Tobacco Transformation By Betaine Aldehyde Dehydrogenase Gene (AnBADH) Of Ammopiptanthus Nanus And Improvement For Drought And Salt Tolerance

Drought, soil salinization and other abiotic stresses, seriously limits crop growth and geographical distribution, and is one of the major reasons to decease crop yield. Therefore, improving crop resistance to abiotic stress has important theoretical and practical significance. Study found that the plant in order to adapt or resist hostile environments, gradually formed a certain resilience mechanisms to alleviate stress injuries. Ammopiptanthus nonus and Ammopiptanthus mongollcus are both belong to Ammopiptanthus., Leguminosae. A. nanus which survived in the western Tarim Basin after the tertiary ancient Mediterranean disappear by gradually adapting the arid climate process. A. nanus living in arid and barren harsh environment, which is the only one of broad-leaved evergreen shrub desert of western China. One of resilience-related genes from A. nanus was cloned, heterologous transformed tobacco to verify expression and study its functions, thus providing genetic resources for breeding stress resistance gene.This study based on the preliminary experiments that the betaine aldehyde dehydrogenase gene have already cloned from A. nanus. A. nanus(AnBADH) dicots expression vector constructed and transformed Strain W38 wild-type tobacco by Agrobacterium-mediated. Transgenic tobacco lines were determined resilience. Experimental results are as follows:1. By Agrobacterium-mediated method, we’ve got 58 resistant transgenic tobacco plants after a preliminary screening of hygromycin.20 plants were positive identified by PCR, amplified bands are the same with positive plasmids. The results showed that the AnBADH gene was inserted into the tobacco genome.2. Three T1 tobacco lines (H13,H16 and H22) seeds germination on MS medium containing hygromycin(20mgL"1). Seedlings were respectively transplanted to MS medium containing NaCl(200 mmol·L-1) and mannitol (200 mmol·L-1) to simulate high salt and drought stress. Determined and morphological changes observed. The results show that seedlings transformed AnBADH under drought and high salinity stress growth normally. Compared with the wild-type control, the transgenic plants grew normally and with well-developed lateral roots and much longer, with a certain degree of ability to drought, high salt stress resistance.3. After antibiotic screening and PCR detection, T1 generation positive strains plants H13, H16 and H22 with 10% polyethylene glycol 6000 and NaCl(300 mmol·L-1) to simulate drought and high salt stress.Betaine,free proline, soluble sugar and MDA content,and relative conductivity in plants were measured. Betaine Content is no significant difference in transgenic plants and wild type under drought stress, and soluble sugar content increased significantly, Free proline content under drought stress in two strains H13 and H16 increased, MDA decreased in strains H22, transgenic lines relative conductivity of drought stress in the first 2 d and 3 d significantly lower than wild-type control.. Betaine Content is no significant difference in transgenic plants and wild type under salt stress, and soluble sugar content increased significantly,Free proline content have risen in three transformed lines after salt stress, while the MDA content significantly less, Transgenic lines relative conductivity of drought stress in the first 2 d significantly lower than wild-type control.4. Under natural drought conditions, the tolerance of transgenic tobacco lines stronger than the wild-type. Drought treatment 28 d, plants upper leaves remain green while the control showing symptoms of severe dehydration. After rehydration transgenic lines can quickly return to normal growth, leaf expansion, while the wild-type plant leaves still wilt state.From the test results of the above aspects, the AnBADH genes transformed lines under drought and salt stress, the cells can rapidly accumulate osmolytes, reduce membrane peroxidation, decreases membrane permeability, plant tolerance enhanced, explained that A. nanus betaine aldehyde dehydrogenase gene (AnBADH) we cloned has good resilience function which can be used in crops genetically modified after further verification.