halide is biosynthesized by an enzymatic reaction: SAM + X- (Cl-, Br-, I-)→CH3X + S-adenosylhomocysteine. This reaction is catalyzed by one kind of methyl transferase (methyl chloride transferase MCT). We cloned AtMCT (AtHOL, from glycophyte- Arabidopsis), ThMCT (from halophyte-Thellungiella halophila), and SsMCT (from halophyte - Suaeda salsa) and overexpressed them in tobacco, respectively.The use of methyl bromide in soil fumigation is to kill pathogens. Pathogens exist as spores or other dormant forms with the absence of host plants and they have high resistance, so high concentration of methyl bromide is required while fumigating. This is likely the main reason of the environmental pollution. If the transgenic tobaccos can generate methyl bromide spontaneously and duratively, and methyl bromide can play a direct role to the pathogens when plants are infecting. This alternative technology for methyl bromide is original.The main results were shown as follows:1) The full length sequences of ThMCT and SsMCT were obtained through RT-PCR and RACE. ThMCT gene, 923 bp, has an ORF of 681 bp, encoding 226 aa, and the molecular weight of it's protein is approximately 25.1 kD. SsMCT gene, 1094 bp, has an ORF of 699 bp, encoding 232 aa, and the molecular weight of it's protein is approximately 25.8 kD.2) The ORFs of AtMCT, ThMCT, and SsMCT were constructed into the vector pINT4 respctively, then transformed into tobacco through the leaf disc method mediated by Agrobacterium. And finally, 18 of AtMCT, 20 of ThMCT and 38 of SsMCT transgenic lines were obtained, repectively. 3) Some transgenic lines of three genes were validated by molecular methods. PCR, Southern and Northern blotting confirmed that the three genes had been integrated into tobacco genomes and had transcripted normally.The translation was tested by Western blotting. Signals were shown in the three kinds of transgenic plants when Western blotting was done using the full protein. However, when Western blotting was done mainly using the soluble protein, only the AtMCT transgenic plants showed signals.4) After treated with the different concentration solution, the content of CH3Cl, CH3Br, and CH3I released by tobaccos were measured by GCMS. Only the AtMCT transgenic a12 line enhanced the content of CH3X than wild type, about 100 times. The others showed almost the same level as the wild type.5) Treated the AtMCT transgenic plants and wild type plants with root-knot nematodes, the AtMCT transgene plant showed enhanced nematode resistance.Chapter 2: The functional analysis of LsNHXs genes from Limonium sinenseThe tonoplast Na+/H+ antiportor, very usefull for plant stress tolerance, can use the H+ gradient as driving force built by tonoplat H+-ATPase and H+-PPase to sequestrate Na+ into the vacuole to eliminate Na+ toxic effect, also it is related to PH regulation, vesicle transport, vacuolar development, and protein sorting. Vacuole, is not only a material storage organelle, under certain physiological conditions, in specific cell types, or at determined developmental stages, the vacuole participates in the export of a variety of solutes ranging from simple sugars and organic acids to amino acids and mineral ions.The early observations of electromicroscope also found that the salt-secreting cells of Limonium contained numerous and well-defined mitochondria and were rich in endoplasmic reticulum, but lacked a conspicuous large central vacuole. Instead, the cells contained a series of smaller"vacuole-like"membrane vesicles, many of which were seen in close proximity to the cell membrane. More important, the tonoplast of the"vacuole-like"vesicles often appeared to fuse with the plasmalemma in agreement with a vesicle-mediated secretion process. So it is speculated that the Na+/H+ antiportor may be related to the salt secretion. Therefore, the LsHNXs genes were cloned in this study, and their function was studied by RNAi. The main results were shown as follows:1) The conservative sequences of three LsHNXs genes (LsHNX1, LsHNX2, and LsHNX3), and the 3'-terminal specific sequences of LsHNX2 and LsHNX3, were cloned by RT-PCR and 3'-RACE.2) The plant RNAi vectors were constructed and the transformation of Limonium sinense was done. The RNAi lines of three single-genes and one double-gene were obtained and were propogated rapidly for the molecular and the stress tolerance analysis.3) To be compared with wild type, the Na+ and K+ contents secreted by the leaves of LsNHX1 transgenic plants were remarkably higher. The Na+ and K+ contents in leaf were little higher and the ratio of K+/Na+ was almost the same as that of wild type.4) To be compared with wild type, the Na+ content secreted by the leaves of LsNHX2 transgenic plants was little lower, and the K+ content was remarkably lower. The Na+ content in leaf was little higher than wild type, while the K+ content and the ratio of K+/Na+ were reduced.5) To be compared with wild type, the Na+ and K+ contents secreted by the leaves of LsNHX3 transgenic plants were higher. The Na+ and K+ contents in leaf were little higher, and the ratio of K+/Na+ was not significantly different than wild type.6) To be compared with wild type, the Na+ content secreted by the leaves of the two genes co-RNAi plants was higher, and the K+ content was not remarkable different. The Na+ content in leaf was little higher, while the K+ content and the ratio of K+/Na+ were lower.It was shown from the above results that LsNHX1, LsNHX2 and LsNHX3 act on different ways. LsNHX2 may locate on the vesicle, and may affect the salt secretion of Limonium sinense by vesicle trafficking, while LsNHX1 and LsNHX3 may locate on the center vacuole, and sequestrate Na+ into vacuole.The main innovations of this research:1. We firstly cloned ThMCT and SsMCT genes form the halophyte, Thellungiella halophila and Suaeda salsa, respectively, and carried out the detailed analysis of their sequences.2. Through the genetic engineering, the transgenic tobacco could emit CH3Br spontaneously and duratively, so that could substitute CH3Br in soil fumigation. This alternative technology for methyl bromide is original.3. Three members of LsHNXs gene family (LsHNX1, LsHNX2, and LsHNX3) were firstly cloned and downexpressed by RNAi, respectively. We found that LsNHX1, LsNHX2 and LsNHX3 act on different ways. LsNHX2 may locate on the vesicle, and may affect the salt secretion of Limonium sinense by vesicle trafficking, while LsNHX1 and LsNHX3 may locate on the center vacuole, and sequestrate Na+ into vacuole.