The Expression And The Vector Construction For RNAi Of The Enzyme Genes Related With Hormone Metabolism About The Coleoptiles Under Submerged Stress

In the long-term rice evolution, it has evolved a mechanism and some strategies to adapt to submerged stress. Furthering study the tolerance mechanism from the molecular level. which can offer a theory for other plants, and other plants also can improve their genes to resistance the submerged stress and increase their crop production. In order to study rice coleoptile from physiology and molecular level, we have done something, such as the rapid growth of rice coleoptile under flooding stress, the difference expression of hormone enzymes and the RNAi vectors construction for the ascent expression genes which belong to the synthesis or degradation key enzyme genes about hormone. The results show that:1. Submerged stress promotes rapid growth of rice coleoptileCompared the length of coleoptile cultured in air and water, it found that submerged stress can promote the rapid growth of coleoptile. Two days after sowing in air and submerged stress, the length of rice coleoptile reached a very significant difference. After sowing four days, five days and six days, the length of rice coleoptile in water was greater significant than the air.2. Submerged stress to the cell structure of rice coleoptileNipponbares were cultured in25℃,dark conditions for four days. Four days later, stripping out the rice coleoptile, and then made it to paraffin to research the crosscutting rice coleoptile cell structure and longitudinal rice coleoptile cell structure. Compared with air groups, in the longitudinal rice coleoptile cell structure, the rice coleoptile’s cell has a phenomenon of elongation under the flooding stress, and in the crosscutting rice coleoptile cell structure, its cell area has been reduced. To adapt to submerged stress environment, the rice coleoptile’s cell can elongation and the cell area decrease in a short time.3. The difference expression of key enzyme genes about hormone synthesis or degradation in rice coleoptile under submerged stressβ-actin as a reference gene, under submerged conditions, the expression difference of the key enzymes gene in the synthesis or degradation about the ethylene (ETH), cytokinin (ZT), and abscisic acid (ABA) in nipponbare’s coleoptile was investigated by RT-PCR. Results showed that during the synthesis of ETH, the expression of ACS-1in the key enzyme ACS gene family was decreased, while ACS-2, ACS-3were increased; the expression of ACO-1, ACO-2, ACO-3in ACO gene family were reduced while ACO-4. ACO-5, ACO-6were improved. During the synthesis of ZT, the expression of IPT-2in the key enzyme IPT gene family was decreased, while IPT-1was increased; and8’-hydroxylase, the key enzyme was increased during the degradation of ABA.4.The difference expression of ACS-2, ACO-6, the key enzyme for ABA degradation-8’-hydroxylase in rice coleoptile by fluorescence quantitative PCR under submerged stressAfter culturing nipponbares four to six days under air condition or submerged stress, the difference expression of ACS-2, ACO-6and8’-hydroxylase gene in rice coleoptiles was monitored by using real-time quantitative PCR. The resusts showed that, after culturing four days, ACS-2was over expressed857.92-fold. After culturing five days, it was over expressed873.16-fold. However, there was no over-expression but a slightly down-expression after culturing six days. After culturing four days, ACO-6was over expressed201.86-fold. After culturing five days, it was over expressed327.01-fold. However, there was no over-expression but a slightly down-expression after culturing six days. After culturing four days,8’-hydroxylase gene was over expressed59.39-fold. After culturing five days, it was over expressed1.17-fold. After culturing six days, it was over expressed1.52-fold. The rapid growth of rice coleoptiles may be involed ACS-2, ACO-6and8’-hydroxylase gene under submerged stress.5. The construction of expression vector for RNA interferenceTo ACS-2, ACO-4, ACO-6, and8’-hydroxylase gene, they were built the construction of expression vector for RNA interference through PCR and MAGIC. From the PCR gel electrophoresis and sequencing blast, the construction of expression vector for RNA interference successed.6. RNAi双元表达载体Conversion the RNAi expression vector of ACO-6and comptent agrobacterium.Through PCR and the double digestion, the construction of dual expression vector for RNA interference successed.