Transformation Of Populus Euphratica Oliv. Genomic DNA Fragment For Arabidopsis Thaliana And Preliminary Identification Of Transgenic Mutant

Since soil desertification and salinization are worldwide problems, the studies on themolecular mechanism of natural salt-tolerant and drought-resistant plants, the selection ofresistance genes and regulatory elements with breeding potentials and the cultivation ofnew salt-tolerant strains are the most efficient approaches to them, which is one of thehotspots in recent scientific research as well.Populus euphratica Oliv. is the unique natural arbor specie of desert and semi-desertareas in Northwest China and Central Asian. In the long term of evolvement, the excellentgenes and exact expression and regulation mechanism were formed. It is a quite preciousgermplasm resource with stress tolerance in wooden plants. In this paper, an BIBAClibrary for Populus euphratica genomic DNA fragment had been constructed and then wastransferred into the model plant Arabidopsis thaliana. Transgenic strains and thephenotypic mutants were obtained. We selected one leaf color mutant strain and studied itfurther more. The main results were as follows:1. In this study，an efficient Agrobacterium-mediated gene transformation systembased on the Floral-dip method was developed for Arabidopsis thaliana. By investigatingthe factors which effected the transformation, the optimum system were Silwet L-77concentration of0.2‰，infected for1min, dark treatment co-culture for8h~10h and inAT medium with50mg L-1Kan. Based on this transformed system and PCR test，205fertile positive transgenic strains were obtained and10visually discernible phenotypicmutant strains were screened out.2. One yellow-color mutant strain y1-1was selected for identification. This yellow-color phenotype is a whole-plant phenotype during the whole period of growth. Mutant y1-1is able for photosynthetic growth and fruiting with a segregating ratio3:1in T2generation.3. Based on the analysis about growth rate, pigment contents of leaves, photosyntheticrate, chlorophyll fluorescence and the ultrastructure of chloroplasts between mutant y1-1and col wild type, the results show that: the kinds of pigments were almost same, but totalchlorophyll content is only49.9%of the WT, which caused this phenotype. In theconditions of low light intensity, high CO2concentration, net photosynthetic rate of theformer was2.47times that of the latter, showing strong adaptability to the environment. After dark adaptation for20min and illumination for1sec, the OJIP curve of the former isalways lower than WT. In JIP-test, that SM, MOof mutant y1-1are respectively47.9%and80.2%and the effective light reaction center Quantity (RC/CSO) is74.6%of WT indicatedthe PS II is hypoplasia. With a higher level at ψOand φEo, photochemical reaction of lightreaction unit is more efficient in mutant y1-1. It can capture85.0%of total absorbed lightenergy and transport52.9%into the electron chain, which are more than that of81.2%and50.6%respectively in WT.4. The ultrastructure of chloroplasts was observed under transmission electronmicroscopy. Compared to WT, the chloroplast of mutant y1-1is slender and the internalstructure is looser, together with more starch grains and osmiophilic particles. At the sametime, to replace fully developed grana, stroma lamellae are almost in a parallel statethroughout the chloroplast chamber of the mutant y1-1.5. The total DNA of the mutant y1-1was extracted from the leaves of T2generationplant for the exogenous gene insertion site analysis by TAIL-PCR technology. The resultsshowed that the exogenous gene is located between6739.7kb and6740.9kb in the5thchromosome of Arabidopsis thaliana, in the open reading frame of gene AT5G19940(TAIR) coding one PAP protein. For few of this gene and PAP protein was reported, thesecondary and tertiary structure of the polypeptide chain is predicted to infer that this PAPprotein is located on the chloroplast membrane thylakoid membrane and associated withstructural molecule activity. The absence of this protein may cause the failure of granastacking and abnormal chloroplast development in mutant y1-1.6. The both end of exogenous DNA have been sequenced, and it was found thatexogenous DNA contains the homologous sequence of disulfurase and formatedehydrogenase.