Detection Of RAPD Markers Linked To Lipoxygenases-null Genes In Rice Embryo (Oryza Sativa L.)

Researches in recent years indicated that the lipoxygenase (LOX) (EC 1.13.11.12) is the key enzyme of the rice lipid deterioration. The absence of LOX may thus, at least in part, reduce degradation, and alleviate seeds aging, and strengthen the storage stability of rice. There were three kinds of LOXs-LOX1, LOX2 and LOX3 in rice chaff and embryo. The absence of LOXs is inherited with a single recessive trait and the absence of LOXs doesn't affect rice growth.The random amplified polymorphic DNA (RAPD) analysis was carried out in wanjian2090, Daw Dam and its near-isogenic lines (NILs). The main results were as follows:1. Compared with the NILs, the absence of LOX doesn't affect rice growth.2. The influencing factors on RAPD were studied and experimental parameters were optimized. They were as follows: 20μL system containing 50ng template, 0.20μ mol/L primer, 1μL 2.5mmol/L dNTP, 1 Units Taq DNA polymerase, 2.0mmol/L MgCl₂ and 13μL ddH₂O. The optimal amplification program was as follows: 2min at 94℃, followed by 94℃ for 30sec, 36.5℃ 30 sec, 72℃ for 90 sec, for 40 cycles and final time of 72℃ for 5min, at last, sustain at 4℃. A high reproducible results were obtained with the optimized experiment conditions.3. A RAPD analysis was made between wanjian2090, Daw Dam and its NILs with 650 arbitrary primers. After experimented by several RAPD repetition, six primers amplified the DNA polymorphic bands in the NILs, linked closely with the rice LOXs-null genes. The heredity background likeness were very consistent between the NILs. Two polymorphic RAPD markers linked to rice embryo LOX 1-null gene were screened, S1302₇₉₇ and S1518₅₃₀; RAPD marker linked to rice embryo LOX2-null gene, S1509₆₈₃; RAPD markers linked to rice embryo LOX3-null gene, S1226₉₂₁, S1380₈₃₃ and S2040₈₄₇.