Abstract:

Objective: To establish a safe and antibiotic-free genetic transformation system of Lespedeza bicolor. Using genetic engineering technology to improve the tolerance to abiotic stress of L. bicolor. Method: The coding sequence of xylose isomerase gene (xylA) was cloned from Escherichia coli DH5α strain by PCR amplification. The pBI121-xylA-BADH plant expression vector with xylA & betaine aldehyde dehydrogenase gene (BADH) was built. The xylose selection system of L. bicolor was built by using xylose instead of a certain amount of sucrose in the mediums. The cotyledonary node explants of L. bicolor were transformed by using the Agrobacterium tumefaciens-mediated method. The transgenic plants were confirmed by PCR amplification and Southern blot analysis. The growth performance of transgenic and wild-type plants under 0.5-2 g·L^-1 NaCl stress was measured. The betaine aldehyde dehydrogenase activity,contents of chlorophyll and conductivity of transgenic and wild-type plants in vitro under 1.0 g·L^-1 NaCl stress were assayed. The total soluble sugars were extracted separately from the leaves of wild-type plants and transgenic plants in vitro. The sugars were assayed by high performance liquid chromatograph. Result: After DNA sequence analysis,the homology is up to 100% between the cloned product and the published xylA DNA sequence from E. coli in GenBank. The PCR products and restriction enzyme digestion assays showed the pBI121-xylA-BADH plant expression vector was correctly constructed. In the transformation of L. bicolor,the selective mediums for regeneration of adventitious buds from cotyledonary nodes should be the medium which contained xylose as the sole carbon resource,while the selective mediums for shoot segment multiplying or rooting should be the mediums which contained 5 g·L^-1 sucrose and 25 g·L^-1 xylose. In Agrobacterium-mediated genetic transformation,the explants were precultured for 1 day,then infected with LBA4404 strain for 20-30 min,and cultured with Agrobacterium tumefaciens for a total of 3 days,relatively more xylose-resistant buds were obtained. Some xylose-resistant buds were assayed by PCR amplification and Southern blot analysis. Two transgenic lines were obtained. The genetic transformation rate of L. bicolor was lower than 4.7%. The growth performance of transgenic and wild-type plants under NaCl stress showed that the transgenic plants grew and rooted normally in the medium containing 2 g·L^-1 NaCl,while the wild-type plants defoliated after leaves turning into yellow,and were unable to root. BADH activity,the contents of chlorophyll and conductivity showed no significant differences between the transgenic and wild-type plants under no NaCl stress. There was no significant change of the BADH activity in wild-type plants when they were treated with NaCl or without NaCl. The BADH activity in transgenic plants increased greatly and up to 8-10 times as high as the wild-type plants when treated with NaCl. The transgenic plants had significant higher contents of chlorophyll and lower conductivity than the wild-type plants under NaCl stress. The assay on total soluble sugars by the high performance liquid chromatograph showed there were fructose and glucose in transgenic plants,while there were glucose and an unlabeled saccharide without standard sample in wild-type plants. Conclusion: The genetic transformation system of L. bicolor under xylose selection system was established. The transgenic plants with stable expression were obtained. The introduction of exogenous betaine aldehyde dehydrogenase gene increased the tolerance of L. bicolor plants to NaCl stress. The introduction of exogenous xylose isomerase gene from E. coli affected the glycometabolism of L. bicolor plants.

Key words: Lespedeza bicolor, genetic transformation, betaine aldehyde dehydrogenase gene, xylose isomerase gene, xylose selection