Creation Of Marker-Free Transgenic Rice With Drought Tolerance

Rice （Oryza sativa） is one of the most important food crop. About half population of the worldhave rice for food.However, as the extreme weather such as drought and floods in recent years appearedfrequently, the production of rice declined greatly. As is reported that the yield of rice caused by droughtclimite reached10²0%of the total rice production in the world every year.The global food security isgetting worse and worse. Therefore, breeding new rice varieties with drought tolerance and reducing theproduction of drought-induced is hot and difficult of the rice studing. Traditional breeding methods dueto lack of germplasm resources and other factors are not well meeting the requirements of the scientists,which makes the molecular breeding increasingly welcomed. In this study, a mixture which containedthe casstte DNA carried the endogenous drought tolerance gene OsAPX2encoded ascorbate peroxidaseand a expression vector carried selected marker gene of hygromycin was transferred into rice callusthrough co-transformation mediated by gene-gun bombardment and marker-free transgenic rice withdrought tolerance was selected thruogh molecular method and phenotype identfication in the T1generation. The main results are as follows:1. Transfor the gene of ascorbate peroxidase （OsAPX2） into japonica Sui8and PCR was used toidentify the co-transformants.The casstte DNA which carried the gene of ascorbate peroxidase （OsAPX2） was digested from aplant expression vector pCAMBIA3301-APX, by two restriction endonuclease EcoRⅠand SphⅠandthen mixed with a plant vector which carried a plant selected marker gene of hygromycin（hyg） in theproportion of1:1. Then the mixture was transferred into japonica Sui8cultivated in the southeast ofchina through gene bombardment mediated co-transformation.846tranformants were obtained afterselection by hygromycin in culture medium with a concentration of50mg/L and32co-transformantswere separated by PCR using primers from OsAPX2.2. PCR was used to identify marker-free transgenic plantsPCR was used to amplify the gene of OsAPX2and hyg in the single plants from T1generationof co-transformants to identify marker-free transgenic plant.44single plants was obtained whichbelongs to8transgenic lines.3. Molecular identification of the marker-free transgenic plantsSouthern blot using the total length of the cds of OsAPX2as probe confirmed that the gene ofOsAPX2was intergrated into the genome of Line6, especially have only one insert site. The result ofRT-PCR and quantitative real time PCR indicated that the gene of OsAPX2got effective expression inLine6and Line7, attained as4-5times as wild type. TAIR-PCR was used to sepreate the flankingsequence of insert site and got one side of the flanking sequence. The result of blast in NCBI indicatedthat the exogenous gene was integrated into chromosome7of Line6and chromosome2of Line7.4. Drought tolerance identification of the marker-free transgenic plants The four-leaf seedlings were exposed to drought stress in the greenhouse.22days’ later, mostseedlings of the wild type turned to be wilting, in contrast the transgenic plant grew normal, whichconcluded that the transgenic plants got enhanced drought tolerance. when count to the percentage ofwilting, wild type showed much higher ratio（85%） than transgenic plant（12%）. The result of analysisingthe content of H₂O₂and MDA of the seedlings under drought stress supported the conclusion that thetransgenic plant had stronger drought tolerance than wild type.3days seedlings after germination werecultivated in the1/2MS medium contained20%polyethylene glycol2000（PEG2000） for3days, thentransfer into normal1/2MS medium for7days for recovery. Analysising the height of the seedlingsfound that the height of transgenic plant were much higher than the wild type, however, the controlplants showed no significant differences.